Aminoalcohol lipidoids and uses thereof

Abstract

Aminoalcohol lipidoids are prepared by reacting an amine with an epoxide-terminated compound are described. Methods of preparing aminoalcohol lipidoids from commercially available starting materials are also provided. Aminoalcohol lipidoids may be prepared from racemic or stereochemically pure epoxides. Aminoalcohol lipidoids or salts forms thereof are preferably biodegradable and biocompatible and may be used in a variety of drug delivery systems. Given the amino moiety of these aminoalcohol lipidoid compounds, they are particularly suited for the delivery of polynucleotides. Complexes, micelles, liposomes or particles containing the inventive lipidoids and polynucleotide have been prepared. The inventive lipidoids may also be used in preparing microparticles for drug delivery. They are particularly useful in delivering labile agents given their ability to buffer the pH of their surroundings.

Claims

1. A method of preparing an aminoalcohol lipidoid compound of the formula: ##STR00373## or a pharmaceutically acceptable salt, wherein: R.sub.A and R.sub.F independently are hydrogen, ##STR00374## ##STR00375## R.sub.Y and R.sub.Z independently are hydrogen, each R.sub.5 is independently selected from: ##STR00376## m is 1; and p is 1; the method comprising the step of reacting one or more equivalents of an amine of the formula: ##STR00377## with one or more epoxide-containing compounds selected from: ##STR00378##

2. The method of claim 1, wherein R.sub.A is: ##STR00379##

3. The method of claim 1, wherein R.sub.F is: ##STR00380##

4. The method of claim 1, wherein: RA is: ##STR00381## and R.sub.F is: ##STR00382##

5. The method of claim 1, wherein: RA is: ##STR00383## and R.sub.F is: ##STR00384##

6. The method of claim 1, wherein R.sub.Y and R.sub.Z independently are hydrogen, or ##STR00385##

7. The method of claim 6, wherein R.sub.Y and R.sub.Z are ##STR00386##

8. The method of claim 1, wherein R.sub.A is: ##STR00387##

9. The method of claim 1, wherein R.sub.F is: ##STR00388##

10. The method of claim 1, wherein the aminoalcohol lipidoid compound is selected from: ##STR00389## ##STR00390## and pharmaceutically acceptable salts thereof.

11. The method of claim 10, wherein the aminoalcohol lipidoid compound is: ##STR00391## or a pharmaceutically acceptable salt thereof.

12. The method of claim 1, wherein the method comprises reacting one or more equivalents of an amine of the formula: ##STR00392## with an epoxide-containing compound of the formula: ##STR00393##

13. The method of claim 12, wherein the aminoalcohol lipidoid compound is: ##STR00394## or a pharmaceutically acceptable salt thereof.

14. The method of claim 12, wherein the epoxide-containing compound is: ##STR00395## and the aminoalcohol lipidoid compound is: ##STR00396## or a pharmaceutically acceptable salt thereof.

15. The method of claim 1, wherein the step of reacting is performed with no solvent present.

16. The method of claim 1, wherein the step of reacting is performed in the presence of an aprotic solvent.

17. The method of claim 1, wherein the step of reacting is performed in the presence of a solvent selected from tetrahydrofuran, diethyl ether, ethyl acetate, DMSO, DMF, methanol, ethanol, and water.

18. The method of claim 1, wherein the step of reacting is performed at a temperature ranging from 25° C. to 100° C.

19. The method of claim 18, wherein the step of reacting is performed at approximately 90° C.

20. The method of claim 1, further comprising the step of purifying the aminoalcohol lipidoid compound.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) FIG. 1 depicts a general synthetic scheme for preparing aminoalcohol lipidoids by combining amines and epoxides, and reacting them at approximately 90° C.

(2) FIG. 2 depicts exemplary amines containing between two and five amine functionalities and racemic epoxides of varying tails, unique functional groups and varying degrees of saturation that may be used for preparing aminoalcohol lipidoids.

(3) FIG. 3 depicts characterization data of aminoalcohol lipidoids derived from amine 114.

(4) FIG. 4 depicts thin layer chromatography (TLC) plates of selected compounds from the aminoalcohol lipidoid library. Plate “A” depicts fully substituted amines, while Plate “B” depicts n−1 substituted amines.

(5) FIG. 5 depicts Firefly luciferase knockdown results relative to untreated cells from complexing RNA (50 ng) with various aminoalcohol lipidoids (at various wt/wt ratios) and incubated with HeLa cells.

(6) FIG. 6 depicts luciferase gene delivery results (Luciferase Expression, “RLU”) in HepG2 cells for various epoxide lipidoid compounds in 10% serum and 0.3 μg of DNA per well.

(7) FIG. 7 depicts Factor VII Knockdown in vivo results and characterization of C57BL/6 mice 48 hours after administration (via tail vein injection at a volume of 0.01 ml/g) of (a) phosphate buffered saline; (b) 1.75 mg/kg entrapped siRNA in lipidoid formulation; and (c) 4 mg/kg entrapped siRNA in lipidoid formulation.

(8) FIG. 8 depicts luciferase knockdown results (measured by relative luceriferase expression, % control) for a library of aminoalcohol lipidoid compounds wherein the ratio of aminoalcohol lipidoid compound to siRNA is 2.5:1 (w/w).

(9) FIG. 9 depicts the luciferase knockdown results (measured by relative luceriferase expression, % control) for fifteen aminoalcohol lipidoid compounds having >90% knockdown wherein the ratio of aminoalcohol lipidoid compound to siRNA is 2.5:1 (w/w).

(10) FIG. 10 depicts: (a) dose (mg/kg) response results (measured by Factor VII knockdown in mice by aminoalcohol lipidoid C14-110 36 hours post-injection) wherein the ratio of aminoalcohol lipidoid compound to siRNA is 10:1 (w/w), the ratio of aminoalcohol lipidoid compound:cholesterol:PEG is 42:48:10, and 44% entrapment of 91 nm particles; and (b) average of % BW change.

(11) FIG. 11 depicts in vitro screening results as luciferase knockdown in HeLa cells by 25 epoxide-based lipidoids at 5:1 w/w ratio.

(12) FIG. 12 depicts in vivo screening results as Factor VII knockdown 48 hours post-injection of formulated epoxide lipidoids in mice.

(13) FIG. 13a depicts dose response results for C16-96B as Factor VII knockdown 48 hours post-injection of formulation C16-96-B in mice.

(14) FIG. 13b depicts corresponding mice body weight loss and/or gain during the experimental that provided the results in FIG. 13.

(15) FIG. 14a depicts dose response results for C14-110B as Factor VII knockdown 72 hours post-injection of formulation C14-110-B in mice.

(16) FIG. 14b depicts corresponding mice body weight loss and/or gain during the experiment that provided the results in FIG. 14a.

(17) FIG. 15 depicts C16-96-B formulation optimization as Factor VII knockdown 48 hours post-injection of formulation C16-96-B in mice at 1 mg/kg dose.

(18) FIG. 16 depicts C16-96-B dose response as Factor VII knockdown 48 hours post-injection of formulation C16-96-B in mice.

(19) FIG. 17a depicts additional in vivo screening and discovery of C12-200 and/or C12-205 as Factor VII knockdown 48 hours post-injection of formulated lipidoids in mice at 0.25 mg/kg dose.

(20) FIG. 17b depicts additional in vivo screening and discovery of C12-200 and/or C12-205 and corresponding mice body weight loss and/or gain during the experiment that provided the results in FIG. 17a.

(21) FIG. 18a depicts dose response results for C12-200 and/or C12-205 and ND98 comparison as Factor VII knockdown 48 hours post-injection of formulated C12-200 and/or C12-205 in mice.

(22) FIG. 18b depicts corresponding mice body weight loss and/or gain during the experiment that provided the results in FIG. 18a.

(23) FIG. 19 depicts formulation optimization of C12-200 and/or C12-205 as Factor VII knockdown 48 hours post-injection of formulated C12-200 and/or C12-205 in mice at 0.01 mg/kg dose.

(24) FIG. 20a depicts a MALDI-TOF mass spectra (intensity vs. m/z ratio) of the crude reaction mixture of technical grade 111 amine and C12 epoxide.

(25) FIG. 20b depicts a MALDI-TOF mass spectra (intensity vs. m/z ratio) of the “purified” product from the crude reaction mixture of technical grade 111 amine and C12 epoxide (from FIG. 20a).

(26) FIG. 21a depicts a MALDI-TOF mass spectra (intensity vs. m/z ratio) of the crude reaction mixture of technical grade 111 amine and C12 epoxide.

(27) FIG. 21b depicts a MALDI-TOF mass spectra (intensity vs. m/z ratio) of the “purified” product from the crude reaction mixture of technical grade 111 amine and C12 epoxide (from FIG. 21a).

(28) FIG. 22 depicts an .sup.1H NMR (400 MHz) spectrum of C12-200 and/or C12-205 (chloroform, room temperature).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

(29) The present invention provides novel aminoalcohol lipidoid compounds and drug delivery systems based on the use of such aminoalcohol lipidoid compounds. The system may be used in the pharmaceutical/drug delivery arts to delivery polynucleotides, proteins, small molecules, peptides, antigen, drugs, etc. to a patient, tissue, organ, cell, etc. These novel compounds may also be used as materials for coating, additives, excipients, materials, bioengineering, etc.

(30) The aminoalcohol lipidoid compounds of the present invention provide for several different uses in the drug delivery art. The amine-containing portion of the aminoalcohol lipidoid compounds may be used to complex polynucleotides, thereby enhancing the delivery of polynucleotide and preventing their degradation. The aminoalcohol lipidoid compounds may also be used in the formation of picoparticles, nanoparticles, microparticles, liposomes, and micelles containing the agent to be delivered. Preferably, the aminoalcohol lipidoid compounds are biocompatible and biodegradable, and the formed particles are also biodegradable and biocompatible and may be used to provide controlled, sustained release of the agent to be delivered. These lipidoids and their corresponding particles may also be responsive to pH changes given that these lipidoids are protonated at lower pH. The lipidoids may also act as proton sponges in the delivery of an agent to a cell to cause endosome lysis.

(31) 1. Aminoalcohol Lipidoid Compounds

(32) The aminoalcohol lipidoid compounds of the present invention are aminoalcohol lipidoid compounds containing primary, secondary, tertiary, and/or quaternary amines, and salts thereof. The amines may be cyclic or acyclic amines. In certain embodiments, the inventive aminoalcohol lipidoid compounds are relatively non-cytotoxic. In another embodiment, the inventive aminoalcohol lipidoid compounds are biocompatible and biodegradable. In certain embodiments, the aminoalcohol lipidoids of the present invention have pK.sub.as in the range of approximately 5.5 to approximately 7.5, more preferably between approximately 6.0 and approximately 7.0. In another embodiment, the aminoalcohol lipidoid compounds may be designed to have a desired pK.sub.a between approximately 3.0 and approximately 9.0, or between approximately 5.0 and approximately 8.0. The inventive aminoalcohol lipidoid compounds are particularly attractive for drug delivery for several reasons: 1) they contain amino groups for interacting with DNA, RNA, other polynucleotides, and other negatively charged agents, for buffering the pH, for causing endosomolysis, for protecting the agent to be delivered, etc.; 2) they can be synthesized from commercially available starting materials; and/or 3) they are pH responsive and can be engineered with a desired pK.sub.a.

(33) In certain embodiments, the aminoalcohol lipidoid compound or composition containing aminoalcohol lipidoid compound(s), are those derived from terminated epoxides of 14 carbons or greater coupled with monomers of three or more amine functional groups. In certain embodiments, the composition containing an aminoalcohol lipidoid compound is about 40-60% lipidoid, about 40-60% cholesterol, and about 5-20% PEG. In certain embodiments, the composition containing an aminoalcohol lipidoid compound is about 50-60% lipidoid, about 40-50% cholesterol, and about 5-10% PEG. In certain embodiments, the composition containing an aminoalcohol lipidoid compound is 52% lipidoid, 48% cholesterol, and 10% PEG. In certain embodiments, the composition containing an aminoalcohol lipidoid is about 50-75% lipidoid, about 20-40% cholesterol, and about 1-10% PEG. In certain embodiments, the composition containing an aminoalcohol lipidoid compound is about 60-70% lipidoid, about 25-35% cholesterol, and about 5-10% PEG.

(34) In certain embodiments, the aminoalcohol lipidoid compounds may be prepared by reacting an amine with a terminal epoxide or an aldehyde according to the following schemes.

