Conjugated lipomers and uses thereof

10933139 · 2021-03-02

Assignee

Massachusetts Institute Of Technology (Cambridge, MA)

Inventors

Cpc classification

International classification

Abstract

The present invention provides inventive conjugated polyethyleneimine (PEI) polymers and conjugated aza-macrocycles (collectively referred to herein as conjugated lipomers or lipomers) containing one or more groups of the formula (iii): ##STR00001##
wherein R.sup.3 and R.sup.4 are as defined herein. Also provided are compositions comprising the inventive conjugated lipomers, and methods of preparation and use.

Claims

1. A conjugated lipomer of the Formula (II):
A private use character Parenopenst L.sup.1 private use character Parenclosest .sub.nZ(II) or a salt thereof, wherein: each instance of L.sup.1 is independently formula: ##STR00315## provided that at least one L.sup.1 is formulae (ii), and at least one L.sup.1 is formulae (iii); n is an integer between 3 and 25, inclusive; each instance of R.sup.2 is independently hydrogen, a substituted or unsubstituted polyethyleneimine, or a group of the formula (iii): ##STR00316## each instance of R.sup.3 is independently substituted or unsubstituted C.sub.8-20 alkyl; each instance of R.sup.4 is hydrogen; A is N(R.sup.5).sub.2, wherein each instance of R.sup.5 is independently hydrogen, or a group of the formula (iii); and Z is hydrogen, or a group of the formula (iii).

2. The conjugated lipomer of claim 1, or a salt thereof, wherein each instance of L.sup.1 is independently selected from formula (ii) and formula (iii).

3. The conjugated lipomer of claim 1, or a salt thereof, wherein n is an integer between 5 and 25, inclusive.

4. The conjugated lipomer of claim 1, or a salt thereof, wherein n is an integer between 10 and 15, inclusive.

5. The conjugated lipomer of claim 1, or a salt thereof, wherein the conjugated lipomer is prepared from a polyethyleneimine polymer having a number average molecule weight (Mn) of less than 1200 g/mol.

6. The conjugated lipomer of claim 5, or a salt thereof, wherein the polyethyleneimine polymer has a number average molecule weight (Mn) of about 600 g/mol.

7. The conjugated lipomer of claim 1, or a salt thereof, wherein each instance of R.sup.2 is independently a substituted or unsubstituted polyethyleneimine, or a group of formula (iii).

8. The conjugated lipomer of claim 1, or a salt thereof, wherein each instance of R.sup.3 is independently unsubstituted C.sub.8-20 alkyl.

9. The conjugated lipomer of claim 8, or a salt thereof, wherein each instance of R.sup.3 is C.sub.13H.sub.27.

10. The conjugated lipomer of claim 1, or a salt thereof, wherein: each instance of L.sup.1 is independently selected from formula (ii) and formula (iii); n is an integer between 10 and 15, inclusive; each instance of R.sup.2 is independently a substituted polyethyleneimine, or a group of formula (iii); R.sup.3 is C.sub.13H.sub.27; R.sup.4 is hydrogen; each R.sup.5 is a group of formula (iii); and Z is a group of formula (iii).

11. A composition comprising the conjugated lipomer of claim 10, or a salt thereof, and an agent.

12. The composition of claim 11, wherein the agent is a cardiovascular active agent, a vasoactive agent, or an antihypertensive agent.

13. The composition of claim 11, wherein the agent is small molecule.

14. The composition of claim 11, wherein the agent is a polynucleotide.

15. The composition of claim 14, wherein the polynucleotide is DNA.

16. The composition of claim 14, wherein the polynucleotide is RNA.

17. The composition of claim 16, wherein the RNA is RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA, or an RNA that encodes a protein or peptide.

18. The composition of claim 17, wherein the RNA is siRNA or miRNA.

19. A method of delivering an agent to a subject, comprising administering to the subject an effective amount of the composition of claim 11.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1U. FIGS. 1A-1R depict PEI and aza-macrocycle precursors (FIG. 1A); a general synthetic method for the conjugation of various epoxide-terminated groups to the precursors (FIG. 1B); exemplary conjugated LPEI.sub.600 polymers synthesized according to the present method (FIG. 1C); exemplary conjugated BPEI.sub.1800 polymers synthesized according to the present method (FIG. 1D); exemplary conjugated aza-macrocycles synthesized according to the present method (FIGS. 1E-1F); and NMR characterization of exemplary conjugated lipomers [FIGS. 1G-1U: .sup.1H NMR (500 MHz) spectra of 2J1 (FIG. 1G), 3I3 (FIG. 1H), 3I7 (FIG. 1I), 5H7 (FIG. 1J), 7C1 (FIG. 1K), 4C4 (FIG. 1L), 4C8 (FIG. 1M), 4D5 (FIG. 1N), 4D9 (FIG. 1O), 6B10 (FIG. 1P), 6C1 (FIG. 1Q), 7H4 (FIG. 1R), 7H8 (FIG. 1S), 7H10 (FIG. 1T), and 7I1 (FIG. 1U)].

(2) FIGS. 2A-2B demonstrate that non-toxic efficacy increases with lipomer:siRNA mass ratios (MR) up to 15 in HeLa cells. A library of 750 compounds was tested, of which a select subset reduced gene expression. FIG. 2A: Target gene expression for 750 lipomers tested at four different mass ratios at a dosage of 45 ng/well (20 nM). More lipomers reduce gene expression at a mass ratio of 10 and 15 than 5 and 2.5, as indicated by a larger curve shoulder. FIG. 2B: Average target (Firefly) and off-target (Renilla) expression for 200 lipomers tested against HeLa cells at a dose of 45 ng/well. Target gene expression decreases from 78% to 63% as mass ratio increases, while off-target expression remains fairly constant, changing from 88% to 86%.

(3) FIG. 3 depicts the gene expression data for seventeen lipomers screened against HeLa, qBEND.3 and HMVEC cells in vitro. The lipomer:siRNA mass ratio was 15 and siRNA dosing was 30 nM. All lipomers studied reduced gene expression in at least one cell line by 80% at this dose, indicating a high degree of in vitro potency, and driving subsequent interest for in vivo testing.

(4) FIGS. 4A-4B depict the dose response of lipomers 7C1, 6C1 and 6C8 in qBEND.3 murine endothelial cells. FIG. 4A: Tie2 expression shows a dose response profile suggesting an IC.sub.50 concentration between 6.0 and 1.5 nM, indicating a high degree of in vitro potency. FIG. 4B: Control gene GapDH expression remains constant at all doses, indicative of negligible off-target effects.

(5) FIGS. 5A-5B. FIG. 5A depicts the size of lipomers 3i7 and 7H8 during the formulation process. Before siRNA is added, lipomer particle size is stable at pH 5.3. Particle size remains stable after addition of siRNA at low pH. After dialysis to pH 7.4, 3i7 lipomer particle size increases while 7H8 lipomer particle size remains constant. Particle size varied from 20 to 130 nm, sizes which are known to increase nanoparticle efficacy (Peer et al. Nature nanotechnology (2007) 2: 751-760). FIG. 5B: 6C1 diameter measured over a period of 18 hours after formulation with cholesterol and conjugation to siRNA. Consistent lipomer particle size indicates that lipomer particles are stable overnight at temperatures ranging from 4 C. to 40 C.

(6) FIG. 6 depicts Factor 7 in vivo expression after tail-vein injection of lipomers 6C1 and 6C8. Lipomers were formulated with cholesterol at a lipomer: cholesterol mole ratio of 100:35, but without C16-PEG. 6C1 was tested at two lipomer: siRNA mass ratios, 15:1 and 10:1. As seen during the in vitro assays, 15:1 mass ratios resulted in higher knockdown. 6C1 reduced gene expression by 64% while 6C8 did not reduced gene expression in vivo. Importantly, these results indicate that lipomers can deliver siRNA effectively in vivo when delivered systemically.

(7) FIG. 7 depicts the relative expression of Factor 7 after systemic dosing with 2 mg/kg siRNA. In this study, lipomers were formulated with cholesterol and C.sub.16-PEG such that the Lipomer:Cholesterol:PEG molar ratio was 100:35:25. The lipomer:siRNA mass ratio was held constant at 15:1. Lipomers were then injected via the tail vein, and Factor 7 expression was measured 48 hours later. Several lipomers were shown to decrease in vivo Factor 7 expression, suggesting that in vivo success was not limited to 6C1 and that other lipomer compounds could reduce gene expression in vivo.

(8) FIG. 8 depicts in vivo gene expression of Factor 7. Lipomer 7H6 reduced gene expression by 66% systemic injection into mice at a dose of 1.5 mg/kg. When 7H6 was combined with a luciferase targeting siRNA, it did not reduce F7 expression, that the gene expression is reduced by siRNA action and not toxicity. This lipomer reduced gene expression by 35% in the study described in FIG. 7.

(9) FIGS. 9A-9B depict the in vivo reduction of tumor gene expression in a subcutaneous tumor model. Mice were injected with luciferase expressing tumor cells subcutaneously and tumors were allowed to grow for two weeks. Before injection of siRNA, luciferase expression was measured, and used as baseline. FIG. 9A: When dosed systemically 1.5 mg/kg of siRNA complexed to lipomer 7H6, luciferase tumor expression was reduced by 51%, indicating that lipomers successfully delivered siRNA into tumor cells. Importantly, the gene expression was reduced after tail vein injection instead of intratumoral injection, since intratumoral injections have limited clinical relevance (if a clinician can inject into a tumor, they often can remove it). FIG. 9B: Tumor before and after injection with 7H6 targeting luciferase expression. Tumor gene expression is markedly reduced.

(10) FIGS. 10A-10B depict the biodistribution of three different successful lipomers (7H6, 7I1 and 3i7) evaluated using Cy5.5 labeled siRNA (AllStars Control siRNA, Qiagen, Valencia, Calif.). Mice were injected with Cy5.5 siRNA (2.5 mg/kg) via the tail vein and sacrificed 1 or 14 hours later. Major organs were harvested and fluorescently imaged to evaluate distribution of Cy5.5 siRNA nanoparticles at 670 (ex. 670 nm, em. 710 nm) using an IVIS imaging system. Lipomers were found in the liver, spleen, kidney lungs and near the injection point. This indicates that they may be used for a variety of ailments, both systemic and within different organs. Since many siRNA delivery vehicles hone primarily to the liver, the delivery to lungs and kidneys may present new clinical targets.

(11) FIG. 11 depicts particle size before and after conjugation of siRNA at a pH of 5.3 (before dialysis). Nanoparticles are kept from aggregating by repulsive factors including steric and electrostatic forces. Upon conjugation of siRNA, electrostatic forces may be reduced, which can lead to aggregation. However, many particles showed stability even after conjugation of siRNA. Note that together, FIGS. 11-15 demonstrate that the lipomer size, surface charge and chemical composition have been characterized. Importantly, different lipomers have different physical properties, making them ideal for different types of applications. As an example, small particles with diameters below 50 nm may be well suited for kidney delivery while larger particles may be better for delivery of genetic material to the lung.

(12) FIGS. 12A-12B depict particle size (FIG. 12A) and surface charge (FIG. 12B) (zeta potential) before and after conjugation of siRNA at pH of 5.3, and after dialysis to a pH of 7.4. FIG. 12A: 7H8 size remained constant after the addition of siRNA and after extrusion, suggesting that electrostatic interactions are less important for particle stability. In contrast, 3I7 size was variable, particularly after dialysis, indicating that this particle could be useful for applications where pH-triggered physical transformations are needed. Both lipomers formed particles under 200 nm, suggesting they would be ideal for biological applications. FIG. 12B: In both cases, the zeta potential (a rough indicator of surface charge) of 7H8 and 3I7 decreased upon the addition of negatively charged siRNA. It became slightly negative after dialysis, indicating these particles may have reduced toxicity owing to high cationic profiles.

(13) FIGS. 13A-13B demonstrate the effect of lipomer: siRNA mass ratio (mr) on the size (FIG. 13A) and zeta potential (FIG. 13B) of particles before and after dialysis. The diameter of 6C1 was found to be constant at mr=10 and 15. The the lipomer: siRNA did not greatly influence size and zeta potential, indicating a high degree of physical stability.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

(14) The present invention provides inventive conjugated polyethyleneimine polymers and conjugated aza-macrocycles (collectively referred to herein as conjugated lipomers or lipomers) containing one or more groups of the formula (iii):

(15) ##STR00016##
wherein R.sup.3 and R.sup.4 are as defined herein.

(16) The conjugated polyethyleneimine polymers are preferably prepared from low molecular weight linear polyethyleneimine (LPEI) and branched polyethyleneimine (BPEI) polymers, i.e., having a number average molar mass (Mn) of 2000 (i.e., approximately 2 kDa).

(17) Also provided are compositions comprising the inventive conjugated lipomers, and methods of preparation and use.

(18) Conjugated Polyethyleneimine Polymers and Preparation Thereof

(19) The present invention provides novel conjugated polyethyleneimine polymers and methods of their preparation.

(20) In one aspect, provided is a conjugated polyethyleneimine polymer of the Formula (II):
A private use character Parenopenst L.sup.1 private use character Parenclosest .sub.nZ(II)
or salt thereof; wherein:

(21) each instance of L.sup.1 is independently selected from formulae:

(22) ##STR00017##
provided that at least one L.sup.1 is selected from formulae (iii);

(23) n is an integer of between 3 to 45, inclusive;

(24) each instance of R.sup.2 is independently hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; a substituted or unsubstituted polyethyleneimine; or a group of the formula (iii):

(25) ##STR00018##
or the two R.sup.2 groups are joined to form a substituted or unsubstituted heterocyclyl;

(26) each instance of R.sup.3 is independently substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; or a hydrophilic polymer;

(27) each instance of R.sup.4 is independently hydrogen, acyl; silyl; a hydroxyl protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; or a substituted or unsubstituted heteroaryl;

(28) A is N(R.sup.5).sub.2, wherein each instance of R.sup.5 is independently hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; or a group of the formula (iii):

(29) ##STR00019##
or two R.sup.5 groups are joined to form a substituted or unsubstituted heterocyclyl; and

(30) Z is hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl, or a group of the formula (iii):

(31) ##STR00020##
or Z and the nitrogen atom to which it is attached form a substituted or unsubstituted heterocyclyl group.

(32) The inventive conjugated polyethyleneimine polymer of Formula (II), or salt thereof, is prepared by contacting a compound of Formula (I), or salt thereof (the precursor polyethyleneimine polymer), with one or more different epoxides of the formula (xii), e.g., provided below in Scheme I:

(33) ##STR00021##
wherein each instance of R.sup.1 is independently selected from hydrogen or a group of the formula (ii):

(34) ##STR00022##
and A, Z, L.sup.1, R.sup.2, R.sup.3, and n is as defined herein; provided that the number average molar mass (Mn) of the precursor polyethyleneimine polymer does not exceed 2000 g/mol (about 2 kDa).

(35) Thus, it is understood that the number average molar mass (Mn) of the compound of Formula (II), as described herein, is approximately 2000 (i.e., approximately 2 kDa) after subtraction of the molecular weight of each instance of the group (iii) attached thereto:

(36) ##STR00023##

(37) In certain embodiments, the conjugated polyethyleneimine polymer is prepared from low molecular weight precursor polyethyleneimine polymer, i.e., having a number average molar mass (Mn) of 2000 (i.e., approximately 2 kDa). In certain embodiments, the conjugated polyethyleneimine polymer is prepared from a precursor polyethyleneimine polymer having an number average molar mass (Mn) of less than 1900, less than 1800, less than 1700, less than 1600, less than 1500, less than 1400, less than 1300, less than 1200, less than 1100, less than 1000, less than 900, less than 800, or less than 700 g/mol. In certain embodiments, the precursor polyethyleneimine polymer has an number average molar mass (Mn) of between about 400 to about 2000, of between about 400 to about 1900, of between about 400 to about 1800, of between about 500 to about 1900, of between about 600 to about 1800 g/mol, of between about 600 to about 800, of between about 600 to about 700, of between about 700 to about 1800, of between about 800 to about 1800, of between about 900 to about 1800, or of between about 1000 to about 1800 g/mol, inclusive.

(38) In certain embodiments, the compound of Formula (I) is a branched polyethyleneimine (BPEI) polymer, and at least one R.sup.1 is of the formula (ii); e.g., for example, 1 to 45, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5, R.sup.1 groups are of the formula (ii). In certain embodiments, the compound of Formula (I) is a linear polyethyleneimine (LPEI) polymer, and each R.sup.1 group is hydrogen.

(39) In certain embodiments of Formula (I), neither R.sup.5, Z, nor R.sup.2 is a group of the formula (iii). For example, in certain embodiments of Formula (I),

(40) each instance of R.sup.2 is independently hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; or a substituted or unsubstituted polyethyleneimine;

(41) A is N(R.sup.5).sub.2, wherein each instance of R.sup.5 is independently hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl; or the two R.sup.5 groups are joined to form a substituted or unsubstituted heterocyclyl; and

(42) Z is hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl; or Z and the nitrogen atom to which it is attached form a substituted or unsubstituted heterocyclyl group.

