COMPOSITIONS COMPRISING MODIFIED PHOSPHOLIPIDS AND USES THEREOF

Abstract

Provided herein are compositions comprising compounds of Formula (I), and salts, co-crystals, tautomers, stereoisomers, solvates, hydrates, polymorphs, and isotopically enriched derivatives thereof; for example, in the form of a particle (e.g., liposome). Also provided are methods, uses, pharmaceutical compositions, and kits involving the compounds and/or compositions described herein, for methods for delivering an agent described herein (e.g., therapeutic agent, diagnostic agent), or for treating and/or preventing a disease in a subject, and methods of synthesizing these compositions.

Claims

1. A composition comprising: a compound of Formula (I): ##STR00083## or a salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, or an isotopically enriched derivative thereof, wherein: x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; y is 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18, 19,20,21,22,23, or 24; R.sup.1 is a phosphoglycerol, phosphocholine, phosphoethanolamine, phosphoinositol, or phosphoserine moiety; R.sup.2 is halogen, optionally substituted acyl, optionally substituted branched or unbranched alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, OR.sup.D1, N(R.sup.D1a).sub.2, SR.sup.D1, SO.sub.2, NO.sub.2, N.sub.3, or CN; R.sup.3 is halogen, optionally substituted acyl, optionally substituted branched or unbranched alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, OR.sup.D1, N(R.sup.D1a).sub.2, SR.sup.D1, SO.sub.2, NO.sub.2, N.sub.3, or CN; wherein R.sup.D1 is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkynyl group, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom; and wherein each occurrence of R.sup.D1a is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted linear alkynyl, optionally substituted cycloalkynyl group, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally two instances of R.sup.D1a are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring.

2. The composition of claim 1, wherein the compound is of Formula (I-A): ##STR00084## or a salt, solvate, hydrate, stereoisomer, polymorph, tautomer, isotopically enriched form, or prodrug thereof, wherein: R.sup.4 is ##STR00085## as valency permits; each occurrence of R.sup.A is independently hydrogen or unsubstituted alkyl; each occurrence of R.sup.A1 is independently hydrogen, unsubstituted alkyl, or an oxygen protecting group; and m is 1, 2, 3, 4, 5, or 6.

3. The composition of claim 1 or 2, wherein the compound is of Formula (I-A-1): ##STR00086## or a salt, solvate, hydrate, stereoisomer, polymorph, tautomer, isotopically enriched form, or prodrug thereof, wherein: R.sup.4 is ##STR00087## as valency permits; each occurrence of R.sup.A is independently hydrogen or unsubstituted alkyl; each occurrence of R.sup.A1 is independently hydrogen, unsubstituted alkyl, or an oxygen protecting group; and m is 1, 2, 3, 4, 5, or 6.

4. The composition of claim 1, wherein R.sup.1 is an unsubstituted phosphoglycerol, unsubstituted phosphocholine, unsubstituted phosphoethanolamine, unsubstituted phosphoinositol, or unsubstituted phosphoserine moiety.

5. The composition of claim any one of claims 2-4, wherein R.sup.4 is ##STR00088##

6. The composition of claim any one of claims 2-5, wherein R.sup.4 is ##STR00089##

7. The composition of any one of claims 2-6, wherein m is 2.

8. The composition of any one of claims 1-7, wherein x is 12, 13, 14, 15, or 16.

9. The composition of any one of claims 1-8, wherein x is 14.

10. The composition of any one of claims 1-9, wherein y is 15.

11. The composition of any one of claims 1-10, wherein R.sup.2 is OR.sup.D1, and R.sup.D1 is optionally substituted acyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkynyl group, or optionally substituted linear alkynyl.

12. The composition of any one of claims 1-11, wherein R.sup.3 is OR.sup.D1, and R.sup.D1 is optionally substituted acyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkynyl group, or optionally substituted linear alkynyl.

13. The composition of any one of claims 1-11, wherein R.sup.3 is methyl and R.sup.2 is OR.sup.D1 and R.sup.D1 is optionally substituted acyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkynyl group, or optionally substituted linear alkynyl.

14. The composition of any one of claims 1-13, wherein R.sup.D1 is ##STR00090## optionally substituted linear alkynyl, optionally substituted phenyl, optionally substituted naphthyl, optionally substituted anthryl, optionally substituted 2H-chromen-2-one (coumarin), optionally substituted cyclooctynyl, optionally substituted dibenzocyclooctyne, or optionally substituted aza-dibenzocyclooctyne; and R.sup.C is hydrogen, optionally substituted alkyl, or optionally substituted alkenyl.

15. The composition of any one of claims 1-14, wherein R.sup.2 is of the formula: ##STR00091##

16. The composition of any one of claims 1-10, wherein: R.sup.2 is N(R.sup.D1a).sub.2, N(R.sup.D1a)C(O)OR.sup.D1, or SR.sup.D1; R.sup.D1 is hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted cycloalkynyl group; and each instance of R.sup.D1a is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, or optionally substituted alkenyl.

17. The composition of claim 16, wherein R.sup.2 is of the formula: ##STR00092## or SH.

18. The composition of any one of claims 1-10, wherein R.sup.2 is Br or N.sub.3.

19. The composition of any one of claims 1-14, wherein R.sup.2 is of the formula: ##STR00093## SH, or N.sub.3.

20. The composition of any one of claims 1-14, wherein R.sup.2 is ##STR00094##

21. The composition of any one of claims 1-20, wherein R.sup.3 is optionally substituted linear alkyl.

22. The composition of any one of claims 1-21, wherein R.sup.3 is optionally substituted linear C.sub.1-4 alkyl.

23. The composition of any one of claims 1-22, wherein R.sup.3 is methyl.

24. The composition of any one of claims 1-20, wherein R.sup.3 is methyl or N.sub.3.

25. The composition of any one of claims 1-20, wherein R.sup.3 is of the formula: ##STR00095##

26. The composition of any one of claims 1-20, wherein R.sup.3 is Br or N.sub.3.

27. The composition of any one of claims 1-26, wherein the compound is of formula: ##STR00096## or a salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, or an isotopically enriched derivative thereof.

28. The composition of any one of claims 1-27, further comprising one or more agents.

29. The composition of claim 28, wherein at least one of the one or more agents is a therapeutic agent or diagnostic agent.

30. The composition of claim 28 or 29, wherein the therapeutic agent is an antibiotic agent, chemotherapeutic agent, anesthetic agent, anti-inflammatory agent, analgesic agent, anti-fibrotic agent, anti-sclerotic agent, or anticoagulant agent.

31. The composition of claim 29 or 30, wherein the therapeutic agent is a local anesthetic.

32. The composition of claim 31, wherein the local anesthetic is a site 1 sodium channel blocker, amino ester local anesthetic, or an amino amide local anesthetic.

33. The composition of any one of claims 29-32, wherein the therapeutic agent is doxorubicin, tetrodotoxin, saxitoxin, neosaxitoxin, bupivacaine, amylocaine, ambucaine, articaine, benzocaine, benzonatate, butacaine, butanilicaine, carbocaine, cepastat, chloraseptic, chloroprocaine, cinchocaine, citanest, cyclomethycaine, dibucaine, diperodon, dimethocaine, eucaine, etidocaine, fomocaine, fotocaine, hydroxyprocaine, isobucaine, levobupivacaine, lidocaine, marcaine, mepivacaine, meprylcaine, metabutoxycaine, nitracaine, orthocaine, orabloc, oxetacaine, oxybuprocaine, paraethoxycaine, phenacaine, piperocaine, piridocaine, polocaine, posimir, pramocaine, prilocaine, primacaine, procaine, procainamide, proparacaine, propoxycaine, pyrrocaine, quinisocaine, ropivacaine, sensorcaine, septocaine, trimecaine, tetracaine, tolycaine, tropacocaine, ulcerease, xylocaine, or zorcaine.

34. The composition of any one of claims 29-33, wherein the therapeutic agent is doxorubicin, tetrodotoxin, or bupivacaine.

35. The composition of any one of claims 28-32, wherein the agent is a small molecule therapeutic agent, a protein therapeutic agent, a nucleic acid therapeutic agent, or small molecule diagnostic agent.

36. The composition of claim 35, wherein the diagnostic agent is a fluorophore.

37. The composition of claim 35 or 36, wherein the diagnostic agent is conjugated to a protein, a polymer, or a small molecule.

38. The composition of any one of claims 33-37, wherein the diagnostic agent is Sulforhodamine B, indocyanine green, fluorescein isothiocyanate, methylene blue, or coumarin.

39. The composition of any one of claims 28-38, comprising two types of therapeutic agents, wherein the therapeutic agents are selected from the group consisting of a local anesthetic, an anti-inflammatory agent, and a sympathomimetic agent.

40. The composition of any one of claims 1-39, wherein the composition comprises dexamethasone and/or epinephrine.

41. The composition of any one of claims 1-40, wherein the composition is in the form of a particle.

42. The composition of any one of claims 1-41, wherein the particle is a liposome, lipid nanoparticle, polymer-lipid hybrid nanoparticle, lipid micelle, micelle-hydrogel hybrid, liposome-entrapped hydrogel, lipid-peptide complex, lipid-nucleic acid complex, lipid-peptide-nucleic acid complex, or lipid coated inorganic nanoparticle.

43. The composition of claim 41 or 42 wherein the composition comprises dexamethasone in the particle; and epinephrine in an injectate.

44. A pharmaceutical composition comprising a composition of any one of claims 1-43, and optionally a pharmaceutically acceptable excipient.

45. A pharmaceutical composition comprising a composition of any one of claims 1-43, a therapeutic agent, and optionally a pharmaceutically acceptable excipient.

46. A method of delivering an agent to a subject or biological sample, comprising administering to the subject or contacting the biological sample with a composition according to any one of claims 28-43, or administering to the subject or contacting the biological sample with the pharmaceutical composition of claim 44 or 45.

47. A method of treating and/or preventing a disease in a subject in need thereof, the method comprising administering to the subject a composition according to any one of claims 28-43 comprising a therapeutically effective amount of a therapeutic agent, or a pharmaceutical composition of claim 44 or 45.

48. Use of a composition delivering an agent to a subject, the use comprising administering to the subject a composition of any one of claims 28-45.

49. Use of a composition to treat and/or prevent a disease in a subject in need thereof, the use comprising administering to the subject a composition according to any one of claims 28-43 comprising a therapeutically effective amount of a therapeutic agent, or a pharmaceutical composition according to claim 44 or 45.

50. A kit for delivering an agent to a subject, comprising a composition of any one of claims 28-45, the agent, and instructions for delivering the agent to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0127] FIG. 1 shows a schematic of liposomes prepared with acyl chain-modified phospholipids.

[0128] FIG. 2 shows the representative synthesis of acyl chain-modified phospholipids.

[0129] FIG. 3 shows the .sup.1H-NMR spectrum of methyl 16-bromohexadecanoate.

[0130] FIG. 4 shows the .sup.1H-NMR spectrum of methyl 16-phenoxyhexadecanoate.

[0131] FIG. 5 shows the .sup.1H-NMR spectrum of 16-phenoxyhexadecanoic acid.

[0132] FIG. 6 shows the .sup.1H-NMR spectrum of 1-palmitoyl-2-(16-phenoxy)palmitoyl-sn-glycero-3-phosphocholine (Ph-DPPC). Peaks as a result of phenoxy-conjugation are highlighted.

[0133] FIG. 7 shows the .sup.1H-NMR spectrum of 1-palmitoyl-2-(16-coumarin)palmitoyl-sn-glycero-3-phosphocholine (CM-DPPC). Peaks as a result of coumarin-conjugation were highlighted.

[0134] FIG. 8 shows the mass spectra of the depicted acyl chain-modified phospholipids.

[0135] FIG. 9 shows the phase transition temperature of acyl chain-modified phospholipids.

[0136] FIGS. 10A-10B show dynamic light scattering and a TEM image of aromatized liposomes (Lip-Phwhich are liposomes containing the synthetic aromatized phospholipid Ph-DPPC). The structures of the Lip-Ph which forms liposomes and NMR characterization thereof are shown in FIG. 6. FIG. 10C is a graph showing the stability of the aromatized liposomes. FIG. 10D is a photograph of a solution of the aromatized liposomes. FIG. 10E is a graph showing the size of the aromatized liposomes after extrusion.

[0137] FIGS. 11A-11H show the encapsulation and cumulative release of Sulforhodamine B (SRho). Data are meansSD, n=4 independent experiments. FIG. 11A shows the chemical structure of sulforhodamine B. FIG. 11B is a graph showing SRho loading in different chemically modified liposomes. FIG. 11C is a graph showing cumulative release of SRho from different formulations at 37 C. (p-values compare groups at 168 h). FIG. 11D is a graph showing the viscosity of different liposomal formulations. FIG. 11E shows the chemical structure of DBCO-modified phospholipids (DBCO-DPPC) and mass spectra of CBO-DPPC. The DBCO group was covalently conjugated to the acyl chain of phospholipid via polar amide bonds. FIG. 11F is a graph showing loading in DBCO-modified liposomes (SRho@Lipo-DBCO). FIG. 11G is a graph showing cumulative release of SRho from SRho@Lipo-DBCO (p-values compare groups at 168 h). FIG. 11H is a graph showing cumulative release of SRho from different formulations. Ph+SRho@Lipo and ICG+SRho@Lipo are liposomes containing physically encapsulated phenol or indocyanine green (ICG). SRho@Lipo-Ph are liposomes containing covalently conjugated phenoxy group (p-values compare groups at 168 h).

[0138] FIGS. 11I-11J are graphs showing the cytotoxicity of aromatic molecules. FIG. 11I shows cell viability of C2C12 cells incubated with free aromatic molecules and aromatic molecule-loaded liposomes; and FIG. 11J shows cell viability of PC12 cells incubated with free aromatic molecules and aromatic molecule-loaded liposomes. The concentration of Phenol and ICG were 5 mM. Data are meanss.d., n=4.

[0139] FIG. 12 is a graph showing pH-dependent partition coefficients of different payloads. Octanol-water partition coefficients were quantified using a miniaturized shake-flask approach.

[0140] FIG. 13A shows the structures of tetrodotoxin, bupivacaine, and doxorubicin.

[0141] FIGS. 13B-13E show the effect of liposome aromatization on the loading of different payloads (tetrodotoxin, bupivacaine, doxorubicin, rhodamine, PEG, albumin). FIG. 13B-C are graphs showing the increase of small molecule loading as a function of molecular weight and hydrophilicity. FIG. 13D-E are graphs showing the increase of macromolecule loading as a function of molecular weight and hydrophilicity.

[0142] FIGS. 13F-13H are graphs showing the cumulative release of tetrodotoxin (TTX, FIG. 13F), bupivacaine hydrochloride (Bup, FIG. 13G), and doxorubicin hydrochloride (Dox, FIG. 13H).

[0143] FIGS. 13I-13J are graphs showing the reduction of the release of small molecular payloads as the function of molecular weight (FIG. 13I) and hydrophilicity (FIG. 13J).

[0144] FIGS. 13K-13M are graphs showing cumulative release of rhodamine-conjugated polyethylene glycol with a molecular weight of 1,000 (SRho-PEG1k, FIG. 13K), rhodamine-conjugated polyethylene glycol with a molecular weight of 10,000 (SRho-PEG10k, FIG. 13L), and albumin-fluorescein isothiocyanate conjugate (FITC-Ab, FIG. 13M).

[0145] FIGS. 13N-13O are graphs showing the reduction of drug release as the function of molecular weight (FIG. 13N) and hydrophilicity (FIG. 13O).

[0146] FIG. 13P is a graph showing cumulative TTX release from different formulations at 37 C. TTX concentrations were quantified by TTX ELISA. Data are meanss.d., n=4. p-values compare groups at 168 h.

[0147] FIGS. 14A-14H show physiochemical characterizations of lipid bilayer-engineered vesicles. FIGS. 14A and 14B show number-weighted diameters of indicated liposomes (liposome (Lip); unmodified liposome with TTX (Lip-TTX); aromatized liposomes encapsulating TTX (Lip-Ph-TTX)) measured by dynamic light scattering. FIG. 14C shows transmission electron micrograph images of liposomes. Scale bar, 600 nm. FIG. 14D shows zeta potential of liposomes. Data are mean s.d., n=4. FIG. 14E, Viscosity of liposomal formulations as a function of shear rate. FIGS. 14F and 14G show cytotoxicity of C2C12 cells (FIG. 14F) and PC12 (FIG. 14G) cells incubated with different formulations. Data are mean s.d., n=4. FIG. 13H shows loading efficiency of tetrodotoxin quantified by enzyme-linked immunosorbent assay (ELISA). Data are mean s.d., n=4.

[0148] FIGS. 15A-15B show cytotoxicity of C2C12 cells and PC12 cells incubated with different formulations (PBS, free TTX, Lip-TTX, Lip-Ph-TTX). Different formulations were directly added into the cell culture media. Data are meansSD, n=4.

[0149] FIG. 16 shows the cumulative release of free TTX as quantified by enzyme-linked immunosorbent assay. Data are meansSD, n=4 independent experiments.

[0150] FIGS. 17A-17B show the retention and localization of the indicated liposomal formulations. FIG. 17A, Representative whole-body image of rats injected with different Cy7-labeled formulations. Fluorescence intensity is represented as radiant efficiency. FIG. 17B, Representative fluorescent confocal photomicrographs 24 hours after administration of different Cy7-labeled formulations, with corresponding hematoxylin-eosin stained sections. The dotted line indicates the nerve perimeter. In FIG. 17 and throughout the Figures, Lip refers to conventional liposomes containing only natural phospholipids (DPPC). Cy7-Lip refers to conventional liposomes with covalently conjugated Cy7 dye. Cy7-Lip-Ph refers to aromatized liposomes with covalently conjugated Cy7 dye.

[0151] FIG. 18 shows the representative whole-body image of rats injected with free Cy7. Fluorescence intensity is represented as radiant efficiency.

[0152] FIG. 19 shows representative fluorescent confocal photomicrographs 0.5 hours after administration of free Cy7, with corresponding hematoxylin-eosin-stained sections. The dotted line indicates the nerve perimeter.

[0153] FIG. 20 is a graph showing quantification of the fluorescence intensity over time (as a percentage of fluorescence at time=0, immediately after injection) for Cy7-conjugated liposomes and free Cy7.

[0154] FIGS. 21A-21H show in vivo performance of TTX formulations. FIG. 19A shows schematic of tetrodotoxin (TTX)-encapsulated liposomes for prolonged duration local anesthesia following sciatic nerve injection. Upon release, TTX blocks the flow of Na+ through voltage-gated sodium channels, thereby providing nerve block. FIG. 21B shows representative time courses of sciatic nerve block following injection of different TTX formulations. FIG. 21C shows duration of sensory nerve blockade of different TTX formulations. Data are meanss.d.; n=8 for each group. FIG. 21D shows duration of sensory nerve blockade from different formulations injected at the sciatic nerve. Daggers indicate 100% mortality. Data are meanss.d. n4. p-values are from unpaired two-tailed t-test. NS, P>0.05 comparing TTX@Lipo at 24.8 g TTX and ICG+TTX@Lipo at 24.4 g; **P=0.007 comparing ICG+TTX@Lipo at 31.0 g TTX and TTX@Lipo-Ph at 32.1 g TTX. FIG. 21E shows thermal latency in the un-injected (contralateral) extremity in the first 12 hours after injection. Data are presented as mean s.d.; n=8 for each group. FIG. 21F shows frequency of block in the contralateral (uninjected) leg. FIG. 21G shows mortality from TTX formulations. FIG. 21H is a graph showing the effect of epinephrine and dexamethasone on the duration of nerve block from TTX@Lipo-Ph.

[0155] FIG. 22 shows a comparison of the durations of sensory and motor blocks of different TTX formulations. Data are meanss.d., n=4. The diagonal dotted line denotes equal durations of sensory and motor block.

[0156] FIGS. 23A-23B show the thermal latency in the un-injected (contralateral) extremity after injection. (FIG. 23A) Lip-Ph-TTX; (FIG. 21B) Lip-Ph-ICG-TTX.

[0157] FIG. 24 shows temperatures of a solution of Lip-Ph-ICG over time (minutes) with continuous irradiation of near infrared laser at different intensity.