(35) ##STR00026##
In certain embodiments, the epoxide is stereochemically pure (e.g., enantiomerically pure). In certain embodiments, the amine is stereochemically pure (e.g., enantiomerically pure). In certain embodiments, the lipidoid is prepared from the reductive amination of an imine which derived from the condensation of an amine and an aldehyde. In certain embodiments, the aminoalcohol lipidoid compounds of the present invention are of one of the formulae:

(36) ##STR00027##
wherein each occurrence of A, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.B, R.sub.C, R.sub.D, R.sub.A, R.sub.F, m, p, x, and y are as defined herein. As will be appreciated by one of skill in the art, the amine may be reacted with an excess of epoxide to form a fully functionalized aminoalcohol lipidoid compound. Or, the lipidoid may have fewer epoxide-derived tails than when fully functionalized. In certain embodiments, each

(37) ##STR00028##
is independently

(38) ##STR00029##
each

(39) ##STR00030##
is independently

(40) ##STR00031##
each

(41) ##STR00032##
is independently

(42) ##STR00033##
and each

(43) ##STR00034##
is independently

(44) ##STR00035##

(45) In certain embodiments, the aminoalcohol lipidoid compound of the present invention is of the formula:

(46) ##STR00036##
wherein:

(47) A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more heteroatoms independently selected from O, S and N, or A is a substituted or unsubstituted, saturated or unsaturated 4-6-membered ring;

(48) R.sub.1 is hydrogen, a substituted, unsubstituted, branched or unbranched C.sub.1-20-aliphatic or a substituted, unsubstituted, branched or unbranched C.sub.1-20 heteroaliphatic, wherein at least one occurrence of R.sub.1 is hydrogen;

(49) R.sub.B, R.sub.C, and R.sub.D are, independently, hydrogen, a substituted, unsubstituted, branched or unbranched C.sub.1-20-aliphatic, or a substituted, unsubstituted, branched or unbranched C.sub.1-20-heteroaliphatic or —CH.sub.2CH(OH)R.sub.E;

(50) R.sub.B and R.sub.D together may optionally form a cyclic structure;

(51) R.sub.C and R.sub.D together may optionally form a cyclic structure; and

(52) R.sub.E is a substituted, unsubstituted, branched or unbranched C.sub.1-20 aliphatic or a substituted, unsubstituted, branched or unbranched C.sub.1-20 heteroaliphatic; or a pharmaceutically acceptable salt thereof.

(53) In certain embodiments, A is an unsubstituted, unbranched, and acyclic C.sub.2-20 alkylene. In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20, alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments, A is a substituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted, by 1 oxygen atom. In certain embodiments, A is of the formula

(54) ##STR00037##
In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted, by 1 or more oxygen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 or more oxygen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 2 oxygen atoms. In certain embodiments, A is of the formula

(55) ##STR00038##
In certain embodiments, A is of the formula

(56) ##STR00039##
wherein n is an integer between 1 and 10, inclusive. In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 2 nitrogen atoms. In certain embodiments, A is of the formula

(57) ##STR00040##
In certain embodiments, A is of the formula

(58) ##STR00041##
wherein n is an integer between 1 and 10, inclusive.

(59) In certain embodiments, A is selected from the following formulae:

(60) ##STR00042## ##STR00043##

(61) In certain embodiments, R.sub.1 is hydrogen. In certain embodiments, R.sub.1 is an unsubstituted and unbranched, C.sub.1-20-aliphatic or C.sub.1-20 heteroaliphatic moiety. In some embodiments, R.sub.1 is an unsubstituted and unbranched, C.sub.10-12-aliphatic group. In some embodiments, R.sub.1 is

(62) ##STR00044##
In some embodiments, R.sub.1 is an unsubstituted and unbranched, C.sub.13 heteroaliphatic group. In some embodiments, R.sub.1 is

(63) ##STR00045##
In so embodiments, R.sub.1 is an unsubstituted and unbranched, C.sub.14 heteroaliphatic group. In some embodiments, R.sub.1 is

(64) ##STR00046##
In certain embodiments, R.sub.1 is selected from the following formulae:

(65) ##STR00047##

(66) In certain embodiments, R.sub.1 is a C.sub.1-20 alkenyl moiety, optionally substituted. In certain embodiments, R.sub.1 is selected from the following formulae:

(67) ##STR00048##

(68) It will be appreciated by one of ordinary skill in the art that the above substituents may have multiple sites of unsaturation, and could be so at any position within the substituent.

(69) In certain embodiments, each

(70) ##STR00049##
is independently

(71) ##STR00050##

(72) In certain embodiments, R.sub.1 is:

(73) ##STR00051##

(74) In certain embodiments, R.sub.1 is selected from the following formulae:

(75) ##STR00052## ##STR00053##

(76) In certain embodiments, R.sub.1 is selected from the following formulae:

(77) ##STR00054## ##STR00055##

(78) In certain embodiments, R.sub.1 is fluorinated. In certain embodiments R.sub.1 is a fluorinated aliphatic moiety. In certain embodiments R.sub.1 is perfluorinated. In certain embodiments R.sub.1 is a perfluorinated aliphatic moiety. In certain embodiments, R.sub.1 is a perfluorinated C.sub.1-20 alkyl group. In certain embodiments, R.sub.1 is selected from the following formulae:

(79) ##STR00056##

(80) In certain embodiments, R.sub.1 is selected from the following formulae:

(81) ##STR00057##

(82) In certain embodiments, R.sub.B is hydrogen. In certain embodiments, R.sub.B is an unsubstituted and unbranched C.sub.1-20-aliphatic. In certain embodiments R.sub.B is C.sub.1-6-alkyl. In certain embodiments R.sub.B is methyl. In certain embodiments R.sub.B is ethyl. In certain embodiments R.sub.B is propyl. In certain embodiments R.sub.B is butyl. In certain embodiments, R.sub.B is an unsubstituted and unbranched C.sub.1-20-heteroaliphatic. In certain embodiments R.sub.B is C.sub.1-6-heteroalkyl. In certain embodiments, R.sub.B is —CH.sub.2CH(OH)R.sub.E.

(83) In certain embodiments, R.sub.C is hydrogen. In certain embodiments, R.sub.C is an unsubstituted and unbranched C.sub.1-20-aliphatic. In certain embodiments R.sub.C is C.sub.1-6-alkyl. In certain embodiments R.sub.C is methyl. In certain embodiments R.sub.C is ethyl. In certain embodiments R.sub.C is propyl. In certain embodiments R.sub.C is butyl. In certain embodiments, R.sub.C is an unsubstituted and unbranched C.sub.1-20-heteroaliphatic. In certain embodiments R.sub.C is C.sub.1-6-heteroalkyl. In certain embodiments, R.sub.C is —CH.sub.2CH(OH)R.sub.E.

(84) In certain embodiments, R.sub.D is hydrogen. In certain embodiments, R.sub.D is an unsubstituted and unbranched C.sub.1-20-aliphatic. In certain embodiments R.sub.D is C.sub.1-6-alkyl. In certain embodiments R.sub.D is methyl. In certain embodiments R.sub.D is ethyl. In certain embodiments R.sub.D is propyl. In certain embodiments R.sub.D is butyl. In certain embodiments, R.sub.D is an unsubstituted and unbranched C.sub.1-20-heteroaliphatic. In certain embodiments R.sub.D is C.sub.1-6-heteroalkyl. In certain embodiments, R.sub.D is —CH.sub.2CH(OH)R.sub.E.

(85) In certain embodiments, R.sub.B, R.sub.C, and R.sub.D are all the same. In certain embodiments, R.sub.B, R.sub.C, and R.sub.D are all hydrogen or all C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.B, R.sub.C, and R.sub.D are all hydrogen. In certain embodiments, R.sub.B, R.sub.C, and R.sub.D are all C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.B, R.sub.C, and R.sub.D are all hydroxyalkyl. In certain embodiments, R.sub.B, R.sub.C, and R.sub.D are all aminoalkyl. In certain embodiments, R.sub.B, R.sub.C, and R.sub.D are hydrogen or methyl. In certain embodiments, at least two of R.sub.B, R.sub.C, and R.sub.D are the same. In certain embodiments, R.sub.B, R.sub.C, and R.sub.D are all different.

(86) In certain embodiments, R.sub.E is hydrogen. In certain embodiments, R.sub.E is an unsubstituted and unbranched C.sub.1-20-aliphatic. In certain embodiments R.sub.E is C.sub.1-6-alkyl. In certain embodiments R.sub.E is methyl. In certain embodiments R.sub.E is ethyl. In certain embodiments R.sub.E is propyl. In certain embodiments R.sub.E is butyl. In certain embodiments, R.sub.E is an unsubstituted and unbranched C.sub.1-20-heteroaliphatic. In certain embodiments R.sub.E is C.sub.6-heteroalkyl.

(87) Particular exemplary compounds include:

(88) ##STR00058##

(89) In certain embodiments, the aminoalcohol lipidoid compound of the present invention is of the formula:

(90) ##STR00059##

(91) A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more heteroatoms independently selected from O, S and N, or A is a substituted or unsubstituted, saturated or unsaturated 4-6-membered ring;

(92) R.sub.1 and R.sub.2 are, independently, hydrogen, a substituted, unsubstituted, branched or unbranched C.sub.1-20-aliphatic or a substituted, unsubstituted, branched or unbranched C.sub.1-20 heteroaliphatic, wherein at least one occurrence of R.sub.1 is hydrogen and at least one occurrence of R.sub.2 is hydrogen;

(93) R.sub.C and R.sub.D are, independently, hydrogen, a substituted, unsubstituted, branched or unbranched C.sub.1-20-aliphatic, or a substituted, unsubstituted, branched or unbranched C.sub.1-20-heteroaliphatic or —CH.sub.2CH(OH)R.sub.E;

(94) R.sub.C and R.sub.D together may optionally form a cyclic structure; and

(95) R.sub.E is a substituted, unsubstituted, branched or unbranched C.sub.1-20 aliphatic or a substituted, unsubstituted, branched or unbranched C.sub.1-20 heteroaliphatic; or a pharmaceutically acceptable salt thereof.

(96) In certain embodiments, A is an unsubstituted, unbranched, and acyclic C.sub.2-20 alkylene. In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is a substituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 oxygen atom. In certain embodiments, A is of the formula

(97) ##STR00060##
In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more oxygen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 or more oxygen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 2 oxygen atoms. In certain embodiments, A is of the formula.

(98) ##STR00061##
In certain embodiments, A is of the formula

(99) ##STR00062##
wherein n is an integer between 1 and 10, inclusive. In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 2 nitrogen atoms. In certain embodiments, A is of the formula,

(100) ##STR00063##
In certain embodiments, A is of the formula

(101) ##STR00064##
wherein n is an integer between 1 and 10, inclusive.

(102) In certain embodiments, A is selected from the following formulae:

(103) ##STR00065## ##STR00066##

(104) In certain embodiments, R.sub.1 and R.sub.2 are hydrogen. In certain embodiments, R.sub.1 and R.sub.2 are, independently, an unsubstituted and unbranched, C.sub.1-20-aliphatic or C.sub.1-20 heteroaliphatic moiety. In some embodiments, R.sub.1 and R.sub.2 are, independently, an unsubstituted and unbranched C.sub.10-12-aliphatic group. In some embodiments, R.sub.1 and R.sub.2 are

(105) ##STR00067##
In some embodiments, R.sub.1 and R.sub.2 are, independently, an unsubstituted and unbranched, C.sub.13 heteroaliphatic group. In some embodiments, R.sub.1 and R.sub.2 are

(106) ##STR00068##
In some embodiments, R.sub.1 and R.sub.2 are, independently, an unsubstituted and unbranched, C.sub.14 heteroaliphatic group. In some embodiments, R.sub.1 and R.sub.2 are

(107) ##STR00069##
In certain embodiments, R.sub.1 and R.sub.2 are, independently, selected from the following formulae:

(108) ##STR00070##

(109) In certain embodiments, R.sub.1 and R.sub.2 are, a C.sub.1-20 alkenyl moiety, optionally substituted. In certain embodiments, R.sub.1 and R.sub.2 are, independently, selected from the following formulae:

(110) ##STR00071##

(111) It will be appreciated by one of ordinary skill in the art that the above substituents may have multiple sites of unsaturation, and could be so at any position within the substituent.

(112) In certain embodiments, R.sub.1 and R.sub.2 are:

(113) ##STR00072##

(114) In certain embodiments, R.sub.1 and R.sub.3 are, independently, selected from the following formulae:

(115) ##STR00073##

(116) In certain embodiments, R.sub.1 and R.sub.2 are fluorinated. In certain embodiments R.sub.1 and R.sub.2 are a fluorinated aliphatic moiety. In certain embodiments R.sub.1 and R.sub.2 are perfluorinated. In certain embodiments R.sub.1 and R.sub.2 are a perfluorinated aliphatic moiety. In certain embodiments, R.sub.1 and R.sub.2 are a perfluorinated C.sub.1-20 alkyl group. In certain embodiments, R.sub.1 and R.sub.2 are selected from the following formulae:

(117) ##STR00074## ##STR00075##

(118) In certain embodiments, R.sub.1 and R.sub.2 are, independently, selected from the following formulae:

(119) ##STR00076##

(120) In certain embodiments, R.sub.1 and R.sub.2 are both the same. In certain embodiments, each of R.sub.1 and R.sub.2 are independently hydrogen or C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.1 and R.sub.2 are both hydrogen. In certain embodiments, R.sub.1 and R.sub.2 are both C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.1 and R.sub.2 are both hydroxyalkyl. In certain embodiments, R.sub.1 and R.sub.2 are both aminoalkyl. In certain embodiments, R.sub.1 and R.sub.2 are different.