(43) In certain embodiments of Formula (I), A is N(R.sup.5).sub.2, wherein at least one R.sup.5 is hydrogen. In certain embodiments of Formula (I) A is N(R.sup.5).sub.2, wherein each R.sup.5 is hydrogen. In certain embodiments of Formula (I), Z is hydrogen.

(44) Alternatively, in certain embodiments of Formula (II), either R.sup.5, Z, and/or R.sup.2 can be a group of the formula (iii) provided that at least one R.sup.2, R.sup.5 and/or Z is hydrogen in the precursor polyethyleneimine polymer. Thus, in certain embodiments of Formula (II), A is N(R.sup.5).sub.2, wherein each instance of R.sup.5 is independently hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; or a group of the formula (iii); or two R.sup.5 groups are joined to form a substituted or unsubstituted heterocyclyl;

(45) and Z is hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl, or a group of the formula (iii); or Z and the nitrogen atom to which it is attached form a substituted or unsubstituted heterocyclyl group.

(46) In certain embodiments of Formula (II), A is N(R.sup.5).sub.2, wherein each instance of R.sup.5 is independently hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted heteroalkyl; or a group of the formula (iii). In certain embodiments of Formula (II), A is N(R.sup.5).sub.2, wherein each instance of R.sup.5 is independently hydrogen; substituted or unsubstituted alkyl; substituted or unsubstituted heteroalkyl; or a group of the formula (iii).

(47) In certain embodiments of Formula (II), Z is hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted heteroalkyl; or a group of the formula (iii). In certain embodiments of Formula (II), Z is hydrogen; substituted or unsubstituted alkyl; substituted or unsubstituted heteroalkyl; or a group of the formula (iii).

(48) As generally defined above, n is an integer of between 3 to 45, inclusive. In certain embodiments, n is an integer of between 3 to 45, between 5 to 45, between 7 to 45, between 9 to 45, between 10 to 45, between 11 to 45, between 12 to 45, between 13 to 45, between 14 to 45, between 5 to 40, between 5 to 35, between 5 to 30, between 5 to 25, between 5 to 20, between 5 to 15, between 10 to 20, between 10 to 15, or between 40 to 45, inclusive. In certain embodiments, n is 14. In certain embodiments, n is 43.

(49) As generally defined above, each instance of R.sup.2 is independently hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; a substituted or unsubstituted polyethyleneimine; or a group of the formula (iii); or the two R.sup.2 groups are joined to form a substituted or unsubstituted heterocyclyl.

(50) In certain embodiments, each instance of R.sup.2 is independently hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; a substituted or unsubstituted polyethyleneimine; or a group of the formula (iii); or the two R.sup.2 groups are joined to form a substituted or unsubstituted heterocyclyl.

(51) In certain embodiments, each instance of R.sup.2 is independently hydrogen; substituted or unsubstituted alkyl; substituted or unsubstituted heteroalkyl; a substituted or unsubstituted polyethyleneimine; or a group of the formula (iii); or the two R.sup.2 groups are joined to form a substituted or unsubstituted heterocyclyl.

(52) In certain embodiments, each instance of R.sup.2 is independently hydrogen; a substituted or unsubstituted polyethyleneimine; or a group of the formula (iii).

(53) In certain embodiments, at least one R.sup.2 is a substituted or unsubstituted polyethyleneimine.

(54) As used herein, a substituted or unsubstituted polyethyleneimine refers to a group of the formula (iv):

(55) ##STR00024##
wherein:

(56) t is an integer of between 1 to 50, inclusive; P is hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; or a group of the formula (iii):

(57) ##STR00025##
or
P, R.sup.6, and the nitrogen atom to which it is attached form a substituted or unsubstituted heterocyclyl group; and

(58) each instance of R.sup.6 is independently selected from hydrogen or a group of the formula (ii) or (iii):

(59) ##STR00026##
wherein R.sup.2, R.sup.3, and R.sup.4 are as defined herein; provided that the number average molar mass (Mn) of the precursor polyethyleneimine polymer does not exceed 2000 g/mol (about 2 kDa).

(60) In certain embodiments, t is an integer of between 1 to 40, between 1 to 30, between 1 to 20, between 1 to 10, between 1 to 5, or between 1 to 3, inclusive.

(61) In certain embodiments of formula (iv), each instance of R.sup.2 is hydrogen or a substituted or unsubstituted polyethyleneimine. For example, each instance of R.sup.6 may comprise any number of branched polyethyleneimine groups, e.g., exemplary non-limiting examples include:

(62) ##STR00027## ##STR00028##
wherein:

(63) each instance of t, t, and t is independently an integer of between 1 to 50, inclusive;

(64) each instance of P, P, and P is independently hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl, or a group of the formula (iii):

(65) ##STR00029##
or
P, P, and P, the respective R.sup.6, R.sup.6, and R.sup.6, and the nitrogen atom to which both are attached independently forms a substituted or unsubstituted heterocyclyl group; and

(66) each instance of R.sup.6, R.sup.6, and R.sup.6, is independently hydrogen or a group of the formula (ii) or (iii):

(67) ##STR00030##
wherein R.sup.2 is as defined as R.sup.2, defined herein; provided that the number average molar mass (Mn) of the precursor polyethyleneimine polymer does not exceed 2000 g/mol (about 2 kDa).

(68) In the instance, wherein at least one R.sup.2 is a substituted or unsubstituted polyethyleneimine, the precursor polyethyleneimine polymer of the Formula (I) is a branched polyethyleneimine (BPEI) polymer, as defined herein, wherein P and R.sup.6 of the the precursor polyethyleneimine polymer of the Formula (I) are not a group of the formula (iii). In this instance, the conjugated polyethyleneimine polymer further comprises at least one L.sup.1 group of the formula (ii); e.g., 1 to 44, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 groups of the formula (ii). In certain embodiments, the conjugated polyethyleneimine polymer is prepared from a branched polyethyleneimine (BPEI) polymer having an number average molar mass (Mn) of less than 2000 g/mol (approximately 2 kDa). In certain embodiments, the conjugated polyethyleneimine polymer is prepared from a branched polyethyleneimine (BPEI) having an number average molar mass (Mn) of less than 1900, less than 1800, less than 1700, less than 1600, less than 1500, less than 1400, less than 1300, less than 1200, less than 1100, less than 1000, less than 900, less than 800, or less than 700 g/mol. In certain embodiments, the BPEI has an number average molar mass (Mn) of between about 400 to about 2000, of between about 400 to about 1900, of between about 400 to about 1800, of between about 500 to about 1900, of between about 600 to about 1800 g/mol, of between about 600 to about 800, of between about 600 to about 700, of between about 700 to about 1800, of between about 800 to about 1800, of between about 900 to about 1800, or of between about 1000 to about 1800 g/mol, inclusive.

(69) In certain embodiments, the conjugated polyethyleneimine polymer is prepared from a branched polyethyleneimine (BPEI) polymer having an number average molar mass (Mn) of about 600 g/mol (BPEI.sub.600). In this instance, in certain embodiments, n is 14.

(70) In certain embodiments, the conjugated polyethyleneimine polymer is prepared from a branched polyethyleneimine (BPEI) polymer having an number average molar mass (Mn) of about 1800 g/mol (BPEI.sub.1800). In this instance, in certain embodiments, n is 43.

(71) In certain embodiments, the precursor polyethyleneimine polymer is a linear polyethyleneimine (LPEI), as defined herein. In this instance, the conjugated polyethyleneimine polymer does not comprise an L.sup.1 group of the formula (ii). In certain embodiments, the conjugated polyethyleneimine polymer is prepared from a linear polyethyleneimine (LPEI) having an number average molar mass (Mn) of less than 2000 g/mol (approximately 2 kDa). In certain embodiments, the conjugated polyethyleneimine polymer is prepared from a linear polyethyleneimine (LPEI) having an number average molar mass (Mn) of less than 1900, less than 1800, less than 1700, less than 1600, less than 1500, less than 1400, less than 1300, less than 1200, less than 1100, less than 1000, less than 900, less than 800, or less than 700 g/mol. In certain embodiments, the LPEI has an number average molar mass (Mn) of between about 400 to about 2000, of between about 400 to about 1900, of between about 400 to about 1800, of between about 500 to about 1900, of between about 600 to about 1800 g/mol, of between about 600 to about 800, of between about 600 to about 700, of between about 700 to about 1800, of between about 800 to about 1800, of between about 900 to about 1800, or of between about 1000 to about 1800 g/mol, inclusive.

(72) In certain embodiments, the LPEI polymer having an number average molar mass (Mn) of about 600 g/mol (LPEI.sub.600). In this instance, in certain embodiments, n is 14.

(73) In certain embodiments, the LPEI polymer having an number average molar mass (Mn) of about 1800 g/mol (LPEI.sub.1800). In this instance, in certain embodiments, n is 43.

(74) As generally defined above, the conjugated polyethyleneimine polymer comprises at least one instance (e.g., 1 to 44, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 instances) of a group of the formula (iii):

(75) ##STR00031##

(76) wherein each instance of R.sup.3 provided in the conjugated polyethyleneimine polymer is independently substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; or a hydrophilic polymer; and

(77) each instance of R.sup.4 is independently hydrogen, acyl; silyl; a hydroxyl protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl.

(78) As generally defined above, each instance of R.sup.3 is independently substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; or a hydrophilic polymer.

(79) In certain embodiments, each instance of R.sup.3 is independently substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; or a hydrophilic polymer.

(80) In certain embodiments, each instance of R.sup.3 is independently substituted or unsubstituted alkyl; substituted or unsubstituted heteroalkyl; or a hydrophilic polymer.

(81) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted alkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.1-50alkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-50alkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-40alkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-30alkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-20alkyl.

(82) In certain embodiments, at least one instance of R.sup.3 is an unsubstituted alkyl. Exemplary unsubstituted alkyl groups include, but are not limited to, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.4H.sub.9, C.sub.5H.sub.11, C.sub.6H.sub.13, C.sub.7H.sub.15, C.sub.8H.sub.17, C.sub.9H.sub.19, C.sub.10H.sub.21, C.sub.11H.sub.23, C.sub.12H.sub.25, C.sub.13H.sub.27, C.sub.14H.sub.29, C.sub.15H.sub.31, C.sub.16H.sub.33, C.sub.17H.sub.35, C.sub.18H.sub.37, C.sub.19H.sub.39, and C.sub.20H.sub.41.

(83) In certain embodiments, at least one instance of R.sup.3 is a substituted alkyl. For example, in certain embodiments, at least one instance of R.sup.3 is an alkyl substituted with one or more fluorine substituents. Exemplary substituted alkyl groups include, but are not limited to:

(84) ##STR00032##

(85) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted alkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.2-50alkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-50 alkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-40 alkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-30 alkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-20 alkenyl. In certain embodiments, at least one instance of R.sup.3 is a substituted C.sub.8-20 alkenyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted alkenyl.

(86) Exemplary unsubstituted alkenyl groups include, but are not limited to:

(87) ##STR00033## Myristoleic (CH.sub.2).sub.7CHCH(CH.sub.2).sub.3CH.sub.3, Palmitoliec (CH.sub.2).sub.7CHCH(CH.sub.2).sub.5CH.sub.3, Sapienic (CH.sub.2).sub.4CHCH(CH.sub.2).sub.8CH.sub.3, Oleic (CH.sub.2).sub.7CHCH(CH.sub.2).sub.7CH.sub.3, Linoleic (CH.sub.2).sub.7CHCHCH.sub.2CHCH(CH.sub.2).sub.4CH.sub.3, -Linolenic (CH.sub.2).sub.7CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CH.sub.3, Arachinodonic (CH.sub.2).sub.3CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CHCH(CH.sub.2).sub.4CH.sub.3, Eicosapentaenoic (CH.sub.2).sub.3CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CH.sub.3, Erucic (CH.sub.2).sub.11CHCH(CH.sub.2).sub.7CH.sub.3, and Docosahexaenoic (CH.sub.2).sub.2CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CH.sub.3.

(88) In embodiments, wherein R.sup.3 is defined as a C.sub.6-50alkyl or C.sub.6-50alkenyl groups, such groups are meant to encompass lipophilic groups (also referred to as a lipid tail). Lipophilic groups comprise a group of molecules that include fats, waxes, oils, fatty acids, and the like. Lipid tails present in these lipid groups can be saturated and unsaturated, depending on whether or not the lipid tail comprises double bonds. The lipid tail can also comprise different lengths, often categorized as medium (i.e., with tails between 7-12 carbons, e.g., C.sub.7-12 alkyl or C.sub.7-12 alkenyl), long (i.e., with tails greater than 12 carbons and up to 22 carbons, e.g., C.sub.13-22 alkyl or C.sub.13-22 alkenyl), or very long (i.e., with tails greater than 22 carbons, e.g., C.sub.23-30 alkyl or C.sub.23-30 alkenyl).

(89) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted alkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.2-50alkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-50 alkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-40 alkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-30 alkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-20 alkynyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted alkynyl. In certain embodiments, at least one instance of R.sup.3 is a substituted alkynyl.

(90) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.1-50 heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-50 heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-40 heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-30 heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-20 heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is a substituted heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted heteroalkyl.

(91) Exemplary unsubstituted heteroalkyl groups include, but are not limited to:

(92) ##STR00034##

(93) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.2-50 heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-50 heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-40 heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-30 heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-20 heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is a substituted heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted heteroalkenyl.

(94) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.2-50 heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-50 heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-40 heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-30 heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-20 heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is a substituted heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted heteroalkynyl.

(95) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted carbocyclyl. In certain embodiments, at least one instance of R.sup.3 is a substituted carbocyclyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted carbocyclyl.

(96) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted heterocyclyl. In certain embodiments, at least one instance of R.sup.3 is a substituted heterocyclyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted heterocyclyl.

(97) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted aryl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted aryl. In certain embodiments, at least one instance of R.sup.3 is a substituted aryl.

(98) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted heteroaryl. In certain embodiments, at least one instance of R.sup.3 is a substituted heteroaryl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted heteroaryl.

(99) In certain embodiments, at least one instance of R.sup.3 is hydrophilic polymer. As used herein, a polymer refers to a compound comprised of at least 3 (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeating covalently bound structural units. By extension, a hydrophilic polymer is a polymer, as defined herein, further comprising at least one group (e.g., an oxygen, nitrogen, and/or sulfur atom) in the repeating structural unit capable of hydrogen bonding. The hydrophilic polymer is preferably biocompatible (i.e., non-toxic). Exemplary hydrophilic polymers include, but are not limited to, polypeptides (e.g., poly-L-lysine), cellulose polymers (e.g., hydroxyethylcellulose, ethylcellulose, carboxymethylcellulose, methylc cellulose, hydroxypropylmethylcellulose (HPMC)), dextran polymers, polymaleic acid polymers, poly(acrylic acid) polymers, poly(vinylalcohol) polymers, polyvinylpyrrolidone (PVP) polymers, and polyethyleneglycol (PEG) polymers.

(100) In certain embodiments, the hydrophilic polymer is a polyethyleneglycol polymer, e.g., of the formula (v):

(101) ##STR00035##
wherein:

(102) R.sup.7 is hydrogen; acyl; silyl; a hydroxyl protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl; and

(103) v is an integer between 3 to 400, inclusive.

(104) In certain embodiments, R.sup.7 is hydrogen. In certain embodiments, R.sup.7 is acyl. In certain embodiments, R.sup.7 is a hydroxyl protecting group. In certain embodiments, R.sup.7 is substituted or unsubstituted alkyl. In certain embodiments, R.sup.7 is a substituted alkyl. In certain embodiments, R.sup.7 is an unsubstituted alkyl. In certain embodiments, R.sup.7 is CH.sub.3 (a polyethyleneglycol monomethylether polymer). In certain embodiments, R.sup.7 is substituted or unsubstituted alkenyl. In certain embodiments, R.sup.7 is substituted or unsubstituted alkynyl. In certain embodiments, R.sup.7 is substituted or unsubstituted heteroalkyl. In certain embodiments, R.sup.7 is substituted or unsubstituted heteroalkenyl. In certain embodiments, R.sup.7 is substituted or unsubstituted heteroalkynyl. In certain embodiments, R.sup.7 is substituted or unsubstituted carbocyclyl. In certain embodiments, R.sup.7 is substituted or unsubstituted heterocyclyl. In certain embodiments, R.sup.7 is substituted or unsubstituted aryl. In certain embodiments, R.sup.7 is and substituted or unsubstituted heteroaryl.

(105) In certain embodiments, v is an integer between 3 to 300, 3 to 200, 3 to 100, 3 to 90, 3 to 80, 3 to 70, 3 to 60, 3 to 50, 5 to 50, 10 to 50, 15 to 50, 20 to 50, 20 to 40, 20 to 30, 20 to 25, 30 to 50, and 40 to 50, inclusive. PEG.sub.1000 corresponds, on average, to a v of about 22.7, wherein R.sup.7 is OCH.sub.3. PEG.sub.2000 corresponds, on average, to a v of about 45.4.