[0158] FIG. 25 shows photo-triggered local anesthesia in the rat footpad. Following injection of 100 of Lip-Ph-ICG-TTX and subsequent irradiation (arrows, 808 nm continuous wave NIR laser at 200 mW/cm2 for 5 minutes), the effect of local anesthesia is represented as a percentage of maximum possible effect.

[0159] FIGS. 26A-26B show the cytotoxicity of formulations to C2C12 (24A) and PC12 (24B) cells. Data are meanss.d., n=4.

[0160] FIGS. 27A-27B show the tissue reaction to TTX-encapsulated liposomes, in rats injected with the indicated TTX formulations (Lip-TTX, Lip-ICG-TTX, Lip-Ph-TTX, and Lip-Ph-ICG-TTX). FIG. 27A: Representative photographs of the site of injection upon dissection 4 days after injection. FIG. 27B: Representative H&E-stained sections of nerves and surrounding tissues, and toluidine blue-stained section of nerves. The scale bar for H&E-stained sections is 100 m; for toluidine blue is 20 m.

[0161] FIG. 28 shows representative photographs of the site of injection upon dissection 14 days after injection of different liposome-TTX formulations, in the rats of FIG. 27. The green color is due to ICG.

[0162] FIG. 29 shows toluidine blue-stained sections of sciatic nerves 4 days and 14 days after injection of different TTX formulations, in the rats of FIG. 27. Scale bar: 100 m.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

[0163] The present invention provides compositions, which form into particles (e.g., liposomes, lipid nanoparticles, polymer-lipid hybrid nanoparticles, lipid coated inorganic nanoparticles), wherein the composition comprises a compound of Formula (I), or a salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, or an isotopically enriched derivative thereof, and methods of use (e.g., delivering therapeutic agents, diagnostic agents) to a subject or biological sample; methods of treating and/or preventing a disease in a subject in need thereof, the method comprising administering to the subject a composition described herein; pharmaceutical compositions comprising the compound of Formula (I), or a salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, or an isotopically enriched derivative thereof and an agent (e.g., therapeutic agents, diagnostic agents); and uses thereof.

[0164] In certain embodiments, the compound is of Formula (I):

##STR00016##

or a salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, or an isotopically enriched derivative thereof, wherein: [0165] x is 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18, 19,20,21,22,23, or 24; [0166] y is 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18, 19,20,21,22,23, or 24; [0167] R.sup.1 is a phosphoglycerol, phosphocholine, phosphoethanolamine, phosphoinositol, or phosphoserine moiety; [0168] R.sup.2 is halogen, optionally substituted acyl, optionally substituted branched or unbranched alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, OR.sup.D1, N(R.sup.D1a).sub.2, SR.sup.D1, SO.sub.2, NO.sub.2, N.sub.3, or CN; [0169] R.sup.3 is halogen, optionally substituted acyl, optionally substituted branched or unbranched alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, OR.sup.D1, N(R.sup.D1a).sub.2, SR.sup.D1, SO.sub.2, NO.sub.2, N.sub.3, or CN; [0170] wherein R.sup.D1 is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkynyl group, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom; and [0171] wherein each occurrence of R.sup.D1a is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted linear alkynyl, optionally substituted cycloalkynyl group, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally two instances of R.sup.D1a are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring.

[0172] In certain embodiments of Formula (I): [0173] x is 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18, 19,20,21,22,23, or 24; [0174] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; [0175] R.sup.1 is a phosphoglycerol, phosphocholine, phosphoethanolamine, phosphoinositol, or phosphoserine moiety; [0176] R.sup.2 is halogen, optionally substituted acyl, optionally substituted branched alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, OR.sup.D1, N(R.sup.D1a).sub.2, SR.sup.D1, SO.sub.2, NO.sub.2, N.sub.3, or CN; [0177] R.sup.3 is halogen, optionally substituted acyl, optionally substituted branched alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, OR.sup.D1, N(R.sup.D1a).sub.2, SR.sup.D1, SO.sub.2, NO.sub.2, N.sub.3, or CN; [0178] wherein R.sup.D1 is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkynyl group, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom; and [0179] wherein each occurrence of R.sup.D1a is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted linear alkynyl, optionally substituted cycloalkynyl group, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally two instances of R.sup.D1a are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring.

[0180] In certain embodiments, the compound is of Formula (I):

##STR00017##

or a salt thereof.

[0181] In certain embodiments, the compound of Formula (I) is of Formula (I-A):

##STR00018##

or a salt, solvate, hydrate, stereoisomer, polymorph, tautomer, isotopically enriched form, or prodrug thereof, wherein: [0182] R.sup.4 is

##STR00019## as valency permits; [0183] each occurrence of R.sup.A is independently hydrogen or unsubstituted alkyl; [0184] each occurrence of R.sup.A1 is independently hydrogen, unsubstituted alkyl, or an oxygen protecting group; and [0185] m is 1, 2, 3, 4, 5, or 6.

[0186] In certain embodiments, the compound of Formula (I) is of Formula (I-A):

##STR00020##

or a salt thereof.

[0187] In certain embodiments, the compound of Formula (I) is of Formula (I-A-1):

##STR00021##

or a salt, solvate, hydrate, stereoisomer, polymorph, tautomer, isotopically enriched form, or prodrug thereof, wherein: [0188] R.sup.4 is

##STR00022## as valency permits; [0189] each occurrence of R.sup.A is independently hydrogen or unsubstituted alkyl; [0190] each occurrence of R.sup.A1 is independently hydrogen, unsubstituted alkyl, or an oxygen protecting group; and [0191] m is 1, 2, 3, 4, 5, or 6.

[0192] In certain embodiments, the compound of Formula (I) is of Formula (I-A-1):

##STR00023## [0193] or a salt thereof.

[0194] In certain embodiments, the compound described herein, e.g., the compound of Formula (I) and/or (I-A), includes substituent R.sup.1. In certain embodiments, R.sup.1 is a phosphoglycerol, phosphocholine, phosphoethanolamine, phosphoinositol, or phosphoserine moiety. In certain embodiments, a phosphoglycerol moiety refers to a compound comprising phosphoglycerol. In certain embodiments, a phosphocholine moiety refers to a compound comprising phosphocholine. In certain embodiments, a phosphoethanolamine moiety refers to a compound comprising phosphoethanolamine. In certain embodiments, a phosphoinositol moiety refers to a compound comprising phosphoinositol. In certain embodiments, a phosphoserine moiety refers to a compound comprising phosphoserine. In certain embodiments, R.sup.1 is an unsubstituted phosphoglycerol, unsubstituted phosphocholine, unsubstituted phosphoethanolamine, unsubstituted phosphoinositol, or unsubstituted phosphoserine moiety. In certain embodiments, R.sup.1 is of the formula:

##STR00024##

wherein m and R.sup.4 are as defined herein. In certain embodiments, m is 1, 2, 3, 4, 5, or 6. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, m is 3. In certain embodiments, m is 4. In certain embodiments, m is 5. In certain embodiments m is 6. In certain embodiments, R.sup.1 is of the formula:

##STR00025##

wherein m is 2, 3, or 4; R.sup.4 is

##STR00026##

as valency permits, and R.sup.A and R.sup.A1 are as defined herein. In certain embodiments, the compound of Formula (I) is of Formula (I-A-1), and m is 2. In certain embodiments, R.sup.4 is

##STR00027##

as valency permits, wherein R.sup.A and R.sup.A1 are as defined herein. In certain embodiments, R.sup.4 is

##STR00028##

In certain embodiments, R.sup.4 is

##STR00029##

In certain embodiments, R.sup.4 is

##STR00030##

In certain embodiments, R.sup.4 is

##STR00031##

In certain embodiments, R.sup.4 is

##STR00032##

In certain embodiments, R.sup.4 is

##STR00033##

In certain embodiments, R.sup.4 is

##STR00034##

R.SUP.4 .is

##STR00035##

In certain embodiments, each occurrence of R.sup.A is independently hydrogen or unsubstituted alkyl. In certain embodiments, at least one instance of R.sup.A is hydrogen. In certain embodiments, at least one instance of R.sup.A is unsubstituted alkyl (e.g., Me). In certain embodiments, each occurrence of R.sup.A1 is independently hydrogen or unsubstituted alkyl. In certain embodiments, at least one instance of R.sup.A1 is hydrogen. In certain embodiments, at least one instance of R.sup.A1 is unsubstituted alkyl (e.g., Me).

[0195] The compound of Formula (I) includes substituent x. In certain embodiments, x is 1, 2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28. In certain embodiments, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In certain embodiments, x is an integer between 1-2, 2-3, 3-4, 4-5, 5-7, 7-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-24, inclusive. In certain embodiments, x is an integer between 12-14, 14-16, 16-17, 17-18, 18-19, 19-20, inclusive. In certain embodiments, x is 1. In certain embodiments, x is 2. In certain embodiments, x is 3. In certain embodiments, x is 4. In certain embodiments, x is 5. In certain embodiments x is 6. In certain embodiments, x is 7. In certain embodiments, x is 8. In certain embodiments, x is 9. In certain embodiments, x is 10. In certain embodiments, x is 11. In certain embodiments x is 12. In certain embodiments x is 12. In certain embodiments x is 13. In certain embodiments, x is 14. In certain embodiments, x is 15. In certain embodiments, x is 16. In certain embodiments, x is 17. In certain embodiments, x is 18. In certain embodiments x is 19. In certain embodiments x is 20. In certain embodiments x is 21. In certain embodiments x is 22. In certain embodiments x is 23. In certain embodiments x is 24. In certain embodiments x is 25. In certain embodiments x is 26.

[0196] The compound of Formula (I) includes substituent y. In certain embodiments, y is 1, 2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28. In certain embodiments, y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In certain embodiments, y is an integer between 1-2, 2-3, 3-4, 4-5, 5-7, 7-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-24, inclusive. In certain embodiments, y is an integer between 12-14, 14-16, 16-17, 17-18, 18-19, 19-20, inclusive. In certain embodiments, y is 1. In certain embodiments, y is 2. In certain embodiments, y is 3. In certain embodiments, y is 4. In certain embodiments, y is 5. In certain embodiments y is 6. In certain embodiments, y is 7. In certain embodiments, y is 8. In certain embodiments, y is 9. In certain embodiments, y is 10. In certain embodiments, y is 11. In certain embodiments y is 12. In certain embodiments y is 12. In certain embodiments y is 13. In certain embodiments, y is 14. In certain embodiments, y is 15. In certain embodiments, y is 16. In certain embodiments, y is 17. In certain embodiments, y is 18. In certain embodiments y is 19. In certain embodiments y is 20. In certain embodiments y is 21. In certain embodiments y is 22. In certain embodiments y is 23. In certain embodiments y is 24. In certain embodiments y is 25. In certain embodiments y is 26. In certain embodiments, x and y are the same. In certain embodiments, x and y are different. In certain embodiments, x and y are both 12, 13, 14, 15, or 16. In certain embodiments, x and y are both an integer between 12-14, 14-16, or 16-18, inclusive. In certain embodiments, x is 12, 13, 14, 15, or 16. In certain embodiments, y is 12, 13, 14, 15, or 16. In certain embodiments, x is an integer between 12-14, 14-16, or 16-18, inclusive. In certain embodiments, y is an integer between 12-14, 14-16, or 16-18, inclusive. In certain embodiments, x is 14. In certain embodiments, x is 15. In certain embodiments, y is 15. In certain embodiments, x is 14, and y is 15.

[0197] In certain embodiments, the compound described herein, e.g., the compound of Formula (I) and/or (I-A), includes substituents R.sup.2 and R.sup.3. In certain embodiments, R.sup.2 and R.sup.3 are the same. In certain embodiments, each of R.sup.2 and R.sup.3 is halogen. In certain embodiments, each of R.sup.2 and R.sup.3 is OR.sup.D1, and R.sup.D1 is optionally substituted acyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkynyl group, or optionally substituted linear alkynyl. In certain embodiments, each of R.sup.2 and R.sup.3 is Br or N.sub.3. In certain embodiments, each of R.sup.2 and R.sup.3 is OPh. In certain embodiments, each of R.sup.2 and R.sup.3 is N.sub.3. In certain embodiments, each of R.sup.2 and R.sup.3 is of the formula:

##STR00036##

In certain embodiments, R.sup.2 and R.sup.3 are different. In certain embodiments, R.sup.3 is linear alkyl (e.g., methyl) and R.sup.2 is OR.sup.D1, and R.sup.D1 is optionally substituted acyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkynyl group, or optionally substituted linear alkynyl. In certain embodiments, R.sup.3 is linear C.sub.1-6 alkyl (e.g., methyl) and R.sup.2 is OR.sup.D1, and R.sup.D1 is optionally substituted acyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkynyl group, or optionally substituted linear alkynyl.

[0198] In certain embodiments, R.sup.2 is optionally substituted acyl, halogen, optionally substituted linear alkyl, optionally substituted carbocyclyl, optionally substituted branched alkyl, optionally substituted heteroaryl, optionally substituted aryl, OR.sup.D, N(R.sup.D1a).sub.2, SR.sup.D1, SO.sub.2, NO.sub.2, N.sub.3, or CN. In certain embodiments, R.sup.2 is not optionally substituted linear alkyl or optionally substituted branched alkyl. In certain embodiments, R.sup.2 and/or R.sup.3 are each optionally substituted linear alkyl, only when the composition further comprises (e.g., physically encapsulates) an agent, for example, a therapeutic agent (e.g., small molecule therapeutic agent, a protein therapeutic agent, a nucleic acid therapeutic agent; diagnostic agent (e.g., fluorophore, for example, organic dyes (e.g., fluorescein, rhodamine, AMCA), or biological fluorophores (e.g., green fluorescent protein, phycoerythrin, allophycocyanin; Sulforhodamine B, indocyanine green, methylene blue, or coumarin). In certain embodiments, R.sup.2 and R.sup.3 are both optionally substituted linear alkyl, only when the composition further comprises (e.g., physically encapsulates) an agent, for example, a therapeutic agent (e.g., small molecule therapeutic agent, a protein therapeutic agent, a nucleic acid therapeutic agent; diagnostic agent (e.g., fluorophore, for example, organic dyes (e.g., fluorescein, rhodamine, AMCA), or biological fluorophores (e.g., green fluorescent protein, phycoerythrin, allophycocyanin; Sulforhodamine B, indocyanine green, methylene blue, or coumarin).

[0199] In certain embodiments, R.sup.2 is halogen, optionally substituted acyl, optionally substituted branched alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, OR.sup.D1, N(R.sup.D1a).sub.2, SR.sup.D1, SO.sub.2, NO.sub.2, N.sub.3, or CN. In certain embodiments, R.sup.2 is halogen (e.g., F, Cl, Br, or I). In certain embodiments, R.sup.2 is Br. In certain embodiments, R.sup.2 is optionally substituted acyl (e.g., C(O)Me). In certain embodiments, R.sup.2 is optionally substituted branched alkyl (e.g., substituted or unsubstituted branched C.sub.3-6 alkyl, for example, isopropyl, t-butyl, sec-butyl, iso-butyl). In certain embodiments, R.sup.2 is optionally substituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 10-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In certain embodiments, R.sup.2 is optionally substituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In certain embodiments, R.sup.2 is optionally substituted aryl (e.g., substituted or unsubstituted, 6- to 10-membered aryl). In certain embodiments, R.sup.2 is benzyl. In certain embodiments, R.sup.2 is substituted or unsubstituted phenyl. In certain embodiments, R.sup.2 is optionally substituted heteroaryl (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, R.sup.2 is OR.sup.D1 (e.g., OH or OMe). In certain embodiments, R.sup.2 is OH. In certain embodiments, R.sup.2 is OR.sup.D1, and R.sup.D1 is optionally substituted acyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkynyl group, or optionally substituted linear alkynyl. In certain embodiments, R.sup.2 is OR.sup.D1, and R.sup.D1 is

##STR00037##

optionally substituted linear alkynyl, optionally substituted phenyl, optionally substituted naphthyl, optionally substituted anthryl, optionally substituted 2H-chromen-2-one (coumarin), optionally substituted cyclooctynyl, optionally substituted dibenzocyclooctyne, or optionally substituted aza-dibenzocyclooctyne; and R.sup.C is hydrogen, optionally substituted alkyl, or optionally substituted alkenyl. In certain embodiments, R.sup.C is hydrogen. In certain embodiments, R.sup.C is optionally substituted alkyl (e.g., substituted or unsubstituted C.sub.1-6 alkyl). In certain embodiments, R.sup.C is optionally substituted alkenyl (e.g., substituted or unsubstituted C.sub.2-6 alkenyl). In certain embodiments, R.sup.2 is OR.sup.D1, and R.sup.D1 is a bulky (e.g., molecular weight between 90-300 g/mol), and/or hydrophobic moiety (e.g., optionally substituted C.sub.1-12 alkyl, optionally substituted C.sub.12-24 alkyl, optionally substituted C.sub.2-12 alkenyl, optionally substituted C.sub.12-24 alkenyl, optionally substituted linear or cyclic C.sub.2-12 alkynyl, optionally substituted linear or cyclic C.sub.12-24 alkynyl, C.sub.3-14 carbocyclyl). In certain embodiments, R.sup.2 is of the formula:

##STR00038##

In certain embodiments, R.sup.2 is of the formula:

##STR00039##

In certain embodiments, R.sup.2 is

##STR00040##

In certain embodiments, R.sup.2 is

##STR00041##

In certain embodiments, R.sup.2 is

##STR00042##

In certain embodiments, R.sup.2 is

##STR00043##

In certain embodiments, R.sup.2 is

##STR00044##

In certain embodiments, R.sup.2 is

##STR00045##

In certain embodiments, R.sup.2 is

##STR00046##

In certain embodiments, R.sup.2 is

##STR00047##

[0200] In certain embodiments, R.sup.2 is N(R.sup.D1a).sub.2 (e.g., NMe.sub.2). In certain embodiments, R.sup.2 is of the formula:

##STR00048##

or SH. In certain embodiments, R.sup.2 is of the formula:

##STR00049##

In certain embodiments, R.sup.2 is of the formula:

##STR00050##

In certain embodiments, R.sup.2 is of the formula:

##STR00051##

In certain embodiments, R.sup.2 is NH.sub.2. In certain embodiments, R.sup.2 is SR.sup.D1 (e.g., SMe). In certain embodiments, R.sup.2 is SH. In certain embodiments, R.sup.2 is SO.sub.2. In certain embodiments, R.sup.2 is NO.sub.2. In certain embodiments, R.sup.2 is N.sub.3. In certain embodiments, R.sup.2 is CN. In certain embodiments, R.sup.2 is Br or N.sub.3. In certain embodiments, R.sup.2 is of the formula:

##STR00052##

or N.sub.3. In certain embodiments, R.sup.2 is

##STR00053##

[0201] In certain embodiments, at least one instance of R.sup.2 or R.sup.3 is OR.sup.D1, N(R.sup.D1a).sub.2 or SR.sup.D1, and R.sup.D1 and R.sup.D1a are as defined herein. In certain embodiments, at least one instance of R.sup.2 or R.sup.3 is N(R.sup.D1a).sub.2, N(R.sup.D1a)C(O)OR.sup.D1, or SR.sup.D1; and R.sup.D1 is hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted cycloalkynyl group; and each instance of R.sup.D1a is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, or optionally substituted alkenyl. In certain embodiments, R.sup.2 is N(R.sup.D1a).sub.2, N(R.sup.D1a)C(O)OR.sup.D1, or SR.sup.D1; and R.sup.D1 is hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted cycloalkynyl group; and each instance of R.sup.D1a is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, or optionally substituted alkenyl. In certain embodiments, R.sup.D1 is hydrogen. In certain embodiments, R.sup.D1 is optionally substituted acyl (e.g., C(O)Me). In certain embodiments, R.sup.D1 is

##STR00054##

and R.sup.C is hydrogen, optionally substituted alkyl, or optionally substituted alkenyl. In certain embodiments, R.sup.D1 is

##STR00055##

In certain embodiments, R.sup.C is hydrogen. In certain embodiments, R.sup.C is optionally substituted alkyl (e.g., substituted or unsubstituted C.sub.1-6 alkyl). In certain embodiments, R.sup.C is optionally substituted alkenyl (e.g., substituted or unsubstituted C.sub.2-6 alkenyl).