(121) In certain embodiments, R.sub.C is hydrogen. In certain embodiments, R.sub.C is an unsubstituted and unbranched C.sub.1-20-aliphatic. In certain embodiments R.sub.C is C.sub.1-6-alkyl. In certain embodiments R.sub.C is methyl. In certain embodiments R.sub.C is ethyl. In certain embodiments R.sub.C is propyl. In certain embodiments R.sub.C is butyl. In certain embodiments, R.sub.C is an unsubstituted and unbranched C.sub.1-20-heteroaliphatic. In certain embodiments R.sub.C is C.sub.1-6-heteroalkyl. In certain embodiments, R.sub.C is —CH.sub.2CH(OH)R.sub.E.

(122) In certain embodiments, R.sub.D is hydrogen. In certain embodiments, R.sub.D is an unsubstituted and unbranched C.sub.1-20-aliphatic. In certain embodiments R.sub.D is C.sub.1-6-alkyl. In certain embodiments R.sub.D is methyl. In certain embodiments R.sub.D is ethyl. In certain embodiments R.sub.D is propyl. In certain embodiments R.sub.D is butyl. In certain embodiments, R.sub.D is an unsubstituted and unbranched C.sub.1-20-heteroaliphatic. In certain embodiments R.sub.D is C.sub.1-6-heteroalkyl. In certain embodiments, R.sub.D is —CH.sub.2CH(OH)R.sub.E.

(123) In certain embodiments, R.sub.C and R.sub.D are both the same. In certain embodiments, each of R.sub.C and R.sub.D are independently hydrogen, or C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.C and R.sub.D are both hydrogen. In certain embodiments, R.sub.C and R.sub.D are both C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.C and R.sub.D are both hydroxyalkyl. In certain embodiments, R.sub.C and R.sub.D are both aminoalkyl. In certain embodiments, R.sub.C and R.sub.D are different.

(124) In certain embodiments, R.sub.E is hydrogen. In certain embodiments, R.sub.E is an unsubstituted and unbranched C.sub.1-20-aliphatic. In certain embodiments R.sub.E is C.sub.1-6-alkyl. In certain embodiments R.sub.E is methyl. In certain embodiments R.sub.E is ethyl. In certain embodiments R.sub.E is propyl. In certain embodiments R.sub.E is butyl. In certain embodiments, R.sub.E is an unsubstituted and unbranched C.sub.1-20-heteroaliphatic. In certain embodiments R.sub.E is C.sub.6-heteroalkyl.

(125) Particular exemplary compounds include:

(126) ##STR00077##

(127) In certain embodiments, the aminoalcohol lipidoid compound of the present invention is of the formula:

(128) ##STR00078##
wherein:

(129) A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more heteroatoms independently selected from O, S and N, or A is a substituted or unsubstituted, saturated or unsaturated 4-6-membered ring;

(130) R.sub.1 and R.sub.3 are, independently, hydrogen, a substituted, unsubstituted, branched or unbranched C.sub.1-20-aliphatic or a substituted, unsubstituted, branched or unbranched C.sub.1-20 heteroaliphatic, wherein at least one occurrence of R.sub.1 is hydrogen and at least one occurrence of R.sub.3 is hydrogen;

(131) R.sub.B and R.sub.D are, independently, hydrogen, a substituted, unsubstituted, branched or unbranched C.sub.1-20-aliphatic, or a substituted, unsubstituted, branched or unbranched C.sub.1-20-heteroaliphatic or —CH.sub.2CH(OH)R.sub.E;

(132) R.sub.B and R.sub.D together may optionally form a cyclic structure; and

(133) R.sub.E is a substituted, unsubstituted, branched or unbranched C.sub.1-20 aliphatic or a substituted, unsubstituted, branched or unbranched C.sub.1-20 heteroaliphatic; or a pharmaceutically acceptable salt thereof.

(134) In certain embodiments, each

(135) ##STR00079##
is independently

(136) ##STR00080##
and each

(137) ##STR00081##
is independently

(138) ##STR00082##

(139) In certain embodiments, A is an unsubstituted, unbranched, and acyclic C.sub.2-20 alkylene. In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is a substituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 oxygen atom. In certain embodiments, A is of the formula

(140) ##STR00083##
In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more oxygen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 or more oxygen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 2 oxygen atoms. In certain embodiments, A is of the formula

(141) ##STR00084##
In certain embodiments, A is of the formula

(142) ##STR00085##
wherein n is an integer between 1 and 10, inclusive. In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 2 nitrogen atoms. In certain embodiments, A is of the formula

(143) ##STR00086##
In certain embodiments, A is of the formula

(144) ##STR00087##
wherein n is an integer between 1 and 10, inclusive.

(145) In certain embodiments, A is selected from the following formulae:

(146) ##STR00088##

(147) In certain embodiments, R.sub.1 and R.sub.3 are hydrogen. In certain embodiments, R.sub.1 and R.sub.3 are, independently, an unsubstituted and unbranched, C.sub.1-20-aliphatic or C.sub.1-20 heteroaliphatic moiety. In some embodiments, R.sub.1 and R.sub.3 are, independently, an unsubstituted and unbranched, C.sub.10-12-aliphatic group. In some embodiments, R.sub.1 and R.sub.3 are

(148) ##STR00089##
In some embodiments, R.sub.1 and R.sub.3 are, independently, an unsubstituted and unbranched, C.sub.13 heteroaliphatic group. In some embodiments, R.sub.1 and R.sub.3 are

(149) ##STR00090##
In some embodiments, R.sub.1 and R.sub.3 are, independently, an unsubstituted and unbranched, C.sub.14 heteroaliphatic group. In some embodiments, R.sub.1 and R.sub.3 are

(150) ##STR00091##
In certain embodiments, R.sub.1 and R.sub.3 are, independently, selected from the following formulae:

(151) ##STR00092##

(152) In certain embodiments, R.sub.1 and R.sub.3 are, a C.sub.1-20 alkenyl moiety, optionally substituted. In certain embodiments, R.sub.1 and R.sub.3 are, independently, selected from the following formulae:

(153) ##STR00093##

(154) It will be appreciated by one of ordinary skill in the art that the above substituents may have multiple sites of unsaturation, and could be so at any position within the substituent.

(155) In certain embodiments, R.sub.1 and R.sub.3 are:

(156) ##STR00094##

(157) In certain embodiments, R.sub.1 and R.sub.3 are, independently, selected from the following formulae:

(158) ##STR00095##

(159) In certain embodiments, R.sub.1 and R.sub.3 are fluorinated. In certain embodiments R.sub.1 and R.sub.3 are a fluorinated aliphatic moiety. In certain embodiments R.sub.1 and R.sub.3 are perfluorinated. In certain embodiments R.sub.1 and R.sub.3 are a perfluorinated aliphatic moiety. In certain embodiments, R.sub.1 and R.sub.3 are a perfluorinated C.sub.1-20 alkyl group. In certain embodiments, R.sub.1 and R.sub.3 are selected from the following formulae:

(160) ##STR00096##

(161) In certain embodiments, R.sub.1 and R.sub.3 are, independently, selected from the following formulae:

(162) ##STR00097##

(163) In certain embodiments, R.sub.1 and R.sub.3 are both the same. In certain embodiments, each of R.sub.1 and R.sub.3 are independently hydrogen, or C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.1 and R.sub.3 are both hydrogen. In certain embodiments, R.sub.1 and R.sub.3 are both C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.1 and R.sub.3 are both hydroxyalkyl. In certain embodiments, R.sub.1 and R.sub.3 are both aminoalkyl. In certain embodiments, R.sub.1 and R.sub.3 are different.

(164) In certain embodiments, R.sub.B is hydrogen. In certain embodiments, R.sub.B is an unsubstituted and unbranched C.sub.1-20-aliphatic. In certain embodiments R.sub.B is C.sub.1-6-alkyl. In certain embodiments R.sub.B is methyl. In certain embodiments R.sub.B is ethyl. In certain embodiments R.sub.B is propyl. In certain embodiments R.sub.B is butyl. In certain embodiments, R.sub.B is an unsubstituted and unbranched C.sub.1-20-heteroaliphatic. In certain embodiments R.sub.B is C.sub.1-6-heteroalkyl. In certain embodiments, R.sub.B is —CH.sub.2CH(OH)R.sub.E.

(165) In certain embodiments, R.sub.D is hydrogen. In certain embodiments, R.sub.D is an unsubstituted and unbranched C.sub.1-20-aliphatic. In certain embodiments R.sub.D is C.sub.1-6-alkyl. In certain embodiments R.sub.D is methyl. In certain embodiments R.sub.D is ethyl. In certain embodiments R.sub.D is propyl. In certain embodiments R.sub.D is butyl. In certain embodiments, R.sub.D is an unsubstituted and unbranched C.sub.1-20-heteroaliphatic. In certain embodiments R.sub.D is C.sub.1-6-heteroalkyl. In certain embodiments, R.sub.D is —CH.sub.2CH(OH)R.sub.E.

(166) In certain embodiments, R.sub.B and R.sub.D are both the same. In certain embodiments, each of R.sub.B and R.sub.D are independently hydrogen, or C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.B and R.sub.D are both hydrogen. In certain embodiments, R.sub.B and R.sub.D are both C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.B and R.sub.D are both hydroxyalkyl. In certain embodiments, R.sub.B and R.sub.D are both aminoalkyl. In certain embodiments, R.sub.B and R.sub.D are different.

(167) In certain embodiments, R.sub.E is hydrogen. In certain embodiments, R.sub.E is an unsubstituted and unbranched C.sub.1-20-aliphatic. In certain embodiments R.sub.E is C.sub.1-6-alkyl. In certain embodiments R.sub.E is methyl. In certain embodiments R.sub.E is ethyl. In certain embodiments R.sub.E is propyl. In certain embodiments R.sub.E is butyl. In certain embodiments, R.sub.E is an unsubstituted and unbranched C.sub.1-20-heteroaliphatic. In certain embodiments R.sub.E is C.sub.6-heteroalkyl.

(168) Particular exemplary compounds include:

(169) ##STR00098##

(170) In certain embodiments, each

(171) ##STR00099##
is independently

(172) ##STR00100##

(173) In certain embodiments, the aminoalcohol lipidoid compound of the present invention is of the formula:

(174) ##STR00101##
wherein:

(175) A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more heteroatoms independently selected from O, S and N, or A is a substituted or unsubstituted, saturated or unsaturated 4-6-membered ring;

(176) R.sub.1, R.sub.2, and R.sub.3 are, independently, hydrogen, a substituted, unsubstituted, branched or unbranched C.sub.1-20-aliphatic or a substituted, unsubstituted, branched or unbranched C.sub.1-20 heteroaliphatic, wherein at least one occurrence of R.sub.1 is hydrogen, at least one occurrence of R.sub.2 is hydrogen and at least one occurrence of R.sub.3 is hydrogen;

(177) R.sub.D is hydrogen, a substituted, unsubstituted, branched or unbranched C.sub.1-20-aliphatic, or a substituted, unsubstituted, branched or unbranched C.sub.1-20-heteroaliphatic or —CH.sub.2CH(OH)R.sub.E; and

(178) R.sub.E is a substituted, unsubstituted, branched or unbranched C.sub.1-20 aliphatic or a substituted, unsubstituted, branched or unbranched C.sub.1-20 heteroaliphatic; or a pharmaceutically acceptable salt thereof.

(179) In certain embodiments, each

(180) ##STR00102##
is independently

(181) ##STR00103##
each

(182) ##STR00104##
is independently

(183) ##STR00105##
and each

(184) ##STR00106##
is independently

(185) ##STR00107##

(186) In certain embodiments, A is an unsubstituted, unbranched, and acyclic C.sub.2-20 alkylene. In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is a substituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 oxygen atom. In certain embodiments, A is of the formula

(187) ##STR00108##
In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more oxygen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 or more oxygen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 2 oxygen atoms. In certain embodiments, A is of the formula

(188) ##STR00109##
In certain embodiments, A is of the formula

(189) ##STR00110##
wherein n is an integer between 1 and 10, inclusive. In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 2 nitrogen atoms. In certain embodiments, A is of the formula

(190) ##STR00111##
In certain embodiments, A is of the formula

(191) ##STR00112##
wherein n is an integer between 1 and 10, inclusive.