(106) In certain embodiments, the number average molar mass (Mn) of the polyethyleneglycol polymer is 10,000. In certain embodiments, the number average molar mass (Mn) of the polyethyleneglycol polymer is 10,000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, or 2000. In certain embodiments, the number average molar mass (Mn) of the polyethyleneglycol polymer is between about 100 to about 10,000, inclusive; e.g., between about 100 to about 5000, between about 100 to about 4000, between about 100 to about 3000, between about 100 to about 2500, between about 100 to about 2000, between about 100 to about 1500, between about 100 to about 1000, between about 100 to about 900, between about 100 to about 800, between about 100 to about 700, between about 100 to about 600, between about 100 to about 500, between about 100 to about 400, between about 100 to about 300, between about 100 to about 200, between about 100 to about 1500, between about 2500 to about 10000, between about 2500 to about 9000, between about 2500 to about 8000, between about 2500 to about 7000, between about 2500 to about 6000, between about 2500 to about 5000, between about 2500 to about 4000, or between about 2500 to about 3000, inclusive. In certain embodiments, the number average molar mass (Mn) of the polyethyleneglycol polymer is 1000 (PEG.sub.1000). In certain embodiments, the number average molar mass (Mn) of the polyethyleneglycol polymer is 2000 (PEG.sub.2000). A 1:1 mixture of PEG.sub.1000 and PEG.sub.2000 is referred to herein as PEG.sub.1.5K.

(107) In certain embodiments, at least one instance of R.sup.3 is a hydrophilic polymer, and at least one instance of R.sup.3 is a substituted or unsubstituted alkyl.

(108) As used herein, when the group R.sup.3 is depicted as bisecting a carbon-carbon bond, e.g., of the group of the formula (iii), it is understood that R.sup.3 may be substituted at either carbon.

(109) Nucleophilic attack of an amino group of the polyethyleneimine polymer at the least sterically hindered carbon of the epoxide provides a group of the formula (iii-a) (route a), while nucleophilic attack at the more sterically hindered carbon of the epoxide provides a group of the formula (iii-b) (route b), wherein R.sup.4 is hydrogen; see, e.g., the conjugation reaction of Scheme II.

(110) It is understood that compounds of the present invention may comprise a mixture of products attached thereto arising from route (a) and route (b) depending on the preference, or lack thereof, of the mode of addition, and that formulae that depict this bisecting bond may comprise a mixture of compounds. The bisecting group R.sup.3 depicted in the formulae seeks to encompasses all contemplated modes of addition.

(111) ##STR00036##

(112) The resulting hydroxyl moiety of the formula (iii-a) or (iii-b), wherein R.sup.4 is hydrogen, can optionally be converted to a substituted hydroxyl, wherein R.sup.4 is a group other than hydrogen, i.e., is independently selected from acyl; silyl; a hydroxyl protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl; using conventional methods. Alkylation, acylation, and/or protection of a hydroxyl moiety are well-known in the art; see, e.g., Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3.sup.rd edition, John Wiley & Sons, 1999; Smith and March, March's Advanced Organic Chemistry, 5.sup.th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3.sup.rd Edition, Cambridge University Press, Cambridge, 1987.

(113) For example, in certain non-limiting embodiments, the hydroxyl moiety is reacted with an electrophile of the formula R.sup.4X wherein R.sup.4 is a group other than hydrogen, and X is a leaving group, to provide a substituted hydroxyl group in formula (iii).

(114) In certain embodiments, each instance of R.sup.4 is independently hydrogen; acyl; silyl; a hydroxyl protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; or substituted or unsubstituted heteroalkenyl. In certain embodiments, each instance of R.sup.4 is independently hydrogen; substituted or unsubstituted alkyl; or substituted or unsubstituted heteroalkyl. In certain embodiments, each instance of R.sup.4 is hydrogen.

(115) It is understood from the present disclosure that the group of formula (iii) represents a group of formula (iii-a) or a group of formula (iii-b):

(116) ##STR00037##

(117) In certain embodiments, the conjugation reaction depicted in Scheme II results in a mixture comprising more lipomers conjugated to a group of formula (iii-a) than to a group of formula (iii-b), e.g., the reaction mixture comprises greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 99%, between about 60% to about 100%, between about 70% to about 100%, between about 80% to about 100%, between about 90% to about 100%, between about 95% to about 100%, or between about 99% to about 100%, of a conjugated lipomer attached to a group of formula (iii-a).

(118) In certain embodiments, the reaction mixture comprises only conjugated lipomers attached to a group formula (iii-a).

(119) In certain embodiments, the epoxide is chiral, i.e., having (R) or (S) stereochemistry. In this instance, in certain embodiments, the conjugation reaction depicted in Scheme II provides a chiral conjugated polyethyleneimine polymer. Chirality in a polymer can be characterized in a variety of ways, e.g., obtaining the optical rotation and/or NMR analysis after chemical modification of the optically active polymer with a chiral derivatizing agent are methods useful in evaluating the chirality of a polymer.

(120) ##STR00038##

(121) In certain embodiments, wherein the epoxide is chiral, the conjugation reaction depicted in Scheme II results in a mixture comprising more lipomers conjugated to a group of formula (R)-(iii-a) than to a group of formula (S)-(iii-a), e.g., the reaction mixture comprises greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 99%, between about 60% to about 100%, between about 70% to about 100%, between about 80% to about 100%, between about 90% to about 100%, between about 95% to about 100%, or between about 99% to about 100%, of a conjugated lipomer attached to a group of formula (R)-(iii-a).

(122) In certain embodiments, reaction mixture comprises only conjugated lipomers attached to a group formula (R)-(iii-a).

(123) In certain embodiments, wherein the epoxide is chiral, the conjugation reaction depicted in Scheme II results in a mixture comprising more lipomers conjugated to a group of formula formula (S)-(iii-a) than formula (R)-(iii-a), e.g., the reaction mixture comprises greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 99%, between about 60% to about 100%, between about 70% to about 100%, between about 80% to about 100%, between about 90% to about 100%, between about 95% to about 100%, or between about 99% to about 100%, of a conjugated lipomer attached to a group of formula (S)-(iii-a).

(124) In certain embodiments, reaction mixture comprises only conjugated lipomers attached to a group formula (S)-(iii-a).

(125) ##STR00039##

(126) In certain embodiments, wherein one epoxide is used in the conjugation reaction, each instance of R.sup.3 is the same in the conjugated polyethyleneimine polymer. For example, in certain embodiments, each instance of R.sup.3 is the same wherein R.sup.3 is a substituted or unsubstituted alkyl. In certain embodiments, each instance of R.sup.3 is the same wherein R.sup.3 is an unsubstituted alkyl. In certain embodiments, each instance of R.sup.3 is the same wherein R.sup.3 is selected from the group consisting of CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.4H.sub.9, C.sub.5H.sub.11, C.sub.6H.sub.13, C.sub.7H.sub.15, C.sub.8H.sub.17, C.sub.9H.sub.19, C.sub.10H.sub.21, C.sub.11H.sub.23, C.sub.12H.sub.25, C.sub.13H.sub.27, C.sub.14H.sub.29, C.sub.15H.sub.31, C.sub.16H.sub.33, C.sub.17H.sub.35, C.sub.18H.sub.37, C.sub.19H.sub.39, and C.sub.20H.sub.41. In certain embodiments, each instance of R.sup.3 is the same wherein R.sup.3 is selected from the group consisting of C.sub.8H.sub.17, C.sub.9H.sub.19, C.sub.10H.sub.21, C.sub.11H.sub.23, C.sub.12H.sub.25, C.sub.13H.sub.27, C.sub.14H.sub.29, C.sub.15H.sub.31, and C.sub.16H.sub.33.

(127) Alternatively, in certain embodiments, wherein more than one epoxide is used in the conjugation reaction (e.g., two, three, four, five, six, seven, eight, nine, or ten different epoxides), the conjugated polyethyleneimine polymer comprises two or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) different R.sup.3 groups.

(128) For example, in certain embodiments, two different epoxides are used in the conjugation reaction. In this instance, in certain embodiments, the conjugated polyethyleneimine polymer comprises two different R.sup.3 groups. For example, in certain embodiments, the conjugated polyethyleneimine polymer comprises a mixture of two different R.sup.3 groups, wherein the first R.sup.3 group is a substituted or unsubstituted alkyl, and the second R.sup.3 group is a hydrophilic polymer (e.g., a polyethyleneglycol polymer). In certain embodiments, the conjugated polyethyleneimine polymer comprises a mixture of two different R.sup.3 groups, wherein the first R.sup.3 group is an unsubstituted alkyl, and the second R.sup.3 group is a polyethyleneglycol polymer. In certain embodiments, the conjugated polyethyleneimine polymer comprises a mixture of two different R.sup.3 groups, wherein the first R.sup.3 group is selected from the group consisting of CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.4H.sub.9, C.sub.5H.sub.11, C.sub.6H.sub.13, C.sub.7H.sub.15, C.sub.8H.sub.17, C.sub.9H.sub.19, C.sub.10H.sub.21, C.sub.11H.sub.23, C.sub.12H.sub.25, C.sub.13H.sub.27, C.sub.14H.sub.29, C.sub.15H.sub.31, C.sub.16H.sub.33, C.sub.17H.sub.35, C.sub.18H.sub.37, C.sub.19H.sub.39, and C.sub.20H.sub.41, and the second R.sup.3 group is PEG.sub.1000. In certain embodiments, the conjugated polyethyleneimine polymer comprises a mixture of two different R.sup.3 groups, wherein the first R.sup.3 group is selected from the group consisting of CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.4H.sub.9, C.sub.5H.sub.11, C.sub.6H.sub.13, C.sub.7H.sub.15, C.sub.8H.sub.17, C.sub.9H.sub.19, C.sub.10H.sub.21, C.sub.11H.sub.23, C.sub.12H.sub.25, C.sub.13H.sub.27, C.sub.14H.sub.29, C.sub.15H.sub.31, C.sub.16H.sub.33, C.sub.17H.sub.35, C.sub.18H.sub.37, C.sub.19H.sub.39, and C.sub.20H.sub.41, and the second R.sup.3 group is PEG.sub.2000.

(129) In certain embodiments, three different epoxides are used in the conjugation reaction. In this instance, in certain embodiments, the conjugated polyethyleneimine polymer comprises three different R.sup.3 groups. For example, in certain embodiments, the conjugated polyethyleneimine polymer comprises a mixture of three different R.sup.3 groups, wherein the first R.sup.3 group is a substituted or unsubstituted alkyl, the second R.sup.3 group is a first hydrophilic polymer (e.g., a polyethyleneglycol polymer, e.g., PEG.sub.1000), and the third R.sup.3 group is a second hydrophilic polymer (e.g., a different polyethyleneglycol polymer, e.g., PEG.sub.2000). In certain embodiments, the conjugated polyethyleneimine polymer comprises a mixture of three different R.sup.3 groups, wherein the first R.sup.3 group is an unsubstituted alkyl, the second R.sup.3 group is PEG.sub.1000, and the third R.sup.3 group is PEG.sub.2000. In certain embodiments, the conjugated polyethyleneimine polymer comprises a mixture of three different R.sup.3 groups, wherein the first R.sup.3 group is selected from the group consisting of CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.4H.sub.9, C.sub.5H.sub.11, C.sub.6H.sub.13, C.sub.7H.sub.15, C.sub.8H.sub.17, C.sub.9H.sub.19, C.sub.10H.sub.21, C.sub.11H.sub.23, C.sub.12H.sub.25, C.sub.13H.sub.27, C.sub.14H.sub.29, C.sub.15H.sub.31, C.sub.16H.sub.33, C.sub.17H.sub.35, C.sub.18H.sub.37, C.sub.19H.sub.39, and C.sub.20H.sub.41, the second R.sup.3 group is PEG.sub.1000, and the third R.sup.3 group is PEG.sub.2000. In certain embodiments a 1:1 mixture of PEG.sub.1000 and PEG.sub.2000 is used. In this instance, the mixture of the second R.sup.3 group and the third R.sup.3 group are referred to herein as PEG.sub.1.5K.

(130) In certain embodiments, the conjugated polymer comprises more of formula (iii) than of formula (i). For example, in certain embodiments, the ratio of groups of the formulae (i) to (iii) is between about 0:10 to about 9:10, inclusive. In certain embodiments, the ratio of groups of the formulae (i) to (iii) is between about 0:10 to about 9:10; between about 1:10 to about 8:10; between about 1:10 to about 7:10; between about 1:10 to about 6:10; between about 1:10 to about 5:10; or between about 2:10 to about 4:10, inclusive. In certain embodiments, the ratio of groups of the formulae (i) to (iii) is between about 3:10 to about 4:10, inclusive.

(131) Alternatively, in certain embodiments, the conjugated polymer comprises more of formula (i) than of formula (iii). For example, in certain embodiments, the ratio of groups of the formulae (iii) to (i) is between about 0:10 to about 9:10, inclusive. In certain embodiments, the ratio of groups of the formulae (iiii) to (i) is between about 0:10 to about 9:10; between about 1:10 to about 8:10; between about 1:10 to about 7:10; between about 1:10 to about 6:10; between about 1:10 to about 5:10; or between about 2:10 to about 4:10, inclusive. In certain embodiments, the ratio of groups of the formulae (iii) to (i) is between about 3:10 to about 4:10, inclusive.

(132) In certain embodiments, wherein the conjugated polyethyleneimine polymer comprises two different R.sup.3 groups, the ratio of the second R.sup.3 group to the first R.sup.3 group is between about 0.01:10 to about 10:10, inclusive. In certain embodiments, the ratio of the second R.sup.3 group to the first R.sup.3 group is between about 0.02:10 to about 10:10; between about 0.03:10 to about 10:10; between about 0.04:10 to about 10:10; between about 0.05:10 to about 10:10; between about 0.06:10 to about 10:10; between about 0.07:10 to about 10:10; between about 0.08:10 to about 10:10; between about 0.08:10 to about 9:10; between about 0.08:10 to about 8:10; between about 0.08:10 to about 7:10; between about 0.08:10 to about 6:10; between about 0.08:10 to about 5:10; between about 0.08:10 to about 4:10; between about 0.08:10 to about 3:10; between about 0.08:10 to about 2:10; or between about 0.08:10 to about 1:10, inclusive. In certain embodiments, the ratio of the second R.sup.3 group to the first R.sup.3 group is about 0.1:10.

(133) In certain embodiments, wherein the conjugated polyethyleneimine polymer comprises three different R.sup.3 groups, the ratio of the sum of the second and third R.sup.3 groups to the first R.sup.3 group is between about 0.01:10 to about 10:10, inclusive. In certain embodiments, the ratio of the sum of the second and third R.sup.3 groups to the first R.sup.3 group is 0.02:10 to about 10:10; between about 0.03:10 to about 10:10; between about 0.04:10 to about 10:10; between about 0.05:10 to about 10:10; between about 0.06:10 to about 10:10; between about 0.07:10 to about 10:10; between about 0.08:10 to about 10:10; between about 0.08:10 to about 9:10; between about 0.08:10 to about 8:10; between about 0.08:10 to about 7:10; between about 0.08:10 to about 6:10; between about 0.08:10 to about 5:10; between about 0.08:10 to about 4:10; between about 0.08:10 to about 3:10; between about 0.08:10 to about 2:10; or between about 0.08:10 to about 1:10, inclusive. In certain embodiments, the ratio of the sum of the second and third R.sup.3 groups to the first R.sup.3 group is about 0.1:10.

(134) Exemplary conjugated polyethyleneimine polymers of the Formula (II) include, but are not limited to, any of the following LPEI conjugated polymers and BPEI conjugated polymers, or salts thereof, provided in Tables 1 and 2, defining the one or more L.sub.1 groups present along the polymer backbone.

(135) TABLE-US-00002 TABLE 1 LPEI conjugated polymers (i) (iii) (iii) 1 0 embedded image 2 3 4 5 6 0 7 8 9 10 11 0 12 13 14 15 16 0 17 18 19 20 21 0 22 23 24 25 26 0 27 28 29 30 31 00 01 32 02 03 33 04 34 05 06 35 07 08 09 36 0

(136) TABLE-US-00003 TABLE 2 BPEI conjugated polymers (i) (ii) (iii) (iii) 1 embedded image 2 3 0 4 5 6 7 0 8 9 10 0 11 12 13 14 0 15 16 17 0 18 19 20 0 21 22 23 0 24 25 26 27 0 28 29 30 00 31 01 02 03 04 32 05 06 07 33 08 09 34 0 35 36
Conjugated Aza-Macrocycles and Preparation Thereof

(137) The present invention further provides conjugated aza-macrocycles; i.e., of the Formula (IV):

(138) ##STR00220##
or salt thereof; wherein:

(139) each instance of L.sup.3 is independently selected from:

(140) ##STR00221##
provided that the aza-macrocycle contains at least one group selected from (vi), (vii) or (viii);

(141) each instance of R.sup.8 is independently hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; or

(142) ##STR00222##
provided the aza-macrocycle contains at least one group of the formula (iii);

(143) each instance of R.sup.3 is independently substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; or a hydrophilic polymer;

(144) each instance of R.sup.4 is independently hydrogen, acyl; silyl; a hydroxyl protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl;

(145) each instance of m and p is independently 0, 1 or 2;

(146) q is an integer selected from 2, 3, or 4; and

(147) the dashed curved line, together with G and Y, is a covalent bond or a group of the formula:

(148) ##STR00223##
wherein s is 0, 1, or 2.