[0202] In certain embodiments, R.sup.D1 is optionally substituted alkyl (e.g., substituted or unsubstituted C.sub.1-6 alkyl). In certain embodiments, R.sup.D1 is substituted or unsubstituted methyl. In certain embodiments, R.sup.D1 is substituted or unsubstituted ethyl. In certain embodiments, R.sup.D1 is substituted or unsubstituted propyl. In certain embodiments, R.sup.D1 is optionally substituted alkenyl (e.g., substituted or unsubstituted C.sub.2-6 alkenyl). In certain embodiments, R.sup.D1 is optionally substituted alkynyl (e.g., substituted or unsubstituted C.sub.2-6 alkynyl). In certain embodiments, R.sup.D1 is optionally substituted linear alkynyl. In certain embodiments, R.sup.D1 is

##STR00056##

In certain embodiments, R.sup.D1 is optionally substituted cyclic alkynyl or optionally substituted cycloalkynyl (e.g., a carbocyclyl group with at least one alkynyl bond). In certain embodiments, R.sup.D1 is optionally substituted cyclooctyne. In certain embodiments, R.sup.D1 is optionally substituted 2H-chromen-2-one (coumarin), optionally substituted cyclooctynyl, optionally substituted dibenzocyclooctyne, or optionally substituted aza-dibenzocyclooctyne. In certain embodiments, R.sup.D1 is

##STR00057##

In certain embodiments, R.sup.D1 is optionally substituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In certain embodiments, R.sup.D1 is optionally substituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In certain embodiments, R.sup.D1 is optionally substituted aryl (e.g., substituted or unsubstituted, 6- to 10-membered aryl). In certain embodiments, R.sup.D1 is benzyl. In certain embodiments, R.sup.D1 is substituted or unsubstituted phenyl. In certain embodiments, R.sup.D1 is phenyl. In certain embodiments, R.sup.D1 is optionally substituted phenyl, optionally substituted naphthyl, optionally substituted anthryl. In certain embodiments, R.sup.D1 is optionally substituted heteroaryl (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, R.sup.D1 is an oxygen protecting group when attached to an oxygen atom (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, benzoyl). In certain embodiments, R.sup.D1 is a sulfur protecting group when attached to a sulfur atom.

[0203] In certain embodiments, at least one instance of R.sup.D1a is hydrogen. In certain embodiments, at least one instance of R.sup.D1a is optionally substituted acyl (e.g., C(O)Me). In certain embodiments, at least one R.sup.D1a is optionally substituted alkyl (e.g., substituted or unsubstituted C.sub.1-6 alkyl). In certain embodiments, at least one instance of R.sup.D1a is substituted or unsubstituted methyl. In certain embodiments, at least one instance of R.sup.D1a is substituted or unsubstituted ethyl. In certain embodiments, at least one instance of R.sup.D1a is substituted or unsubstituted propyl. In certain embodiments, at least one instance of R.sup.D1a is optionally substituted alkenyl (e.g., substituted or unsubstituted C.sub.2-6 alkenyl). In certain embodiments, at least one instance of R.sup.D1a is optionally substituted alkynyl (e.g., substituted or unsubstituted C.sub.2-6 alkynyl). In certain embodiments, at least one instance of R.sup.D1a is optionally substituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In certain embodiments, at least one instance of R.sup.D1a is optionally substituted cyclic alkynyl or optionally substituted cycloalkynyl (e.g., a carbocyclyl group with at least one alkynyl bond). In certain embodiments, at least one instance of R.sup.D1a is optionally substituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of R.sup.D1a is optionally substituted aryl (e.g., substituted or unsubstituted, 6- to 10-membered aryl). In certain embodiments, at least one instance of R.sup.D1a is benzyl. In certain embodiments, at least one instance of R.sup.D1a is substituted or unsubstituted phenyl. In certain embodiments, at least one instance of R.sup.D1a is optionally substituted heteroaryl (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of R.sup.D1a is a nitrogen protecting group (e.g., benzyl (Bn), t-butyl carbonate (BOC or Boc), benzyl carbamate (Cbz), 9-fluorenylmethyl carbonate (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl, or p-toluenesulfonamide (Ts)). In certain embodiments, two instances of R.sup.D1a are taken together with their intervening atoms to form a optionally substituted heterocyclic ring (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur) or optionally substituted heteroaryl ring (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, one instance of R.sup.D1a is

##STR00058##

In certain embodiments, one instance of R.sup.D1a is hydrogen and one instance of R.sup.D1a is

##STR00059##

[0204] In certain embodiments, R.sup.3 is optionally substituted acyl, halogen, optionally substituted linear alkyl, optionally substituted carbocyclyl, optionally substituted branched alkyl, optionally substituted heteroaryl, optionally substituted aryl, OR.sup.D, N(R.sup.D1a).sub.2, SR.sup.D1, SO.sub.2, NO.sub.2, N.sub.3, or CN. In certain embodiments, R.sup.3 is halogen, optionally substituted acyl, optionally substituted branched alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, OR.sup.D1, N(R.sup.D1a).sub.2SR.sup.D1, SO.sub.2, NO.sub.2, N.sub.3, or CN. In certain embodiments, R.sup.3 is halogen, optionally substituted acyl, optionally substituted linear alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, OR.sup.D1, N(R.sup.D1a).sub.2, SR.sup.D1, SO.sub.2, NO.sub.2, N.sub.3, or CN. In certain embodiments, R.sup.3 is optionally substituted linear alkyl. In certain embodiments, R.sup.3 is optionally substituted C.sub.1-4 linear alkyl. In certain embodiments, R.sup.3 is methyl. In certain embodiments, x is 15 and R.sup.3 is methyl. In certain embodiments, R.sup.3 is halogen (e.g., F, Cl, Br, or I). In certain embodiments, R.sup.3 is Br. In certain embodiments, R.sup.3 is optionally substituted acyl (e.g., C(O)Me). In certain embodiments, R.sup.3 is optionally substituted branched alkyl (e.g., substituted or unsubstituted branched C.sub.3-6 alkyl, for example, isopropyl, t-butyl, sec-butyl, iso-butyl). In certain embodiments, R.sup.3 is optionally substituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 10-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In certain embodiments, R.sup.3 is optionally substituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In certain embodiments, R.sup.3 is optionally substituted aryl (e.g., substituted or unsubstituted, 6- to 10-membered aryl). In certain embodiments, R.sup.3 is benzyl. In certain embodiments, R.sup.3 is substituted or unsubstituted phenyl. In certain embodiments, R.sup.3 is optionally substituted heteroaryl (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, R.sup.3 is OR.sup.D1 (e.g., OH or OMe). In certain embodiments, R.sup.3 is OH. In certain embodiments, R.sup.2 is OR.sup.D1, and R.sup.D1 is optionally substituted acyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkynyl group, or optionally substituted linear alkynyl. In certain embodiments, R.sup.3 is OR.sup.D1, and R.sup.D1 is

##STR00060##

optionally substituted linear alkynyl, optionally substituted phenyl, optionally substituted naphthyl, optionally substituted anthryl, optionally substituted 2H-chromen-2-one (coumarin), optionally substituted cyclooctynyl, optionally substituted dibenzocyclooctyne, or optionally substituted aza-dibenzocyclooctyne; and R.sup.C is hydrogen, optionally substituted alkyl, or optionally substituted alkenyl. In certain embodiments, R.sup.C is hydrogen. In certain embodiments, R.sup.C is optionally substituted alkyl (e.g., substituted or unsubstituted C.sub.1-6 alkyl). In certain embodiments, R.sup.C is optionally substituted alkenyl (e.g., substituted or unsubstituted C.sub.2-6 alkenyl). In certain embodiments, R.sup.3 is of the formula:

##STR00061##

In certain embodiments, R.sup.3 is

##STR00062##

In certain embodiments, R.sup.3 is

##STR00063##

In certain embodiments, R is

##STR00064##

In certain embodiments, R.sup.3 is

##STR00065##

In certain embodiments, R.sup.3 is

##STR00066##

In certain embodiments, R.sup.3 is

##STR00067##

In certain embodiments, R.sup.3 is

##STR00068##

In certain embodiments, R.sup.3 is

##STR00069##

[0205] In certain embodiments, R.sup.3 is N(R.sup.D1a).sub.2 (e.g., NMe.sub.2). In certain embodiments, R.sup.3 is of the formula:

##STR00070##

or SH. In certain embodiments, R.sup.3 is of the formula:

##STR00071##

or SH. In certain embodiments, R.sup.3 is of the formula:

##STR00072##

In certain embodiments, R.sup.3 is of the formula:

##STR00073##

In certain embodiments, R.sup.3 is of the formula:

##STR00074##

In certain embodiments, R.sup.3 is NH.sub.2. In certain embodiments, R.sup.3 is SR.sup.D1 (e.g., SMe). In certain embodiments, R.sup.3 is SO.sub.2. In certain embodiments, R.sup.3 is NO.sub.2. In certain embodiments, R.sup.3 is N.sub.3. In certain embodiments, R.sup.3 is CN. In certain embodiments, R.sup.3 is Br or N.sub.3. In certain embodiments, R.sup.3 is methyl or N.sub.3. In certain embodiments, R.sup.3 is N(R.sup.D1a).sub.2, N(R.sup.D1a)C(O)OR.sup.D1, or SR.sup.D1; and R.sup.D1 is hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted cycloalkynyl group; and each instance of R.sup.D1a is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, or optionally substituted alkenyl.

[0206] In certain embodiments, the compound of Formula (I) is of formula:

##STR00075## [0207] or a salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, or an isotopically enriched derivative thereof. In certain embodiments, the compound of Formula (I) is of formula:

##STR00076## ##STR00077##

or a salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, or an isotopically enriched derivative thereof.

[0208] In certain embodiments, the compound of Formula (I) is of formula:

##STR00078##

or a salt thereof. In certain embodiments, the compound of Formula (I) is of formula:

##STR00079## ##STR00080##

or a salt thereof.

[0209] In another aspect, provided is a composition in the form of a particle (e.g., liposome), wherein a compound comprising an aromatic group (e.g., fluorophore, or dye, such as indocyanine green) is physically, non-covalently incorporated by the particle, which also comprises a local anesthetic agent (e.g., TTX). In certain embodiments, provided is a liposome comprising indocyanine green physically, non-covalently incorporated by the liposome, which also comprises TTX. In certain embodiments, provided is the liposome of Lip-ICG-TTX (as shown in FIG. 21 and described in Example 1).

[0210] In certain embodiments, the composition described herein further comprises one or more agents, for example, an agent (e.g., therapeutic agent, diagnostic agent). In certain embodiments, at least one of the one or more agents is a therapeutic agent or diagnostic agent. In certain embodiments, the therapeutic agent is an antibiotic agent, chemotherapeutic agent, anesthetic agent, anti-inflammatory agent, analgesic agent, anti-fibrotic agent, anti-sclerotic agent, or anticoagulant agent. Therapeutic agents, in certain embodiments, may include, but are not limited to, antimicrobial agents, antibiotics, anesthetics, anti-inflammatories, chemotherapeutic agents, analgesics, anti-fibrotics, anti-sclerotics, and anticoagulants. Therapeutic agents may include, but are not limited to, antibiotics, anesthetics, anti-inflammatories, analgesics, anti-fibrotics, anti-sclerotics, and anticoagulants. In certain embodiments, the therapeutic agent is an antimicrobial agent. In certain embodiments, the therapeutic agent is an antibiotic agent. In certain embodiments, the therapeutic agent is a chemotherapeutic agent. In certain embodiments, the therapeutic agent is an anesthetic agent. In certain embodiments, the therapeutic agent is an anti-inflammatory agent. In certain embodiments, the therapeutic agent is an analgesic agent. In certain embodiments, the therapeutic agent is an anti-fibrotic agent. In certain embodiments, the therapeutic agent is an anti-sclerotic agent. In certain embodiments, the therapeutic agent is an anticoagulant agent. In certain embodiments, the therapeutic agent is present as a pharmaceutically acceptable salt or a free base of the active agent. In certain embodiments, the therapeutic agent is present as a pharmaceutically acceptable salt of the active agent.

[0211] In certain embodiments, the therapeutic agent is an antimicrobial agent. In certain embodiments, the therapeutic agent is an antibiotic. Any antibiotic may be used in the system. In certain embodiments the antibiotic is approved for use in humans or other animals. In certain embodiments the antibiotic is approved for use by the U.S. Food & Drug Administration. In certain embodiments, the antibiotic may be selected from the group consisting of cephalosporins, quinolones, polypeptides, macrolides, penicillins, and sulfonamides. Exemplary antibiotics may include, but are not limited to, ciprofloxacin, cefuroxime, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftobiprole, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, bacitracin, colistin, polymyxin B, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, meticillin, nafcillin, oxacillin, penicillin, piperacillin, ticarcillin, mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole, trimethoprim, and trimethoprim-sulfamethoxazole. In certain embodiments, the therapeutic agent is doxorubicin.

[0212] In certain embodiments, the therapeutic agent is an antibiotic agent, anesthetic agent, anti-inflammatory agent, analgesic agent, anti-fibrotic agent, anti-sclerotic agent, anticoagulant agent, or diagnostic agent. In certain embodiments, the antibiotic is a quinolone, for example, a fluoroquinolone. In certain embodiments, the antibiotic is a carbapenem. In certain embodiments, the antibiotic is a quinolone (e.g., fluoroquinolone) or a beta lactam antibiotic (e.g., penicillin, cephalosporin (e.g., ceftriaxone)). In certain embodiments, the antibiotic is amoxicillin, azithromicicn, cefuroxime, ceftriaxone, trimethoprim, levofloxacin, moxifloxacin, meropenem, or ciprofloxacin. In some embodiments, the antibiotic is ciprofloxacin. In some embodiments, the antibiotic is ciprofloxacin and pharmaceutically acceptable salts thereof. In some embodiments, the antibiotic is ciprofloxacin hydrochloride. In some embodiments, the antibiotic is levofloxacin. In some embodiments, the antibiotic is ceftriaxone. In some embodiments, the antibiotic is ciprofloxacin, gatifloxacin, ceftriaxone, gemifloxacin, moxalactam, levofloxacin, meropenem, or ampicillin; or pharmaceutically acceptable salts thereof. In some embodiments, the antibiotic is ciprofloxacin, gatifloxacin, ceftriaxone, gemifloxacin, moxalactam, levofloxacin, meropenem, or ampicillin. In some embodiments, the antibiotic (e.g., fluoroquinolone or a beta lactam antibiotic, for example, ciprofloxacin, ceftriaxone) is formulated in the composition from a powder form of the antibiotic. In some embodiments, the antibiotic (e.g., fluoroquinolone or a beta lactam antibiotic, for example, ciprofloxacin, ceftriaxone) is formulated in the composition from a liquid form of the antibiotic.

[0213] Exemplary antibiotics, include, but are not limited to: Abamectin, Actinomycin (e.g., Actinomycin A, Actinomycin C, Actinomycin D, Aurantin), Alatrofloxacin mesylate, Amikacin sulfate, Aminosalicylic acid, Anthracyclines (e.g., Aclarubicin, Adriamycin, Doxorubicin, Epirubicin, Idarubicin), Antimycin (e.g., Antimycin A), Avermectin, BAL 30072, Bacitracin, Bleomycin, Cephalosporins (e.g., 7-Aminocephalosporanic acid, 7-Aminodeacetoxycephalosporanic acid, Cefaclor, Cefadroxil, Cefamandole, Cefazolin, Cefepime, Cefixime, Cefmenoxime, Cefmetazole, Cefoperazone, Cefotaxime, Cefotetan, Cefotiam, Cefoxitin, Cefpirome, Cefpodoxime proxetil, Cefsulodin, Cefsulodin sodium, Ceftazidime, Ceftizoxime, Ceftriaxone, Cefuroxime, Cephalexin, Cephaloridine, Cephalosporin C, Cephalothin, Cephalothin sodium, Cephapirin, Cephradine), Ciprofloxacin, Enrofloxacin, Clarithromycin, Clavulanic acid, Clindamycin, Colicin, Cyclosporin (e.g. Cyclosporin A), Dalfopristin/quinupristin, Daunorubicin, Doxorubicin, Epirubicin, GSK 1322322, Geneticin, Gentamicin, Gentamicin sulfate, Gramicidin (e.g. Gramicidin A), Grepafloxacin hydrochloride, Ivermectin, Kanamycin (e.g. Kanamycin A), Lasalocid, Leucomycin, Levofloxacin, Linezolid, Lomefloxacin, Lovastatin, MK 7655, Meropenem, Mevastatin, Mithramycin, Mitomycin, Monomycin, Natamycin, Neocarzinostatin, Neomycin (e.g. Neomycin sulfate), Nystatin, Oligomycin, Olivomycin, Pefloxacin, Penicillin (e.g. 6-Aminopenicillanic acid, Amoxicillin, Amoxicillin-clavulanic acid, Ampicillin, Ampicillin sodium, Azlocillin, Carbenicillin, Cefoxitin, Cephaloridine, Cloxacillin, Dicloxacillin, Mecillinam, Methicillin, Mezlocillin, Nafcillin, Oxacillin, Penicillin G, Penicillin G potassium, Penicillin G procaine, Penicillin G sodium, Penicillin V, Piperacillin, Piperacillin-tazobactam, Sulbactam, Tazobactam, Ticarcillin), Phleomycin, Polymyxin (e.g., Colistin, Polymyxin B), Pyocin (e.g. Pyocin R), RPX 7009, Rapamycin, Ristocetin, Salinomycin, Sparfloxacin, Spectinomycin, Spiramycin, Streptogramin, Streptovaricin, Tedizolid phosphate, Teicoplanin, Telithromycin, Tetracyclines (e.g. Achromycin V, Demeclocycline, Doxycycline, Doxycycline monohydrate, Minocycline, Oxytetracycline, Oxytetracycline hydrochloride Tetracycline, Tetracycline hydrochloride), Trichostatin A, Trovafloxacin, Tunicamycin, Tyrocidine, Valinomycin, ()-Florfenicol, Acetylsulfisoxazole, Actinonin, Amikacin sulfate, Benzethonium chloride, Cetrimide, Chelerythrine, Chlorhexidine (e.g., Chlorhexidine gluconate), Chlorhexidine acetate, Chlorhexidine gluconate, Chlorothalonil, Co-Trimoxazole, Dichlorophene, Didecyldimethylammonium chloride, Dihydrostreptomycin, Enoxacin, Ethambutol, Fleroxacin, Furazolidone, Methylisothiazolinone, Monolaurin, Oxolinic acid, Povidone-iodine, Spirocheticides (e.g., Arsphenamine, Neoarsphenamine), Sulfaquinoxaline, Thiamphenicol, Tinidazole, Triclosan, Trovafloxacin, Tuberculostatics (e.g., 4-Aminosalicylic acid, AZD 5847, Aminosalicylic acid, Ethionamide), Vidarabine, Zinc pyrithione, and Zirconium phosphate.