(192) In certain embodiments, A is selected from the following formulae:

(193) ##STR00113##

(194) In certain embodiments, R.sub.1, R.sub.2 and R.sub.3 are hydrogen. In certain embodiments, R.sub.1, R.sub.2 and R.sub.3 are, independently, an unsubstituted and unbranched, C.sub.1-20-aliphatic or C.sub.1-20 heteroaliphatic moiety. In some embodiments, R.sub.1, R.sub.2 and R.sub.3 are, independently, an unsubstituted and unbranched, C.sub.12-aliphatic group. In some embodiments, R.sub.1, R.sub.2 and R.sub.3 are

(195) ##STR00114##
In some embodiments, R.sub.1, R.sub.2 and R.sub.3 are, independently, an unsubstituted and unbranched, C.sub.13 heteroaliphatic group. In some embodiments, R.sub.1, R.sub.2 and R.sub.3 are

(196) ##STR00115##
In some embodiments, R.sub.1, R.sub.2 and R.sub.3 are, independently, an unsubstituted and unbranched, C.sub.14 heteroaliphatic group. In some embodiments, R.sub.1, R.sub.2 and R.sub.3 are

(197) ##STR00116##
In certain embodiments, R.sub.1, R.sub.2 and R.sub.3 are, independently, selected from the following formulae:

(198) ##STR00117##

(199) In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are, a C.sub.1-20 alkenyl moiety, optionally substituted. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are, independently, selected from the following formulae:

(200) ##STR00118##

(201) It will be appreciated by one of ordinary skill in the art that the above substituents may have multiple sites of unsaturation, and could be so at any position within the substituent.

(202) In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are:

(203) ##STR00119##

(204) In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are, independently, selected from the following formulae:

(205) ##STR00120##

(206) In certain embodiments, R.sub.1, R.sub.2 and R.sub.3 are fluorinated. In certain embodiments R.sub.1, R.sub.2 and R.sub.3 are a fluorinated aliphatic moiety. In certain embodiments R.sub.1, R.sub.2 and R.sub.3 are perfluorinated. In certain embodiments R.sub.1, R.sub.2 and R.sub.3 are a perfluorinated aliphatic moiety. In certain embodiments, R.sub.1, R.sub.2 and R.sub.3 are a perfluorinated C.sub.1-20 alkyl group. In certain embodiments, R.sub.1, R.sub.2 and R.sub.3 are selected from the following formulae:

(207) ##STR00121##

(208) In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are, independently, selected from the following formulae:

(209) ##STR00122##

(210) In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are all the same. In certain embodiments, at least two of R.sub.1, R.sub.2, and R.sub.3 are the same. In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are all different.

(211) In certain embodiments, R.sub.D is hydrogen. In certain embodiments, R.sub.D is an unsubstituted and unbranched C.sub.1-20-aliphatic. In certain embodiments R.sub.D is C.sub.1-6-alkyl. In certain embodiments R.sub.D is methyl. In certain embodiments R.sub.D is ethyl. In certain embodiments R.sub.D is propyl. In certain embodiments R.sub.D is butyl. In certain embodiments, R.sub.D is an unsubstituted and unbranched C.sub.1-20-heteroaliphatic. In certain embodiments R.sub.D is C.sub.1-6-heteroalkyl. In certain embodiments, R.sub.D is —CH.sub.2CH(OH)R.sub.E.

(212) In certain embodiments, R.sub.E is hydrogen. In certain embodiments, R.sub.E is an unsubstituted and unbranched C.sub.1-20-aliphatic. In certain embodiments R.sub.E is C.sub.1-6-alkyl. In certain embodiments R.sub.E is methyl. In certain embodiments R.sub.E is ethyl. In certain embodiments R.sub.E is propyl. In certain embodiments R.sub.E is butyl. In certain embodiments, R.sub.E is an unsubstituted and unbranched C.sub.1-20-heteroaliphatic. In certain embodiments R.sub.E is C.sub.1-6-heteroalkyl.

(213) Particular exemplary compounds include:

(214) ##STR00123##

(215) In certain embodiments, each

(216) ##STR00124##
is independently

(217) ##STR00125##

(218) In certain embodiments, the aminoalcohol lipidoid compound of the present invention is of the formula:

(219) ##STR00126##
wherein:

(220) A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more heteroatoms independently selected from O, S and N, or A is a substituted or unsubstituted, saturated or unsaturated 4-6-membered ring; and

(221) R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are, independently, hydrogen, a substituted, unsubstituted, branched or unbranched C.sub.1-20-aliphatic or a substituted, unsubstituted, branched or unbranched C.sub.1-20 heteroaliphatic, wherein at least one occurrence of R.sub.1 is hydrogen, at least one occurrence of R.sub.2 is hydrogen, at least one occurrence of R.sub.3 is hydrogen and at least one occurrence of R.sub.4 is hydrogen; or a pharmaceutically acceptable salt thereof.

(222) In certain embodiments, each

(223) ##STR00127##
is independently

(224) ##STR00128##
each

(225) ##STR00129##
is independently

(226) ##STR00130##
each

(227) ##STR00131##
is independently

(228) ##STR00132##
and each

(229) ##STR00133##
is independently

(230) ##STR00134##

(231) In certain embodiments, A is an unsubstituted, unbranched, and acyclic C.sub.2-20 alkylene. In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is a substituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 oxygen atom. In certain embodiments, A is of the formula

(232) ##STR00135##
In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more oxygen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 or more oxygen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 2 oxygen atoms. In certain embodiments, A is of the formula

(233) ##STR00136##
In certain embodiments, A is of the formula

(234) ##STR00137##
wherein n is an integer between 1 and 10, inclusive. In certain embodiments, A is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic C.sub.2-20 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 1 or more nitrogen atoms. In certain embodiments A is an unsubstituted, unbranched, and acyclic C.sub.2-10 alkylene, optionally interrupted by 2 nitrogen atoms. In certain embodiments, A is of the formula

(235) ##STR00138##
In certain embodiments, A is of the formula

(236) ##STR00139##
wherein n is an integer between 1 and 10, inclusive.

(237) In certain embodiments, A is selected from the following formulae:

(238) ##STR00140##

(239) In certain embodiments, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are hydrogen. In certain embodiments, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are, independently, an unsubstituted and unbranched, C.sub.1-20-aliphatic or C.sub.1-20 heteroaliphatic moiety. In some embodiments, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are, independently, an unsubstituted and unbranched, C.sub.12-aliphatic group. In some embodiments, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are

(240) ##STR00141##
In some embodiments, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are, independently, an unsubstituted and unbranched, C.sub.13 heteroaliphatic group. In some embodiments, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are

(241) ##STR00142##
In some embodiments, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are, independently, an unsubstituted and unbranched, C.sub.14 heteroaliphatic group. In some embodiments, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are

(242) ##STR00143##
In certain embodiments, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are, independently, selected from the following formulae:

(243) ##STR00144##

(244) In certain embodiments, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are, a C.sub.1-20 alkenyl moiety, optionally substituted. In certain embodiments, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are, independently, selected from the following formulae:

(245) ##STR00145##

(246) It will be appreciated by one of ordinary skill in the art that the above substituents may have multiple sites of unsaturation, and could be so at any position within the substituent.

(247) In certain embodiments, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are:

(248) ##STR00146##

(249) In certain embodiments, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are, independently, selected from the following formulae:

(250) ##STR00147##

(251) In certain embodiments, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are fluorinated. In certain embodiments R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are a fluorinated aliphatic moiety. In certain embodiments R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are perfluorinated. In certain embodiments R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are a perfluorinated aliphatic moiety. In certain embodiments, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are a perfluorinated C.sub.1-20 alkyl group. In certain embodiments, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are selected from the following formulae:

(252) ##STR00148##

(253) In certain embodiments, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are, independently, selected from the following formulae:

(254) ##STR00149##

(255) In certain embodiments, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are all the same. In certain embodiments, at least two of R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are the same. In certain embodiments, at least three of R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are the same. In certain embodiments, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are all different.

(256) Particular exemplary compounds include:

(257) ##STR00150##

(258) In certain embodiments, each

(259) ##STR00151##
is independently

(260) ##STR00152##

(261) In certain embodiments the aminoalcohol lipidoid compound or composition containing a mixture of aminoalcohol lipidoid compounds is prepared by reacting amine 120 with the epoxide-terminated compound C14. In certain embodiments, the aminoalcohol lipidoid compound C14-120 is one of the formulae below:

(262) ##STR00153##

(263) In certain embodiments, each

(264) ##STR00154##
is independently

(265) ##STR00155##
In certain embodiments the aminoalcohol lipidoid composition, is a composition containing one or more of the above aminoalcohol lipidoid compounds.

(266) In certain embodiments the aminoalcohol lipidoid compound or composition containing a mixture of aminoalcohol lipidoid compounds is prepared by reacting amine 120 with the epoxide-terminated compound C16. In certain embodiments, the aminoalcohol lipidoid compound C16-120 is of one of the formulae below:

(267) ##STR00156##
In certain embodiments, each

(268) ##STR00157##
is independently

(269) ##STR00158##
In certain embodiments the aminoalcohol lipidoid composition, is a composition containing one or more of the above aminoalcohol lipidoid compounds.

(270) In certain embodiments the aminoalcohol lipidoid compound or composition containing a mixture of aminoalcohol lipidoid compounds is prepared by reacting amine 98 with the epoxide-terminated compound C14. In certain embodiments, the aminoalcohol lipidoid compound C14-98 is of one of the formulae below:

(271) ##STR00159##
In certain embodiments, each

(272) ##STR00160##
is independently

(273) ##STR00161##
In certain embodiments the aminoalcohol lipidoid composition, is a composition containing one or more of the above aminoalcohol lipidoid compounds.

(274) In certain embodiments the aminoalcohol lipidoid compound or composition containing a mixture of aminoalcohol lipidoid compounds is prepared by reacting amine 113 with the epoxide-terminated compound C14. In certain embodiments, the aminoalcohol lipidoid compound C14-113 is of one of the formulae below:

(275) ##STR00162##
In certain embodiments, each

(276) ##STR00163##
is independently

(277) ##STR00164##
In certain embodiments the aminoalcohol lipidoid composition, is a composition containing one or more of the above aminoalcohol lipidoid compounds.

(278) In certain embodiments the aminoalcohol lipidoid compound or composition containing a mixture of aminoalcohol lipidoid compounds is prepared by reacting amine 96 with the epoxide-terminated compound C18. In certain embodiments, the aminoalcohol lipidoid compound is of one of the formulae below:

(279) ##STR00165##
In certain embodiments, each

(280) ##STR00166##
p is independently

(281) ##STR00167##
In certain embodiments the aminoalcohol lipidoid composition, is a composition containing one or more of the above aminoalcohol lipidoid compounds.

(282) In certain embodiments the aminoalcohol lipidoid compound or composition containing a mixture of aminoalcohol lipidoid compounds is prepared by reacting amine 96 with the epoxide-terminated compound C14. In certain embodiments, the aminoalcohol lipidoid compound C14-96 is of one of the formulae below:

(283) ##STR00168##

(284) In certain embodiments, each

(285) ##STR00169##
is independently

(286) ##STR00170##
In certain embodiments the aminoalcohol lipidoid composition, is a composition containing one or more of the above aminoalcohol lipidoid compounds.

(287) In certain embodiments the aminoalcohol lipidoid compound or composition containing a mixture of aminoalcohol lipidoid compounds is prepared by reacting amine 110 with the epoxide-terminated compound C14. In certain embodiments, the aminoalcohol lipidoid compound C14-110 is of one of the formulae below:

(288) ##STR00171## ##STR00172##
In certain embodiments, each

(289) ##STR00173##
is independently

(290) ##STR00174##
In certain embodiments the aminoalcohol lipidoid composition, is a composition containing one or more of the above aminoalcohol lipidoid compounds.

(291) In certain embodiments, the aminoalcohol lipidoid compound of the present invention is of the formula:

(292) ##STR00175##
wherein:

(293) p is an integer between 1 and 3, inclusive;

(294) m is an integer between 1 and 3, inclusive;

(295) R.sub.A is hydrogen; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl;

(296) ##STR00176##

(297) R.sub.F is hydrogen; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl;

(298) ##STR00177##

(299) each occurrence of R.sub.5 is independently hydrogen; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl;

(300) wherein, at least one of R.sub.A, R.sub.F, R.sub.Y, and R.sub.Z is

(301) ##STR00178##

(302) each occurrence of x is an integer between 1 and 10, inclusive;

(303) each occurrence of y is an integer between 1 and 10, inclusive;

(304) each occurrence of R.sub.Y is hydrogen; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl;

(305) ##STR00179##

(306) each occurrence of R.sub.Z is hydrogen; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl;

(307) ##STR00180##

(308) or a pharmaceutically acceptable salt thereof.