(149) The conjugated aza-macrocyle, or salt thereof, is prepared similarly to the above described method of preparing a conjugated polyethyleneimine polymer, i.e., by contacting a compound of Formula (III), or salt thereof (an aza-macrocycle precursor) with one or more different epoxides; e.g. as provided below in Scheme IV.

(150) Scheme IV.

(151) ##STR00224##
wherein each instance of L.sup.2 is independently selected from:

(152) ##STR00225##
provided that the aza-macrocycle precursor contains at least one group selected from (vi), (vii) or (viii); and wherein R.sup.8, m, p, G, Y, and the dashed curved line, are as defined herein, and further provided that the aza-macrocycle precursor has at least one R.sup.8 group which is hydrogen.

(153) Exemplary aza-macrocycle precursors are depicted in FIG. 1A. In certain embodiments, the aza-macrocycle precursor is a cyclic structure having from 10 to 30 ring members, inclusive; e.g., from 10 to 20 ring members, inclusive, or from 12 to 18 ring members, inclusive. In certain embodiments, the aza-macrocycle precursor is a 10-membered ring, an 11-membered ring, a 12-membered ring, a 13-membered ring, a 14-membered ring, a 15-membered ring, a 16-membered ring, a 17-membered ring, or an 18-membered ring. By extension, the conjugated aza-macrocycle, prepared therefrom, is also a cyclic structure having from 10 to 30 ring members, inclusive; e.g., from 10 to 20 ring members, inclusive, or from 12 to 18 ring members, inclusive. In certain embodiments, the conjugated aza-macrocycle is a 10-membered ring, an 11-membered ring, a 12-membered ring, a 13-membered ring, a 14-membered ring, a 15-membered ring, a 16-membered ring, a 17-membered ring, or an 18-membered ring.

(154) In certain embodiments of Formula (III), R.sup.8 is not a group of the formula (iii). Thus, in certain embodiments of Formula (III), each instance of R.sup.8 is independently hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl; provided that at least one R.sup.8 group is hydrogen. In certain embodiments of Formula (III), each instance of R.sup.8 is independently hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; or substituted or unsubstituted heteroalkyl. In certain embodiments of Formula (III), each instance of R.sup.8 is hydrogen.

(155) Alternatively, the conjugated aza-macrocycle of Formula (IV) requires that at least one R.sup.8 group is of the formula (iii). Thus, in certain embodiments, each instance of R.sup.8 is independently hydrogen; acyl; silyl; sulfonyl; an amino protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; or a group of the formula (iii), provided the aza-macrocycle contains at least one group of the formula (iii). In certain embodiments, each instance of R.sup.8 is independently selected from hydrogen; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; or a group of the formula (iii), provided the aza-macrocycle contains at least one group of the formula (iii). In certain embodiments, each instance of R.sup.8 is independently selected from hydrogen or a group of the formula (iii), provided the aza-macrocycle contains at least one group of the formula (iii).

(156) In certain embodiments, at least one R.sup.8 group of the aza-macrocycle is hydrogen. In certain embodiments, one R.sup.8 group of the aza-macrocycle is hydrogen. In certain embodiments, two R.sup.8 groups of the aza-macrocycle are hydrogen. In certain embodiments, three R.sup.8 groups of the aza-macrocycle are hydrogen. In certain embodiments, four R.sup.8 groups of the aza-macrocycle are hydrogen.

(157) In certain embodiments, 1 to 9 R.sup.8 groups provided in the aza-macrocycle is a group of the formula (iii); e.g., 1 to 9; 1 to 8; 1 to 7; 1 to 6; 1 to 5; 1 to 4; 1 to 3; 1 to 2 R.sup.8 groups, inclusive, are of the formula (iii). In certain embodiments, 1 R.sup.8 group of the aza-macrocycle is of the formula (iii). In certain embodiments, 2 R.sup.8 groups of the aza-macrocycle are groups of the formula (iii). In certain embodiments, 3 R.sup.8 groups of the aza-macrocycle are groups of the formula (iii). In certain embodiments, 4 R.sup.8 groups of the aza-macrocycle are groups of the formula (iii). In certain embodiments, all of the R.sup.8 groups of the aza-macrocycle are groups of the formula (iii).

(158) In certain embodiments, the ratio of NH to NR.sup.8 groups provided in the aza-macrocycle, wherein R.sup.8 is a group of the formula (iii), is between about 90:10 to about 0:100.

(159) As generally defined above, each instance of R.sup.3 is independently selected from substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; or a hydrophilic polymer. In certain embodiments, each instance of R.sup.3 is independently selected from substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; or a hydrophilic polymer. In certain embodiments, each instance of R.sup.3 is independently selected from substituted or unsubstituted alkyl; substituted or unsubstituted heteroalkyl; or a hydrophilic polymer.

(160) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted alkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.1-50alkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-50alkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-40alkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-30alkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-20alkyl.

(161) In certain embodiments, at least one instance of R.sup.3 is an unsubstituted alkyl. Exemplary unsubstituted alkyl groups include, but are not limited to, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.4H.sub.9, C.sub.5H.sub.11, C.sub.6H.sub.13, C.sub.7H.sub.15, C.sub.8H.sub.17, C.sub.9H.sub.19, C.sub.10H.sub.21, C.sub.11H.sub.23, C.sub.12H.sub.25, C.sub.13H.sub.27, C.sub.14H.sub.29, C.sub.15H.sub.31, C.sub.16H.sub.33, C.sub.17H.sub.35, C.sub.18H.sub.37, C.sub.19H.sub.39, and C.sub.20H.sub.41.

(162) In certain embodiments, at least one instance of R.sup.3 is a substituted alkyl. For example, in certain embodiments, at least one instance of R.sup.3 is an alkyl substituted with one or more fluorine substituents. Exemplary substituted alkyl groups include, but are not limited to:

(163) ##STR00226##

(164) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted alkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.2-50alkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-50 alkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-40 alkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-30 alkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-20 alkenyl. In certain embodiments, at least one instance of R.sup.3 is a substituted C.sub.8-20 alkenyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted alkenyl.

(165) Exemplary unsubstituted alkenyl groups include, but are not limited to:

(166) ##STR00227## Myristoleic (CH.sub.2).sub.7CHCH(CH.sub.2).sub.3CH.sub.3, Palmitoliec (CH.sub.2).sub.7CHCH(CH.sub.2).sub.5CH.sub.3, Sapienic (CH.sub.2).sub.4CHCH(CH.sub.2).sub.8CH.sub.3, Oleic (CH.sub.2).sub.7CHCH(CH.sub.2).sub.7CH.sub.3, Linoleic (CH.sub.2).sub.7CHCHCH.sub.2CHCH(CH.sub.2).sub.4CH.sub.3, -Linolenic (CH.sub.2).sub.7CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CH.sub.3, Arachinodonic (CH.sub.2).sub.3CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CHCH(CH.sub.2).sub.4CH.sub.3, Eicosapentaenoic (CH.sub.2).sub.3CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CH.sub.3, Erucic (CH.sub.2).sub.11CHCH(CH.sub.2).sub.7CH.sub.3, and Docosahexaenoic (CH.sub.2).sub.2CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CHCHCH.sub.2CH.sub.3.

(167) In embodiments, wherein R.sup.3 is defined as a C.sub.6-50alkyl or C.sub.6-50alkenyl groups, such groups are meant to encompass lipophilic groups (also referred to as a lipid tail). Lipophilic groups comprise a group of molecules that include fats, waxes, oils, fatty acids, and the like. Lipid tails present in these lipid groups can be saturated and unsaturated, depending on whether or not the lipid tail comprises double bonds. The lipid tail can also comprise different lengths, often categorized as medium (i.e., with tails between 7-12 carbons, e.g., C.sub.7-12 alkyl or C.sub.7-12 alkenyl), long (i.e., with tails greater than 12 carbons and up to 22 carbons, e.g., C.sub.13-22 alkyl or C.sub.13-22 alkenyl), or very long (i.e., with tails greater than 22 carbons, e.g., C.sub.23-30 alkyl or C.sub.23-30 alkenyl).

(168) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted alkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.2-50alkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-50 alkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-40 alkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-30 alkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-20 alkynyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted alkynyl. In certain embodiments, at least one instance of R.sup.3 is a substituted alkynyl.

(169) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.1-50 heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-50 heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-40 heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-30 heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-20 heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is a substituted heteroalkyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted heteroalkyl.

(170) Exemplary unsubstituted heteroalkyl groups include, but are not limited to:

(171) ##STR00228##

(172) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.2-50 heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-50 heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-40 heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-30 heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-20 heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is a substituted heteroalkenyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted heteroalkenyl.

(173) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.2-50 heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-50 heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-40 heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-30 heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted C.sub.8-20 heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is a substituted heteroalkynyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted heteroalkynyl.

(174) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted carbocyclyl. In certain embodiments, at least one instance of R.sup.3 is a substituted carbocyclyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted carbocyclyl.

(175) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted heterocyclyl. In certain embodiments, at least one instance of R.sup.3 is a substituted heterocyclyl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted heterocyclyl.

(176) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted aryl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted aryl. In certain embodiments, at least one instance of R.sup.3 is a substituted aryl.

(177) In certain embodiments, at least one instance of R.sup.3 is substituted or unsubstituted heteroaryl. In certain embodiments, at least one instance of R.sup.3 is a substituted heteroaryl. In certain embodiments, at least one instance of R.sup.3 is an unsubstituted heteroaryl.

(178) In certain embodiments, at least one instance of R.sup.3 is hydrophilic polymer. As used herein, a polymer refers to a compound comprised of at least 3 (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeating covalently bound structural units. By extension, a hydrophilic polymer is a polymer, as defined herein, further comprising at least one group (e.g., an oxygen, nitrogen, and/or sulfur atom) in the repeating structural unit capable of hydrogen bonding. The hydrophilic polymer is preferably biocompatible (i.e., non-toxic). Exemplary hydrophilic polymers include, but are not limited to, polypeptides (e.g., poly-L-lysine), cellulose polymers (e.g., hydroxyethylcellulose, ethylcellulose, carboxymethylcellulose, methylc cellulose, hydroxypropylmethylcellulose (HPMC)), dextran polymers, polymaleic acid polymers, poly(acrylic acid) polymers, poly(vinylalcohol) polymers, polyvinylpyrrolidone (PVP) polymers, and polyethyleneglycol (PEG) polymers.

(179) In certain embodiments, the hydrophilic polymer is a polyethyleneglycol polymer, e.g., of the formula (V):

(180) ##STR00229##
wherein:

(181) R.sup.7 is selected from hydrogen; acyl; silyl; a hydroxyl protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl; and

(182) v is an integer between 3 to 400, inclusive.

(183) In certain embodiments, R.sup.7 is selected from hydrogen. In certain embodiments, R.sup.7 is acyl. In certain embodiments, R.sup.7 is a hydroxyl protecting group. In certain embodiments, R.sup.7 is substituted or unsubstituted alkyl. In certain embodiments, R.sup.7 is a substituted alkyl. In certain embodiments, R.sup.7 is an unsubstituted alkyl. In certain embodiments, R.sup.7 is CH.sub.3 (a polyethyleneglycol monomethylether polymer). In certain embodiments, R.sup.7 is substituted or unsubstituted alkenyl. In certain embodiments, R.sup.7 is substituted or unsubstituted alkynyl. In certain embodiments, R.sup.7 is substituted or unsubstituted heteroalkyl. In certain embodiments, R.sup.7 is substituted or unsubstituted heteroalkenyl. In certain embodiments, R.sup.7 is substituted or unsubstituted heteroalkynyl. In certain embodiments, R.sup.7 is substituted or unsubstituted carbocyclyl. In certain embodiments, R.sup.7 is substituted or unsubstituted heterocyclyl. In certain embodiments, R.sup.7 is substituted or unsubstituted aryl. In certain embodiments, R.sup.7 is and substituted or unsubstituted heteroaryl.

(184) In certain embodiments, v is an integer between 3 to 300, 3 to 200, 3 to 100, 3 to 90, 3 to 80, 3 to 70, 3 to 60, 3 to 50, 5 to 50, 10 to 50, 15 to 50, 20 to 50, 20 to 40, 20 to 30, 20 to 25, 30 to 50, and 40 to 50, inclusive. PEG.sub.1000 corresponds, on average, to a v of about 22.7, wherein R.sup.7 is OCH.sub.3. PEG.sub.2000 corresponds, on average, to a v of about 45.4.

(185) In certain embodiments, the number average molar mass (Mn) of the polyethyleneglycol polymer is 10,000. In certain embodiments, the number average molar mass (Mn) of the polyethyleneglycol polymer is 10,000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, or 2000. In certain embodiments, the number average molar mass (Mn) of the polyethyleneglycol polymer is between about 100 to about 10,000, inclusive; e.g., between about 100 to about 5000, between about 100 to about 4000, between about 100 to about 3000, between about 100 to about 2500, between about 100 to about 2000, between about 100 to about 1500, between about 100 to about 1000, between about 100 to about 900, between about 100 to about 800, between about 100 to about 700, between about 100 to about 600, between about 100 to about 500, between about 100 to about 400, between about 100 to about 300, between about 100 to about 200, between about 100 to about 1500, between about 2500 to about 10000, between about 2500 to about 9000, between about 2500 to about 8000, between about 2500 to about 7000, between about 2500 to about 6000, between about 2500 to about 5000, between about 2500 to about 4000, or between about 2500 to about 3000, inclusive. In certain embodiments, the number average molar mass (Mn) of the polyethyleneglycol polymer is 1000 (PEG.sub.1000). In certain embodiments, the number average molar mass (Mn) of the polyethyleneglycol polymer is 2000 (PEG.sub.2000). A 1:1 mixture of PEG.sub.1000 and PEG.sub.2000 is referred to herein as PEG.sub.1.5K.

(186) In certain embodiments, at least one instance of R.sup.3 is a hydrophilic polymer, and at least one instance of R.sup.3 is a substituted or unsubstituted alkyl.

(187) As used herein, when the group R.sup.3 is depicted as bisecting a carbon-carbon bond, e.g., of the group of the formula (iii), it is understood that R.sup.3 may be substituted at either carbon. It is understood from the present disclosure that the group of formula (iii) represents a group of formula (iii-a) or a group of formula (iii-b). Nucleophilic attack of an amino group of the aza-macrocycle at the least sterically hindered carbon of the epoxide provides a group of the formula (iii-a) (route a), while nucleophilic attack at the more sterically hindered carbon of the epoxide provides a group of the formula (iii-b) (route b), wherein R.sup.4 is hydrogen; see, e.g., the conjugation reaction of Scheme V. It is thus understood that compounds of the present invention may comprise a mixture of products attached thereto arising from route (a) and route (b) depending on the preference, or lack thereof, of the mode of addition, and that formulae that depict this bisecting bond may comprise a mixture of compounds. The bisecting group R.sup.3 depicted in the formulae seeks to encompasses all contemplated modes of addition.

(188) ##STR00230##

(189) The resulting hydroxyl moiety of the formula (iii-a) or (iii-b), wherein R.sup.4 is hydrogen, can optionally be converted to a substituted hydroxyl, wherein R.sup.4 is a group other than hydrogen, i.e., is independently acyl; silyl; a hydroxyl protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; substituted or unsubstituted heteroalkenyl; substituted or unsubstituted heteroalkynyl; substituted or unsubstituted carbocyclyl; substituted or unsubstituted heterocyclyl; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl; using conventional methods. Alkylation, acylation, and/or protection of a hydroxyl moiety are well-known in the art; see, e.g., Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3.sup.rd edition, John Wiley & Sons, 1999; Smith and March, March's Advanced Organic Chemistry, 5.sup.th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3.sup.rd Edition, Cambridge University Press, Cambridge, 1987. For example, in certain non-limiting embodiments, the hydroxyl moiety is reacted with an electrophile of the formula R.sup.4X wherein R.sup.4 is a group other than hydrogen and X is a leaving group to provide a substituted hydroxyl group in formula (iii).

(190) In certain embodiments, each instance of R.sup.4 is independently hydrogen; acyl; silyl; a hydroxyl protecting group; substituted or unsubstituted alkyl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heteroalkyl; or substituted or unsubstituted heteroalkenyl. In certain embodiments, each instance of R.sup.4 is independently hydrogen; substituted or unsubstituted alkyl; or substituted or unsubstituted heteroalkyl. In certain embodiments, each instance of R.sup.4 is hydrogen.