[0214] In certain embodiments, the therapeutic agent is a Food and Drug Administration (FDA) approved drug for treating infections or infectious diseases. Exemplary FDA approved agents include, but are not limited to: Avycaz (ceftazidime-avibactam), Cresemba (isavuconazonium sulfate), Evotaz (atazanavir and cobicistat, Prezcobix (darunavir and cobicistat), Dalvance (dalbavancin), Harvoni (ledipasvir and sofosbuvir), Impavido (miltefosine), Jublia (efinaconazole), Kerydin (tavaborole), Metronidazole, Orbactiv (oritavancin), Rapivab (peramivir injection), Sivextro (tedizolid phosphate), Triumeq (abacavir, dolutegravir, and lamivudine), Viekira Pak (ombitasvir, paritaprevir, ritonavir and dasabuvir), Xtoro (finafloxacin), Zerbaxa (ceftolozane+tazobactam), Luzu (luliconazole), Olysio (simeprevir), Sitavig (acyclovir), Sovaldi (sofosbuvir), Abthrax (raxibacumab), Afinitor (everolimus), Cystaran (cysteamine hydrochloride), Dymista (azelastine hydrochloride and fluticasone propionate), Fulyzaq (crofelemer), Jetrea (ocriplasmin), Linzess (linaclotide), Qnasl (beclomethasone dipropionate) nasal aerosol, Sirturo (bedaquiline), Sklice (ivermectin), Stribild (elvitegravir, cobicistat, emtricitabine, tenofovir disoproxil fumarate), Tudorza Pressair (aclidinium bromide inhalation powder), Complera (emtricitabine/rilpivirine/tenofovir disoproxil fumarate), Dificid (fidaxomicin), Edurant (rilpivirine), Eylea (aflibercept), Firazyr (icatibant), Gralise (gabapentin), Incivek (telaprevir), Victrelis (boceprevir), Egrifta (tesamorelin), Teflaro (ceftaroline fosamil), Zymaxid (gatifloxacin), Bepreve (bepotastine besilate), Vibativ (telavancin), Aptivus (tipranavir), Astepro (azelastine hydrochloride nasal spray), Intelence (etravirine), Patanase (olopatadine hydrochloride), Viread (tenofovir disoproxil fumarate), Isentress (raltegravir), Selzentry (maraviroc), Veramyst (fluticasone furoate), Xyzal (levocetirizine dihydrochloride), Eraxis (anidulafungin), Noxafil (posaconazole), Prezista (darunavir), Tyzeka (telbivudine), Veregen (kunecatechins), Baraclude (entecavir), Fuzeon (enfuvirtide), Lexiva (fosamprenavir calcium), Reyataz (atazanavir sulfate), Clarinex, Hepsera (adefovir dipivoxil), Pegasys (peginterferon alfa-2a), Sustiva, Vfend (voriconazole), Zelnorm (tegaserod maleate), Avelox (moxifloxacin hydrochloride), Cancidas, Invanz, Peg-Intron (peginterferon alfa-2b), Rebetol (ribavirin), Spectracef, Tavist (clemastine fumarate), Twinrix, Valcyte (valganciclovir HCl), Xigris (drotrecogin alfa), ABREVA (docosanol), Cefazolin, Kaletra, Lamisil (terbinafine hydrochloride), Lotrisone (clotrimazole/betamethasone diproprionate), Lotronex (alosetron HCL), Trizivir (abacavir sulfate, lamivudine, zidovudine AZT), Synercid, Synagis, Viroptic, Aldara (imiquimod), Bactroban, Ceftin (cefuroxime axetil), Combivir, Condylox (pokofilox), Famvir (famciclovir), Floxin, Fortovase, INFERGEN (interferon alfacon-1), Intron A (interferon alfa-2b, recombinant), Mentax (butenafine HCl), Norvir (ritonavir), Omnicef, Rescriptor (delavirdine mesylate), Taxol, Timentin, Trovan, VIRACEPT (nelfinavir mesylate), Zerit (stavudine), AK-Con-A (naphazoline ophthalmic), Allegra (fexofenadine hydrochloride), Astelin nasal spray, Atrovent (ipratropium bromide), Augmentin (amoxicillin/clavulanate), Crixivan (Indinavir sulfate), Elmiron (pentosan polysulfate sodium), Havrix, Leukine (sargramostim), Merrem (meropenem), Nasacort AQ (triamcinolone acetonide), Tavist (clemastine fumarate), Vancenase AQ, Videx (didanosine), Viramune (nevirapine), Zithromax (azithromycin), Cedax (ceftibuten), Clarithromycin (Biaxin), Epivir (lamivudine), Invirase (saquinavir), Valtrex (valacyclovir HCl), Zyrtec (cetirizine HCl), Acyclovir, Penicillin (penicillin g potassium), Cubicin (Daptomycin), Factive (Gemifloxacin), Albenza (albendazole), Alinia (nitazoxanide), Altabax (retapamulin), AzaSite (azithromycin), Besivance (besifloxacin ophthalmic suspension), Biaxin XL (clarithromycin extended-release), Cayston (aztreonam), Cleocin (clindamycin phosphate), Doribax (doripenem), Dynabac, Flagyl ER, Ketek (telithromycin), Moxatag (amoxicillin), Rapamune (sirolimus), Restasis (cyclosporine), Tindamax (tinidazole), Tygacil (tigecycline), and Xifaxan (rifaximin). In certain embodiments, the antibiotic agent is selected from the group consisting of ciprofloxacin, cefuroxime, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftobiprole, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, bacitracin, colistin, polymyxin B, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, meticillin, nafcillin, oxacillin, penicillin, piperacillin, ticarcillin, mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole, trimethoprim, and trimethoprim-sulfamethoxazole. In certain embodiments, the antibiotic agent is ciprofloxacin, gatifloxacin, ceftriaxone, gemifloxacin, moxalactam, levofloxacin, meropenem, or ampicillin. In certain embodiments, the antibiotic agent is ciprofloxacin or ceftriaxone. In certain embodiments, the antibiotic agent is ciprofloxacin. In certain embodiments, the antibiotic agent is ceftriaxone.

[0215] In certain embodiments, the therapeutic agent is a local anesthetic (e.g., tetrodotoxin, saxitoxin, neosaxitoxin, bupivacaine, amylocaine, ambucaine, articaine, benzocaine, benzonatate, butacaine, butanilicaine, carbocaine, cepastat, chloraseptic, chloroprocaine, cinchocaine, citanest, cyclomethycaine, dibucaine, diperodon, dimethocaine, eucaine, etidocaine, fomocaine, fotocaine, hydroxyprocaine, isobucaine, levobupivacaine, lidocaine, marcaine, mepivacaine, meprylcaine, metabutoxycaine, nitracaine, orthocaine, orabloc, oxetacaine, oxybuprocaine, paraethoxycaine, phenacaine, piperocaine, piridocaine, polocaine, posimir, pramocaine, prilocaine, primacaine, procaine, procainamide, proparacaine, propoxycaine, pyrrocaine, quinisocaine, ropivacaine, sensorcaine, septocaine, trimecaine, tetracaine, tolycaine, tropacocaine, ulcerease, xylocaine, or zorcaine). In certain embodiments, the therapeutic agent is a local anesthetic compound, for example, but not limited to, an amino ester compound (e.g., procaine, tetracaine, chloroprocaine, benzocaine, butacaine, dimethocaine) or an amino amide compound (e.g., procainamide, lidocaine). In certain embodiments, the local anesthetic is a sodium channel blocker, for example, a site 1 sodium channel blocker (e.g., tetrodotoxin, saxitoxins (saxitoxin, neosaxitoxin), gonyautoxins (gonyautoxin V, gonyautoxin VI), -conotoxins) or an amino amide local anesthetic. In certain embodiments, the local anesthetic is tetrodotoxin, saxitoxin, neosaxitoxin, bupivacaine, amylocaine, ambucaine, articaine, benzocaine, benzonatate, butacaine, butanilicaine, carbocaine, cepastat, chloraseptic, chloroprocaine, cinchocaine, citanest, cyclomethycaine, dibucaine, diperodon, dimethocaine, eucaine, etidocaine, fomocaine, fotocaine, hydroxyprocaine, isobucaine, levobupivacaine, lidocaine, marcaine, mepivacaine, meprylcaine, metabutoxycaine, nitracaine, orthocaine, orabloc, oxetacaine, oxybuprocaine, paraethoxycaine, phenacaine, piperocaine, piridocaine, polocaine, posimir, pramocaine, prilocaine, primacaine, procaine, procainamide, proparacaine, propoxycaine, pyrrocaine, quinisocaine, ropivacaine, sensorcaine, septocaine, trimecaine, tetracaine, tolycaine, tropacocaine, ulcerease, xylocaine, or zorcaine. In certain embodiments, the local anesthetic is tetrodotoxin, saxitoxin, neosaxitoxin, lidocaine, procaine, or bupivacaine. In certain embodiments, the local anesthetic is bupivacaine.

[0216] In certain embodiments, the agent is an immunostimulatory agent, for example, an immunostimulatory agent is an agent that stimulates an immune response (including enhancing a pre-existing immune response) in a subject to whom it is administered, whether alone or in combination with another agent. Examples include antigens, adjuvants (e.g., TLR ligands such as imiquimod and residuimod, imidazoquino lines, nucleic acids comprising an unmethylated CpG dinucleotide, monophosphoryl lipid A (MPLA) or other lipopolysaccharide derivatives, single-stranded or double Stranded RNA, flagellin, muramyl dipeptide), cytokines including interleukins (e.g., IL-2, IL-7, IL-15 (or superagonist/mutant forms of these cytokines), IL-12, IFN-gamma, IFN-alpha, GM-CSF, FLT3-ligand, etc.), immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3, or single chain/antibody fragments of these molecules), and the like.

[0217] In certain embodiments, the agent is an antigen. The antigen may be without limitation a cancer antigen, a self or autoimmune antigen, a microbial antigen, an allergen, or an environmental antigen. The antigen may be peptide, lipid, or carbohydrate in nature, but it is not so limited. In certain embodiments, the antigen agent is a cancer antigen. A cancer antigen is an antigen that is expressed preferentially by cancer cells (i.e., it is expressed at higher levels in cancer cells than on non-cancer cells) and in Some instances it is expressed solely by cancer cells. The cancer antigen may be expressed within a cancer cell or on the surface of the cancer cell. The cancer antigen may be MART-1/Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectal associated antigen (CRC)C0171A/GAT33, carcinoembryonic antigen (CEA), CAP-1, CAP2, etv6, AMLI, prostate specific antigen (PSA), PSA-1, PSA2, PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor/CD3-Zeta chain, and CD20. The cancer antigen may be selected from the group consisting of MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5). The cancer antigen may be selected from the group consisting of GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9. The cancer antigen may be selected from the group consisting of BAGE, RAGE, LAGE-1, NAG, GnTV. MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21 ras, RCAS1, C.-fetoprotein, E-cadherin, C-catenin, B-catenin, Y-catenin, p120ctn, gp1007, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37. Ig-idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papilloma virus proteins, Smad family of tumor antigens, Imp-1, PlA, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20, and c-erbB-2.

[0218] In certain embodiments, the agent is an anti-cancer agent. An anti-cancer agent is an agent that at least partially inhibits the development or progression of a cancer, including inhibiting in whole or in part symptoms associated with the cancer even if only for the short term. Several anti-cancer agents can be categorized as DNA damaging agents and these include topoisomerase inhibitors, DNA alkylating agents, DNA strand break inducing agents, anti-microtubule agents, anti-metabolic agents, anthracyclines, Vinca alkaloids, or epipodophyllotoxins. The anti-cancer agent may be an enzyme inhibitor including without limitation tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinase inhibitor, or an EGFR inhibitor. The anti-cancer agent may be a VEGF inhibitor including without limitation bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), Sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and semaphorin. The anti-cancer agent may be an antibody or an antibody fragment including without limitation an antibody or an anti body fragment including but not limited to bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), alemtuzumab (CAMPATH, indicated for B cell chronic lymphocytic leukemia.), gemtuzumab (MYLOTARG, hP67.6, anti-CD33, indicated for leukemia Such as acute myeloid leukemia), rituximab (RITUXAN), to situmomab (BEXXAR, anti-CD20, indicated for B cell malignancy), MDX-210 (bispecific antibody that binds simultaneously to HER-2/neu oncogene protein product and type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI)). oregovomab (OVAREX, indicated for ovarian cancer), edrecolomab (PANOREX), daclizumab (ZENAPAX), palivizumab (SYNAGIS, indicated for respiratory conditions such as RSV infection), ibritumomab tiuxetan (ZEVALIN, indicated for Non-Hodgkin's lymphoma), cetuximab (ERBITUX), MDX-447, MDX-22, MDX-220 (antiTAG-72), IORC5, IOR-T6 (anti-CD1), IOR EGF/R3, celogovab (ONCOSCINT OV103), epratuzumab (LYMPHOCIDE), pemtumomab (THERAGYN), and Gliomab-H (indicated for brain cancer, melanoma). In certain embodiments, the agent is doxorubicin.

[0219] In certain embodiments, the therapeutic agent is a chemotherapeutic agent or an antibiotic agent. In certain embodiments, the therapeutic agent is an anesthetic agent, chemotherapeutic agent, or an antibiotic agent. In certain embodiments, the therapeutic agent is an anesthetic agent or a chemotherapeutic agent. In certain embodiments, the therapeutic agent is doxorubicin, tetrodotoxin, saxitoxin, neosaxitoxin, bupivacaine, amylocaine, ambucaine, articaine, benzocaine, benzonatate, butacaine, butanilicaine, carbocaine, cepastat, chloraseptic, chloroprocaine, cinchocaine, citanest, cyclomethycaine, dibucaine, diperodon, dimethocaine, eucaine, etidocaine, fomocaine, fotocaine, hydroxyprocaine, isobucaine, levobupivacaine, lidocaine, marcaine, mepivacaine, meprylcaine, metabutoxycaine, nitracaine, orthocaine, orabloc, oxetacaine, oxybuprocaine, paraethoxycaine, phenacaine, piperocaine, piridocaine, polocaine, posimir, pramocaine, prilocaine, primacaine, procaine, procainamide, proparacaine, propoxycaine, pyrrocaine, quinisocaine, ropivacaine, sensorcaine, septocaine, trimecaine, tetracaine, tolycaine, tropacocaine, ulcerease, xylocaine, or zorcaine. In certain embodiments, the agent is doxorubicin, bupivacaine, or tetrodotoxin. In certain embodiments, the agent is bupivacaine or tetrodotoxin. In certain embodiments, the agent is bupivacaine. In certain embodiments, the agent is tetrodotoxin.

[0220] In certain embodiments, the diagnostic agent is an imaging agent. An imaging agent is an agent that emits signal directly or indirectly thereby allowing its detection in vivo. Imaging agents such as contrast agents and radioactive agents that can be detected using medical imaging techniques, such as nuclear medicine scans, magnetic resonance imaging (MRI), positron-emission tomography (PET), and in vivo fluorescence imaging. Examples of such imaging agents are fluorophores (e.g., Cy7, fluorescein, rhodamine, AMCA), or biological fluorophores (e.g., green fluorescent protein, phycoerythrin, allophycocyanin). In certain embodiments, the diagnostic agent is Sulforhodamine B, indocyanine green, fluorescein isothiocyanate, methylene blue, or coumarin. In certain embodiments, the diagnostic agent is Sulforhodamine B, indocyanine green, methylene blue, or coumarin. In certain embodiments, the diagnostic agent is conjugated to a protein, a polymer, or a small molecule. In certain embodiments, the diagnostic agent is conjugated to a protein (e.g., albumin). In certain embodiments, the diagnostic agent is conjugated to albumin. In certain embodiments, the diagnostic agent is fluorescein isothiocyanate conjugated to albumin. In certain embodiments, the diagnostic agent is conjugated to a polymer (e.g., PEG). In certain embodiments, the diagnostic agent is conjugated to PEG having a molecular weight of about 1000 g/mol. In certain embodiments, the diagnostic agent is conjugated to PEG having a molecular weight of about 10000 g/mol. In certain embodiments, the diagnostic agent is Sulforhodamine B conjugated to PEG having a molecular weight of about 1000 g/mol. In certain embodiments, the diagnostic agent is Sulforhodamine B conjugated to PEG having a molecular weight of about 10000 g/mol.

[0221] In certain embodiments, the composition comprises at least two types of therapeutic agents selected from the group consisting of a local anesthetic (e.g., tetrodotoxin or TTX)), an anti-inflammatory agent, and a sympathomimetic or vasoconstrictor agent (such as alpha- and beta-adrenergic agonists (sympathomimetic or vasoconstrictor agent), for example, epinephrine; vasopressin analogs, norepinephrine, phenylephrine, dopamine, dobutamine). In certain embodiments, the composition comprises an anti-inflammatory agent (e.g., glucocorticoid receptor agonist, such as dexamethasone, corticosterone, glucocorticoids, mifepristone, hydrocortisone, corticotropin) and/or and a sympathomimetic or vasoconstrictor agent (e.g., epinephrine). In certain embodiments, the one or more agents (e.g., local anesthetic; anti-inflammatory (e.g., glucocorticoid receptor agonist, such as dexamethasone, corticosterone, glucocorticoids, mifepristone, hydrocortisone, corticotropin)) in the particle (e.g., liposome) is delivered together with one or more compounds or therapeutic agents (e.g., alpha- and beta-adrenergic agonists (sympathomimetic or vasoconstrictor agent), for example, epinephrine; vasopressin analogs, norepinephrine, phenylephrine, dopamine, dobutamine) in an injectate (liquid vehicle). In certain embodiments, in the composition, the injectate comprises an adjuvant agent such as alpha- and beta-adrenergic agonists (sympathomimetic or vasoconstrictor agent), for example, epinephrine; vasopressin analogs, norepinephrine, phenylephrine, dopamine, dobutamine); and the particle (e.g. liposome) comprises the anti-inflammatory (e.g., glucocorticoid receptor agonist, such as dexamethasone, corticosterone, glucocorticoids, mifepristone, hydrocortisone, corticotropin), and/or the local anesthetic (e.g., tetrodotoxin or TTX). In certain embodiments, the one or more agents (e.g., anti-inflammatory (e.g., glucocorticoid receptor agonist, such as dexamethasone, corticosterone, glucocorticoids, mifepristone, hydrocortisone, corticotropin)) in the particle is delivered together with one or more compounds or therapeutic agents (e.g., alpha- and beta-adrenergic agonists (sympathomimetic or vasoconstrictor agent), for example, epinephrine; vasopressin analogs, norepinephrine, phenylephrine, dopamine, dobutamine) in an injectate (liquid vehicle). In certain embodiments, the one or more agents (e.g., local anesthetic; anti-inflammatory (e.g., glucocorticoid receptor agonist, such as dexamethasone, corticosterone, glucocorticoids, mifepristone, hydrocortisone, corticotropin)) in the particle is delivered together with one or more compounds (e.g., alpha- and beta-adrenergic agonists (sympathomimetic or vasoconstrictor agent), for example, epinephrine; vasopressin analogs, norepinephrine, phenylephrine, dopamine, dobutamine) in an injectate (liquid vehicle), for prolonging a nerve blockade. In certain embodiments, an anti-inflammatory (e.g., glucocorticoid receptor agonist, such as dexamethasone, corticosterone, glucocorticoids, mifepristone, hydrocortisone, corticotropin) in the particle is delivered together with one or more alpha- and beta-adrenergic agonists (sympathomimetic or vasoconstrictor agent), e.g., epinephrine; vasopressin analogs, norepinephrine, phenylephrine, dopamine, dobutamine) in an injectate (liquid vehicle), for prolonging a nerve blockade. In certain embodiments, the composition comprises dexamethasone and a local anesthetic (e.g., TTX) in the particle; and epinephrine in an injectate. In certain embodiments, the composition comprises dexamethasone in the particle; and epinephrine in an injectate. In certain embodiments, the composition comprises tetrodotoxin and epinephrine in a particle and dexamethasone in another particle. In certain embodiments, the composition comprises dexamethasone in a liposome; and tetrodotoxin and epinephrine in an injectate. In certain embodiments, the composition comprises tetrodotoxin in a liposome; and epinephrine in an injectate. In certain embodiments, the composition comprises tetrodotoxin in a liposome; and epinephrine in a liposome.

[0222] In certain embodiments, the agent is a therapeutic agent, which is a small molecule therapeutic agent, a protein therapeutic agent, a nucleic acid therapeutic agent. In certain embodiments, the agent is a small molecule therapeutic agent, a protein therapeutic agent, a nucleic acid therapeutic agent, or small molecule diagnostic agent (e.g., fluorophore). In certain embodiments, the diagnostic agent is a fluorophore, for example, organic dyes (e.g., fluorescein, rhodamine, AMCA), or biological fluorophores (e.g., green fluorescent protein, phycoerythrin, allophycocyanin), including hydrophobic dyes and/or photosensitizers comprising aromatic groups. In certain embodiments, the diagnostic agent is a fluorophore, for example, organic dyes (e.g., fluorescein, rhodamine, AMCA), or biological fluorophores (e.g., green fluorescent protein, phycoerythrin, allophycocyanin). In certain embodiments, the diagnostic agent is Sulforhodamine B, indocyanine green, methylene blue, or coumarin. In certain embodiments, the diagnostic agent is Sulforhodamine B, indocyanine green, fluorescein isothiocyanate, methylene blue, or coumarin. In certain embodiments, the agent is physically incorporated into the particle. In certain embodiments, the agent is not physically incorporated into the particle. In certain embodiments, the agent is conjugated as part of Formula (I), as described herein.