(309) In certain embodiments, R.sub.A is hydrogen. In certain embodiments, R.sub.A is hydrogen. In certain embodiments, R.sub.A is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic. In certain embodiments, R.sub.A is C.sub.1-C.sub.6 aliphatic. In certain embodiments, R.sub.A is C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.A is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic. In certain embodiments, R.sub.A is substituted or unsubstituted aryl. In certain embodiments, R.sub.A is substituted or unsubstituted heteroaryl. In certain embodiments, R.sub.A is

(310) ##STR00181##
In certain embodiments, each

(311) ##STR00182##
is independently

(312) ##STR00183##
In certain embodiments, R.sub.A is

(313) ##STR00184##
In certain embodiments, R.sub.A is

(314) ##STR00185##
In certain embodiments R.sub.A is

(315) ##STR00186##
In certain embodiments R.sub.A is

(316) ##STR00187##
In certain embodiments R.sub.A is

(317) ##STR00188##

(318) In certain embodiments, R.sub.F is hydrogen. In certain embodiments, no R.sub.F is hydrogen. In certain embodiments, R.sub.F is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic. In certain embodiments, R.sub.F is C.sub.1-C.sub.6 aliphatic. In certain embodiments, R.sub.F is C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.F is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic. In certain embodiments, R.sub.F is substituted or unsubstituted aryl. In certain embodiments, R.sub.F is substituted or unsubstituted heteroaryl. In certain embodiments, R.sub.F is

(319) ##STR00189##
In certain embodiments, each

(320) ##STR00190##
In certain embodiments, each

(321) ##STR00191##
In certain embodiments, R.sub.F is

(322) ##STR00192##
In certain embodiments, R.sub.F is

(323) ##STR00193##
In certain embodiments R.sub.F is

(324) ##STR00194##
In certain embodiments R.sub.F is

(325) ##STR00195##
In certain embodiments R.sub.F is

(326) ##STR00196##
In certain embodiments R.sub.F is

(327) ##STR00197##
In certain embodiments R.sub.F

(328) ##STR00198##

(329) In certain embodiments, no R.sub.A is hydrogen and no R.sub.F is hydrogen. In certain embodiments, R.sub.A is

(330) ##STR00199##
and R.sub.F is

(331) ##STR00200##
In certain embodiments, R.sub.A is

(332) ##STR00201##
and R.sub.F is

(333) ##STR00202##
In certain embodiments, each

(334) ##STR00203##
In certain embodiments, each

(335) ##STR00204##
In certain embodiments, R.sub.A is

(336) ##STR00205##
and R.sub.F is

(337) ##STR00206##
In certain embodiments, each

(338) ##STR00207##
In certain embodiments, each

(339) ##STR00208##
In certain embodiments, R.sub.A is

(340) ##STR00209##
and R.sub.F is

(341) ##STR00210##
In certain embodiments, R.sub.A is

(342) ##STR00211##
and R.sub.F is

(343) ##STR00212##

(344) In certain embodiments, each

(345) ##STR00213##
In certain embodiments, each

(346) ##STR00214##
In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, m is 3.

(347) In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3.

(348) In certain embodiments, R.sub.5 is hydrogen. In certain embodiments, R.sub.5 is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic. In certain embodiments, R.sub.5 is C.sub.8-C.sub.16 aliphatic. In certain embodiments, R.sub.5 is C.sub.8-C.sub.16 alkyl. In some embodiments, R.sub.5 is an unsubstituted and unbranched, C.sub.10-12-aliphatic group. In some embodiments, R.sub.5 is

(349) ##STR00215##
In some embodiments, R.sub.5 is

(350) ##STR00216##
In some embodiments, R.sub.5 is

(351) ##STR00217##
In certain embodiments, R.sub.5 is selected from the following formulae:

(352) ##STR00218##

(353) In certain embodiments, R.sub.5 is a C.sub.1-20 alkenyl moiety, optionally substituted. In certain embodiments, R.sub.5 is selected from the following formulae:

(354) ##STR00219##
In certain embodiments, R.sub.5 is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic. In some embodiments, R.sub.5 is an unsubstituted and unbranched, C.sub.13 heteroaliphatic group. In some embodiments, R.sub.5 is an unsubstituted and unbranched, C.sub.14 heteroaliphatic group. In certain embodiments, R.sub.5 is:

(355) ##STR00220##

(356) In certain embodiments, R.sub.5 is, independently, selected from the following formulae:

(357) ##STR00221##

(358) It will be appreciated by one of ordinary skill in the art that the above substituents may have multiple sites of unsaturation, and could be so at any position within the substituent.

(359) In certain embodiments, R.sub.5 is substituted or unsubstituted aryl. In certain embodiments, R.sub.5 is or substituted or unsubstituted heteroaryl.

(360) In certain embodiments, R.sub.5 is fluorinated. In certain embodiments R.sub.5 is a fluorinated aliphatic moiety. In certain embodiments R.sub.5 is perfluorinated. In certain embodiments R.sub.5 is a perfluorinated aliphatic moiety. In certain embodiments, R.sub.5 is a perfluorinated C.sub.1-20 alkyl group. In certain embodiments, R.sub.5 is selected from the following formulae:

(361) ##STR00222##

(362) In certain embodiments, R.sub.5 is selected from the following formulae:

(363) ##STR00223##

(364) In certain embodiments, each R.sub.5 is independently hydrogen, or C.sub.1-C.sub.6 alkyl. In certain embodiments, each R.sub.5 is hydrogen. In certain embodiments, R.sub.1 and R.sub.3 each R.sub.5 is C.sub.1-C.sub.6 alkyl. In certain embodiments, each R.sub.5 is hydroxyalkyl. In certain embodiments, each R.sub.5 is aminoalkyl. In certain embodiments, two R.sub.5 variables are the same. In certain embodiments, three R.sub.5 variables are the same. In certain embodiments, each R.sub.5 variable is different from the other.

(365) In certain embodiments, x is 1. In certain embodiments, x is 2. In certain embodiments, x is 3. In certain embodiments, x is 4. In certain embodiments, x is 5. In certain embodiments, x is 6. In certain embodiments, x is 7. In certain embodiments, x is 8. In certain embodiments, x is 9. In certain embodiments, x is 10.

(366) In certain embodiments, y is 1. In certain embodiments, y is 2. In certain embodiments, y is 3. In certain embodiments, y is 4. In certain embodiments, y is 5. In certain embodiments, y is 6. In certain embodiments, y is 7. In certain embodiments, y is 8. In certain embodiments, y is 9. In certain embodiments, y is 10.

(367) In certain embodiments, x is 1 and y is 2. In certain embodiments, x is 1 and y is 3. In certain embodiments, x is 1 and y is 4. In certain embodiments, x is 1 and y is 5. In certain embodiments, x is 2 and y is 2. In certain embodiments, x is 2 and y is 3.

(368) In certain embodiments, R.sub.Y is hydrogen. In certain embodiments, R.sub.Y is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic. In certain embodiments, R.sub.Y is C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.Y is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic. In certain embodiments, R.sub.Y is substituted or unsubstituted aryl. In certain embodiments, R.sub.Y is substituted or unsubstituted heteroaryl. In certain embodiments, R.sub.Y is

(369) ##STR00224##
In certain embodiments, each

(370) ##STR00225##
is independently

(371) ##STR00226##
In certain embodiments, R.sub.Y is

(372) ##STR00227##

(373) In certain embodiments, R.sub.Z is hydrogen. In certain embodiments, R.sub.Z is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic. In certain embodiments, R.sub.Y is C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.Z is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic. In certain embodiments, R.sub.Z is substituted or unsubstituted aryl. In certain embodiments, R.sub.Z is substituted or unsubstituted heteroaryl. In certain embodiments, R.sub.Z is

(374) ##STR00228##
In certain embodiments, each

(375) ##STR00229##
is independently

(376) ##STR00230##
In certain embodiments, R.sub.Z is

(377) ##STR00231##

(378) Particular exemplary compounds include:

(379) ##STR00232##

(380) In certain embodiments, the aminoalcohol lipidoid compounds of the present invention comprises a mixture of formulae:

(381) ##STR00233##

(382) In certain embodiments, each

(383) ##STR00234##
is independently

(384) ##STR00235##

(385) In certain embodiments, the aminoalcohol lipidoid compound of the present invention is of the formula:

(386) ##STR00236##
wherein:

(387) each occurrence of R.sub.A is independently hydrogen; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl;

(388) ##STR00237##
wherein at least one R.sub.A is

(389) ##STR00238##

(390) each occurrence of R.sub.5 is independently hydrogen; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic; substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl;

(391) each occurrence of x is an integer between 1 and 10, inclusive;

(392) each occurrence of y is an integer between 1 and 10, inclusive;

(393) or a pharmaceutically acceptable salt thereof.

(394) In certain embodiments, R.sub.A is hydrogen. In certain embodiments, no R.sub.A is hydrogen. In certain embodiments, at least one R.sub.A is hydrogen. In certain embodiments, R.sub.A is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic. In certain embodiments, R.sub.A is substituted or unsubstituted aryl. In certain embodiments, R.sub.A is substituted or unsubstituted heteroaryl. In certain embodiments, two R.sub.A's together may form a cyclic structure. In certain embodiments, at least one R.sub.A is

(395) ##STR00239##
In certain embodiments, each

(396) ##STR00240##
In certain embodiments, each

(397) ##STR00241##
is independently

(398) ##STR00242##
In certain embodiments, at least one R.sub.A is

(399) ##STR00243##
In certain embodiments, R.sub.A is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic. In certain embodiments, at least one R.sub.A is an alkenyl group. In certain embodiments, at least one R.sub.A is

(400) ##STR00244##
In certain embodiments, at least one R.sub.A is

(401) ##STR00245##
In certain embodiments, at least one R.sub.A is

(402) ##STR00246##
In certain embodiments, at least one R.sub.A is

(403) ##STR00247##
In certain embodiments, at least one R.sub.A is

(404) ##STR00248##
In certain embodiments, at least one R.sub.A is

(405) ##STR00249##
In certain embodiments, at least one R.sub.A is an alkynyl group. In certain embodiments, at least one R.sub.A is

(406) ##STR00250##
In certain embodiments, at least one R.sub.A is a substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic group. In certain embodiments, at least one R.sub.A is a heteroaliphatic group. In certain embodiments, at least one R.sub.A is

(407) ##STR00251##
In certain embodiments, at least one R.sub.A is

(408) ##STR00252##
In certain embodiments, at least one R.sub.A is

(409) ##STR00253##
In certain embodiments, at least one R.sub.A is

(410) ##STR00254##
In certain embodiments, two R.sub.A variables are the same. In certain embodiments, three R.sub.A variables are the same. In certain embodiments, each R.sub.A variable is different from the other.

(411) In certain embodiments, R.sub.5 is hydrogen. In certain embodiments, R.sub.5 is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 aliphatic. In certain embodiments, R.sub.5 is C.sub.8-C.sub.16 aliphatic. In certain embodiments, R.sub.5 is C.sub.8-C.sub.16 alkyl. In some embodiments, R.sub.5 is an unsubstituted and unbranched, C.sub.10-12-aliphatic group. In some embodiments, R.sub.5 is

(412) ##STR00255##
In some embodiments, R.sub.5 is

(413) ##STR00256##
In some embodiments, R.sub.5 is

(414) ##STR00257##
In certain embodiments, R.sub.5 is selected from the following formulae:

(415) ##STR00258##

(416) In certain embodiments, R.sub.5 is a C.sub.1-20 alkenyl moiety, optionally substituted. In certain embodiments, R.sub.5 is selected from the following formulae:

(417) ##STR00259##

(418) In certain embodiments, R.sub.5 is substituted or unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20 heteroaliphatic. In some embodiments, R.sub.5 is an unsubstituted and unbranched, C.sub.13 heteroaliphatic group. In some embodiments, R.sub.5 is an unsubstituted and unbranched, C.sub.14 heteroaliphatic group. In certain embodiments, R.sub.5 is:

(419) ##STR00260##

(420) In certain embodiments, R.sub.5 is, independently, selected from the following formulae:

(421) ##STR00261##

(422) It will be appreciated by one of ordinary skill in the art that the above substituents may have multiple sites of unsaturation, and could be so at any position within the substituent.