(191) In certain embodiments, the conjugation reaction depicted in Scheme V results in a mixture comprising more lipomers conjugated to a group of formula (iii-a) than to a group of formula (iii-b), e.g., the reaction mixture comprises greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 99%, between about 60% to about 100%, between about 70% to about 100%, between about 80% to about 100%, between about 90% to about 100%, between about 95% to about 100%, or between about 99% to about 100%, of a conjugated lipomer attached to a group of formula (iii-a).

(192) In certain embodiments, the reaction mixture comprises only conjugated lipomers attached to a group formula (iii-a).

(193) In certain embodiments, the epoxide is chiral, i.e., having (R) or (S) stereochemistry. In this instance, in certain embodiments, the conjugation reaction depicted in Scheme V provides a chiral conjugated aza-macrocycle.

(194) In certain embodiments, wherein the epoxide is chiral, the conjugation reaction depicted in Scheme V results in a mixture comprising more lipomers conjugated to a group of formula (R)-(iii-a) than to a group of formula (S)-(iii-a), e.g., the reaction mixture comprises greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 99%, between about 60% to about 100%, between about 70% to about 100%, between about 80% to about 100%, between about 90% to about 100%, between about 95% to about 100%, or between about 99% to about 100%, of conjugated lipomer attached to a group of formula (R)-(iii-a).

(195) In certain embodiments, the reaction mixture comprises only conjugated lipomers attached to a group formula (R)-(iii-a).

(196) In certain embodiments, wherein the epoxide is chiral, the conjugation reaction depicted in Scheme V results in a mixture comprising more lipomers conjugated to a group of formula (S)-(iii-a) than to a group of formula (R)-(iii-a), e.g., the reaction mixture comprises greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 99%, between about 60% to about 100%, between about 70% to about 100%, between about 80% to about 100%, between about 90% to about 100%, between about 95% to about 100%, or between about 99% to about 100%, of a conjugated lipomer attached to a group of formula (S)-(iii-a).

(197) In certain embodiments, the reaction mixture comprises only conjugated lipomers attached to a group formula (S)-(iii-a).

(198) ##STR00231##

(199) In certain embodiments, wherein one epoxide is used in the conjugation reaction, each instance of R.sup.3 is the same in the conjugated aza-macrocycle. For example, in certain embodiments, each instance of R.sup.3 is the same wherein R.sup.3 is a substituted or unsubstituted alkyl. In certain embodiments, each instance of R.sup.3 is the same wherein R.sup.3 is an unsubstituted alkyl. In certain embodiments, each instance of R.sup.3 is the same wherein R.sup.3 is selected from the group consisting of CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.4H.sub.9, C.sub.5H.sub.11, C.sub.6H.sub.13, C.sub.7H.sub.15, C.sub.8H.sub.17, C.sub.9H.sub.19, C.sub.10H.sub.21, C.sub.11H.sub.23, C.sub.12H.sub.25, C.sub.13H.sub.27, C.sub.14H.sub.29, C.sub.15H.sub.31, C.sub.16H.sub.33, C.sub.17H.sub.35, C.sub.18H.sub.37, C.sub.19H.sub.39, and C.sub.20H.sub.41. In certain embodiments, each instance of R.sup.3 is the same wherein R.sup.3 is selected from the group consisting of C.sub.8H.sub.17, C.sub.9H.sub.19, C.sub.10H.sub.21, C.sub.11H.sub.23, C.sub.12H.sub.25, C.sub.13H.sub.27, C.sub.14H.sub.29, C.sub.15H.sub.31, and C.sub.16H.sub.33.

(200) Alternatively, in certain embodiments, wherein more than one epoxide is used in the conjugation reaction (e.g., two, three, four, five, six, seven, eight, nine, or ten different epoxides), the conjugated aza-macrocycle comprises two or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) different R.sup.3 groups.

(201) For example, in certain embodiments, two different epoxides are used in the conjugation reaction. In this instance, in certain embodiments, the conjugated aza-macrocycle comprises two different R.sup.3 groups. For example, in certain embodiments, the conjugated aza-macrocycle comprises a mixture of two different R.sup.3 groups, wherein the first R.sup.3 group is a substituted or unsubstituted alkyl, and the second R.sup.3 group is a hydrophilic polymer (e.g., a polyethyleneglycol polymer, as defined herein). In certain embodiments, the conjugated aza-macrocycle comprises a mixture of two different R.sup.3 groups, wherein the first R.sup.3 group is an unsubstituted alkyl, and the second R.sup.3 group is a polyethyleneglycol polymer. In certain embodiments, the conjugated aza-macrocycle comprises a mixture of two different R.sup.3 groups, wherein the first R.sup.3 group is selected from the group consisting of CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.4H.sub.9, C.sub.5H.sub.11, C.sub.6H.sub.13, C.sub.7H.sub.15, C.sub.8H.sub.17, C.sub.9H.sub.19, C.sub.10H.sub.21, C.sub.11H.sub.23, C.sub.12H.sub.25, C.sub.13H.sub.27, C.sub.14H.sub.29, C.sub.15H.sub.31, C.sub.16H.sub.33, C.sub.17H.sub.35, C.sub.18H.sub.37, C.sub.19H.sub.39, and C.sub.20H.sub.41, and the second R.sup.3 group is PEG.sub.1000. In certain embodiments, the conjugated aza-macrocycle comprises a mixture of two different R.sup.3 groups, wherein the first R.sup.3 group is selected from the group consisting of CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.4H.sub.9, C.sub.5H.sub.11, C.sub.6H.sub.13, C.sub.7H.sub.15, C.sub.8H.sub.17, C.sub.9H.sub.19, C.sub.10H.sub.21, C.sub.11H.sub.23, C.sub.12H.sub.25, C.sub.13H.sub.27, C.sub.14H.sub.29, C.sub.15H.sub.31, C.sub.16H.sub.33, C.sub.17H.sub.35, C.sub.18H.sub.37, C.sub.19H.sub.39, and C.sub.20H.sub.41, and the second R.sup.3 group is PEG.sub.2000.

(202) In certain embodiments, three different epoxides are used in the conjugation reaction. In this instance, in certain embodiments, the conjugated aza-macrocycle comprises three different R.sup.3 groups. For example, in certain embodiments, the conjugated aza-macrocycle comprises a mixture of three different R.sup.3 groups, wherein the first R.sup.3 group is a substituted or unsubstituted alkyl, the second R.sup.3 group is a first hydrophilic polymer (e.g., a polyethyleneglycol polymer, as defined herein), and the third R.sup.3 group is a second hydrophilic polymer (e.g., a different polyethyleneglycol polymer). In certain embodiments, the conjugated aza-macrocycle comprises a mixture of three different R.sup.3 groups, wherein the first R.sup.3 group is an unsubstituted alkyl, the second R.sup.3 group is PEG.sub.1000, and the third R.sup.3 group is PEG.sub.2000. In certain embodiments, the conjugated aza-macrocycle comprises a mixture of three different R.sup.3 groups, wherein the first R.sup.3 group is selected from the group consisting of CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.4H.sub.9, C.sub.5H.sub.11, C.sub.6H.sub.13, C.sub.7H.sub.15, C.sub.8H.sub.17, C.sub.9H.sub.19, C.sub.10H.sub.21, C.sub.11H.sub.23, C.sub.12H.sub.25, C.sub.13H.sub.27, C.sub.14H.sub.29, C.sub.15H.sub.31, C.sub.16H.sub.33, C.sub.17H.sub.35, C.sub.18H.sub.37, C.sub.191439, and C.sub.20H.sub.41, the second R.sup.3 group is PEG.sub.1000, and the third R.sup.3 group is PEG.sub.2000.

(203) In certain embodiments, wherein the conjugated aza-macrocycle comprises two different R.sup.3 groups, the ratio of the second R.sup.3 group to the first R.sup.3 group is between about 0.01:10 to about 10:10, inclusive. In certain embodiments, the ratio of the second R.sup.3 group to the first R.sup.3 group is between about 0.02:10 to about 10:10; between about 0.03:10 to about 10:10; between about 0.04:10 to about 10:10; between about 0.05:10 to about 10:10; between about 0.06:10 to about 10:10; between about 0.07:10 to about 10:10; between about 0.08:10 to about 10:10; between about 0.08:10 to about 9:10; between about 0.08:10 to about 8:10; between about 0.08:10 to about 7:10; between about 0.08:10 to about 6:10; between about 0.08:10 to about 5:10; between about 0.08:10 to about 4:10; between about 0.08:10 to about 3:10; between about 0.08:10 to about 2:10; or between about 0.08:10 to about 1:10, inclusive. In certain embodiments, the ratio of the second R.sup.3 group to the first R.sup.3 group is about 0.1:10.

(204) In certain embodiments, wherein the conjugated aza-macrocycle comprises three different R.sup.3 groups, the ratio of the sum of the second and third R.sup.3 groups to the first R.sup.3 group is between about 0.01:10 to about 10:10, inclusive. In certain embodiments, the ratio of the sum of the second and third R.sup.3 groups to the first R.sup.3 group is 0.02:10 to about 10:10; between about 0.03:10 to about 10:10; between about 0.04:10 to about 10:10; between about 0.05:10 to about 10:10; between about 0.06:10 to about 10:10; between about 0.07:10 to about 10:10; between about 0.08:10 to about 10:10; between about 0.08:10 to about 9:10; between about 0.08:10 to about 8:10; between about 0.08:10 to about 7:10; between about 0.08:10 to about 6:10; between about 0.08:10 to about 5:10; between about 0.08:10 to about 4:10; between about 0.08:10 to about 3:10; between about 0.08:10 to about 2:10; or between about 0.08:10 to about 1:10, inclusive. In certain embodiments, the ratio of the sum of the second and third R.sup.3 groups to the first R.sup.3 group is about 0.1:10.

(205) As generally defined above, each instance of m and p is independently 0, 1 or 2. In certain embodiments, each instance of m is is independently 0, 1 or 2. In certain embodiments, each instance of m is 1. In certain embodiments, each instance of m is 2. In certain embodiments, each instance of m is independently 1 or 2. In certain embodiments, each instance of p is independently 0, 1 or 2. In certain embodiments, each instance of p is 1. In certain embodiments, each instance of p is 2. In certain embodiments, each instance of p is independently 1 or 2.

(206) As generally defined above, q is an integer 2, 3, or 4. In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 2 or 3. In certain embodiments, q is 4.

(207) As generally defined above, the dashed curved line, together with G and Y, is a covalent bond or a group of the formula:

(208) ##STR00232##
wherein s is 0, 1, or 2.

(209) In certain embodiments, the dashed curved line, together with G and Y, is a covalent bond.

(210) In certain embodiments, the dashed curved line, together with G and Y, a group of the formula:

(211) ##STR00233##
wherein s is 0, 1, or 2. In certain embodiments, s is 1 or 2. In certain embodiments, s is 1.

(212) In certain embodiments, the dashed curved line, together with G and Y, a group of the formula:

(213) ##STR00234##
wherein s is 0, 1, or 2. In certain embodiments, s is 1 or 2. In certain embodiments, s is 1.

(214) As generally defined above, each instance of L.sup.3 is independently:

(215) ##STR00235##
provided that the conjugated aza-macrocycle contains at least one group (vi), (vii) or (viii);

(216) In certain embodiments, the conjugated aza-macrocycle comprises at least one instance of the group of the formula (vi). In certain embodiments, each instance of L.sup.3 is a group of the formula (vi). For example, in this instance, the conjugated aza-macrocycle of the Formula (IV) is of the Formula (V), (VI), or (VII):

(217) ##STR00236##
or salt thereof, wherein R.sup.8 is as defined herein.

(218) In certain embodiments, the conjugated aza-macrocycle comprises at least one instance of the group of the formula (vii), (viii), or (ix). In certain embodiments, the conjugated aza-macrocycle comprises at least one instance of the group of the formula (vii). In certain embodiments, the conjugated aza-macrocycle comprises at least one instance of the group of the formula (viii). In certain embodiments, the conjugated aza-macrocycle comprises at least one instance of the group of the formula (ix). In these instances, in certain embodiments, the conjugated aza-macrocycle of the Formula (IV) is of the Formula (VIII) or (IX):

(219) ##STR00237##
or salt thereof, wherein R.sup.8 is as defined herein.

(220) Exemplary conjugated aza-macrocycles of the Formula (V) include, but are not limited to:

(221) ##STR00238## ##STR00239## ##STR00240## ##STR00241## ##STR00242## ##STR00243## ##STR00244## ##STR00245## ##STR00246##
and salts thereof, wherein R.sup.3 is defined herein. In certain embodiments, R.sup.3 is a polyethyleglycol polymer. In certain embodiments, R.sup.3 is PEG.sub.1000. In certain embodiments, R.sup.3 is PEG.sub.2000. In certain embodiments, R.sup.3 is PEG.sub.1.5K.

(222) Exemplary conjugated aza-macrocycles of the Formula (VI) include, but are not limited to:

(223) ##STR00247## ##STR00248## ##STR00249## ##STR00250## ##STR00251## ##STR00252## ##STR00253## ##STR00254## ##STR00255## ##STR00256## ##STR00257## ##STR00258## ##STR00259## ##STR00260## ##STR00261## ##STR00262## ##STR00263## ##STR00264##
and salts thereof, wherein R.sup.3 is defined herein. In certain embodiments, R.sup.3 is a polyethyleglycol polymer. In certain embodiments, R.sup.3 is PEG.sub.1000. In certain embodiments, R.sup.3 is PEG.sub.2000. In certain embodiments, R.sup.3 is PEG.sub.1.5K.

(224) Exemplary conjugated aza-macrocycles of the Formula (VII) include, but are not limited to:

(225) ##STR00265## ##STR00266## ##STR00267## ##STR00268## ##STR00269## ##STR00270## ##STR00271## ##STR00272## ##STR00273## ##STR00274## ##STR00275## ##STR00276## ##STR00277## ##STR00278## ##STR00279## ##STR00280## ##STR00281## ##STR00282## ##STR00283## ##STR00284## ##STR00285## ##STR00286## ##STR00287## ##STR00288## ##STR00289## ##STR00290## ##STR00291## ##STR00292## ##STR00293## ##STR00294## ##STR00295## ##STR00296## ##STR00297## ##STR00298## ##STR00299## ##STR00300## ##STR00301## ##STR00302## ##STR00303## ##STR00304## ##STR00305##
and salts thereof, wherein R.sup.3 is defined herein. In certain embodiments, R.sup.3 is a polyethyleglycol polymer. In certain embodiments, R.sup.3 is PEG.sub.1000. In certain embodiments, R.sup.3 is PEG.sub.2000. In certain embodiments, R.sup.3 is PEG.sub.1.5K.

(226) Exemplary conjugated aza-macrocycles of the Formula (VII) include, but are not limited to:

(227) ##STR00306## ##STR00307## ##STR00308## ##STR00309## ##STR00310##
and salts thereof, wherein R.sup.3 is defined herein. In certain embodiments, R.sup.3 is a polyethyleglycol polymer. In certain embodiments, R.sup.3 is PEG.sub.1000. In certain embodiments, R.sup.3 is PEG.sub.2000. In certain embodiments, R.sup.3 is PEG.sub.1.5K.

(228) Exemplary conjugated aza-macrocycles of the Formula (VIII) include, but are not limited to:

(229) ##STR00311## ##STR00312## ##STR00313## ##STR00314##
and salts thereof, wherein R.sup.3 is defined herein. In certain embodiments, R.sup.3 is a polyethyleglycol polymer. In certain embodiments, R.sup.3 is PEG.sub.1000. In certain embodiments, R.sup.3 is PEG.sub.2000. In certain embodiments, R.sup.3 is PEG.sub.1.5K.
Additional Methods of Preparation

(230) As described herein, preparation of both conjugated polyethyleneimine complexes and conjugated macrocycles (also referred to herein as conjugated lipomers or lipomers) is achieved using similar reaction conditions and reagents. In particular, the precursors are treated with one or more epoxide agents to provide the inventive conjugated lipomer. Each of the precursors is dissolved in an organic solvent (e.g., THF, CH.sub.2Cl.sub.2, MeOH, EtOH, CHCl.sub.3, hexanes, toluene, benzene, CCl.sub.4, glyme, diethyl ether, etc.), the one or more epoxides are added, and the reaction mixture is heated to yield the desired conjugated lipomer.

(231) In certain embodiments, the reaction mixture is heated to between about 50 C. to about 150 C. In certain embodiments, the reaction mixture is heated to about 90 C.

(232) In certain embodiments, the epoxide is chiral. The chiral epoxides useful in the invention can be obtained from a variety of sources which are familiar to those skilled in the art of organic synthesis. In some embodiments, the chiral epoxides useful in the invention can be obtained commercially. In some embodiments, the chiral epoxides useful in the invention can be synthesized according to methods known to those of skill in the art, such as, but not limited to the Sharpless epoxidation of primary and secondary allylic alcohols into 2,3-epoxyalcohols (Katsuki et al., J. Am. Chem. Soc. 1980, 102, 5974; Hill et al., Org. Syn., Coll. Vol. 7, p. 461 (1990); Vol. 63, p. 66 (1985); Katsuki et al., Org. React. 1996, 48, 1-300; incorporated herein by reference.) In some embodiments, the chiral epoxides useful in the invention are obtained from the resolution of racemic epoxides. In some embodiments, the chiral epoxides useful in the invention are obtained by the separation of enantiomers or diastereoisomers using chiral chromatography.