[0223] In certain embodiments, the composition described herein further comprises water. In certain embodiments, the composition described herein forms a particle (e.g., liposome, lipid nanoparticle, polymer-lipid hybrid nanoparticle, lipid micelle, micelle-hydrogel hybrid, liposome-entrapped hydrogel, lipid-peptide complex, lipid-nucleic acid complex, lipid-peptide-nucleic acid complex, or lipid coated inorganic nanoparticle) upon addition of water (hydration). In certain embodiments, the composition described herein is in the form of a particle (e.g., liposome, nanoparticle, for example, lipid nanoparticle, polymer-lipid hybrid nanoparticle, lipid coated inorganic nanoparticle). In certain embodiments, the composition described herein is in the form of a liposome or nanoparticle (e.g., lipid nanoparticle, polymer-lipid hybrid nanoparticle, or lipid coated inorganic nanoparticle). In certain embodiments, the particle is a liposome. In certain embodiments, the liposome comprises dipalmitoylphosphatidylcholine. In certain embodiments, the particle is a liposome, lipid nanoparticle, polymer-lipid hybrid nanoparticle, lipid micelle, micelle-hydrogel hybrid, liposome-entrapped hydrogel, lipid-peptide complex, lipid-nucleic acid complex, lipid-peptide-nucleic acid complex, or lipid coated inorganic nanoparticle. In certain embodiments, the particle is a liposome, lipid nanoparticle, polymer-lipid hybrid nanoparticle, or lipid coated inorganic nanoparticle.

Pharmaceutical Compositions, Kits, and Administration

[0224] In certain embodiments, provided herein is a pharmaceutical composition comprising a composition described herein, a therapeutic agent described herein, and optionally a pharmaceutically acceptable excipient. As used herein, the term excipient means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as excipients include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The compositions of this invention can be administered to subjects (e.g., humans and/or to animals), orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), bucally, or as an oral or nasal spray.

[0225] In certain embodiments, the subject is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject described herein is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal, such as a dog or cat. In certain embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal, such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic animal (e.g., transgenic mice and transgenic pigs). In certain embodiments, the subject is a fish or reptile.

[0226] In certain embodiments, the cell being contacted with a compound or composition described herein is in vitro. In certain embodiments, the cell being contacted with a compound or composition described herein is in vivo.

[0227] Liquid dosage forms for oral administration include emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredients (i.e., microparticles, nanoparticles, liposomes, micelles, polynucleotide/lipid complexes), the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0228] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In certain embodiments, the particles are suspended in a carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween 80.

[0229] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0230] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the particles with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the particles.

[0231] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the particles are mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents.

[0232] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0233] The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

[0234] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0235] Dosage forms for topical or transdermal administration of an inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The particles are admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this invention.

[0236] The ointments, pastes, creams, and gels may contain, in addition to the particles of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0237] Powders and sprays can contain, in addition to the particles of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.

[0238] Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the microparticles or nanoparticles in a proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the particles in a polymer matrix or gel.

[0239] The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, any two doses of the multiple doses include different or substantially the same amounts of a compound described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, or biological sample (e.g., tissue, or cell). In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject or biological sample (e.g., tissue, or cell). In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 g and 1 g, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a compound described herein. In certain embodiments, the therapeutic agent, for example, local anesthetic (e.g., TTX) comprises between approximately 0-100 g therapeutic agent, (0-0.1% in mass percentage of the formulations) per 0.45 kg rat, or an equivalent amount in a human dosage, for example, between approximately 0 to 1.0 mg/kg human (e.g., 0-0.1 mg/kg, 0.1-0.2 mg/kg, 0.2-0.3 mg/kg, 0.2-0.3 mg/kg, 0.3-0.4 mg/kg, 0.4-0.5 mg/kg, 0.5-0.6 mg/kg, 0.6-0.7 mg/kg, 0.8-0.9 mg/kg, 0.9-1.0 mg/kg, 1.0-1.2 mg/kg). Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult. The amount to be administered to, for example, a human, can be calculated from the amount administered to a rat, as determined using standard calculations by a person of ordinary skill in the art.

Kits

[0240] Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form.

[0241] Thus, in one aspect, provided are kits including a first container comprising a compound, composition, or pharmaceutical composition described herein. In certain embodiments, the kits are useful for delivering an agent to a subject, comprising a composition described herein, the agent, and instructions for delivering the agent to a subject in need thereof. In certain embodiments, the kits comprise a container, a composition of a compound described herein, and instructions for administering the composition or pharmaceutical composition thereof to a subject in need thereof. In certain embodiments, the kits comprise a container, a composition of a compound described herein, and instructions for administering the composition or pharmaceutical composition thereof to a subject in need thereof. In certain embodiments, a kit described herein further includes instructions for using the compound or pharmaceutical composition included in the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for assembling the composition described herein. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition. In certain embodiments, a kit described herein further includes a dropper, syringe, or catheter. In certain embodiments, a kit described herein further includes a syringe.

Methods of Treatment and Uses

[0242] In one aspect, provided are methods for delivering an agent described herein (e.g., therapeutic agent (such as antibiotic agent, chemotherapeutic agent, anesthetic agent, anti-inflammatory agent, analgesic agent, anti-fibrotic agent, anti-sclerotic agent, or anticoagulant agent) or diagnostic agent, such as a fluorophore, e.g., Sulforhodamine B, indocyanine green, methylene blue, coumarin) to a subject or biological sample (e.g., cell, tissue), comprising administering to the subject or contacting the biological sample with a composition described herein (e.g., composition comprising a compound of Formula (I), or administering to the subject or contacting the biological sample with the pharmaceutical composition described herein. In certain embodiments, the composition is in the form of a particle, e.g., a liposome, lipid nanoparticle, polymer-lipid hybrid nanoparticle, lipid micelle, micelle-hydrogel hybrid, liposome-entrapped hydrogel, lipid-peptide complex, lipid-nucleic acid complex, lipid-peptide-nucleic acid complex, or lipid coated inorganic nanoparticle. In certain embodiments, the composition is in the form of a particle, e.g., a liposome, lipid nanoparticle, polymer-lipid hybrid nanoparticle, or lipid coated inorganic nanoparticle. In certain embodiments, the composition is in the form of a liposome. In certain embodiments, the composition is in the form of a nanoparticle. In certain embodiments, the composition is in the form of a nanoparticle, for example, a lipid nanoparticle, polymer-lipid hybrid nanoparticle, or lipid coated inorganic nanoparticle. In certain embodiments, the methods for delivering an agent (e.g., therapeutic agent, diagnostic agent) described herein are used for local anesthesia, photodynamic therapy, inflammation, molecular imaging, photothermal therapy, and/or fluorescence imaging. In certain embodiments, the methods for delivering an anesthetic agent (e.g., local anesthetic) described herein elicit prolonged peripheral nerve blockade for up to three or more days following a single application (e.g., injection) of the composition described herein.

[0243] In certain embodiments, in methods described herein, in therapeutic applications, a drug may be incorporated into the aqueous core of the liposomes or interior of the lipid bilayers. In certain embodiments, in methods described herein, in diagnostic applications, a labeling moiety may be conjugated to the phospholipids and/or incorporated into the interior of the lipid bilayers.

[0244] For example, in one aspect, provided is a method of treating and/or preventing a disease in a subject in need thereof, the method comprising administering to the subject a composition described herein (e.g., composition comprising a compound of Formula (I) (e.g., in the form of a particle, for example, a liposome, lipid nanoparticle, polymer-lipid hybrid nanoparticle, or lipid coated inorganic nanoparticle) comprising a therapeutically effective amount of a therapeutic agent (e.g., a small molecule therapeutic agent, a protein therapeutic agent, a nucleic acid therapeutic agent; for example, antibiotic agent, chemotherapeutic agent, anesthetic agent, anti-inflammatory agent, analgesic agent, anti-fibrotic agent, anti-sclerotic agent, anticoagulant agent), or a pharmaceutical composition described herein.

[0245] The present disclosure also provides uses of a composition described herein (e.g., composition comprising a compound of Formula (I), for delivering an agent described herein (e.g., therapeutic agent (such as antibiotic agent, chemotherapeutic agent, anesthetic agent, anti-inflammatory agent, analgesic agent, anti-fibrotic agent, anti-sclerotic agent, or anticoagulant agent) or diagnostic agent, such as a fluorophore, e.g., Sulforhodamine B, indocyanine green, methylene blue, coumarin) to a subject or biological sample (e.g., cell, tissue), comprising administering to the subject or contacting the biological sample with a composition described herein (e.g., composition comprising a compound of Formula (I), or administering to the subject or contacting the biological sample with the pharmaceutical composition described herein. In certain embodiments, the composition is in the form of a particle, e.g., a liposome, lipid nanoparticle, polymer-lipid hybrid nanoparticle, or lipid coated inorganic nanoparticle.

[0246] The present disclosure also provides uses of a composition described herein (e.g., composition comprising a compound of Formula (I), for treating and/or preventing a disease in a subject in need thereof, the method comprising administering to the subject a composition described herein (e.g., composition comprising a compound of Formula (I) (e.g., in the form of a particle, for example, a liposome, lipid nanoparticle, polymer-lipid hybrid nanoparticle, or lipid coated inorganic nanoparticle) comprising a therapeutically effective amount of a therapeutic agent (e.g., a small molecule therapeutic agent, a protein therapeutic agent, a nucleic acid therapeutic agent; for example, antibiotic agent, chemotherapeutic agent, anesthetic agent, anti-inflammatory agent, analgesic agent, anti-fibrotic agent, anti-sclerotic agent, anticoagulant agent), or a pharmaceutical composition described herein.

Methods of Synthesis

[0247] In one aspect, provided are methods of synthesizing the particles (e.g., liposomes) described herein, comprising the compounds of Formula (I) (e.g., modified phospholipids) and/or other unmodified phospholipids (e.g., phosphoglycerol, phosphocholine, phosphoethanolamine, phosphoinositol, phosphoserine; such as, DOPC, DSPC), and lipids (e.g., cholesterol) described herein, wherein the ratio of the lipids to each other is variable (e.g., ratio of 3:3:2:3). In certain embodiments, the particle (e.g., liposome) is synthesized using Ph-DPPC, DOPC, DSPG, and/or cholesterol. In certain embodiments, the particle (e.g., liposome) is synthesized using phenoxy-conjugated DPPC (Ph-DPPC depicted in FIG. 6), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPG), and cholesterol, wherein the ratio of the lipids to each other is variable (e.g., ratio of 3:3:2:3).

[0248] In certain embodiments, the particle (e.g., liposome) is synthesized by combining a compound of Formula (I) (e.g., Ph-DPPC), one or more unmodified phospholipids (e.g., phosphoglycerol, phosphocholine, phosphoethanolamine, phosphoinositol, phosphoserine; such as, DOPC, DSPC), and/or lipids (e.g., cholesterol); hydrating, and mixing, which subsequently forms the particle (e.g., liposome). In certain embodiments, the particle (e.g., liposome) is synthesized by combining Ph-DPPC, DOPC, DSPG, and/or cholesterol, hydrating, and mixing, which subsequently forms the particle (e.g., liposome). In certain embodiments, the particle (e.g., liposome) is synthesized by combining Ph-DPPC, DOPC, DSPG, and cholesterol, and hydrated (e.g., with a buffer, such as phosphate buffered saline (PBS)) while mixing (e.g., agitating), which subsequently forms the particle (e.g., liposome). In certain embodiments, the particle (e.g., liposome) is synthesized by combining Ph-DPPC, DOPC, DSPG, and cholesterol, and hydrated while mixing, which subsequently forms the particle (e.g., liposome), as described in Example 1 below (Preparation and characterization of aromatized liposomes). In certain embodiments, the particle (e.g., liposome) is synthesized using 1-100% molar percentage (e.g., at least 10%, for example at least 15%, or at least 25%) of the compounds of Formula (I) (e.g., modified phospholipids, for example, Ph-DPPC, or a modified phospholipid of Formula (I) described herein). In certain embodiments, the particle (e.g., liposome) is synthesized using about 0-99% molar percentage (e.g., about 40-75%) of the unmodified phospholipids (e.g., phosphoglycerol, phosphocholine, phosphoethanolamine, phosphoinositol, phosphoserine; such as, DOPC, DSPC). In certain embodiments, the particle (e.g., liposome) is synthesized using 1-100% molar percentage (e.g., at least 10%, for example at least 15%, or at least 25%) of the compounds of Formula (I) (e.g., Ph-DPPC, or a modified phospholipid of Formula (I) described herein); and about 0-99% molar percentage (e.g., about 40-75%) of the unmodified phospholipids (e.g., phosphoglycerol, phosphocholine, phosphoethanolamine, phosphoinositol, phosphoserine; such as, DOPC, DSPC). In certain embodiments, provided are particles (e.g., liposomes) described herein, comprising the compounds of Formula (I) (e.g., modified phospholipids) and/or other unmodified phospholipids (e.g., phosphoglycerol, phosphocholine, phosphoethanolamine, phosphoinositol, phosphoserine; such as, DOPC, DSPC), and lipids (e.g., cholesterol) described herein, wherein the ratio of the lipids to each other is variable (e.g., ratio of 3:3:2:3), synthesized by the methods described herein. In certain embodiments, the particle (e.g., liposome) comprises an agent (e.g., therapeutic agent, diagnostic agent; e.g., TTX, Cy7) as described herein. In certain embodiments, the particle comprising unmodified phospholipids (e.g., phosphoglycerol, phosphocholine, phosphoethanolamine, phosphoinositol, phosphoserine; such as, DOPC, DSPC), and lipids (e.g., cholesterol) described herein, wherein the ratio of the lipids to each other is variable (e.g., ratio of 3:3:2:3), is as described in Example 1.

EXAMPLES

[0249] In order that the present disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope. The compounds provided herein can be prepared from readily available starting materials using the following general methods and procedures or methods known in the art. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by those skilled in the art by routine optimization procedures.

[0250] In certain embodiments, the phospholipid compounds disclosed herein are synthesized as outlined in Examples 1 and 2, and in the scheme of FIG. 2. DPPC is dipalmitoylphosphatidylcholine. In certain examples, DPPC is modified with various functional groups, e.g., Ph-DPPC is DPPC modified with a phenoxy group and CM-DPPC is DPPD modified with a coumarin group as shown in FIG. 8. Lip-Ph or Lipo-Ph are liposomes containing Ph-DPPC. Lip-Ph or Lipo-Ph are liposomes containing Ph-DPPC. Lipo-CM are liposomes containing CM-DPPC. Lipo-DBCO are liposomes containing DPPC modified with a DBCO group (e.g., FIG. 11E). Unmodified liposome is referred to as Lip or Lipo. Unmodified liposome is DPPC.

[0251] Nomenclature in the application includes naming the agent encapsulated in a liposome by use of agent name @liposome (e.g., TTX@Lipo-Ph is tetrodotoxin encapsulated in liposomes containing Ph-DPPC) or the agent name after the liposome (e.g., Lip-Ph-Bup is bupivacaine encapsulated in liposomes containing Ph-DPPC).

Example 1: Acyl Chain-Modified Phospholipids and Lipids Incorporating TTX and Dyes

[0252] The engineering of synthetic lipid vesicles that incorporate aromatic groups within lipid bilayers for improved drug loading and sustained drug release, using a strategy for the synthesis of acyl chain-modified phospholipids with terminal aromatic groups are described. Formulation of phospholipids conjugated to aromatic groups and of natural phospholipids allowed the formation of aromatized liposomes which may have similar physiochemical properties to conventional liposomes but have significantly decreased membrane permeability and leakage of encapsulated cargos.

[0253] This data shows an example in an area where liposome products are used clinically: local anesthesia..sup.2 The anesthetic tetrodotoxin (TTX) was selected as the principal model compound as it is it has many of the properties that are most problematic in encapsulation in liposomes: low molecular weight, extreme hydrophilicity, high potency, and narrow therapeutic window. Aromatized liposomes enabled increased TTX loading, prolonged therapeutic duration 20-30-fold compared to free TTX and by more than 3-fold compared to unmodified liposomes, expanded the therapeutic window, and mitigated systemic toxicity. These rationally designed phospholipids could create a new approach to the delivery of a variety of therapeutics.

[0254] Taking inspiration from the physiochemistry of naturally-occurring lipid bilayers and the passive diffusion of molecules across cell membranes, it is believed that additional stabilization forces that can tighten the packing of lipid bilayers may help retain entrapped molecules and improve drug loading. The incorporation of aromatic groups into lipid bilayers may stabilize the liposomes via p-p stacking interactions. (FIG. 1) Aromatic rings are attracted to one another by a combination of dispersion forces and dipole-induced interactions, known as - stacking interactions.sup.17. - stacking interactions are considered a special type of van der Waals forces.sup.18. As with hydrophobic interactions, the role of - stacking interactions has been explored in the fabrication and stabilization of supramolecular structures.sup.19, including drug delivery systems.sup.20.

Synthesis of Acyl Chain-Modified Phospholipids.

[0255] A strategy to conjugate aromatic groups onto the acyl chains of phospholipids was developed (FIG. 2). The process began with the synthesis of phenoxy-conjugated phospholipids because the benzene ring was the smallest aromatic group that could enable n-n stacking interactions.sup.21. The phenoxy groups were selected because their ether bond was much less polar than amide and ester bonds, minimizing the disruption of the hydrophobic network within lipid bilayers.sup.22. Methyl 16-bromohexadecanoate, phenol, and potassium carbonate were mixed in anhydrous acetonitrile at 60 C. overnight to create methyl 16-phenoxyhexadecanoate. Deprotection of the carboxylic acid yielded 16-phenoxy-palmitic acid, which then reacted with 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPC) via a coupling reaction to create phenoxy-conjugated DPPC (Ph-DPPC). In the .sup.1H-NMR spectrum of Ph-DPPC, the representative signals of aromatic rings at 7.2 and 6.9 ppm clearly demonstrated the successful conjugation of a phenoxy group to the acyl chains of the phospholipid (Ph-DPPC) (FIGS. 3-7), which was also confirmed by LC-MS (FIG. 8). Coumarin conjugated DPPC (CM-DPPC) was synthesized following similar procedures. The coupling of lysoPC and functionalized fatty acids represents a general strategy for the synthesis of acyl chain-modified phospholipids.

[0256] The yield of the above coupling reaction was extremely low with commonly used coupling reagents such as N,N-dicyclohexylcarbodiimide (DCC), N,N-diisopropylcarbodiimide (DIC), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), probably due to the poor solubility of lysoPC in organic solvents like chloroform. After screening multiple coupling reagents, 2,6-dichlorobezoyl chloride and 1-methylimidazole were used in the synthesis due to their high potency in activating fatty acid, which lead to rapid reaction rates and over 75% yield.sup.23.

[0257] The phase transition temperatures (Tm) of Ph-DPPC and CM-DPPC were 47.4 and 66.1 C., respectively (FIG. 9). Preparation of liposomes containing CM-DPPC required high temperatures due to the high Tm, which may limit their use as carriers for temperature-sensitive cargos like proteins and nucleic acids. Therefore, the focus was on liposomes incorporating Ph-DPPC for drug encapsulation and on release investigations.

Preparation and Characterization of Aromatized Liposomes.

[0258] Aromatized liposomes were prepared following a thin-film hydration method.sup.24. In a typical procedure, a dried lipid thin film containing Ph-DPPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPG), and cholesterol in a 3:3:2:3 molar ratio was hydrated with phosphate buffered saline (PBS). Both phosphocholine (PC) and phosphoglycerol (PG) are needed to maintain the physical stability of liposomes. DOPC is used in these formulations to facilitate membrane fusion and fine tune membrane permeability. DOPC was used for Exparel and Depodur, two clinically approved liposome products for pain relief. DSPG was used because it was found that negatively charged DSPG can enhance the encapsulation of positively charged TTX. DPPC, DSPG and cholesterol (Chol) were used for sciatic nerve injection, showing no nerve damage.