(423) In certain embodiments, R.sub.5 is substituted or unsubstituted aryl. In certain embodiments, R.sub.5 is or substituted or unsubstituted heteroaryl. In certain embodiments, R.sub.5 is

(424) ##STR00262##
In certain embodiments, each

(425) ##STR00263##
is independently

(426) ##STR00264##
In certain embodiments, R.sub.5 is

(427) ##STR00265##

(428) In certain embodiments, R.sub.5 is fluorinated. In certain embodiments R.sub.5 is a fluorinated aliphatic moiety. In certain embodiments R.sub.5 is perfluorinated. In certain embodiments R.sub.5 is a perfluorinated aliphatic moiety. In certain embodiments, R.sub.5 is a perfluorinated C.sub.1-20 alkyl group. In certain embodiments, R.sub.5 is selected from the following formulae:

(429) ##STR00266## ##STR00267##

(430) In certain embodiments, R.sub.5 is selected from the following formulae:

(431) ##STR00268##

(432) In certain embodiments, each R.sub.5 is independently hydrogen, or C.sub.1-C.sub.6 alkyl. In certain embodiments, each R.sub.5 is hydrogen. In certain embodiments, R.sub.1 and R.sub.3 each R.sub.5 is C.sub.1-C.sub.6 alkyl. In certain embodiments, each R.sub.5 is hydroxyalkyl. In certain embodiments, each R.sub.5 is aminoalkyl. In certain embodiments, two R.sub.5 variables are the same. In certain embodiments, three R.sub.5 variables are the same. In certain embodiments, each R.sub.5 variable is different from the other.

(433) In certain embodiments, x is 1. In certain embodiments, x is 2. In certain embodiments, x is 3. In certain embodiments, x is 4. In certain embodiments, x is 5. In certain embodiments, x is 6. In certain embodiments, x is 7. In certain embodiments, x is 8. In certain embodiments, x is 9. In certain embodiments, x is 10.

(434) In certain embodiments, y is 1. In certain embodiments, y is 2. In certain embodiments, y is 3. In certain embodiments, y is 4. In certain embodiments, y is 5. In certain embodiments, y is 6. In certain embodiments, y is 7. In certain embodiments, y is 8. In certain embodiments, y is 9. In certain embodiments, y is 10.

(435) In certain embodiments, an aminoalcohol lipidoid compound or composition containing aminoalcohol lipidoid compound(s) is prepared by reacting an amine of one of the formulae:

(436) ##STR00269## ##STR00270## ##STR00271## ##STR00272## ##STR00273##
with an epoxide-containing compound of one of the formulae:

(437) ##STR00274## ##STR00275##

(438) In certain embodiments, the epoxide-terminated compounds are of the formula:

(439) ##STR00276## ##STR00277##

(440) In certain embodiments, the epoxide-containing compound is of the formula:

(441) ##STR00278## ##STR00279##

(442) In certain embodiments, the epoxide contains one or more chiral centers, such as those shown below for amine C8b:

(443) ##STR00280##

(444) In certain embodiments, one equivalent of an amine is reacted with one equivalent of an epoxide-terminated compound. In certain embodiments, one equivalent of an amine is reacted with one, two, three, four, five, six or more equivalents of an epoxide-terminated compound. In certain embodiments, the amount of epoxide-terminated compound is limiting to prevent the functionalization of all amino groups. The resulting aminoalcohol lipidoid or aminoalcohol lipidoid composition in these instances contain secondary amino groups and/or primary amino groups. Aminoalcohol lipidoid compounds having secondary amines are particular useful in certain instances. In certain embodiments, amine-containing aminoalcohol lipidoid compounds that have not been fully functionalized are further reacted with another electrophile (e.g., terminal epoxide, alkyl halide, etc.). Such further functionalization of the amines of the aminoalcohol lipidoid compound results in aminoalcohol lipidoid compounds with different epoxide-compound derived tails. One, two, three, four, five, or more tails may be different from the other tails of the aminoalcohol lipidoid compounds.

(445) In certain embodiments, it will be appreciated by one skilled in the art that the amine and the epoxide will react at the unsubstituted carbon of the epoxide resulting in an aminoalcohol lipidoid compound as shown in the following schemes.

(446) ##STR00281##

(447) ##STR00282##

(448) ##STR00283##

(449) ##STR00284##

(450) In certain embodiments, the epoxide is stereochemically pure (e.g., enantiomerically pure). In certain embodiments, the amine is stereochemically pure (e.g., enantiomerically pure). In certain embodiments, the lipidoid is prepared from the reductive amination of an imine which derived from the condensation of an amine and an aldehyde. The compounds of the invention can have an enantiomeric excess or a diastereomeric excess up to and including 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%.

(451) In other embodiments, it will be appreciated by one skilled in the art that the amine and the epoxide will react at the substituted carbon of the epoxide resulting in an aminoalcohol lipidoid compound as shown in the following scheme.

(452) ##STR00285##
While the above reaction may be less preferred, it is likely to occur at least to some degree and may be more favored under certain reaction conditions. An aminoalcohol lipidoid compound may have amines that have reacted in one or both manners.

(453) In certain embodiments, the amine and epoxide-terminated compound are reacted together neat. In other embodiments, the reaction is done in a solvent (e.g., THF, CH.sub.2Cl.sub.2, MeOH, EtOH, CHCl.sub.3, hexanes, toluene, benzene, CCl.sub.4, glyme, diethyl ether, etc.). In certain embodiments, the reaction mixture is heated. In certain embodiments, the reaction mixture is heated to temperature ranging from 30° C.-100° C. In another embodiment, the reaction mixture is heated to approximately 90° C. The reaction may also be catalyzed. For example, the reaction may be catalyzed by the addition of an acid, base, or metal (e.g., Lewis acid). The reaction may be allowed to proceed for hours, days, or weeks. In certain embodiments, the reaction is allowed to proceed for 1-7 days. In certain embodiments, the reactions were run from about 1 to about 3 days. The resulting composition may be used with or without purification. In certain embodiments, the lipidoids are subsequently subjected to an alkylation step (e.g., reaction with methyl iodide) to form quaternary amine salts. Optionally, various salt forms of the lipidoids may be prepared. In certain embodiments, the salts are pharmaceutically acceptable salts.

(454) In certain embodiments the aminoalcohol lipidoid compound or composition containing a mixture of aminoalcohol lipidoid compounds is prepared by reacting amine 200

(455) ##STR00286##
with an epoxide-terminated compound. In certain embodiments, the amine 200-derived aminoalcohol lipidoid compounds (i.e., C12-200) and its various possible isomers are of the formulae below:

(456) ##STR00287## ##STR00288##

(457) In certain embodiments, each

(458) ##STR00289##
is independently

(459) ##STR00290##

(460) In certain embodiments the aminoalcohol lipidoid composition, is a composition containing one or more of the above aminoalcohol lipidoid compounds.

(461) In certain embodiments the aminoalcohol lipidoid compound or composition containing a mixture of aminoalcohol lipidoid compounds is prepared by reacting amine 205

(462) ##STR00291##
with an epoxide-terminated compound C12. In certain embodiments, the amine 205-derived aminoalcohol lipidoid compounds (i.e., C12-205) and its various possible isomers are of the formulae below:

(463) ##STR00292## ##STR00293## ##STR00294##

(464) In certain embodiments, each

(465) ##STR00295##
is independently

(466) ##STR00296##

(467) In certain embodiments the aminoalcohol lipidoid composition, is a composition containing one or more of the above aminoalcohol lipidoid compounds.

(468) In certain embodiments, the aminoalcohol lipidoid is of the formula

(469) ##STR00297##
In certain embodiments, the aminoalcohol lipidoid compound is of the formula

(470) ##STR00298##
In certain embodiments, the aminoalcohol lipidoid compound is a mixture of

(471) ##STR00299##
In certain embodiments, each

(472) ##STR00300##
is independently

(473) ##STR00301##

(474) In certain embodiments the aminoalcohol lipidoid composition, is a composition containing one or more of the above aminoalcohol lipidoid compounds.

(475) In certain embodiments the aminoalcohol lipidoid compound or composition containing a mixture of aminoalcohol lipidoid compounds is prepared by reacting amine 96

(476) ##STR00302##
with an epoxide-terminated compound C16. In certain embodiments, the amine 96-derived aminoalcohol lipidoid compounds (i.e., C16-96) and its various possible isomers are of the formulae below:

(477) ##STR00303##

(478) In certain embodiments the aminoalcohol lipidoid compound or composition containing a mixture of aminoalcohol lipidoid compounds is prepared by reacting amine 210

(479) ##STR00304##
with the epoxide-terminated compound C12. In a similar manner as illustrated above, one skilled in the art will readily be able to determine the various possible 210-derived aminoalcohol lipidoid compounds (i.e., C12-210) isomeric structures that are possible from this reaction.

(480) In certain embodiments the aminoalcohol lipidoid composition, is a composition containing one or more of the above aminoalcohol lipidoid compounds.

(481) In certain embodiments the aminoalcohol lipidoid compound or composition containing a mixture of aminoalcohol lipidoid compounds is prepared by reacting amine 220

(482) ##STR00305##
with the epoxide-terminated compound C12. In a similar manner as illustrated above, one skilled in the art will readily be able to determine the various possible 220-derived aminoalcohol lipidoid compounds (i.e., C12-220) isomeric structures that are possible from this reaction.

(483) In certain embodiments the aminoalcohol lipidoid composition, is a composition containing one or more of the above aminoalcohol lipidoid compounds.

(484) In certain embodiments, the aminoalcohol lipidoid compound or composition containing a mixture of aminoalcohol lipidoid compounds is prepared by reacting amine 111

(485) ##STR00306##
with the epoxide-terminated compound C12. In a similar manner as illustrated above, one skilled in the art will readily be able to determine the various possible 111-derived aminoalcohol lipidoid compounds (i.e., C12-111) isomeric structures that are possible from this reaction.

(486) In certain embodiments the aminoalcohol lipidoid composition, is a composition containing one or more of the above aminoalcohol lipidoid compounds.

(487) 2. Synthesis of Aminoalcohol Lipidoid Compounds

(488) The inventive aminoalcohol lipidoid compounds may be prepared by any method known in the art. Preferably the aminoalcohol lipidoid compounds are prepared from commercially available starting materials, such as terminal-epoxide compounds, interior epoxide compounds, and amines. In another embodiment, the aminoalcohol lipidoid compounds are prepared from easily and/or inexpensively prepared starting materials. As would be appreciated by one of skill in the art, the inventive aminoalcohol lipidoid compounds can be prepared by total synthesis starting from commercially available starting materials. A particular aminoalcohol lipidoid compound may be the desired final product of the synthesis, or a mixture of aminoalcohol lipidoid compounds may be the desired final product.

(489) In certain embodiments, the inventive aminoalcohol lipidoid compound is prepared by reacting an amine with an epoxide-terminated compound. An exemplary reaction scheme is shown in FIG. 1.

(490) Any amine containing between one, two, and five amine functionalities is useful in preparing inventive aminoalcohol lipidoid compounds. Primary amines useful in this invention include, but are not limited to, methylamine, ethylamine, isopropylamine, aniline, substituted anilines, ethanolamine, decylamine, undecylamine, dodecylamine, tetradecylamine, hexadecylamine, and octadecylamine. The amine may be a bis(primary amine) including, but not limited to, ethylenediamine, 1,3 diaminopropane, 1,4 diamino butane, 1,5 diaminopentane, 1,6 diaminohexane, 2,2′(ethylenedioxy)bis(ethylamine). The amine may be a bis(secondary amine). Secondary amines useful in this invention include, but are not limited to dipropylamine and methylpentylamine. The amine may include both primary and secondary amines including, but not limited to, (2-aminoethyl) ethanolamine, diethylenetriamine and triethylenetetramine. Preferably, the amine is commercially available. In certain embodiments, the amine is stereochemically pure (e.g., enantiomerically pure).