(233) As would be appreciated by one of skill in this art, the degree of conjugation may be controlled by the reaction conditions (e.g., temperature, starting materials, concentration, solvent, etc.) used in the synthesis.

(234) The synthesized conjugated lipomer may be purified by any technique known in the art including, but not limited to, precipitation, crystallization, chromatography, distillation, etc. In certain embodiments, the conjugated lipomer is purified through repeated precipitations in organic solvent (e.g., diethyl ether, hexane, etc.).

(235) In certain embodiments, the conjugated lipomer is isolated as a salt. For example, in certain embodiments, the conjugated lipomer is reacted with an acid (e.g., an organic acid or inorganic acid) to form the corresponding salt. In other embodiments, the tertiary amine is alkylated to form a quaternary ammonium salt of the conjugated lipomer. The tertiary amines may be alkylated with any alkylating agent, for example, alkyl halides such as methyl iodide may be used to from the quaternary amino groups. The anion associated with the quaternary amine may be any organic or inorganic anion. Preferably, the anion is a pharmaceutically acceptable anion.

(236) The invention also provides libraries of the inventive conjugated lipomers prepared by the inventive methods. These conjugated lipomers may be prepared and/or screened using high-throughput techniques involving liquid handlers, robots, microtiter plates, computers, etc. In certain embodiments, the conjugated lipomers are screened for their ability to transfect polynucleotides or other agents (e.g., proteins, peptides, small molecules) into the cell.

(237) In one embodiment, a library of different conjugated lipomers is prepared in parallel. A different precursor and/or epoxide is added to each vial in a set of vials or to each well of a multi-well plate used to prepare the library. The array of reaction mixtures is incubated at a temperature and length of time sufficient to allow formation of the conjugated lipomer. In one embodiment, the vials are incubated at approximately 90 C. overnight. In certain embodiments, the vials are incubated from 1 to 7 days at approximately 90 C. In certain embodiments, the vials are incubated from 3 to 4 days at approximately 90 C. In certain embodiments, the vials are incubated from 1 to 2 days at approximately 90 C. The conjugated lipomer may then be isolated and purified using techniques known in the art. The conjugated lipomer may then be screened using high-throughput techniques to identify conjugated lipomers with a desired characteristic (e.g., solubility in water, solubility at different pH, ability to bind polynucleotides, ability to bind heparin, ability to bind small molecules, ability to bind protein, ability to form microparticles, ability to increase transfection efficiency, etc.). In certain embodiments the conjugated lipomers may be screened for properties or characteristics useful as coatings, additives, materials, and excipients in biotechnology and biomedical applications such as the coating of medical devices or implants with films or multilayer films, as non-biofouling agents, micropatterning agents, and cellular encapsulation agents. In certain embodiments the conjugated lipomer may be screened for properties or characteristics useful in gene therapy (e.g., ability to bind polynucleotides, increase in transfection efficiency) or the administration and/or delivery of therapeutic agents (e.g., polynucleotide, small molecule, antigen, drug, protein, peptide, etc.) to a subject, patient, tissue, organ, or cell, etc.

(238) Polynucleotide Complexes

(239) The inventive conjugated lipomers are particularly useful in the administration of polynucleotides. For example, the inventive conjugated lipomers possess tertiary amines, and although these amines are hindered, they are available to interact with a polynucleotide (e.g., DNA, RNA, synthetic analogs of DNA and/or RNA, DNA/RNA hydrids, etc.). Polynucleotides or derivatives thereof are contacted with the inventive conjugated lipomers under conditions suitable to form polynucleotide/lipomer complexes. The interaction of the lipomer with the polynucleotide is thought to at least partially prevent the degradation of the polynucleotide. By neutralizing the charge on the backbone of the polynucleotide, the neutral or slightly-positively-charged complex is also able to more easily pass through the hydrophobic membranes (e.g., cytoplasmic, lysosomal, endosomal, nuclear) of the cell. In certain embodiments, the complex is slightly positively charged. In certain embodiments, the complex has a positive -potential, more preferably the -potential is between 0 and +30.

(240) The conjugated lipomer is preferably at least partially provided as a salt (i.e., is protonated) so as to form a complex with the negatively charged polynucleotide. In certain embodiments, the polynucleotide/lipomer complexes form particles that are useful in the delivery of polynucleotides to cells. In certain embodiments, more than one conjugated lipomer may be associated with a polynucleotide molecule. For example, the complex may include 1-100 conjugated lipomers, 1-1000 conjugated lipomers, 10-1000 conjugated lipomers, or 100-10,000 conjugated lipomers.

(241) In certain embodiments, the complex may form a particle. In certain embodiments, the diameter of the particles ranges from 10-500 micrometers. In certain embodiments, the diameter of the particles ranges from 10-1200 micrometers. In certain embodiments, the diameter of the particles ranges from 50-150 micrometers. In certain embodiments, the diameter of the particles ranges from 10-500 nm, more preferably the diameter of the particles ranges from 10-1200 nm, and most preferably from 50-150 nm. The particles may be associated with a targeting agent as described below. In certain embodiments, the diameter of the particles ranges from 10-500 pm, more preferably the diameter of the particles ranges from 10-1200 pm, and most preferably from 50-150 pm. The particles may be associated with a targeting agent as described below. The film architecture is precisely designed and can be controlled to 1 nm precision with a range from 1 to 150000 nm and with a definite knowledge of its molecular composition.

(242) The polynucleotide may be complexed, encapsulated by the inventive conjugated lipomers, or included in a composition comprising the inventive conjugated lipomers. The polynucleotide may be any nucleic acid including, but not limited to, RNA and DNA. In certain embodiments, the polynucleotide is DNA. In certain embodiments, the polynucleotide is RNA.

(243) In certain embodiments, the polynucleotide is an RNA that carries out RNA interference (RNAi). The phenomenon of RNAi is discussed in greater detail, for example, in the following references, each of which is incorporated herein by reference: Elbashir et al., 2001, Genes Dev., 15:188; Fire et al., 1998, Nature, 391:806; Tabara et al., 1999, Cell, 99:123; Hammond et al., Nature, 2000, 404:293; Zamore et al., 2000, Cell, 101:25; Chakraborty, 2007, Curr. Drug Targets, 8:469; and Morris and Rossi, 2006, Gene Ther., 13:553. In certain embodiments, the polynucleotide is a dsRNA (double-stranded RNA). In certain embodiments, the polynucleotide is an siRNA (short interfering RNA). In certain embodiments, the polynucleotide is an shRNA (short hairpin RNA).

(244) In certain embodiments, the polynucleotide is an miRNA (micro RNA). micro RNAs (miRNAs) are genomically encoded non-coding RNAs of about 21-23 nucleotides in length that help regulate gene expression, particularly during development (see, e.g., Bartel, 2004, Cell, 116:281; Novina and Sharp, 2004, Nature, 430:161; and U.S. Patent Publication 2005/0059005; also reviewed in Wang and Li, 2007, Front. Biosci., 12:3975; and Zhao, 2007, Trends Biochem. Sci., 32:189; each of which are incorporated herein by reference).

(245) In certain embodiments, the polynucleotide is an antisense RNA.

(246) In some embodiments, dsRNA, siRNA, shRNA, miRNA and/or antisense RNA can be designed and/or predicted using one or more of a large number of available algorithms. To give but a few examples, the following resources can be utilized to design and/or predict dsRNA, siRNA, shRNA, and/or miRNA: algorithms found at Alnylum Online, Dharmacon Online, OligoEngine Online, Molecula Online, Ambion Online, BioPredsi Online, RNAi Web Online, Chang Bioscience Online, Invitrogen Online, LentiWeb Online GenScript Online, Protocol Online; Reynolds et al., 2004, Nat. Biotechnol., 22:326; Naito et al., 2006, Nucleic Acids Res., 34:W448; Li et al., 2007, RNA, 13:1765; Yiu et al., 2005, Bioinformatics, 21:144; and Jia et al., 2006, BMC Bioinformatics, 7: 271; each of which is incorporated herein by reference).

(247) The polynucleotides may be of any size or sequence, and they may be single- or double-stranded. In certain embodiments, the polynucleotide is greater than 100 base pairs long. In certain embodiments, the polynucleotide is greater than 1000 base pairs long and may be greater than 10,000 base pairs long. The polynucleotide is optionally purified and substantially pure. Preferably, the polynucleotide is greater than 50% pure, more preferably greater than 75% pure, and most preferably greater than 95% pure. The polynucleotide may be provided by any means known in the art. In certain embodiments, the polynucleotide has been engineered using recombinant techniques (for a more detailed description of these techniques, please see Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, Inc., New York, 1999); Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989); each of which is incorporated herein by reference). The polynucleotide may also be obtained from natural sources and purified from contaminating components found normally in nature. The polynucleotide may also be chemically synthesized in a laboratory. In certain embodiments, the polynucleotide is synthesized using standard solid phase chemistry.

(248) The polynucleotide may be modified by chemical or biological means. In certain embodiments, these modifications lead to increased stability of the polynucleotide. Modifications include methylation, phosphorylation, end-capping, etc.

(249) Derivatives of polynucleotides may also be used in the present invention. These derivatives include modifications in the bases, sugars, and/or phosphate linkages of the polynucleotide. Modified bases include, but are not limited to, those found in the following nucleoside analogs: 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine. Modified sugars include, but are not limited to, 2-fluororibose, ribose, 2-deoxyribose, 3-azido-2,3-dideoxyribose, 2,3-dideoxyribose, arabinose (the 2-epimer of ribose), acyclic sugars, and hexoses. The nucleosides may be strung together by linkages other than the phosphodiester linkage found in naturally occurring DNA and RNA. Modified linkages include, but are not limited to, phosphorothioate and 5-N-phosphoramidite linkages. Combinations of the various modifications may be used in a single polynucleotide. These modified polynucleotides may be provided by any means known in the art; however, as will be appreciated by those of skill in this art, the modified polynucleotides are preferably prepared using synthetic chemistry in vitro.

(250) The polynucleotides to be delivered may be in any form. For example, the polynucleotide may be a circular plasmid, a linearized plasmid, a cosmid, a viral genome, a modified viral genome, an artificial chromosome, etc.

(251) The polynucleotide may be of any sequence. In certain embodiments, the polynucleotide encodes a protein or peptide. The encoded proteins may be enzymes, structural proteins, receptors, soluble receptors, ion channels, pharmaceutically active proteins, cytokines, interleukins, antibodies, antibody fragments, antigens, coagulation factors, albumin, growth factors, hormones, insulin, etc. The polynucleotide may also comprise regulatory regions to control the expression of a gene. These regulatory regions may include, but are not limited to, promoters, enhancer elements, repressor elements, TATA box, ribosomal binding sites, stop site for transcription, etc. In certain embodiments, the polynucleotide is not intended to encode a protein. For example, the polynucleotide may be used to fix an error in the genome of the cell being transfected.

(252) The polynucleotide may also be provided as an antisense agent or RNA interference (RNAi) (Fire et al., Nature 391:806-811, 1998; incorporated herein by reference). Antisense therapy is meant to include, e.g., administration or in situ provision of single- or double-stranded oligonucleotides or their derivatives which specifically hybridize, e.g., bind, under cellular conditions, with cellular mRNA and/or genomic DNA, or mutants thereof, so as to inhibit expression of the encoded protein, e.g., by inhibiting transcription and/or translation (Crooke Molecular mechanisms of action of antisense drugs Biochim. Biophys. Acta 1489(1):31-44, 1999; Crooke Evaluating the mechanism of action of antiproliferative antisense drugs Antisense Nucleic Acid Drug Dev. 10(2):123-126, discussion 127, 2000; Methods in Enzymology volumes 313-314, 1999; each of which is incorporated herein by reference). The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix (i.e., triple helix formation) (Chan et al., J. Mol. Med. 75(4):267-282, 1997; incorporated herein by reference).

(253) In certain embodiments, the polynucleotide to be delivered comprises a sequence encoding an antigenic peptide or protein. Nanoparticles containing these polynucleotides can be delivered to an individual to induce an immunologic response sufficient to decrease the chance of a subsequent infection and/or lessen the symptoms associated with such an infection. The polynucleotide of these vaccines may be combined with interleukins, interferon, cytokines, and adjuvants such as cholera toxin, alum, Freund's adjuvant, etc. A large number of adjuvant compounds are known; a useful compendium of many such compounds is prepared by the National Institutes of Health (see Allison Dev. Biol. Stand. 92:3-11, 1998; Unkeless et al., Annu. Rev. Immunol. 6:251-281, 1998; and Phillips et al., Vaccine 10:151-158, 1992; each of which is incorporated herein by reference).

(254) The antigenic protein or peptides encoded by the polynucleotide may be derived from such bacterial organisms as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter jejuni, and the like; from such viruses as smallpox, influenza A and B, respiratory syncytial virus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and the like; and from such fungal, protozoan, and parasitic organisms such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and the like.

(255) Table 3 of the Examples provides the nitrogen: phosphate ratio of conjugated lipomers of the present invention. The nitrogen:phosphate ratio (i.e., the ratio between the amino groups present in the lipomer, and the phosphate groups present in the polynucleotide) is between about 10:1 to about 50:1, inclusive. In certain embodiments, the nitrogen phosphate ratio is between about 10:1 to about 45:1, between about 15:1 to about 45:1, or between about 20:1 to about 40:1, inclusive. Increasing nitrogen:phosphate ratios have been shown to positively influence delivery of genetic material by increasing nucleic acid binding and negatively influence delivery by increasing toxicity (see, e.g., Incani et al., Soft Matter (2010) 6:2124-2138).

(256) Table 3 of the Examples also provides the lipomer:polynucleotide mass ratio and molar ratios measured from complexes of polynucleotide and conjugated lipomers of the present invention. The conjugated lipomer:polynucleotide mass ratio is between about 10:1 to about 20:1, inclusive. In certain embodiments, the conjugated lipomer:polynucleotide mass ratio is about 15:1. The conjugated lipomer:polynucleotide molar ratio is between about 10:1 to about 400:1, inclusive. In certain embodiments, the conjugated lipomer:polynucleotide molar ratio is between about 10:1 to about 350:1, between about 15:1 to about 300:1, or between about 20:1 to about 250:1, inclusive.

(257) Particles

(258) The conjugated lipomers of the present invention may also be used to form drug delivery devices. The inventive conjugated lipomers have several properties that make them particularly suitable in the preparation of drug delivery devices. These include: 1) the ability of the lipomer to complex and protect labile agents; 2) the ability to buffer the pH in the endosome; 3) the ability to act as a proton sponge and cause endosomolysis; and 4) the ability to neutralize the charge on negatively charged agents.

(259) In certain embodiments, the conjugated lipomers are used to form particles containing the agent to be delivered. The inventive conjugated lipomers may be used to encapsulate agents including, but not limited to, organic molecules (e.g., cholesterol), inorganic molecules, nucleic acids, proteins, peptides, polynucleotides, targeting agents, isotopically labeled organic or inorganic molecules, vaccines, immunological agents, etc. Other exemplary agents are described in greater detail herein. These particles may include other materials such as polymers (e.g., synthetic polymers (e.g., PEG, PLGA), natural polymers (e.g., phospholipids)). In certain embodiments, the conjugated lipomers are mixed with one or more agents (e.g., cholesterol) and/or one or more other materials (e.g., polymers). For example, as shown in FIGS. 5A-5B and described in the Examples, a conjugated lipomer was mixed with an agent and a polymer, or just mixed with an agent, to provide inventive particles.

(260) In certain embodiments, the diameter of the particles range from between 1 micrometer to 1,000 micrometers. In certain embodiments, the diameter of the particles range from between from 1 micrometer to 100 micrometers. In certain embodiments, the diameter of the particles range from between from 1 micrometer to 10 micrometers. In certain embodiments, the diameter of the particles range from between from 10 micrometer to 100 micrometers. In certain embodiments, the diameter of the particles range from between from 100 micrometer to 1,000 micrometers. In certain embodiments, the particles range from 1-5 micrometers. In certain embodiments, the diameter of the particles range from between 1 nm to 1,000 nm. In certain embodiments, the diameter of the particles range from between from 1 nm to 100 nm. In certain embodiments, the diameter of the particles range from between from 1 nm to 10 nm. In certain embodiments, the diameter of the particles range from between from 10 nm to 100 nm. In certain embodiments, the diameter of the particles range from between from 100 nm to 1,000 nm. In certain embodiments, the particles range from 1-5 nm. In certain embodiments, the diameter of the particles range from between 1 pm to 1,000 pm. In certain embodiments, the diameter of the particles range from between from 1 pm to 100 pm. In certain embodiments, the diameter of the particles range from between from 1 pm to 10 pm. In certain embodiments, the diameter of the particles range from between from 10 pm to 100 pm. In certain embodiments, the diameter of the particles range from between from 100 pm to 1,000 pm. In certain embodiments, the particles range from 1-5 pm.