[0259] Mechanical agitation was applied during the hydration to facilitate membrane fusion and formation of multilamellar vesicles.sup.10. Combination of Ph-DPPC and natural phospholipids afford aromatized liposomes similar to conventional liposomes as confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) (FIGS. 10A-10B). The liposomes displayed similar size and did not aggregate after storage at 4 C. for several weeks, indicating good stability (FIGS. 10C, 10D). Nanoscale aromatized liposomes can be produced after extrusion through polycarbonate filters (FIG. 10E), which is favorable for systemic administration.

[0260] The hydrophilic small molecule fluorophore sulforhodamine B (SRho, log P=0.53, FIG. 11A) was loaded into liposomes by hydrating the lipid cake with a 10 mg/mL SRho solution in PBS. The loading of SRho in aromatized liposomes (SRho@Lipo-Ph) was 19% greater than in unmodified liposomes (FIG. 11B), showing slightly increased drug loading efficiency as a result of aromatization. The release kinetics of SRho was evaluated in vitro. Free (unencapsulated) dye was released within 6 hours. In SRho@Lipo, more than 12% SRho was released in 24 h. In comparison, less than 6.0% and 5.5% of SRho was released in 24 h from SRho@Lipo-Ph and SRho@Lipo-CM (FIG. 11C). Compared to Lipo-Ph, Lipo-CM did not considerably increase the drug loading or reduce the cumulative drug release in first 24 h. Viscosity is an important factor for injectability. The viscosity of different formulations was characterized at a range of angular frequencies. The aqueous solution of Lipo-CM was notably more viscous than Lipo-Ph (FIG. 11D). Lipo-Ph was chosen for subsequent studies.

[0261] The acyl chain of phospholipids was modified with dibenzocyclooctyne (DBCO) (DBCO-DPPC, FIG. 11E), a group with three rings including two aromatic rings. The loading of SRho in DBCO-modified liposomes was 2.30.3%, comparable to that in unmodified liposomes (FIG. 11F). 21% of SRho was released from SRho@Lipo-DBCO in 24 h, which was faster than from SRho@Lipo (FIG. 11G). The bulky DBCO group may interfere with the packing of hydrocarbon chains, making the liposome leakier.

[0262] Since the covalently conjugated phenyl group slowed the release of payload, the extent to which physical (non-covalent) encapsulation of phenol into lipid bilayers would stabilize liposomes was studied. To compare the effects of covalent conjugation and physical encapsulation, SRho was co-encapsulated with 1 mM phenol into liposomes (Ph+SRho@Lipo) (FIG. 11H). Ph+SRho@Lipo released 7.1% of SRho in 24 h, 42% less than from unmodified liposomes, but 20% more than from covalently aromatized liposomes.

[0263] Phenol is intrinsically toxic, as confirmed by cytotoxicity data (FIG. 11I-J). Therefore, in order to be able to assess the effect of non-covalently incorporated aromatic groups in vivo, SRho was co-encapsulated with 1 mM indocyanine green (ICG), an FDA approved fluorophore containing four aromatic rings. SRho release from ICG+SRho@Lipo was less rapid than from unmodified liposomes, but more than from covalently aromatized liposomes (FIG. 11H). Since there was no statistically significant difference between the effect of Ph and ICG on drug release, the latter was used in downstream experiments.

Effect of Liposome Aromatization on Different Payloads

[0264] The effect of liposome aromatization to include payload molecules with different characteristics such as hydrophilicity and molecular weight was studied. The hydrophilicity of different payloads was measured by their octanol-water partition coefficients (FIG. 12). In addition to SRho described above, the following small molecule compounds were encapsulated (FIG. 13A), using the same encapsulation and analytical techniques: tetrodotoxin (TTX, log P=0.13), a hydrophilic ultrapotent local anesthetic; bupivacaine hydrochloride (Bup, log P=0.27), an amphiphilic amino-amide local anesthetic in current clinical use; doxorubicin hydrochloride (Dox, log P=0.22), a chemotherapeutic drug that has been used clinically in liposomes to treat cancer.

[0265] Loading of small molecule compounds was increased by 19-60% by aromatization of liposomes (Table 1). Neither molecular weight (R.sup.2=0.32) nor hydrophilicity (R.sup.2=0.00) correlated with the degree of increase in drug loading (FIG. 13B-E). Release in the first 24 h was reduced by 30-60% by aromatization of liposomes (FIGS. 13F-H, Table 2). Although molecular weight (FIG. 13I; R.sup.2=0.01) did not correlate strongly with the degree of reduction, hydrophilicity showed some correlation with the degree of reduction (FIG. 13J; R.sup.2=0.64).

TABLE-US-00001 TABLE 1 Effect of aromatization on the loading of different payloads. Drug loading Increase Payload Lipo Lipo-Ph in loading P values SRho 2.6 0.3% 3.1 0.2% 19.2% P = 0.009 TTX 0.11 0.01% 0.13 0.01% 25.5% P = 0.004 Bup 3.5 0.7% 5.6 0.8% 60.0% P = 0.006 Dox 0.8 0.1 1.1 0.1% 30.4% P = 0.004 SRho-PEG1k 0.78 0.04 0.87 0.02.sup. 11.5% P = 0.011 SRho-PEG10k 1.64 0.13 1.71 0.02.sup. 4.3% P > 0.05 FITC-Ab 3.63 0.20 3.78 0.16.sup. 4.0% P > 0.05

TABLE-US-00002 TABLE 2 Effect of aromatization on the release of different payloads. Cumulative release in first 24 h Decrease Payload Lipo Lipo-Ph in release P values SRho 12.2 0.5% 5.9 0.7% 51.6% P < 0.001 TTX 17.1 0.3% 11.3 0.4% 34.0% P < 0.001 Bup 70.7 1.9% 37.0 2.7% 47.7% P < 0.001 Dox 26.8 1.1% 17.6 1.0% 34.3% P < 0.001 SRho-PEG1k 28.2 2.5% 19.6 2.0% 30.5% P = 0.002 SRho-PEG10k 26.2 2.3% 14.5 2.1% 44.6% P < 0.001 FITC-Ab 14.2 1.2% 6.3 0.8% 55.6% P < 0.001

[0266] Similar loading and release experiments were done with liposomes loaded with the macromolecules rhodamine-conjugated polyethylene glycol (1 kDa and 10 kDa, Log P=2.05 and 2.19, respectively) and fluorescein isothiocyanate-conjugated albumin (Log P=2.39, FIG. 12). Although aromatization had a modest (or no) effect on drug loading of macromolecules (Table 1), aromatization decreased the release of macromolecules in the first 24 h from liposomes (FIG. 13K-M). The effect of liposomal aromatization on drug release increased with increasing molecular weight (FIG. 13N; R.sup.2=0.81) and increasing hydrophilicity (FIG. 13O; R.sup.2=0.97).

[0267] These data showed that aromatized liposomes had benefits over conventional liposomes for delivery of a wide range of small and large molecules.

[0268] In anticipation of in vivo experimentation, TTX was also co-encapsulated in unmodified liposomes with ICG (ICG+TTX@Lipo). 13% of TTX was released from ICG+TTX@Lipo within 24 h, which was less rapid than from unmodified liposomes, but more than from aromatized liposomes in the same period (FIG. 13P).

[0269] Tetrodotoxin (TTX) was used to assess the effect of aromatized liposomes on sustained release of lower molecular weight drugs. The high potency and minimal cardiac and tissue toxicity of TTX made it an appealing local anesthetic.sup.25. However, systemic toxicity can be dose-limiting.sup.26, even when it is encapsulated.sup.27. The extremely high hydrophilicity (Log P=6.2), low molecular weight (319), and narrow therapeutic window make the encapsulation and controlled delivery of TTX challenging.sup.28.

[0270] Liposomes encapsulating TTX were prepared by the same hydration method. Liposomes fabricated with or without Ph-DPPC showed similar median number-weighted diameters around 1 m (FIGS. 14A-14B; Table 3). TEM confirmed the formation of spherical vesicles (FIG. 14C). All liposomes showed similar negative zeta potentials around 30 mV, indicating that the incorporation of aromatized phospholipids or encapsulation of TTX did not change their surface charge (FIG. 14D). Incorporation of aromatic groups within lipid bilayers did not significantly alter the size, zeta potential, or polydispersity of liposomes. All liposomal solutions showed very low viscosity of less than 10 mPa.Math.s (FIG. 14E), which was not significantly higher than that of PBS (1.9 mPa.Math.s), suggesting easy injectability.

TABLE-US-00003 TABLE 3 Characterization of liposomal formulations. Diameter Zeta Potential Polydispersity Formulations (mm) (mV) Index Lipo 1.21 0.19 29.2 2.3 0.192 0.061 TTX@Lipo 1.26 0.14 26.5 1.2 0.122 0.037 ICG + TTX@Lipo 1.38 0.31 29.9 2.5 0.125 0.070 TTX@Lipo-Ph 1.14 0.17 27.7 3.5 0.159 0.046 Data are means s.d., n = 4.

[0271] The cytotoxicity of aromatized liposomes was evaluated in myoblast C2C12.sup.29 and pheochromocytoma PC12.sup.30 cell lines to assess potential cytotoxicity to muscle and nerve cells, respectively. Cell viability, assessed by MTS assays, was very high in all tested groups (FIGS. 14F-14G and FIGS. 15A-15B), indicating that incorporation of synthetic Ph-DPPC did not compromise the biocompatibility of the liposomes.

[0272] Aromatized liposomes encapsulating TTX (Lip-Ph-TTX) showed increased TTX loading compared to unmodified ones (Lip-TTX) (FIG. 14H). Release of TTX from liposomes was quantified by enzyme-linked immunosorbent assay (ELISA). More than 95% TTX was released after 8 hours in the experimental set-up (FIG. 16). Physical-Encapsulation into Lip-TTX enabled sustained release of TTX over 168 hours. However, rapid burst release was still observed, where nearly 20% of encapsulated TTX was released in 24 hours (FIG. 13F). Lip-Ph-TTX significantly reduced the burst release of TTX compared to Lip-TTX.

Localization and Retention of Liposomal Formulations.

[0273] To assess whether lipid bilayer engineering affected the local retention of liposomes, liposomes were covalently conjugated with Cy7 and injected at the sciatic nerve site. Fluorescence images of rats were then taken at different time points using an in vivo imaging system (IVIS) (FIG. 17A). While free Cy7 (fluorophore is not covalently conjugated to any compound or phospholipid) rapidly diffused away from the injection site within hours (FIG. 17A and FIG. 18), both unmodified liposomes (Cy7-Lip) and aromatized liposomes (Cy7-Lip-Ph) remained there for several weeks, consistent with previous studies.sup.32. Fluorescent confocal microscopy confirmed the localization of Cy7-conjugated liposomes as well as free dyes in the connective tissue between muscles and nerves (FIGS. 17B, 19, and 20). These findings suggested that differences in anesthetic effect between formulations would be due to differences in drug release, not tissue retention. Cy7-Lip-Ph refers to Cy7

##STR00081##

that is covalently conjugated to the phospholipid.

In Vivo Therapeutic Efficacy and Toxicity.

[0274] To assess whether the sustained release of drug with aromatized liposomes could translate into improved duration of effect and reduced toxicity in vivo, the liposomes were used in a rat model of peripheral nerve block. Rats were injected at the left sciatic nerve with 300 L of different TTX formulations (FIG. 21A). Neurobehavioral testing based on a modified hot-plate test was applied in both hind paws to assess the duration of functional deficits.sup.24. Local anesthesia was assessed by the length of time a rat would leave its hind paw on a hotplate (thermal latency). The duration of deficits on the injected (left) side reflected the duration of nerve block, whereas deficits on the contralateral un-injected (right) side indicated numbness from systemic distribution of TTX.

[0275] Rats receiving sciatic nerve injection of free TTX showed dose-dependent nerve blockade, where 2 g of TTX in PBS buffer showed no detectable block while 4 g of free TTX in PBS buffer enabled a median duration of sensory block of 2.50.6 hours (FIGS. 21B-21D). However, 4 g of free TTX induced systemic toxicity, as reflected by a sensory deficit lasting 2.10.5 hours in the contralateral leg (FIG. 21E-21F). Injection of 5 g of free TTX was uniformly fatal (FIG. 21G).

[0276] Nerve block duration and safety were improved by encapsulation of TTX in liposomes (Lip-TTX) (FIG. 21B). The actual dose in each formulation varied slightly due to differences in TTX loading efficiency (Table 4). The onset of nerve blockade occurred 10-15 minutes after injection of Lip-TTX, and resulted in a duration of 19.94.4 hours (FIG. 21C). In addition, Lip-TTX improved the safety of TTX as evidenced by the decrease in contralateral deficits (FIG. 21E). The contralateral block occurred in 62.5% of animals (FIG. 21F). The peak latency in the contralateral leg was 7.51.0 seconds, shorter than 12 seconds (maximum thermal latency, rats were removed manually after 12 seconds to prevent potential thermal injury) with free TTX. 3 out of 4 rats died when TTX@Lipo containing 24.8 g TTX was injected. Higher loadings of TTX (29.6 g) were fatal. Contralateral deficits were not detectable 6 hours after injection, indicating that sustained release of TTX from liposomes significantly decreased systemic toxicity.

TABLE-US-00004 TABLE 4 Comparison of loading efficiency and loading in different TTX formulations. Loading Drug Formulations Payload Efficiency (%) Loading (%) Lipo TTX@Lipo TTX 22.6 1.9 0.106 0.009 ICG + TTX@Lipo ICG 84.3 3.8 0.524 0.024 TTX 27.1 1.7 0.127 0.008 TTX@Lipo-Ph TTX 28.5 2.0 0.133 0.009 Data are means s.d., n = 4.

[0277] Aromatized liposomes (Lip-Ph-TTX) further improved the therapeutic duration and safety of TTX (FIG. 21B). The onset of nerve blockade occurred 20-30 minutes after the injection of Lip-Ph-TTX, and produced 100% successful block in all animals. Nerve block from TTX@Lipo-Ph containing 25.3 g TTX (FIG. 21D) lasted 36.94.6 h (2-fold the duration obtained with 20.4 g TTX in TTX@Lipo), there was no contralateral block (FIG. 21F), and there were no animal deaths (FIG. 21G). Lip-Ph-TTX containing 32.1 g of TTX achieved a median duration of sensory block to 57.111.6 h, 23-fold greater that from 4 g free TTX and 2.9 times longer than that from Lip-TTX (FIG. 21C). This dose was higher than a dose that was uniformly fatal in animals receiving TTX@Lipo (29.6 g TTX). TTX@Lipo-Ph containing 37.5 g TTX enabled nerve block lasting 64.58.1 h. Increasing the TTX loading to 44.2 g further increased the duration of block to 70.79.8 h (Table 5). Even at that loading, there were no animal deaths, and contralateral block only occurred in 50% of animals (lower than the percentage with 20.4 g TTX in TTX@Lipo). The peak latency in the contralateral leg was further decreased to 4.10.4 seconds and the contralateral deficits could not be detected after 4 hours (FIG. 21E). These data were consistent with the slower release kinetics of TTX@Lipo-Ph and demonstrated that liposome aromatization resulted in prolonged block and reduced toxicity.

TABLE-US-00005 TABLE 5 In vivo assessment of TTX formulations. Duration of Contralateral TTX dose nerve block peak latency Formulation (g) (h) (s) Free TTX 4.0 2.5 0.6 12 0.0 TTX@Lipo 20.4 19.9 4.4 7.5 1.0 ICG + TTX@Lipo 24.4 26.9 5.5 6.7 0.9 TTX@Lipo-Ph 25.3 36.9 4.6 3.3 0.4 32.1 57.1 11.6 4.2 0.5 37.5 64.5 8.1 5.0 0.4 44.2 70.7 9.8 7.5 1.2 Data are means s.d., n = 4.

[0278] Stabilization of liposomes by n-n stacking could also be achieved by physical encapsulation of hydrophobic aromatic molecules into lipid bilayers. ICG, which contains four aromatic rings, could stabilize lipid bilayers via additional hydrophobic interactions with fatty acids and - stacking interactions with each other.sup.33. To compare the effect of covalent conjugation and physical encapsulation, hydrophobic indocyanine green (ICG, log P=6.05).sup.3, an FDA approved fluorophore, was encapsulated into liposomes. Lip-ICG-TTX, in which ICG and TTX were co-encapsulated into unmodified liposomes containing 24.4 g TTX (structure of tetrodotoxin or TTX which is shown below), achieved sensory nerve block lasting 26.95.5 hours (FIGS. 21C-D), 10.8-fold that with 4 g free TTX, but 2.1 times less than that achieved by Lip-Ph-TTX. Encapsulation of ICG (structure of indocyanine green or ICG which is shown below) also decreased the peak latency for contralateral leg to 6.60.9 seconds (FIG. 21E). Contralateral block occurred in 37.5% of animals (FIG. 21F; similar to that from TTX@Lipo) and there were no animal deaths (FIG. 21G). 2 of 4 animals administered 31 g TTX in ICG+TTX@Lipo died. All animals receiving 38 g TTX in ICG+TTX@Lipo died.

##STR00082##

[0279] Since the performance of liposomal TTX could be improved by both encapsulation and covalent modification with aromatic compounds, it was assessed whether combining the two approaches within the same liposome formulation (Lip-Ph-ICG-TTX) would further improve the therapeutic duration and mitigate the toxicity of TTX. Lip-Ph-ICG-TTX containing 35.8 g TTX achieved the most prolonged nerve blockade, 73.517.4 hours (FIG. 21C). Moreover, Lip-Ph-ICG-TTX also displayed the least systemic TTX distribution as evidenced by the peak contralateral latency of 3.90.4 seconds (FIG. 21E).

[0280] Whether the codelivery of encapsulated adjuvant compounds with aromatized liposomes can further enhance the nerve block duration was investigated. Previous studies have combined S1SCBs like TTX with adjuvants with -adrenergic activity like epinephrine and glucocorticoid receptor agonists like dexamethasone for prolonged nerve blockade. TTX@Lipo-Ph containing 45.1 g TTX combined with epinephrine (Epi, 3.6 g) in the injectate (Epi+TTX@Lipo-Ph) and dexamethasone (Dex, 290 g) in the liposome (Dex@Lipo-Ph+TTX@Lipo-Ph) markedly increased the nerve block duration to 128.822.3 h and 186.523.8 h respectively (FIG. 21H). No contralateral block was observed in these groups. Prolonged duration local anesthesia (PDLA) lasting weeks is desirable for treating prolonged pain like cancer pain.

[0281] Motor nerve blockade was assessed by a weight-bearing test to determine the motor strength of the rat's hind paw. Aromatized liposomes also significantly prolonged the duration of the motor block (FIG. 22). For free TTX, there was no statistically significant difference between the duration of sensory and motor nerve block. For the liposome formulations, the durations of motor block were 8-13% longer than those of sensory block.

[0282] Systemic toxicity limits the dosing of TTX. A low dose (<2 g) of TTX did not induce detectable nerve block in rats while a high dose (>5 g) was uniformly lethal (FIG. 21G). With Lip-TTX, death did not occur when the TTX dose was 20 g (FIG. 21G). Lip-ICG-TTX increased the maximum dose of TTX to 24.4 g. For Lip-Ph-TTX, no animal death was observed even when 45 g of TTX were administered, and none with 54 g TTX in Lip-Ph-ICG-TTX. However, increasing the dose of TTX in Lip-Ph-TTX and Lip-Ph-ICG-TTX led to significantly increased contralateral deficits in uninjected legs (FIG. 23), suggesting dose-dependent systemic toxicity.

[0283] Encapsulation of ICG also enabled triggerable drug release as results of photothermal effect of ICG (FIG. 24) Neurobehavior test showed that TTX-loaded ICG indeed significantly prolonged anesthesia duration and enabled repeatable light-triggered nerve blockade (FIG. 25), indicating incorporation of additional stabilization forces within lipid bilayers have great potential for on-demand pain relief.

Cytotoxicity and Tissue Reaction

[0284] The in vitro cytotoxicity to muscle and nerve of TTX formulations was evaluated in myoblast C2C1229 and pheochromocytoma PC1230 (FIGS. 26A-B) cell lines, that are used to assess myo- and neuro-toxicity respectively. After 48 h of incubation with different TTX formulations (TTX, TTX@Lipo, ICG+TTX@Lipo and TTX@Lipo-Ph), cell viabilities as assessed by MTS assays were similar to that of PBS, indicating that liposomes containing Ph-DPPC did not cause cytotoxicity.