(491) In certain embodiments, the amine used in the synthesis of the aminoalcohol lipidoid compound is of the formula:

(492) ##STR00307## ##STR00308## ##STR00309## ##STR00310## ##STR00311##

(493) Epoxide-terminated compounds that are useful in the present invention include any epoxide-terminated compounds that are racemic or stereoisomers thereof, all of varying chain lengths and feature unique functional groups having varying degrees of saturation. In certain embodiments, the epoxide is stereochemically pure (e.g., enantiomerically pure). In certain embodiments, the epoxide contains one or more chiral centers. In certain embodiments, the epoxide-terminated compounds are of the formula:

(494) ##STR00312##

(495) In certain embodiments, the epoxide-terminated compounds are of the formula:

(496) ##STR00313##

(497) In certain embodiments, the epoxide-terminated compounds are of the formula:

(498) ##STR00314##

(499) In certain embodiments, the epoxide contains one or more chiral centers, such as those shown below:

(500) ##STR00315##

(501) In certain embodiments, the enantiomeric epoxide

(502) ##STR00316##
is resolved from the racemic mixture of epoxides using hydrolytic kinetic resolution (HKR) catalyzed with the (R,R)-HKR catalyst of the formula:

(503) ##STR00317##
In further embodiments, the enantiomeric epoxide

(504) ##STR00318##
is resolved from the racemic mixture of epoxides using hydrolytic kinetic resolution (HKR) catalyzed with the (S,S)-HKR catalyst of the formula:

(505) ##STR00319##
In certain embodiments, the aminoalcohol lipidois of the invention are prepared from a process comprising steps of:
(a) converting the epoxide primary alcohol of the formula:

(506) ##STR00320##
into the corresponding protected primary alcohol derivative of the formula:

(507) ##STR00321##
(b) reacting the protected primary alcohol derivative of the formula:

(508) ##STR00322##
with a carbon-based nucleophile to produce the secondary alcohol of the formula:

(509) ##STR00323##
(c) converting the secondary alcohol of the formula:

(510) ##STR00324##
into the corresponding protected secondary alcohol derivative of the formula

(511) ##STR00325##
(d) deprotecting the protected secondary alcohol derivative of the formula

(512) ##STR00326##
into the corresponding primary alcohol of the formula

(513) ##STR00327##
(e) oxidizing the primary alcohol of the formula

(514) ##STR00328##
into the corresponding aldehyde of the formula

(515) ##STR00329##
(f) condensing the aldehyde of the formula:

(516) ##STR00330##
with an amine of the formula:

(517) ##STR00331##
to produce an imine of the formula:

(518) ##STR00332##
and (g) reducing an imine of the formula:

(519) ##STR00333##
to produce the corresponding amine of the formula:

(520) ##STR00334##
wherein R.sub.1 is hydrogen, a substituted, unsubstituted, branched or unbranched C.sub.1-20-aliphatic or a substituted, unsubstituted, branched or unbranched C.sub.1-20 heteroaliphatic, wherein at least one occurrence of R.sub.1 is hydrogen; R.sub.B, R.sub.C, and R.sub.D are, independently, hydrogen, a substituted, unsubstituted, branched or unbranched C.sub.1-20-aliphatic, or a substituted, unsubstituted, branched or unbranched C.sub.1-20-heteroaliphatic or —CH.sub.2CH(OH)R.sub.E; R.sub.B and R.sub.D together may optionally form a cyclic structure; R.sub.C and R.sub.D together may optionally form a cyclic structure; R.sub.E is a substituted, unsubstituted, branched or unbranched C.sub.1-20 aliphatic or a substituted, unsubstituted, branched or unbranched C.sub.1-20 heteroaliphatic; and PG.sub.1 and PG.sub.2 are O-protecting groups as described herein.
In certain embodiments, the epoxide primary alcohol of step (a) is

(521) ##STR00335##
and the amine of step (f) is

(522) ##STR00336##
In certain embodiments, the epoxide primary alcohol of step (a) is

(523) ##STR00337##
and the amine of step (f) is

(524) ##STR00338##

(525) 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) and 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 on chiral columns.

(526) In certain embodiments, the reaction is performed neat without the use of a solvent. In other embodiments, a solvent is used for the reaction. Both or one of the starting amine or epoxide-terminated compound 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 resulting solutions are combined, and the reaction mixture is heated to yield the desired aminoalcohol lipidoid compound. In certain embodiments, the reaction mixture is heated to a temperature ranging from 25° C. to 100° C., preferably at approximately 90° C. The reaction may also be catalyzed. For example, the reaction may be catalyzed by the addition of an acid, base, or metal. The reagents may be allowed to react for hours, days, or weeks. Preferably, the reaction is allowed to proceed from overnight (e.g., 8-12 hours) to 7 days.

(527) The synthesized aminoalcohol lipidoid compounds may be purified by any technique known in the art including, but not limited to, precipitation, crystallization, chromatography, distillation, etc. In certain embodiments, the aminoalcohol lipidoid compound is purified through repeated precipitations in organic solvent (e.g., diethyl ether, hexane, etc.). In certain embodiments, the aminoalcohol lipidoid compound is isolated as a salt. The aminoalcohol lipidoid compound is reacted with an acid (e.g., an organic acid or inorganic acid) to form the corresponding salt. In certain embodiments, the tertiary amine is alkylated to form a quaternary ammonium salt of the aminoalcohol lipidoid compound. 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.

(528) In certain embodiments, the reaction mixture results in a mixture of isomers with varying numbers and positions of epoxide-derived compound tails. Such mixtures of products or compounds may be used as is, or a single isomer, or compound, may be purified from the reaction mixture. When an amine is not exhaustively alkylated, the resulting primary, secondary, or tertiary amines may be further reacted with another aminoalcohol lipidoid compound, epoxide-terminated compound, or other electrophile. The resulting aminoalcohol lipidoid compound may then be optionally purified.

(529) In certain embodiments, a desired aminoalcohol lipidoid compound is prepared by traditional total synthesis. In certain embodiments, a commercially available amine is the starting material. One or more amino groups of the amine are optionally protected. The unprotected amino groups are reacted with an epoxide-terminated compound. The product is optionally purified. Protecting groups are removed, and the free amino groups are optionally reacted with another aminoalcohol lipidoid compound, epoxide-terminated compound, or other electrophile. Such a sequence may be repeated depending on the desired complexity of the inventive product being prepared. The final product may then be optionally purified.

(530) In one embodiment, a library of different aminoalcohol lipidoid compounds is prepared in parallel. A different amine and/or epoxide-terminated compound 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 aminoalcohol lipidoid compounds to occur. 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 aminoalcohol lipidoid compounds may then be isolated and purified using techniques known in the art. The aminoalcohol lipidoid compounds may then be screened using high-throughput techniques to identify aminoalcohol lipidoid compounds 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 aminoalcohol lipidoid compounds may be screened for properties or characteristics useful in gene therapy (e.g., ability to bind polynucleotides, increase in transfection efficiency).

(531) 3. Polynucleotide Complexes

(532) The ability of cationic compounds to interact with negatively charged polynucleotides through electrostatic interactions is well known. Cationic lipids such as Lipofectamine have been prepared and studied for their ability to complex and transfect polynucleotides. The interaction of the lipid 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 ζ-potential, more preferably the ζ-potential is between 0 and +30.

(533) The aminoalcohol lipidoid compounds of the present invention possess tertiary amines. 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 aminoalcohol lipidoid compounds under conditions suitable to form polynucleotide/lipidoid complexes. The lipidoid is preferably at least partially protonated so as to form a complex with the negatively charged polynucleotide. In certain embodiments, the polynucleotide/lipidoid complexes form particles that are useful in the delivery of polynucleotides to cells. In certain embodiments, multiple aminoalcohol lipidoid molecules may be associated with a polynucleotide molecule. The complex may include 1-100 aminoalcohol lipidoid molecules, 1-1000 aminoalcohol lipidoid molecules, 10-1000 aminoalcohol lipidoid molecules, or 100-10,000 aminoalcohol lipidoid molecules.

(534) 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.

(535) 4. Polynucleotide

(536) The polynucleotide to be complexed, encapsulated by the inventive aminoalcohol lipidoid compounds, or included in a composition with the inventive aminoalcohol lipidoid compounds 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.

(537) 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.

(538) In certain embodiments, the polynucleotide is a dsRNA (double-stranded RNA).

(539) In certain embodiments, the polynucleotide is an siRNA (short interfering RNA).

(540) In certain embodiments, the polynucleotide is an shRNA (short hairpin RNA).

(541) 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).

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

(543) In some embodiments, a 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).

(544) 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.

(545) 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.

(546) 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.

(547) 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.

(548) 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.

(549) 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).

(550) 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 and can be found on the internet (www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf, incorporated herein by reference; see also 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).

(551) The antigenic protein or peptides encoded by the polynucleotide may be derived from such bacterial organisms as Streptococccus 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.

(552) 5. Particles

(553) The aminoalcohol lipidoid compounds of the present invention may also be used to form drug delivery devices. The inventive aminoalcohol lipidoid compounds may be used to encapsulate agents including polynucleotides, small molecules, proteins, peptides, metals, organometallic compounds, etc. The inventive aminoalcohol lipidoid compounds have several properties that make them particularly suitable in the preparation of drug delivery devices. These include: 1) the ability of the lipid 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. In certain embodiments, the aminoalcohol lipidoid compounds are used to form particles containing the agent to be delivered. These particles may include other materials such as proteins, carbohydrates, synthetic polymers (e.g., PEG, PLGA), and natural polymers.

(554) 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.

(555) 6. Methods of Preparing Particles

(556) 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.

(557) 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).

(558) 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).

(559) 7. Micelles and Liposomes

(560) The aminoalcohol lipidoid compounds of the invention may be used to prepare micelles or liposomes. Many techniques for preparing micelles and liposomes are known in the art, and any method may be used with the inventive aminoalcohol lipidoid compounds to make micelles and liposomes. In addition, any agent including polynucleotides, small molecules, proteins, peptides, metals, organometallic compounds, etc. may be included in a micelle or liposome. Micelles and liposomes are particularly useful in delivering hydrophobic agents such as hydrophobic small molecules.

(561) In certain embodiments, liposomes (lipid or aminoalcohol lipidoid compound vesicles) 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 lipsomes involves preparing the aminoalcohol lipidoid compounds for hydration, hydrating the aminoalcohol lipidoid compounds with agitation, and sizing the vesicles to achieve a homogenous distribution of liposomes. Aminoalcohol lipidoid compounds are first dissolved in an organic solvent to assure a homogeneous mixture of aminoalcohol lipidoid compounds. The solvent is then removed to form a lipidoid film. This film is thoroughly dried to remove residual organic solvent by placing the vial or flask on a vacuum pump overnight. Hydration of the lipidoid film/cake is accomplished by adding an aqueous medium to the container of dry lipidoid 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 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 vesicles (LUV) with a mean diameter of 120-140 nm.

(562) 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 aminoalcohol lipidoid is C14-120. In certain embodiments, the aminoalcohol lipidoid is C16-120. In certain embodiments, the aminoalcohol lipidoid is C14-98. In certain embodiments, the aminoalcohol lipidoid is C14-113. In certain embodiments, the aminoalcohol lipidoid is C18-96. In certain embodiments, the aminoalcohol lipidoid is C14-96. In certain embodiments, the aminoalcohol lipidoid is C14-110. In certain embodiments, the amount of aminoalcohol lipidoid compound 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.

(563) Certain aminoalcohol lipidoid compounds 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 aminoalcohol lipidoid compounds allows for simple assembly of liposomes without the need for additional steps or devices such as an extruder.

(564) 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 Structure-Activity 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.

(565) 8. Agent

(566) The agents to be delivered by the system 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 a small molecule, organometallic compound, nucleic acid, protein, peptide, polynucleotide, metal, an isotopically labeled chemical compound, drug, vaccine, immunological agent, etc.

(567) In certain embodiments, the agents are organic compounds with pharmaceutical activity. In another embodiment of the invention, the agent is a clinically used drug. In certain embodiments, the drug is an antibiotic, anti-viral agent, anesthetic, steroidal agent, anti-inflammatory agent, anti-neoplastic agent, antigen, vaccine, antibody, decongestant, antihypertensive, sedative, birth control agent, progestational agent, anti-cholinergic, analgesic, anti-depressant, anti-psychotic, β-adrenergic blocking agent, diuretic, cardiovascular active agent, vasoactive agent, non-steroidal anti-inflammatory agent, nutritional agent, etc.

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

(569) 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.

(570) 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 Streptococccus 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.

(571) 9. Targeting Agents

(572) The inventive complexes, liposomes, micelles, microparticles, picoparticles and nanoparticles 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.

(573) 10. Pharmaceutical Compositions

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

(575) Pharmaceutical compositions of the present invention and for use in accordance with the present invention may include a pharmaceutically acceptable excipient or carrier. As used herein, the term “pharmaceutically acceptable carrier” 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 pharmaceutically acceptable carriers are 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 pharmaceutical 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.

(576) Liquid dosage forms for oral administration include pharmaceutically acceptable 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.

(577) 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.

(578) 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.

(579) 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.

(580) 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.

(581) 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.

(582) 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.

(583) 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.

(584) 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.

(585) 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.

(586) 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.

(587) 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.

(588) These and other aspects of the present invention will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the invention but are not intended to limit its scope, as defined by the claims.

EXAMPLES

Example 1

(589) Synthesis and Characterization of 1,2-aminoalcohols

(590) These lipidoids were synthesized by combining amines and epoxides in a glass vial equipped with a stirbar and heated to 90° C., as shown in FIG. 1. The amines chosen contain between two and five amine functionalities, while the epoxides are racemic, of varying chain lengths and feature unique functional groups and varying degrees of saturation (FIG. 2). The reaction times varied from 24-72 hours at this temperature. Mixtures generally remained clear throughout the reaction and became noticeably viscous as the reaction progressed. Upon cooling, many became waxy solids. The extent of the reaction could be controlled by the number of equivalents of epoxide added to the reaction mixture. For example, in the examples shown, amine 114 has a maximum of five points for substitution. Addition of five equivalents of epoxide would yield an amine core with five alkane chains linked by a 1,2-aminoalcohol. Addition of four equivalents of epoxide would yield only four chains linked by the same structure. This was verified by thin layer chromatography (TLC), which showed primarily one product existing in the crude reaction mixtures set up as described.