(261) The inventive particles may be prepared using any method known in this art. These include, but are not limited to, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, simple and complex coacervation, and other methods well known to those of ordinary skill in the art. In certain embodiments, methods of preparing the particles are the double emulsion process and spray drying. The conditions used in preparing the particles may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology, stickiness, shape, etc.). The method of preparing the particle and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may also depend on the agent being encapsulated and/or the composition of the matrix.

(262) Methods developed for making particles for delivery of encapsulated agents are described in the literature (for example, please see Doubrow, M., Ed., Microcapsules and Nanoparticles in Medicine and Pharmacy, CRC Press, Boca Raton, 1992; Mathiowitz and Langer, J. Controlled Release 5:13-22, 1987; Mathiowitz et al., Reactive Polymers 6:275-283, 1987; Mathiowitz et al., J. Appl. Polymer Sci. 35:755-774, 1988; each of which is incorporated herein by reference).

(263) If the particles prepared by any of the above methods have a size range outside of the desired range, the particles can be sized, for example, using a sieve. The particle may also be coated. In certain embodiments, the particles are coated with a targeting agent. In other embodiments, the particles are coated to achieve desirable surface properties (e.g., a particular charge).

(264) Micelles and Liposomes and Lipoplexes

(265) The conjugated lipomers of the invention may also be used to prepare micelles liposomes. In addition, any agent may be included in a micelle or liposome. Micelles and liposomes are particularly useful in delivering hydrophobic agents such as hydrophobic small molecules. When the micelle or liposome is complexed with (e.g., encapsulates or covers) a polynucleotide it is referred to as a lipoplex. Many techniques for preparing micelles, liposomes, and lipoplexes are known in the art, and any method may be used with the inventive conjugated lipomers to make micelles and liposomes.

(266) In certain embodiments, liposomes are formed through spontaneous assembly. In other embodiments, liposomes are formed when thin lipid films or lipid cakes are hydrated and stacks of lipid crystalline bilayers become fluid and swell. The hydrated lipid sheets detach during agitation and self-close to form large, multilamellar vesicles (LMV). This prevents interaction of water with the hydrocarbon core of the bilayers at the edges. Once these particles have formed, reducing the size of the particle can be modified through input of sonic energy (sonication) or mechanical energy (extrusion). See Walde, P. Preparation of Vesicles (Liposomes) In Encylopedia of Nanoscience and Nanotechnology; Nalwa, H. S. Ed. American Scientific Publishers: Los Angeles, 2004; Vol. 9, pp. 43-79; Szoka et al., Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes) Ann. Rev. Biophys. Bioeng. 9:467-508, 1980; each of which is incorporated herein. The preparation of liposomes involves preparing the conjugated lipomers for hydration, hydrating the conjugated lipomers with agitation, and sizing the vesicles to achieve a homogenous distribution of liposomes. Conjugated lipomers are first dissolved in an organic solvent to assure a homogeneous mixture of conjugated lipomers. The solvent is then removed to form a polymer-derived film. This polymer-derived film is thoroughly dried to remove residual organic solvent by placing the vial or flask on a vacuum pump overnight. Hydration of the polymer-derived film is accomplished by adding an aqueous medium and agitating the mixture. Disruption of LMV suspensions using sonic energy typically produces small unilamellar vesicles (SUV) with diameters in the range of 15-50 nm. Lipid extrusion is a technique in which a lipid/polymer suspension is forced through a polycarbonate filter with a defined pore size to yield particles having a diameter near the pore size of the filter used. Extrusion through filters with 100 nm pores typically yields large, unilamellar polymer-derived vesicles (LUV) with a mean diameter of 120-140 nm.

(267) In certain embodiments, the polynucleotide is an RNA molecule (e.g., an RNAi molecule). In other embodiments, the polynucleotide is a DNA molecule. In certain embodiments, the amount of poly(beta-amino alcohol) in the liposome ranges from 30-80 mol %, preferably 40-70 mol %, more preferably 60-70 mol %. These liposomes may be prepared using any method known in the art. In certain embodiments (e.g., liposomes containing RNAi molecules), the liposomes are prepared by lipid extrusion.

(268) Certain conjugated lipomers can spontaneously self assemble around certain molecules, such as DNA and RNA, to form liposomes. In some embodiments, the application is the delivery of polynucleotides. Use of these conjugated lipomers allows for simple assembly of liposomes without the need for additional steps or devices such as an extruder.

(269) The following scientific papers described other methods for preparing liposomes and micelles: Narang et al., Cationic Lipids with Increased DNA Binding Affinity for Nonviral Gene Transfer in Dividing and Nondividing Cells Bioconjugate Chem. 16:156-68, 2005; Hofland et al., Formation of stable cationic lipid/DNA complexes for gene transfer Proc. Natl. Acad. Sci. USA 93:7305-7309, July 1996; Byk et al., Synthesis, Activity, and StructureActivity Relationship Studies of Novel Cationic Lipids for DNA Transfer J. Med. Chem. 41(2):224-235, 1998; Wu et al., Cationic Lipid Polymerization as a Novel Approach for Constructing New DNA Delivery Agents Bioconjugate Chem. 12:251-57, 2001; Lukyanov et al., Micelles from lipid derivatives of water-soluble polymers as delivery systems for poorly soluble drugs Advanced Drug Delivery Reviews 56:1273-1289, 2004; Tranchant et al., Physicochemical optimisation of plasmid delivery by cationic lipids J. Gene Med. 6:S24-S35, 2004; van Balen et al., Liposome/Water Lipophilicity: Methods, Information Content, and Pharmaceutical Applications Medicinal Research Rev. 24(3):299-324, 2004; each of which is incorporated herein by reference.

(270) Agents

(271) The agents to be delivered by the systems of the present invention may be therapeutic, diagnostic, or prophylactic agents. Any chemical compound to be administered to an individual may be delivered using the inventive complexes, picoparticles, nanoparticles, microparticles, micelles, or liposomes. The agent may be an organic molecule (e.g., cholesterol, a drug), inorganic molecule, nucleic acid, protein, peptide, polynucleotide, targeting agent, isotopically labeled organic or inorganic molecule, vaccine, immunological agent, etc.

(272) In certain embodiments, the agents are organic molecules with pharmaceutical activity, e.g., a drug. In certain embodiments, the drug is an antibiotic, anti-viral agent, anesthetic, steroidal agent, anti-inflammatory agent, anti-neoplastic agent, anti-cancer agent, antigen, vaccine, antibody, decongestant, antihypertensive, sedative, birth control agent, progestational agent, anti-cholinergic, analgesic, anti-depressant, anti-psychotic, -adrenergic blocking agent, diuretic, cardiovascular active agent, vasoactive agent, non-steroidal anti-inflammatory agent, nutritional agent, etc.

(273) In certain embodiments of the present invention, the agent to be delivered may be a mixture of agents.

(274) Diagnostic agents include gases; metals; commercially available imaging agents used in positron emissions tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents. Examples of suitable materials for use as contrast agents in MRI include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium. Examples of materials useful for CAT and x-ray imaging include iodine-based materials.

(275) Prophylactic agents include, but are not limited to, antibiotics, nutritional supplements, and vaccines. Vaccines may comprise isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, and cell extracts. Prophylactic agents may be combined with interleukins, interferon, cytokines, and adjuvants such as cholera toxin, alum, Freund's adjuvant, etc. Prophylactic agents include antigens of such bacterial organisms as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter jejuni, and the like; antigens of such viruses as smallpox, influenza A and B, respiratory syncytial virus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and the like; antigens of fungal, protozoan, and parasitic organisms such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and the like. These antigens may be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof.

(276) Targeting Agents

(277) The inventive conjugated lipomers, and the complexes, liposomes, micelles, microparticles, picoparticles and nanoparticles, prepared therefrom, may be modified to include targeting agents since it is often desirable to target a particular cell, collection of cells, or tissue. A variety of targeting agents that direct pharmaceutical compositions to particular cells are known in the art (see, for example, Cotten et al., Methods Enzym. 217:618, 1993; incorporated herein by reference). The targeting agents may be included throughout the particle or may be only on the surface. The targeting agent may be a protein, peptide, carbohydrate, glycoprotein, lipid, small molecule, nucleic acids, etc. The targeting agent may be used to target specific cells or tissues or may be used to promote endocytosis or phagocytosis of the particle. Examples of targeting agents include, but are not limited to, antibodies, fragments of antibodies, low-density lipoproteins (LDLs), transferrin, asialycoproteins, gp120 envelope protein of the human immunodeficiency virus (HIV), carbohydrates, receptor ligands, sialic acid, aptamers, etc. If the targeting agent is included throughout the particle, the targeting agent may be included in the mixture that is used to form the particles. If the targeting agent is only on the surface, the targeting agent may be associated with (i.e., by covalent, hydrophobic, hydrogen bonding, van der Waals, or other interactions) the formed particles using standard chemical techniques.

(278) Compositions

(279) The present invention contemplates an inventive conjugated lipomer as a component of a composition which may be useful in a variety of medical and non-medical applications. For example, pharmaceutical compositions comprising an inventive conjugated lipomer may be useful in the delivery of an effective amount of an agent to a subject in need thereof. Nutraceutical compositions comprising an inventive conjugated lipomer may be useful in the delivery of an effective amount of a nutraceutical, e.g., a dietary supplement, to a subject in need thereof. Cosmetic compositions comprising an inventive conjugated lipomer may be formulated as a cream, ointment, balm, paste, film, or liquid, etc., and may be useful in the application of make-up, hair products, and materials useful for personal hygiene, etc. Compositions comprising an inventive conjugated lipomer may be useful for non-medical applications, e.g., such as an emulsion or emulsifier, useful, for example, as a food component, for extinguishing fires, for disinfecting surfaces, for oil cleanup, etc.

(280) In certain embodiments, the composition comprises one or more conjugated lipomers of the present invention. One or more conjugated lipomers refers to one or more different types of conjugated lipomers included in the composition, and encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different types of conjugated lipomers.

(281) In certain embodiments, the inventive conjugated lipomers are useful as compositions, either for delivery of an effective amount of an agent to a subject in need thereof (e.g., a pharmaceutical composition, a cosmetic composition) or for use as an excipient. For example, cosmetic compositions may further use the inventive lipomers as excipients rather than as a delivery system encapsulating an agent to be delivered. In certain embodiments, the composition is a pharmaceutical composition. In certain embodiments, the composition is a cosmetic composition.

(282) In certain embodiments, the composition further comprises an agent, as described herein. For example, in certain embodiments, the agent is a small molecule, organometallic compound, nucleic acid, protein, peptide, polynucleotide, metal, targeting agent, an isotopically labeled chemical compound, drug, vaccine, or immunological agent. In certain embodiments, the agent is a polynucleotide. In certain embodiments, the polynucleotide is DNA or RNA. In certain embodiments, the RNA is RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA.

(283) In certain embodiments, the polynucleotide and the one or more conjugated lipomers are not covalently attached.

(284) In certain embodiments, the one or more conjugated lipomers are in the form of a particle. In certain embodiments, the particle is a nanoparticle or microparticle. In certain embodiments, the one or more conjugated lipomers are in the form of liposomes or micelles. It is understood that, in certain embodiments, these conjugated lipomers self-assemble to provide a particle, micelle or liposome. In certain embodiments, the particle, liposome, or micelle encapsulates an agent. The agent to be delivered by the particles, liposomes, or micelles may be in the form of a gas, liquid, or solid. The inventive conjugated lipomers may be combined with polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids etc. to form the particles. These particles may be combined with an excipient to form pharmaceutical and cosmetic compositions.

(285) Once the complexes, micelles, liposomes, or particles have been prepared, they may be combined with one or more excipients to form a composition that is suitable to administer to animals including humans.

(286) As would be appreciated by one of skill in this art, the excipients may be chosen based on the route of administration as described below, the agent being delivered, time course of delivery of the agent, etc.

(287) In certain embodiments, provided is a composition comprising an inventive conjugated lipomer and, optionally, an 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 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.

(288) 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.

(289) 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.

(290) 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.

(291) 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.

(292) 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.

(293) 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.

(294) 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.

(295) 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.

(296) 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.

(297) 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.

(298) 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.

(299) 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.

(300) Methods of Use

(301) In another aspect, provided are methods of using the inventive conjugated lipomers, e.g., for the treatment of a disease, disorder or condition from which a subject suffers. It is contemplated that the inventive conjugated lipomers will be useful in the treatment of a variety of diseases, disorders or conditions, especially as a system for delivering agents useful in the treatment of that particular disease, disorder or condition.

(302) For example, in one aspect, provided is a method of treating cancer comprising administering to a subject in need thereof an effective amount of a conjugated lipomer of the present invention, e.g., a conjugated lipomer of the Formula (II) or (IV), or salt thereof, or a composition thereof. In certain embodiments, the method further comprises administering an anti-cancer agent. In certain embodiments, the conjugated lipomer encapsulates the anti-cancer agent. In certain embodiments, the conjugated lipomer and the anti-cancer agent form a particle (e.g., a nanoparticle, a microparticle, a micelle, a liposome, a lipoplex).

(303) A subject to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g, infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or other non-human animals, for example mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs), birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys), reptiles, amphibians, and fish. In certain embodiments, the non-human animal is a mammal. The non-human animal may be a male or female and at any stage of development. A non-human animal may be a transgenic animal.

(304) As used herein, and unless otherwise specified, the terms treat, treating and treatment contemplate an action that occurs while a subject is suffering from the specified disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or retards or slows the progression of the disease, disorder or condition (therapeutic treatment), and also contemplates an action that occurs before a subject begins to suffer from the specified disease, disorder or condition (prophylactic treatment).

(305) In general, the effective amount of a compound refers to an amount sufficient to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound of the invention may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, health, and condition of the subject. An effective amount encompasses therapeutic and prophylactic treatment.

(306) As used herein, and unless otherwise specified, a therapeutically effective amount of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder or condition, or to delay or minimize one or more symptoms associated with the disease, disorder or condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the disease, disorder or condition. The term therapeutically effective amount can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.

(307) As used herein, and unless otherwise specified, a prophylactically effective amount of a compound is an amount sufficient to prevent a disease, disorder or condition, or one or more symptoms associated with the disease, disorder or condition, or prevent its recurrence. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the disease, disorder or condition. The term prophylactically effective amount can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

(308) Exemplary cancers include, but are not limited to, acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma), Ewing sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrm's macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia/lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), nonsmall cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer and vulvar cancer (e.g., Paget's disease of the vulva).

(309) Anti-cancer agents encompass biotherapeutic anti-cancer agents as well as chemotherapeutic agents.

(310) Exemplary biotherapeutic anti-cancer agents include, but are not limited to, interferons, cytokines (e.g., tumor necrosis factor, interferon , interferon ), vaccines, hematopoietic growth factors, monoclonal serotherapy, immunostimulants and/or immunodulatory agents (e.g., IL-1, 2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF) and antibodies (e.g. HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab), ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR (tositumomab)).

(311) Exemplary chemotherapeutic agents include, but are not limited to, anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g. goserelin and leuprolide), anti-androgens (e.g. flutamide and bicalutamide), photodynamic therapies (e.g. vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g. carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g. busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide), platinum containing compounds (e.g. cisplatin, carboplatin, oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine, and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalent such as nanoparticle albumin-bound paclitaxel (ABRAXANE), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel, e.g., 2-paclitaxel methyl 2-glucopyranosyl succinate; docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR inhibitors (e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonucleotide reductase inhibitors (e.g. hydroxyurea and deferoxamine), uracil analogs (e.g. 5-fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g. cytarabine (ara C), cytosine arabinoside, and fludarabine), purine analogs (e.g. mercaptopurine and Thioguanine), Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g. 1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g. staurosporine), actinomycin (e.g. actinomycin D, dactinomycin), bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline (e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g. verapamil), Ca.sup.2+ ATPase inhibitors (e.g. thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN, AZD2171), dasatinib (SPRYCEL, BMS-354825), erlotinib (TARCEVA), gefitinib (IRESSA), imatinib (Gleevec, CGP57148B, STI-571), lapatinib (TYKERB, TYVERB), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA), semaxanib (semaxinib, SU5416), sunitinib (SUTENT, SU11248), toceranib (PALLADIA), vandetanib (ZACTIMA, ZD6474), vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN), bevacizumab (AVASTIN), rituximab (RITUXAN), cetuximab (ERBITUX), panitumumab (VECTIBIX), ranibizumab (Lucentis), nilotinib (TASIGNA), sorafenib (NEXAVAR), everolimus (AFINITOR), alemtuzumab (CAMPATH), gemtuzumab ozogamicin (MYLOTARG), temsirolimus (TORISEL), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/or XL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTOR inhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone, dexamethasone, campathecin, plicamycin, asparaginase, aminopterin, methopterin, porfiromycin, melphalan, leurosidine, leurosine, chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin aminopterin, and hexamethyl melamine.

EXAMPLES

(312) In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.