[0285] Rats injected with different TTX formulations were euthanized 4 days and 14 days after sciatic nerve injections. At dissection, liposomes could be seen at the sciatic nerve, indicating the accurate injection of TTX-encapsulated liposomes (FIG. 27A). Small amounts of residual liposomes could still be identified at the injection site after 14 days (FIG. 28), consistent with the IVIS results.

[0286] Myotoxicity, neurotoxicity, and inflammation are well established side effects of local anesthetics.sup.35, although with site 1 sodium channel blockers tissue reaction is due to the vehicle, not the anesthetic. Drug delivery systems themselves are known to cause inflammation that can outlast the duration of nerve block and may enhance local anesthetic myotoxicity.

[0287] Animals injected with liposome-TTX formulations were euthanized 4 days or 14 days after injection. The sciatic nerves and surrounding tissues were harvested, sectioned, and stained. Tissue reaction was assessed by hematoxylin and eosin (H&E) staining (FIG. 27B). There was no statistically significant difference between the scores in Lip-TTX, Lip-ICG-TTX, Lip-Ph-TTX, and Lip-Ph-ICG-TTX (Table 6). No myotoxicity in animals in any group 4 days or 14 days after injection (Table 7). Mild to moderate inflammation was seen around the sciatic nerves, and there was no statistically significant difference in myotoxicity scores between these groups and the untreated groups. Inflammation is also commonly found in a broad range of delivery systems, including those for local anesthetics.sup.36, and is generally considered to be safe.sup.37. The inflammation and myotoxicity scores of the formulations are comparable or better than those of Exparel in the same animal model, suggesting the observed tissue reactions are acceptable for potential clinical use.

[0288] Sciatic nerves were stained with toluidine blue due to the low sensitivity of H&E staining for nerve damage (FIG. 27B and FIG. 29). No neurotoxicity or nerve injury was observed in any group, regardless of the time point. The perineural tissues were normal. Compared to untreated sciatic nerves, no changes were observed in axonal density or myelin structure in any TTX formulations.

TABLE-US-00006 TABLE 6 Inflammation 4 and 14 days after injection of formulations. Liposomal Inflammation Scores formulation Day 4 P value Day 14 P value Untreated 0 (0-0) 0 (0-0) Free TTX 0 (0-1) >0.05 0 (0-0) >0.05 TTX@Lipo 3 (3-3) <0.001 2 (2-2) <0.001 ICG + TTX@Lipo 2 (2-2) <0.001 1 (0-2) 0.040 TTX@Lipo-Ph 2.5 (2-3) <0.001 2 (1-3) 0.003 Inflammation scores: 0-4. P values result from the comparison of liposomal formulations to the untreated group (Mann Whitney U test). There were no statistically significant differences between liposomal formulations. Data are medians with 25th and 75th percentiles in parentheses; n = 4.

TABLE-US-00007 TABLE 7 Myotoxicity 4 and 14 days after injection of formulations. Liposomal Myotoxicity Scores formulation Day 4 P value Day 14 P Value Untreated 0 (0-0) 0 (0-0) Free TTX 0 (0-0) >0.05 0 (0-0) >0.05 TTX@Lipo 0.5 (0-4) >0.05 0.5 (0-1) >0.05 ICG + TTX@Lipo 0 (0-0) >0.05 0 (0-0) >0.05 TTX@Lipo-Ph 0 (0-0) >0.05 0.5 (0-4) >0.05 Myotoxicity scores: 0-6. P values result from the comparison of liposomal formulations to the untreated group (Mann Whitney U test). There was no statistically significant difference in myotoxicity scores between different liposome groups and the untreated groups. Data are medians with 25th and 75th percentiles in parentheses from four independent rats.

Analysis

[0289] By targeting the rate-limiting step in the release of encapsulated molecules from liposomes, it was shown here that the aromatized liposomes which incorporating aromatic groups within lipid bilayers for drug loading can sustain drug delivery. A chemical strategy was conceived to conjugate aromatic groups to the acyl chains of phospholipids. This synthetic route can be used for the preparation of a variety of acyl chain-modified phospholipids. Combination of aromatic groups-conjugated phospholipids and natural phospholipids formulates aromatized liposomes with similar size, zeta potential, and polydispersity to conventional liposomes. Release kinetics revealed that aromatized liposomes enabled increased drug loading and decreased release rate of hydrophilic payloads with different water solubility, molecular weight, and charge state. In vivo assessment revealed that aromatized liposomes significantly prolonged the therapeutic duration of encapsulated TTX, a local anesthetic with low molecular weight, high potency and narrow therapeutic window. Lip-Ph-TTX achieved more than 57 hours of continuous nerve block, compared to that of free TTX (2.5 hours) and unmodified liposomes (19.9 hours). Combination of aromatized liposomes and physical encapsulation of hydrophobic ICG into lipid bilayers further extended the duration of action to 73.5 h, a marked improvement over previous TTX formulations.sup.39. Aromatized liposomes also drastically mitigated the toxicity and expanded the therapeutic window of TTX, addressing three major challenges in applying neurotoxins like TTX as local anesthetics for clinical use. In current animal models, it is difficult to achieve such long nerve block without sustained drug delivery systems as evidenced by the fact that 5 g was fatal for rats.

[0290] The decreased permeability of aromatized liposomes was attributed to the incorporation of aromatic groups within lipid bilayers. First, n-n stacking interactions between aromatic groups noncovalently crosslinked lipids from opposite layers and adjacent lipids from the same layer, providing additional stabilization forces to rigidify lipid bilayers.sup.40. Incorporation of phenoxy and coumarin groups increased the Tm of phospholipids from 41.1 C. to 47.4 and 66.1 C., respectively. The coumarin group was slightly less hydrophobic than phenoxy group due to the existence of lactone structure, indicating the additional stabilization of lipid bilayers resulted from n-n stacking interactions instead of hydrophobic interactions. These observations indeed suggested that incorporation of aromatic groups within lipid bilayers was a practical and powerful approach to decrease the permeability of liposomes. The effect of n-n stacking interactions on stabilizing lipid bilayers was also validated by the fact that simply encapsulating aromatic ICG into lipid bilayers also enabled prolonged nerve block in vivo. The effect of aromatic groups modification in stabilizing drug delivery systems and preventing premature drug release was also found in polymeric micelles.sup.41 and hydrogels.sup.42, indicating broad applicability in applying n-n stacking interactions for sustained drug delivery. Second, terminal aromatic groups on acyl chains likely fill the free space within lipid bilayers and serve as extra physical barriers to slow down the diffusion of hydrophilic molecules.sup.43. Thirdly, the presence of aromatic groups is unfavorable for the lateral motion of phospholipids and transient formation of free spaces within lipid bilayers.sup.44 due to the intrinsic rigidity of ring structures.sup.43, which probably decreases the fluidity and permeability of lipid bilayers.

[0291] Acute perioperative and chronic pain are among the most prevalent medical conditions.sup.45. Limited therapeutic alternatives to pain management have produced an overreliance on opioid anesthetics worldwide.sup.46. Conventional anesthetics are short in duration and the associated neurotoxicity and myotoxicity are severe.sup.39. A slight overdose of drugs like fentanyl may lead to severe side effects or even death.sup.47. TTX and other neurotoxins have emerged as appealing non-opioid alternatives for prolonged pain relief due to their high sodium channel sensitivity, extremely high potency, minimal cardiovascular toxicity, and long-lasting effect.sup.26. However, the dosing of these anesthetics is limited because their severe systemic toxicity and narrow therapeutic window.sup.17. This work is of direct relevance to the extensive efforts underway to exploit alternative approaches in addressing the growing epidemic of opioid abuse and overdose. Aromatized liposomes may provide new opportunities for prolonged nonopioid local pain relief.

[0292] Aromatized liposomes, as previously described above, may also be attractive for the encapsulation and sustained release of therapeutics with aromatic rings, which account for more than 80% of globally approved drugs.sup.48. Beyond drug delivery, membrane-modified liposomes could serve as good model systems for the investigation of biological membranes such as structural organizations and the interrelation between different components at the molecular level, which remain largely elusive.sup.49. The aromatized liposomes can be prepared following existing FDA guidelines with aromatic groups-conjugated phospholipids, which favors their potential clinical translations.sup.9.

[0293] As discussed above, herein is described the improvement of sustained drug release by the aromatization of the inner aspect of liposomal lipid bilayers. Aromatized liposomes increased the drug loading and considerably decreased the release of payloads with different water solubilities and molecular weights. These changes had an impact in vivo: aromatization prolonged the duration of local anesthesia from TTX liposomes to more than 3 days, and curtailed systemic toxicity.

[0294] Aromatic groups were incorporated within the lipid bilayers of liposomes to target the rate-limiting step in the passive diffusion of molecules from liposomes. There are a number of mechanisms by which this might occur. n-n stacking interactions, non-covalent crosslinked adjacent phospholipids, decreased lateral motion of lipids within the liposomal membranes, stabilized lipid bilayers, decreased their fluidity and permeability. The rigid ring structures of aromatic groups may reduce the fluidity and permeability of lipid bilayers as observed in some bacteria. The aromatized liposomes may find broad application in the encapsulation and release of a broad range of drugs, including chemotherapeutics, macromolecular drugs, and proteins drugs. The release kinetics suggested the reduced release of small molecules due to aromatization likely correlates to multiple factors. Neither molecular weight nor hydrophilicity correlated strongly with the degree of reduction. The reduced release of macromolecules as result of aromatization correlated with their molecular weight and hydrophilicity.

[0295] There were constraints on the effect of aromatization on the loading of liposomes and the resulting release kinetics. While the addition of one ring enhanced performance, a second ring (coumarin) had minimal effect on liposome performance, and greatly increased viscositywhich would adversely affect removal of free drug from the formulation, and injectability in the clinical. Addition of a bulky three-member ring (DBCO) actually impaired performance. That impairment may be due to the bulky group interfering with the packing of hydrocarbon chains and creating free space within lipid bilayers, which could increase the permeability of liposomes. The DBCO group was conjugated to the acyl chain via two amide bonds, which may also make the liposome leakier. It remains to be seen whether further hydrophobic non-bulky modifications of aromatic groups would further enhance liposome performance.

[0296] Conventional anesthetics are short in duration and can have associated neurotoxicity and myotoxicity. Limited therapeutic alternatives in pain management have produced an overreliance on opioid anesthetics worldwide. A long-acting formulation of bupivacaine liposome (Exparel) has been approved for clinical use in local anesthesia. However, a recent study revealed that Exparel did not demonstrate significant pain relief compared to standard bupivacaine in 74.58% of randomized clinical trials. Developing new formulations for prolonged non-addictive pain relief has long been of research and clinical interest. Herein is described a formulation that provides three days of nerve block from a single injection. Addition of dexamethasone further extended the duration of analgesia to over a week. One important consideration in that context is that use in larger animals (such as humans) will allow for the use of larger doses, enabling longer blocks. There would also be less systemic toxicity because it tracks relatively linearly with the compound's volume of distribution (i.e., the animal's size) while the duration of nerve block does not. Nerve block lasting 2-3 days would cover the duration of most postoperative pain. Longer durations would be useful for severe localized chronic pain like cancer pain.

[0297] In summary, lipid bilayer-engineered vesicles were developed for sustained drug delivery. Incorporation of additional physical barrier and stabilization forces within lipids bilayers increased the drug loading, reduced the burst release of payloads, prolonged the action time, expanded the therapeutic window, and mitigated the systemic toxicity of encapsulated drugs.

Example 2. Methods for Example 1

Materials

[0298] 16-bromohexadecanoic acid, acetyl chloride, anhydrous methanol, phenol, potassium carbonate, anhydrous acetonitrile, anhydrous tetrahydrofuran, hydrochloric acid, 2,6-dichlorobenzoyl chloride, 1-methylimidazole and cholesterol were purchased from Sigma-Aldrich (St. Louis, MO, USA). 16:0 DPPC, 18:1 DOPC, 18:0 DSPG, 16:0 lyso PC, Cy7-DOPC were purchased from Avanti Polar Lipids. Cyanine 7 carboxylic acid was purchased from Lumiprobe Corporation. Tetrodotoxin (TTX) was obtained from Abcam. TTX ELISA kits were purchased from Reagen LLC. Dulbecco's phosphate buffered saline (PBS) were purchased from Thermo Fisher Scientific.

Synthesis of Aromatic Group-Modified Phospholipids

[0299] To a 100 mL round-bottom flask was added 16-bromohexadanoic acid (4 mmol), acetyl chloride (10 mmol) and anhydrous methanol. The solution was stirred overnight at room temperature. After removing of solvent, the resulting solids were resuspended in dichloromethane, then washed with NaHCO.sub.3 (2200 mL) and brine (2200 mL). The organic phase was separated, dried over Na.sub.2SO.sub.4 and concentrated to afford the methyl 16-bromohexadecanoate in excellent yield (90-95%). .sup.1H NMR (CDCl.sub.3, 400 MHz, ppm, ): 3.66 (s, 3H), 3.44-3.36 (t, 2H), 2.33-2.25 (t, 2H), 1.89-1.79 (m, 2H), 1.66-1.55 (m, 2H), 1.47-1.36 (m, 2H), 1.34-1.21 (m, 20H). .sup.13C NMR (CDCl.sub.3, 400 MHz, ppm, ): 174.09, 51.33, 34.06, 33.78, 32.86, 29.63, 29.61, 29.58, 29.54, 29.45, 29.26, 29.15, 28.78, 28.19, 24.95. ESI-MS: m/z calculated for C.sub.17H.sub.33BrO.sub.2 [M+H].sup.+: 349.2; observed: 349.2.

[0300] The methyl 16-bromohexadecanoate (4 mmol) and Na.sub.2CO.sub.3 (10 mmol) was added in 30 mL MeCN in 100 mL round-bottom bottle flask and the solution was heated to 60 C. Phenol (5 mmol) was dissolved in 1 mL MeCN and added to the reaction mixture. The reaction mixture was stirred for 16 hours at 60 C. The reaction mixture was cooled to room temperature, filtrated to remove the salt and concentrated. The crude material was resuspended with in dichloromethane, then washed with NaHCO.sub.3 (2200 mL) and brine (2200 mL). The organic phase was separated, dried over Na.sub.2SO.sub.4 and concentrated. The obtained crude materials was purified by silica gel flash chromatography eluting with 10:1 hexanes:ethyl acetate (EtOAc) to afford methyl 16-phenoxyhexadecanoate in good yield (69-75%). .sup.1H NMR (CDCl.sub.3, 400 MHz, ppm, ): 7.34-7.22 (m, 2H), 6.96-6.85 (m, 3H), 3.99-3.91 (t, 2H), 3.66 (s, 3H), 2.35-2.25 (t, 2H), 1.83-1.71 (m, 2H), 1.68-1.56 (m, 2H), 1.50-1.39 (m, 2H), 1.39-1.20 (m, 20H). .sup.13C NMR (CDCl.sub.3, 400 MHz, ppm, ): 174.11, 159.28, 129.52, 120.58, 114.66, 68.05, 51.34, 34.21, 29.78, 29.77, 29.72, 29.57, 29.55, 29.45, 29.38, 29.20, 26.21, 24.82. ESI-MS: m/z calculated for C.sub.23H.sub.38O.sub.3[M+H].sup.+: 363.3; observed: 363.3.

[0301] To a 100 mL round-bottom flask were added the methyl 16-phenoxyhexadecanoate (4 mmol), THF (20 mL) and deionized water (20 mL). 1 M NaOH (20 mmol) was then added dropwise and reaction mixture was stirred for 16 hours at room temperature. The reaction mixture was diluted with EtOAc (100 mL), washed with 10% HCl (50 mL) washed with deionized water (2100 mL) and brine (100 mL). The organic phase was separated, dried over Na.sub.2SO.sub.4 and concentrated to afford the 16-phenoxyhexadecanoic acid in good yield (70-80%). .sup.1H NMR (CDCl.sub.3, 400 MHz, ppm, ): 7.34-7.22 (m, 2H), 6.96-6.85 (m, 3H), 3.99-3.91 (t, 2H), 2.39-2.30 (t, 2H), 1.84-1.72 (m, 2H), 1.70-1.58 (m, 2H), 1.52-1.41 (m, 2H), 1.39-1.20 (m, 20H). .sup.13C NMR (CDCl.sub.3, 400 MHz, ppm, ): 180.31, 159.28, 129.52, 120.58, 114.66, 68.05, 34.21, 29.78, 29.77, 29.72, 29.57, 29.55, 29.45, 29.38, 29.20, 26.21, 24.82. ESI-MS: m/z calculated for C.sub.17H.sub.33BrO.sub.2 [M+H].sup.+: 349.3; observed: 349.3.

[0302] To a solution of 16-phenoxyhexadecanoic acid (1.0 mmol), 16:0 lyso-PC (0.9 mmol) and 1-methylimidazole (3.0 mmol) in CHCl.sub.3 (6 mL) was added 2,6-dichlorobenzoyl chloride (2.0 mmol) and the resulting mixture was stirred for 16 hours at room temperature. The reaction mixture was then concentrated under reduced pressure. The crude was purified by silica gel chromatography (1:1 MeOH/CH.sub.2Cl.sub.2) affording 1-palmitoyl-2-(16-phenoxy) palmitoyl-sn-glycero-3-phosphocholine (Ph-DPPC) as a white wax in moderate yield (50-60%). .sup.1H NMR (CD.sub.3OD, 400 MHz, ppm, ): 7.28-7.20 (m, 2H), 6.94-6.85 (m, 3H), 5.29-5.20 (m, 1H), 4.48-4.39 (m, 1H), 4.32-4.22 (m, 2H), 4.21-4.13 (m, 1H), 4.04-3.98 (t, 2H), 3.97-3.92 (t, 2H), 3.66-3.61 (t, 2H), 3.22 (s, 9H), 2.38-2.28 (m, 4H), 1.80-1.72 (m, 2H), 1.66-1.55 (m, 4H), 1.52-1.42 (m, 4H), 1.41-1.22 (m, 44H) 0.93-0.86 (t, 3H). .sup.13C NMR (CDCl.sub.3, 400 MHz, ppm, ): 174.93, 173.62, 160.61, 130.39, 121.49, 115.53, 71.79, 68.88, 67.50, 64.92, 63.69, 60.49, 54.75, 35.12, 34.94, 33.08, 30.81, 30.78, 30.71, 30.66, 30.52, 30.49, 30.46, 30.45, 30.23, 30.20, 27.19, 26.05, 26.03, 23.74. ESI-MS: m/z calculated for C.sub.46H.sub.84NO.sub.9P [M+H].sup.+: 826.6; observed: 826.6.

Liposome Preparation and Characterization

[0303] The liposomes were prepared using the thin-film hydration method. The lipid formulation (Ph-DPPC, DOPC, DSPG and cholesterol at a molar ratio of 3:3:2:3) was dissolved in a solution of chloroform and methanol (ratio: 9:1). The solvent was evaporated under reduced pressure, and the lipid was redissolved in tert-butanol, followed by freeze-drying. The lipid cake was hydrated with SRho, Bup, TTX, ICG, polyethylene glycol, FITC-Albumin in PBS buffer (pH=7.4) or hydrated with bupivacaine hydrochloride, or doxorubicin hydrochloride in saline. After ten freeze-thaw cycles, the solution was dialyzed against PBS for 48 hours in a dialysis tube with a molecular mass cut-off of 1,000 kDa. The dialysis media were changed with fresh PBS at least twice a day. Drugs and dye in all formulations were quantitated after disruption of the liposome with octyl--D-glucopyranoside (100 mM, volume ratio of 2:1 to formulations).

Instruments and Characterization of Materials

[0304] .sup.1H and .sup.13C NMR experiments were measured on a Varian 400 M NMR spectrometer. Dynamic light scattering (DLS) (Zetasizer Pro; Malvern Panalytical, USA) was used to determine the hydrodynamic diameter and zeta potential of liposomes. The liposome solutions were also characterized by transmission electron microscopy (TEM, Tecnai G2 T20; FEI company, OR, USA) using a negative staining method with uranyl acetate (1.0% w/w). An Agilent 1260 series high-performance liquid chromatography (HPLC) with a UV-vis detector was used for analyzing the Bupivacaine hydrochloride in the releasing experiments. An Agilent Infinity lab LC/MSD XT single quadrupole mass analyzer was used for molecular weight measurement. Viscosity was tested on a TA DHR-2 rheometer with a frequency of 1 Hz, shear strain of 1%, and a temperature of 25 C.

Partition Coefficient Quantification

[0305] Octanol-water partition coefficients (Log P) were quantified using a miniaturized shake-flask approach. To investigate the analytes in either fully ionized or neutral state as well as their pH-dependent partitioning behavior, measurements were performed using sodium-based buffer solutions at a constant ionic strength of 0.1 M covering three different pH values: pH 4.0 (citrate buffer), pH 7.4 (phosphate buffer), and pH 10.2 (carbonate buffer). All buffer solutions were saturated with octanol prior to analysis and vice versa. All octanol-water mixtures were vortexed for 5 min and then stirred for 24 h at room temperature to reach equilibrium and phase distribution. Kinetic measurements were performed to confirm the equilibrium conditions in the setup. After equilibration and phase separation, all samples were analyzed on an Agilent HPLC 1260 or a plate reader (BioTek, Winooski, VT).

[0306] As described in the liposome preparation section, the lipid cake was hydrated with SRho, TTX, polyethylene glycol (PEG), FITC-albumin in PBS buffer (pH=7.4) or hydrated with bupivacaine hydrochloride, doxorubicin hydrochloride in saline. The pH of bupivacaine hydrochloride and doxorubicin hydrochloride in physiological saline at the concentration tested is close to 4 as measured by a Mettler Toledo Seven Easy pH meter. Thus, Log P values of bupivacaine hydrochloride and doxorubicin hydrocholoride at pH 4.0 were used for the comparison of loading and release. Log P values at pH 7.4 of other payloads were used for the comparison of loading and release.

Cell Culture

[0307] Cell culture of C2C12 mouse myoblasts (American Type Culture Collection (ATCC), Manassas, VA, USA) and PC12 rat adrenal gland pheochromocytoma cells (ATCC, Manassas, VA, USA) were performed. C2C12 cells were cultured in DMEM with 20% FBS and 1% Penicillin Streptomycin. Cells were seeded onto a 24-well plate at 50,000 cells mL-1 and incubated for 10-14 days in DMEM with 2% horse serum and 1% Penicillin Streptomycin to differentiate into myotubules. PC12 cells were grown in DMEM with 12.5% horse serum, 2.5% FBS, and 1% Penicillin Streptomycin. Cells were seeded onto a 24-well-plate, and 50 ng mL-1 nerve growth factor was added 24 hours after seeding.

Cell Viability

[0308] To determine the cytotoxicity, cells were exposed to different TTX formulations using a 24-well Transwell membrane system (Costar 3495, pore size 0.4 m) (Corning Incorporated, ME, USA). Cells were incubated in 0.9 mL of media in the cell culture wells, and 100 L of test samples were added above the Transwell membranes, which were immersed in the media in the wells. Cell viability was evaluated by the MTS assay (Promega, WI, USA) 96 hours after incubation.

[0309] Cytotoxicity of the materials to muscle and nerve cells was also assessed by a direct contact setup. Different formulations were directly added into the cell culture media and incubated in the media bathing the cells (i.e., in direct contact with them) in conventional cell culture wells (lipid concentration: 20 mg/mL). After 24 hours, cell viabilities were evaluated with the MTS assay, and their survival expressed as percentages of results in untreated cells.

In Vitro Drug Release

[0310] Cumulative release of small molecules (e.g., Sulforhodamine B, Tetrodotoxin, Bupivacaine hydrochloride, and doxorubicin hydrochloride) were performed by placing 200 L of samples into a Slide-A-Lyzer MINI dialysis device (Thermo Fisher Scientific, Waltham, MA) with a 10,000 MW cut-off, further dialyzed with 14 mL release media and incubated at 37 C. on a platform shaker (New Brunswick Innova 40, 60 rpm). At predetermined intervals, the dialysis solution was exchanged with fresh, pre-warmed release media. The release media of Sulforhodamine B, Tetrodotoxin was PBS (pH=7.4). The release media of Bupivacaine hydrochloride, and doxorubicin hydrochloride was physiological saline. The concentration of TTX in release media was quantified by an enzyme-linked immunosorbent assay (ELISA, Reagen LLC). The concentration of Sulforhodamine B in release media was determined by a plate reader (BioTek, Winooski, VT) with excitation and emission wavelengths of 560 nm and 580 nm. The concentration of bupivacaine hydrochloride (Bup) in release media was determined by high-performance liquid chromatography. The concentrations of SRho, TTX and Bup in release studies were 4 mM, 0.3 mM and 20 mM as reported previously.

[0311] The maximum cut-off of commercially available Slide-A-Lyzer MINI dialysis device was 20,000 MW. Thus, the cumulative release of macromolecules was performed by placing 500 L of samples into a Float-A-Lyzer G2 dialysis devices (Spectrum Laboratories Inc, Piscataway, NJ) with a 1000,000 MW cut-off, further dialyzed with 14 mL PBS and incubated at 37 C. on a platform shaker (New Brunswick Innova 40, 60 rpm). At predetermined intervals, the dialysis solution was exchanged with fresh, pre-warmed release media. The concentration of dye-conjugated macromolecules was determined by a plate reader (BioTek, Winooski, VT). The concentrations of SRho-PEG1k, SRho-PEG10k and FITC-Ab in release studies were 1 mM, 0.5 mM and 0.5 mM, respectively.

Animal Studies

[0312] Animal studies were conducted following protocols approved by the Boston Children's Hospital Animal Care and Use Committee in accordance with the guidelines of the International Association for the Study of Pain. Adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA) weighing 350-400 g were housed in groups under a 12-hours/12-hours light/dark cycle with lights on at 6:00 AM.

[0313] Sciatic nerve injections were performed with a 23 G needle at the left sciatic nerve under brief isoflurane-oxygen anesthesia. The needle was introduced posteromedial to the greater trochanter, pointing in the anteromedial direction, and upon contact with bone, the formulations were injected onto the sciatic nerve.

[0314] Neurobehavioural testing was conducted on both hindquarters. Deficits in the right (uninjected) extremity served as a metric of systemic drug distribution. Sensory nerve blockade was assessed by modified hotplate testing. The hind paws were exposed in sequence (left then right) to a 56 C. hot plate (Stoelting, Wood Dale, IL, USA), and the time the animal allowed its paw to remain on the hotplate (thermal latency) was measured. A thermal latency of 2 seconds indicated no nerve blockade (baseline), and a thermal latency of 12 seconds was maximal latency. Successful nerve blockade was defined as achieving a thermal latency above 7 seconds. Hind paws were removed from the hotplate after 12 seconds to prevent thermal injury. Measurements were repeated three times in each animal at each time point and the median was used for further data analysis.

[0315] Motor nerve block was assessed by a weight-bearing test to determine the motor strength of the rat's hind paw. The rat was positioned with one hind paw on a digital balance and was allowed to bear its own weight. The maximum weight that the rat could bear without the ankle touching the balance was recorded, and motor block was considered achieved when the motor strength was less than half maximal. Measurements were repeated three times at each time point and the median was used for further data analysis.

[0316] Duration of sensory block were calculated as the time required for thermal latency to return to 7 seconds (halfway between the baseline and maximal latencies). The duration of motor block was defined as the time it took for the weight-bearing to return to halfway between normal and maximal block.

Laser Scanning Confocal Microscopy (LSCM) Imaging

[0317] Cy7-conjugated DOPC was used in the preparation of Cy7-labeled liposomes following the same procedures as described previously with Cy7 molar concentration at 0.2 mg/mL. Under brief isoflurane-oxygen anesthesia, rats were injected with 0.3 mL of different Cy7 labeled liposomal formulations. Sciatic nerves together with surrounding tissues were harvested and embedded into OCT compound (VWR, PA, USA), then frozen and stored at 20 C. Sections (10 m) were prepared using a cryostat microtome (Leica CM3050 S, Wetzlar, Germany) and mounted onto glass slides. Afterward, slides were fixed with 4% paraformaldehyde for 20 minutes at room temperature, washed in PBS (pH 7.4) 3 times. Nuclei were stained with Hoechst 33342. The slices were imaged using a Zeiss LSM 710 multi-photon confocal microscopy (Carl Zeiss AG, Oberkochen, Germany).

In Vivo Imaging System (IVIS) Imaging

[0318] Cy7-conjugated DOPC was used in the preparation of Cy7-labeled liposomes following the same procedures as described previously with Cy7 molar concentration at 0.2 mg/mL. Under isoflurane-oxygen anesthesia, rats were shaved and injected with 0.3 mL of different Cy7-labeled liposomal formulation. The in vivo fluorescence images were captured, and the fluorescence intensity was evaluated at predetermined time points post-injection (under brief isoflurane-oxygen anesthesia) using a Spectrum IVIS (PerkinElmer, MA, USA). Whole-body animal images were recorded non-invasively. The 745 nm excitation filter and the 800 nm emission filter were used for the imaging. Quantitative analysis was carried out using the Live Imaging software of the IVIS.

Tissue Harvesting and Histology

[0319] Rats were sacrificed at 4 days and 14 days after the injection (it was determined that these time points were useful in evaluating both acute and chronic inflammation and myotoxicity), and the sciatic nerve was harvested together with surrounding tissues. The samples were scored for inflammation (0-4) and myotoxicity (0-6). All scoring and other histological assessments were performed by an observer blinded as to the nature of the individual samples.

[0320] The inflammation score was a subjective quantification of severity in which 0 was normal and 4 was severe inflammation. The myotoxicity score was determined by the nuclear internalization and regeneration of myocytes, two representative characteristics of local anesthetics' myotoxicity. Nuclear internalization was characterized by myocytes having nuclei located away from their usual location at the periphery of the cell. Regeneration was characterized by the presence of shrunken myocytes with basophilic cytoplasm. The scoring scale was as follows: 0=normal; 1=perifascicular internalization; 2=deep internalization (more than five cell layers); 3=perifascicular regeneration; 4=deep tissue regeneration (more than five cell layers); 5=hemifascicular regeneration; 6=holofascicular regeneration.

[0321] To evaluate the neurotoxicity of formulations, the sciatic nerves were fixed in Karnovsky's KII solution, processed and Epon-embedded for toluidine blue staining. They were assessed by optical microscopy in a masked fashion.

Statistical Analysis.

[0322] Statistical comparisons were performed using the Student t-test (one-sided) unless stated otherwise. Thermal latency, inflammation and myotoxicity scores were reported as medians and quartiles due to their ordinal or non-Gaussian character. Data are presented as meansSD (n=4) in release kinetics, cell work, neurobehavioral, and histology studies. Data were considered statistically significant if P<0.05 (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05).

REFERENCES

[0323] 1. Torchilin, V. P. Recent advances with liposomes as pharmaceutical carriers. Nature reviews Drug discovery 4, 145 (2005). [0324] 2. Bulbake, U., Doppalapudi, S., Kommineni, N. & Khan, W. Liposomal formulations in clinical use: an updated review. Pharmaceutics 9, 12 (2017). [0325] 3. Xing, H., Hwang, K. & Lu, Y. Recent developments of liposomes as nanocarriers for theranostic applications. Theranostics 6, 1336 (2016). [0326] 4. Allen, T. M. & Cullis, P. R. Liposomal drug delivery systems: from concept to clinical applications. Advanced drug delivery reviews 65, 36-48 (2013). [0327] 5. Bozzuto, G. & Molinari, A. Liposomes as nanomedical devices. International journal of nanomedicine 10, 975 (2015). [0328] 6. Barenholz, Y. Liposome application: problems and prospects. Current opinion in colloid & interface science 6, 66-77 (2001). [0329] 7. Kakkar, A., Traverso, G., Farokhzad, O. C., Weissleder, R. & Langer, R. Evolution of macromolecular complexity in drug delivery systems. Nature Reviews Chemistry 1, 1-17 (2017). [0330] 8. Papahadjopoulos, D., Nir, S. & Ohki, S. Permeability properties of phospholipid membranes: effect of cholesterol and temperature. Biochimica et Biophysica Acta (BBA)-Biomembranes 266, 561-583 (1972). [0331] 9. Pattni, B. S., Chupin, V. V. & Torchilin, V. P. New developments in liposomal drug delivery. Chemical reviews 115, 10938-10966 (2015). [0332] 10. Moon, J. J., et al. Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses. Nature materials 10, 243 (2011). [0333] 11. Storck, E. M., Ozbalci, C. & Eggert, U. S. Lipid cell biology: a focus on lipids in cell division. Annual review of biochemistry 87, 839-869 (2018). [0334] 12. Harayama, T. & Riezman, H. Understanding the diversity of membrane lipid composition. Nature reviews Molecular cell biology 19, 281 (2018). [0335] 13. Yang, N. J. & Hinner, M. J. Getting across the cell membrane: an overview for small molecules, peptides, and proteins. in Site-specific protein labeling 29-53 (Springer, 2015). [0336] 14. Lodish, H., et al. Diffusion of small molecules across phospholipid bilayers. Molecular Cell Biology (2000). [0337] 15. Barba-Bon, A., Nilam, M. & Hennig, A. Supramolecular chemistry in the biomembrane. ChemBioChem (2020). [0338] 16. Sezgin, E., Levental, I., Mayor, S. & Eggeling, C. The mystery of membrane organization: composition, regulation and roles of lipid rafts. Nature reviews Molecular cell biology 18, 361 (2017). [0339] 17. Martinez, C. R. & Iverson, B. L. Rethinking the term pi-stacking. Chemical Science 3, 2191-2201 (2012). [0340] 18. Krieg, E., Bastings, M. M., Besenius, P. & Rybtchinski, B. Supramolecular polymers in aqueous media. Chemical reviews 116, 2414-2477 (2016). [0341] 19. Deng, J.-H., et al. - stacking interactions: Non-negligible forces for stabilizing porous supramolecular frameworks. Science advances 6, eaax9976 (2020). [0342] 20. Zhuang, W.-R., et al. Applications of - stacking interactions in the design of drug-delivery systems. Journal of Controlled Release 294, 311-326 (2019). [0343] 21. Morin, C., Simon, D. & Sautet, P. Chemisorption of benzene on Pt (111), Pd (111), and Rh (111) metal surfaces: a structural and vibrational comparison from first principles. The Journal of Physical Chemistry B 108, 5653-5665 (2004). [0344] 22. Li, L., Vorobyov, I. & Allen, T. W. The different interactions of lysine and arginine side chains with lipid membranes. The Journal of Physical Chemistry B 117, 11906-11920 (2013). [0345] 23. Acharya, H. P. & Kobayashi, Y. A rapid condensation between lysophosphorylcholine and fatty acids with an easily separable amine base. Synlett 2005, 2015-2018 (2005). [0346] 24. Rwei, A. Y., et al. Ultrasound-triggered local anaesthesia. Nature biomedical engineering 1, 644-653 (2017). [0347] 25. Lahaye, L. A. & Butterworth, J. F. Site-1 Sodium Channel Blockers as Local AnestheticsWill Neosaxitoxin Supplant the Need for Continuous Nerve Blocks?Anesthesiology: The Journal of the American Society of Anesthesiologists 123, 741-742 (2015). [0348] 26. Kohane, D. S., et al. A re-examination of tetrodotoxin for prolonged duration local anesthesia. Anesthesiology: The Journal of the American Society of Anesthesiologists 89, 119-131 (1998). [0349] 27. Kohane, D. S., et al. Prolonged duration local anesthesia from tetrodotoxin-enhanced local anesthetic microspheres. Pain 104, 415-421 (2003). [0350] 28. Bane, V., Lehane, M., Dikshit, M., O'Riordan, A. & Furey, A. Tetrodotoxin: Chemistry, toxicity, source, distribution and detection. Toxins 6, 693-755 (2014). [0351] 29. Lomonte, B., et al. Comparative study of the cytolytic activity of myotoxic phospholipases A2 on mouse endothelial (tEnd) and skeletal muscle (C2C12) cells in vitro. Toxicon 37, 145-158 (1999). [0352] 30. Slotkin, T. A., MacKillop, E. A., Ryde, I. T., Tate, C. A. & Seidler, F. J. Screening for developmental neurotoxicity using PC12 cells: comparisons of organophosphates with a carbamate, an organochlorine, and divalent nickel. Environmental health perspectives 115, 93-101 (2007). [0353] 31. Kohane, D. S. Microparticles and nanoparticles for drug delivery. Biotechnology and bioengineering 96, 203-209 (2007). [0354] 32. McAlvin, J. B., et al. Multivesicular liposomal bupivacaine at the sciatic nerve. Biomaterials 35, 4557-4564 (2014). [0355] 33. Wang, Y.-W., et al. Dye-enhanced graphene oxide for photothermal therapy and photoacoustic imaging. Journal of Materials Chemistry B 1, 5762-5767 (2013). [0356] 34. Reinhart, M. B., Huntington, C. R., Blair, L. J., Heniford, B. T. & Augenstein, V. A. Indocyanine green: historical context, current applications, and future considerations. Surgical innovation 23, 166-175 (2016). [0357] 35. Neal, J. M., Salinas, F. V. & Choi, D. S. Local anesthetic-induced myotoxicity after continuous adductor canal block. Regional Anesthesia & Pain Medicine 41, 723-727 (2016). [0358] 36. Jia, X., Colombo, G., Padera, R., Langer, R. & Kohane, D. S. Prolongation of sciatic nerve blockade by in situ cross-linked hyaluronic acid. Biomaterials 25, 4797-4804 (2004). [0359] 37. Padera, R. F., Tse, J. Y., Bellas, E. & Kohane, D. S. Tetrodotoxin for prolonged local anesthesia with minimal myotoxicity. Muscle & Nerve: Official Journal of the American Association of Electrodiagnostic Medicine 34, 747-753 (2006). [0360] 38. Tibbitt, M. W., Dahlman, J. E. & Langer, R. Emerging frontiers in drug delivery. Journal of the American Chemical Society 138, 704-717 (2016). [0361] 39. Santamaria, C. M., Woodruff, A., Yang, R. & Kohane, D. S. Drug delivery systems for prolonged duration local anesthesia. Materials Today 20, 22-31 (2017). [0362] 40. Fleming, S. & Ulijn, R. V. Design of nanostructures based on aromatic peptide amphiphiles. Chemical Society Reviews 43, 8150-8177 (2014). [0363] 41. Shi, Y., et al. Complete regression of xenograft tumors upon targeted delivery of paclitaxel via HH stacking stabilized polymeric micelles. ACS nano 9, 3740-3752 (2015). [0364] 42. Li, F., et al. Smart Hydrogels Co-switched by Hydrogen Bonds and n-R Stacking for Continuously Regulated Controlled-Release System. Advanced Functional Materials 20, 669-676 (2010). [0365] 43. Monteiro, N., Martins, A., Reis, R. L. & Neves, N. M. Liposomes in tissue engineering and regenerative medicine. Journal of the Royal Society Interface 11, 20140459 (2014). [0366] 44. Vaz, W. L., Goodsaid-Zalduondo, F. & Jacobson, K. Lateral diffusion of lipids and proteins in bilayer membranes. FEBS letters 174, 199-207 (1984). [0367] 45. Wu, C. L. & Raja, S. N. Treatment of acute postoperative pain. The Lancet 377, 2215-2225 (2011). [0368] 46. Califf, R. M., Woodcock, J. & Ostroff, S. A proactive response to prescription opioid abuse. New England Journal of Medicine 374, 1480-1485 (2016). [0369] 47. Schmid, C. L., et al. Bias factor and therapeutic window correlate to predict safer opioid analgesics. Cell 171, 1165-1175. e1113 (2017). [0370] 48. Mao, F., et al. Chemical structure-related drug-like criteria of global approved drugs. Molecules 21, 75 (2016). [0371] 49. Siontorou, C. G., Nikoleli, G.-P., Nikolelis, D. P. & Karapetis, S. K. Artificial lipid membranes: past, present, and future. Membranes 7, 38 (2017).

EQUIVALENTS AND SCOPE

[0372] In the claims articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0373] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the disclosure, or aspects described herein, is/are referred to as comprising particular elements and/or features, certain embodiments described herein or aspects described herein consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms comprising and containing are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments described herein, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0374] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment described herein can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0375] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.