(313) Library Synthesis

(314) The inventive lipomers were synthesized using (i) a polymer PEI backbone a linear PEI with a molecular weight of 600 (LPEI.sub.600); a branched PEI with a molecular weight of 600 (BPEI.sub.600); a branched PEI with a molecular weight of 1800 (BPEI.sub.1800); or (ii) macrocycles comprising amino groups (e.g., aza-crown macrocycles). In each instance, the backbone was chemically modified with alkyl tails and/or PEG polymers by direct alkylation of one or more amino groups. Four structural parameters were varied in the screen: the number of alkyl tails and/or PEG polymers per backbone, and the length of the alkyl groups and/or PEG polymer per backbone. More specifically, the number of alkyl tails per backbone were varied from 0 to 43 alkyl groups per backbone or from 0 to 4 alkyl groups per macrocycle, while the number of PEG polymer per molecule ranged from 0.1 to 0.3 per backbone or macrocycle. The resulting lipomer library was screened for in vitro efficacy against one murine cancer cell line (qBEND.3), two human cancer cell lines (HeLa or HCT-116), and one primary human cell line (HMVEC). Seventeen lipomers were found to reduce gene expression in vitro by more than 80% at doses of 30 nM with negligible toxicity. These lipomers were then tested for systemic in vivo delivery in two animal models: a Factor 7 liver delivery model and a hepatocellular carcinoma tumor model. The in vivo data demonstrated that these new lipomers can deliver siRNA effectively in animals.

(315) PEI polymers and aza-macrocycles (FIG. 1A) were conjugated to alkyl and PEG polymers via an epoxide ring-opening reaction shown in FIG. 1B. LPEI.sub.600, BPEI.sub.600, BPEI.sub.1800 or aza-macrocycles were reacted with epoxides with alkyl chains having lengths between 10 and 18 carbons, inclusive. The reaction took place in 100% EtOH for 48-72 hours in capped glass vials at a temperature of about 90 C. After 48-72 hours, the solution was removed from the hotplate and the ethanol was removed via vacuum evaporation. In some of the reactions, LPEI.sub.600, BPEI.sub.600, BPEI.sub.1800 or the aza-macrocycles were also simultaneously alkylated with PEG.sub.1000 (PEG.sub.1K), PEG.sub.2000 (PEG.sub.2K) or a 1:1 mixture of PEG.sub.1K and PEG.sub.2K (PEG.sub.1.5K). This synthesis is ideal for high-throughput screening since it does not require protection/deprotection steps, organic solvents or complex environmental conditions (Love et al. Proc Natl Acad Sci USA (2010) 107:1864-1869).

(316) Table 3 lists the theoretical molecular weight, molar ratios, number of backbone nitrogens and nitrogen: phosphate ratios of compounds.

(317) TABLE-US-00004 TABLE 3 Lipomer: Lipomer: siRNAMass siRNA Mole Backbone N:P Molar Mass Ratio Ratio Nitrogens Ratio 7C1 3764.00 15.00 53.00 14.00 17.67 7H6 2667.00 15.00 74.80 14.00 24.93 7H4 2524.00 15.00 79.04 14.00 26.35 2C6 4603.28 15.00 43.34 43.00 44.37 7h8 2960.00 15.00 67.40 14.00 22.47 7G2 808.00 15.00 246.91 4.00 23.51 7B2 9620.00 15.00 20.74 43.00 21.23 4D1 750.00 15.00 266.00 4.00 25.33 4C8 934.00 15.00 213.60 4.00 20.34 7I1 4102.00 15.00 48.63 14.00 16.21 6c8 1056.00 15.00 188.92 4.00 17.99 7F10 890.00 15.00 224.16 4.00 21.35 6B10 888.00 15.00 224.66 4.00 21.40 4c4 850.00 15.00 234.71 4.00 22.35 4C5 1062.00 15.00 187.85 4.00 17.89 4C3 638.00 15.00 312.70 4.00 29.78
Reagents

(318) BPEI with a number molecular weight (M.sub.n) of 600 and a weight molecular weight (M.sub.w) of 800 of (BPEI.sub.600) was purchased from Sigma Aldrich (catalog number 408719). BPEI with a M.sub.n of 1800 and M.sub.w of 2000 (BPEI.sub.1800) was purchased from Alfa Aesar (catalog number 40528).

(319) Aza-macrocycles were purchased from Sigma Aldrich and Alfa Aesar (1,4,8,12 Tetraazacyclopentadecane TCI Catalog number 1691 or Sigma Aldrich Catalog Number 259512, 1,4,10,13-Tetraoxa-7,16-Diaazacyclooctade Sigma Aldrich Catalog Number 295809, 1,4,8,11 Tetraazacyclotetradecane TCI Catalog Number T1597, 1,4,7,10-tetraazacyclododecane TCI Catalog Number T1874.

(320) The following epoxides were purchased from Sigma Aldrich or TCI America: 1,2-epoxydecane TCI Catalog number E0315 or Sigma Aldrich Catalog Number 260339, 1,2-epoxydodecane TCI Catalog number D1984 or Sigma Aldrich Catalog Number 260207, 1,2-epoxytetradecane TCI Catalog number E0314 or Sigma Aldrich Catalog Number 260266, 1,2-epoxyhexadecane TCI Catalog number E0316 or Sigma Aldrich Catalog Number 260215, 1,2-epoxyoctadecane TCI Catalog number E0313 or Sigma Aldrich Catalog Number 260231.

(321) PEG.sub.1000 and PEG.sub.2000 were purchased from Creative Pegworks.

(322) In Vitro Screening

(323) Following synthesis, the ability to specifically reduce gene expression in vitro was tested against four cell lines; HeLa cells (ATCC, Manassas, Va.) transfected by Alnylam Pharmaceuticals to express both Renilla and Firefly luciferase, HCT-116 human colorectal cells (Caliper Life Sciences) transfected to express Firefly luciferase, and qBEND.3 murine endothelial cells and HMVEC primary human endothelial cells, both expressing the endothelial marker Tie2 (Akine et al. Nat Biotechnol (2008) 26:561-569). Lipomers were complexed with anti-Firefly luciferase siRNA (siLuc) (Dharmacon, Boulder, Colo.) or anti-Tie2 siRNA (siTie2) (Alnylam, Cambridge, Mass.) by incubating the lipomer with siRNA in 25 mMol NaAc with a pH of 5.2. The siRNA and lipomers were bound via electrostatic interactions between the negative phosphate backbone of the siRNA and the protonated amine backbone. GapDH siRNA (siGap) (Alnylam, Cambridge, Mass.) was used as a control for Tie2 expression. More specifically, lipomers were conjugated with siTie2 or siGap. Tie2 expression was measured using both complexes and successful compounds were those that reduced Tie2 expression Both Tie2 and GapDH More specifically, and compounds that reduced Tie2 expression even when conjugated to GapDH were considered toxic. Similarly, HeLa cell toxicity was tested by measuring both (targeted) Firefly luciferase and (control) Renilla Luciferase. Successful compounds reduced target gene expression and did not influence control gene expression.

(324) The in vitro screen was initiated by seeding cells onto a 96 well plate at a density of 15,000 cells/well. Twenty-four hours later, 30 nM lipomer-siRNA solution was placed in each well. The mixture was incubated overnight at 37 C. and 5% CO.sub.2. Luciferase and Renilla gene expression was then tested using luminescence (Dual Glow Assay, Promega, Madison, Wis.) while Tie2 expression was measured using a Quantigene 2.0 BDNA assay (Panomics, Santa Clara, Calif.). Compounds were considered successful if they reduced target gene expression by more than 80% while off-target gene knockdown was less than 20%. In most cases, successful candidates reduced off-target gene expression by less than 10%.

(325) Since polymer: siRNA mass ratio has been shown to influence the delivery of genetic material, the library was screened at lipomer: siRNA mass ratios of 2.5, 5, 10 and 15 (FIG. 2). During mass ratio studies, the amount of lipomer changed while the amount of siRNA remained constant such that the siRNA molarity was 30 nM. Compounds effective against HeLa cells were tested for efficacy against endothelial cells at a dose of 30 nM, as shown in FIG. 3. Following the initial screening of the library, a subset of effective compounds was tested for dose response. The lipomer:siRNA mass ratio was maintained at 15:1 while the amount of molarity of the solution decreased from 30 nM to 0.4 nM. These results are shown in FIG. 4.

(326) The mechanism of cellular uptake was then studied using fluorescently labeled siRNA. Briefly, cells can envelop external particles via a variety of different mechanisms. Each uptake mechanism results in different physiological processes. For this reason, identifying the specific uptake mechanism is important for nanoparticle delivery (Sahay et al., J. Controlled Release (2010) 145:182-195). Fortunately, most pathways are identifiable via canonical molecules present during their activation. In this case, the caveolae-mediated pathway was identified by the presence of a molecule termed cholera toxin B. Specifically, HeLa nuclei (blue), cholera toxin B (green) and Cysteine-5 tagged siRNA (siCy5) (red) were stained concurrently after Cy5 tagged siRNA was conjugated to compound 6B10. It was confirmed by the overlap between the red Cy5 siRNA and cholera toxin B that some molecules were taken up via caveolae-mediated endocytosis.

(327) In Vivo Formulation and Physical Characterization

(328) All in vivo experiments were reviewed and approved by the MIT Institutional Animal Care and Welfare Committee and were conducted in the MIT division of comparative medicine. Using in vitro screening described above, approximately 20 compounds were identified as leading candidates for in vivo analysis. To test the compounds in vivo, lipomers were diluted in 100% EtOH and mixed with C.sub.16-PEG (Aniara, Wilmington, Ohio) and cholesterol (Sigma Aldrich, St. Louis, Mo.) such that Lipomer:PEG:cholesterol molar ratios were 100:25:35. In another study, lipomers were diluted in 100% EtOH and combined with cholesterol, but not C.sub.16-PEG. Particles, in 25 mMol buffered sodium acetate (pH 5.3), were extruded using a 10 mL Lipex extruder (Northern Lipids, Canada) at 40 C. through 50 nm pore-size polycarbonate membranes until size distribution was uniform. siRNA was then added to the lipomer dispersion for 30 minutes, facilitating electrostatic interactions that led to siRNA complexation. Then, lipomers were dialyzed against 1PBS (pH 7.4) for 75 minutes to remove unbound siRNA and excess acetate. Particle size and charge were measured immediately after mixing with PEG and cholesterol, after conjugation to siRNA, and after dialysis. Particle size was measured with dynamic light scattering (DLS) and surface charge was estimated using zeta potential analysis (Zetapals, Brookhaven Instruments, Holtsville, N.Y.). Finally, to measure the stability of particles after conjugation of siRNA, compound size was measured 1 hour, 3 hours, 6 hours and 18 hours after formulation at 37 C., 25 C., 4 C. and 20 C. Both size and stability are shown in FIG. 5A-5B.

(329) After dialysis, samples were injected into one of two animal models. In the first model, lipomers with anti-Factor 7 siRNA were injected to 8-wk-old Fox Chase female mice (Charles River, Boston, Mass.). 48 hours after injection, blood serum was collected and analyzed for Factor 7 expression (Biophen, Aniara, Mason, Ohio). Serum Factor 7 levels were compared to mice injected with PBS (see FIGS. 6, 7, and 8).

(330) In a second study, 8 to 12-week-old Fox Chase female mice, bearing a 1 cm luciferase-expressing HEPg2 tumor in the flank, were injected with anti-LUC siRNA. Before, and 72 hr after intravenous siRNA administration (dosed at 2.0 mg/kg). Luciferase expression was measured by injecting 150 mg/kg D-luciferin ip (XenoLight Rediject D-Luciferin Ultra, Caliper Life Sciences, Hopkinton, Mass.) and recording luminescence with an IVIS Spectrum (Xenogen-Calipers Life Science). Reduction in luciferase expression was determined by comparing luciferase expression pre- and post-siRNA treatment (see FIGS. 9A-9B).

(331) Finally, the biodistribution of three different successful compounds (7H6, 7I1 and 3I7) was evaluated using Cy5.5 labeled siRNA (AllStars Control siRNA, Qiagen, Valencia, Calif.). Mice were injected with Cy5.5 siRNA (2.5 mg/kg) via the tail vein and sacrificed 1 or 14 hours later. Major organs were harvested and fluorescently imaged at 670 (ex. 670 nm, em. 710 nm) using an IVIS imaging system (see FIGS. 10A-10B).

(332) The Factor 7 siRNA was provided by Alnylam Pharmaceuticals and had a sense sequence of 5-GGAucAucucAAGucuuAcT*T-3 (SEQ ID NO 1) as previously reported (Akine, A. et al. Nature Biotechnology 26, 561-569). The Cy5-tagged siRNA was purchased from Qiagen and had a proprietary sequence (Product Number 1027297). The Luciferase targeting siRNA was purchased from Dharmacon and had a proprietary sequence (Product Number D-002050-0120).

(333) Results

(334) To identify compounds that effectively transfect multiple cell lines in vitro, 750 lipomers were screened against HeLa and HCT-116 cells. Since lipomer: siRNA mass ratios have been related in other studies to both efficacy and toxicity, the library was screened at lipomer: siRNA mass ratios of 2.5, 5, 10 and 15. Slightly less than 5% of the compounds transfected HeLa cells effectively, as shown qualitatively in FIG. 2A and quantitatively in FIG. 2B. Furthermore, lipomer: siRNA mass ratios of 10 and 15 were found to be most efficacious (FIG. 2A), while FIG. 2B indicates that at these ratios off-target effects (i.e., any decrease in Renilla luciferase expression) are negligible.

(335) Compounds found to be efficacious (>80% knockdown and <20% toxicity) against HeLa cells were then screened for knockdown against primary human endothelial cells and murine endothelial cells. FIG. 3 presents compounds that were found to be highly efficacious in multiple cell lines. Interestingly, the reduction in gene expression varied from one cell line to another, where, in general, primary endothelial cells were easier to transfect.

(336) Compounds were then tested for in vitro siRNA dose response against qBEND.3 cells. Increasing siRNA dosing led to decreased gene expression in vitro (FIG. 4A). At a dose of 30 nM, gene expression was reduced by up to 95% while GapDH (i.e., control gene) expression remained constant. Compounds reduced expression by nearly 80% at a dose of 6 nM, and by 25% at 1.2 nM, suggesting a 3.4 nM siRNA (400 nM lipomer) dose would reduce gene expression by 50%. Furthermore, off-target effects were abrogated, as shown by the constant gene expression of the GapDH control (FIG. 4B).

(337) Compounds were then tested for in vivo efficacy against the expression of Factor 7, a serum protein produced by the liver. In an initial study, lipomers formulated with cholesterol (but without Cm-PEG) were complexed with anti-Factor 7 siRNA (siF7, Alnylam Pharmaceuticals) and injected to the tail vein, at an siRNA dose of 2 mg/kg and a lipomer:siRNA mass ratio of 15:1 or 10:1 (FIG. 6). Analyzing Factor 7 in the blood demonstrated more than 64% knockdown and that a 15:1 mass ratio resulted in higher knockdown. Adding C.sub.16-PEG to the formulation (lipomer:cholesterol:C.sub.16-PEG mole ratio 100:35:25) reduced knockdown slightly (FIGS. 7 and 8). No change in animal weight was measured in the several days post administration, pointing to the good in vivo tolerability of the compounds.

(338) In vivo efficacy was further evaluated in a tumor model. Fox Chase mice bearing subcutaneous HepG2 tumors (1 cm diameter) that express Firefly-luciferase were injected with anti-Luc siRNA (1.5 mg/kg). Tumor luminescence was measured before, and 72 hr after, siRNA administration. A 51% decrease in tumor luminescence, was measured when using a representative efficacious compound7H6 (FIG. 9A).

(339) Finally, siRNA delivery systems 3I7, 7H6, and 7I1B complexing fluorescently labeled siRNA were injected to the tail vein. The resulting biodistribution showed that siRNA accumulates in the liver, spleen, kidney and lungs and the tail of mice (FIG. 10). All compounds had high selectivity to the liver. A significant amount of 7H6 accumulated in the lungs while 7I1B also tended to accumulate in the kidneys. This suggests that different compounds can be optimized for delivery in different organs.

Other Embodiments

(340) All patents, patent applications, and literature references cited herein are incorporated herein by reference.

(341) Having now described some illustrative embodiments of the invention, it should be apparent to those skilled in the art that the foregoing is merely illustrative and not limiting, having been presented by way of example only. Numerous modifications and other illustrative embodiments are within the scope of one of ordinary skill in the art and are contemplated as falling within the scope of the invention. In particular, although many of the examples presented herein involve specific combinations of method acts or system elements, it should be understood that those acts and those elements may be combined in other ways to accomplish the same objectives. Acts, elements, and features discussed only in connection with one embodiment are not intended to be excluded from a similar role in other embodiments. Further, for the one or more means-plus-function limitations recited in the following claims, the means are not intended to be limited to the means disclosed herein for performing the recited function, but are intended to cover in scope any means, known now or later developed, for performing the recited function. Use of terms such as first, second, third, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc., or i), ii), etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

(342) The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

Conjugated lipomers and uses thereof

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