(591) To verify the identity of the molecules, a few test reactions were set up and purified by silica gel chromatography. The components of the crude reaction mixture were separated and tested by NMR and mass spectrometry. Again, in the case of amine 114, three products were identified: three, four and five tailed products. The molecular weight was confirmed by mass spectrometry, and the structure was verified by NMR (FIG. 3, which shows characterization data of epoxide lipidoids derived from amine 114). These isolated compounds were then used as standards versus selected members of the library for TLC analysis. Reactions set up to fully substitute the amine had similar R.sub.f and staining profiles to the fully substituted standard. Reactions set up to occupy n−1 positions of the amine had similar R.sub.f and staining profiles to n−1 standard (FIG. 4).

Example 2

(592) In Vitro Screening for RNA Delivery

(593) Epoxide lipidoids were tested for their ability to deliver siRNA to a HeLa cell line that stably expresses both firefly and Renilla luciferase. Efficacy was determined by complexing the lipidoid with siRNA specific for firefly luciferase, adding this mixture to cells and measuring the subsequent ratio of firefly to Renilla expression. This procedure was performed in 96-well microtiter plates to enable high throughput testing of the materials. In this assay, reduction of both firefly and Renilla expression indicates toxicity, while reduction of only firefly expression is an indication of specific knockdown due to siRNA. Initial screening results of selected members of the library are shown in FIG. 5. Many members of this sampling showed some ability to transfect cells and give rise to some knockdown of firefly luciferase. Of these the best performers were generally lipidoids derived from epoxides of 14 carbons or longer coupled with monomers of three or more amine functional groups. A few show nearly complete ablation of firefly expression, even at the lowest dose of lipidoid tested.

Example 3

(594) RNA Encapsulation Efficiency

(595) Formulation for in vitro experiments is a simple mixing of RNA with lipidoid at a set ratio in buffer prior to addition to cells. In vivo formulation requires the addition of extra ingredients to facilitate circulation throughout the body. To test the ability of these lipidoids to form particles suitable for in vivo work, we followed a standard formulation procedure utilized in the lab. These particles consisted of 42% lipidoid, 48% cholesterol and 10% PEG. After formation of the particle, RNA was added and allowed to integrate with the complex. The encapsulation efficiency was determined using a standard Ribogreen assay. As shown in the table below, these particles were on the order of 100 nm after extrusion, with some achieving encapsulation efficiency of over 90%.

(596) Particle Size and Entrapment Efficiency of Selected Epoxide Lipidoids

(597) TABLE-US-00001 Compound Size (nm) Entrapment (%) C14-120-B 95.2 92.75 C16-120-B 128.4 67.22 C14-98-B 126.9 44.84 C14-113-B 92.7 96.42

Example 4

(598) HepG2 cells were seeded at a density of 15,000 cells per well into opaque white 96-well plates (Corning-Costar, Kennebunk, Me.) 24 hours prior to transfection to allow for growth and confluence. Working dilutions of lipidoids were made in 25 mM sodium acetate (pH 5) at a concentration of 0.5 mg/ml. For gene delivery experiments pCMV-Luc firefly luciferase DNA (ElimBiopharmaceuticals, South San Francisco, Calif.) was used. Lipidoid:DNA complexes were formed by electrostatic interaction between positively charged lipidoid molecules and negatively charged nucleic acids. By varying the volume of lipidoid solution added to a constant amount of DNA, varying weight:weight ratios of lipidoid to DNA were tested. Lipidoid solution (75 μl) was added to DNA solution (75 μl) and mixed well. Mixtures were then incubated at room temperature for 20 minutes to allow for complexation. These complexes (30 μl) were then added to serum containing medium (200 μl) and mixed well. Growth medium was then removed from the cells and lipidoid:DNA complex containing medium was immediately added. Total DNA loading was 300 ug DNA per well. Lipofectamine 2000 transfection was performed as described by the vendor. Complexes were allowed to incubate with cells for 48 hours. Luciferase expression was then quantified by Bright-Glo assay (Promega, Madison, Wis.). Briefly, 48 hours post-transfection, the lipidoid:DNA complex containing growth medium was removed from cells using a 12-channel aspirating wand. 200 ul of a 1:1 mixture of Bright-Glo reagent and non-phenol red containing DMEM was added to each well of the 96-well plate with cells. After 10 minute incubation at room temperature, luminescence was measured using a luminometer. (n=3). Exemplary results are depicted in FIG. 6.

Example 5

(599) Lipidoid-based siRNA formulations comprised lipidoid, cholesterol, polyethylene glycol-lipid (PEG-lipid) and siRNA. Stock solutions of Lipidoid, mPEG2000-DMG MW 2660 (synthesized by Alnylam), and cholesterol MW 387 (Sigma-Aldrich) were prepared in ethanol and mixed to yield a molar ratio of 42:10:48. Mixed lipids were added to 200 mM sodium acetate buffer pH 5.2 to yield a solution containing 35% ethanol, resulting in spontaneous formation of empty lipidoid nanoparticles. Resulting nanoparticles were extruded through an 80 nm membrane (three passes). siRNA in 35% ethanol and 50 mM sodium acetate pH 5.2 was added to the nanoparticles at 10:1 (wt/wt) total lipids:siRNA and incubated at 37° C. for 30 min. Ethanol removal and buffer exchange of siRNA-containing lipidoid nanoparticles was achieved by dialysis against PBS using a 3,500 MWCO membrane. Particle size was determined using a Malvern Zetasizer NanoZS (Malvern). siRNA content and entrapment efficiency was determined by Ribogreen assay.

(600) C57BL/6 mice (Charles River Labs) received either saline or siRNA in lipidoid formulations via tail vein injection at a volume of 0.01 ml/g. Mice were dosed at either 1.75 or 4 mg/kg entrapped siRNA. At 48 hours after administration, animals were anesthetized by isofluorane inhalation and blood was collected into serum separator tubes by retroorbital bleed. Serum levels of Factor VII protein were determined in samples using a chromogenic assay (Biophen FVII, Aniara Corporation) according to the manufacturer's protocols. A standard curve was generated using serum collected from saline-treated animals. Exemplary results are depicted in FIG. 7.

Example 6

(601) In Vitro Screening of Epoxide Library

(602) Compounds of the epoxide-based lipidoid library were synthesized according to the procedures described herein. The compounds were then screened for siRNA delivery efficacy to a cancer cell line, using a Hela-derived cell line genetically engineered to express luciferase reporter proteins. In these experiments, the ability of each material to facilitate sequence-specific gene silencing was evaluated by comparison of protein levels in treated groups to untreated controls. For each compound, delivery experiments were performed using varying weight ratios of lipidoid:siRNA. In the original disclosure, knockdown results for the entire library were shown. An abbreviated data set is shown in FIG. 11 demonstrating the results for the top 25 performing compounds in the in vitro screen, including C16-96-B, C14-200 and/or C14-205, and C12-200 and/or C12-205.

Example 7

(603) In Vivo Screening of Top Performing Epoxide Lipidoids

(604) To test siRNA delivery efficacy in vivo, a mouse model for liver delivery was used. Factor VII, a hepatocyte-specific blood clotting factor, served as a model protein for knockdown studies. Once produced by hepatocytes, Factor VII is released into the bloodstream, and a baseline level of expression can be determined by simple blood draw and quantification of protein levels by colorimetric assay. By delivering anti-Factor VII siRNA to hepatocytes, knockdown of this model protein can be achieved and a percentage of silencing can be determined by comparison to an untreated control.

(605) Following the in vitro screen, compounds were purified as detailed in Part 1 (see Example 14). For in vivo testing, the compounds were formulated with cholesterol and a PEG-lipid for serum stability and siRNA packaging. In these experiments, lipidoids were formulated at a 42:48:10 molar ratio of lipidoid:cholesterol:PEG. The weight ratio of total lipids (lipidoid+cholesterol+PEG) to siRNA was 10:1. After each formulation, the particles were characterized for size and siRNA entrapment efficiency using dynamic light scattering and Ribogreen assay, respectively. The total dose of siRNA administered in the initial screen varies from group to group due to the differences in entrapment efficiency of the lipidoid particles. In all experiments, the dose of siRNA administered to each mouse is consistent according to body weight. The knockdown results from the in vivo screen are shown in FIG. 12. B1 and B2 nomenclature signify different compounds visualized by TLC and isolated during purification. As shown, C14-11-B and C16-96-B were the lead compounds from the initial screen. It should be noted that while some compounds did not show efficacy in this screen, a simple adjustment of formulation composition may greatly improve the results.

Example 8

(606) Following the initial in vivo screening experiments, two compounds were used to conduct a dose response. In these experiments and all subsequent experiments, the siRNA dose is based on total siRNA content in the formulation, not entrapped siRNA. The dose response results are shown in FIG. 13a and FIG. 14a. In addition to Factor VII measurement, the change in mouse body weight is recorded and a loss in weight is generally considered formulation induced toxicity (See FIG. 13b and FIG. 14b). Tables 1 and 2 tabulate the formulation parameters and characterization data from these experiments.

(607) TABLE-US-00002 TABLE 1 Formulation parameters and characterization data for C16-96-B dose response formulation Formulation Total Characterization Lipidoid Lipid:Chol:PEG Lipid:siRNA Entrapment Size C16-96-B 65:29:6 10:1 81% 107.8 nm

(608) TABLE-US-00003 TABLE 2 Formulation parameters and characterization data for C14-110-B dose response formulation Formulation Total Characterization Lipidoid Lipid:Chol:PEG Lipid:siRNA Entrapment Size C14-110-B 42:48:10 10:1 44% 115 nm

Example 9

(609) After completing the dose response, C16-96-B was chosen for further investigation and optimization. In the next experiments, the percent composition of the formulations was varied to observe the effect of composition on particle size, entrapment, and efficacy. The compositions investigated are shown in Table 3. FIG. 15 shows the knockdown results from the formulations tested. Where the formulation in red was the previous best, it is shown that efficacy can be improved by formulating particles at different compositions.

(610) TABLE-US-00004 TABLE 3 Formulation parameters and characterization data for C16-96-B formulation optimization experiment Formulation Parameters Total Formulation Lipidoid Chol PEG Lipid:siRNA Entrapment 1 63 31 6 8.5:1 80% 2 65 29 6 8.5:1 80% 3 67 27 6 8.5:1 80% 4 69 25 6 8.5:1 84% 5 71 23 6 8.5:1 85% 6 63 33 4 8.5:1 85% 7 65 31 4 8.5:1 85% 8 67 29 4 8.5:1 84% 9 69 27 4 8.5:1 83% 10 71 25 4 8.5:1 85%

Example 10

(611) A second dose response was conducted with the new percent composition parameters. The knockdown results and particle formulation/characteristics are shown in FIG. 16 and Table 4, respectively. By formulating at this composition, approximately 40% knockdown was achieved at a dose of 0.25 mg/kg. Using this result as the new benchmark, the library was revisited and previously untested materials were screened at 0.25 mg/kg in attempt to find other compounds which could give similar or better results.

(612) TABLE-US-00005 TABLE 4 Formulation Total Characterization Lipidoid Lipid:Chol:PEG Lipid:siRNA Entrapment Size C16-96-B 71:23:6 20:1 83% 205 nm

Example 11

(613) In the revisited in vivo screen, compound C12-200 and/or C12-205 was identified as giving nearly complete silencing at a dose of 0.25 mg/kg. As depicted in FIG. 12, the slightly longer tailed version of this compound, C12-200 and/or C12-205 was previously identified as a highly efficient delivery agent, showing complete silencing at a much higher dose of 7.5 mg/kg total siRNA. It is quite possible that this compound could facilitate complete silencing at much lower doses, but this has not yet been fully explored as the focus has been on C12-200 and/or C12-205. Following the discovery of the efficacy of this compound, the characterization experiments detailed in Part 1 (see Example 14) were initiated.

(614) The knockdown and body weight change results of this screen are depicted in FIG. 17b, and Table 5 tabulates the formulation parameters and characteristics. FIG. 17a depicts knockdown results for a second batch of C16-96-B. In previous experiments, this compound had resulted in approximately 40% knockdown at a dose of 0.25 mg/kg. From mass spec analysis, it was shown that this batch is two-tailed as opposed to the more efficacious three-tailed version that had been used in the previous studies.

(615) TABLE-US-00006 TABLE 5 Formulation parameters and characterization data for revisited in vivo screening Formulation Parameters Total Entrapment Size Formulation Lipid Chol PEG Lipid:siRNA (%) (nm) C14-96-B 73.1 21.3 5.6 8.5 87 81.7 C16-96-B 71.0 23.0 6.0 8.5 55 170.2 C12-200 and/or 45.0 45.5 9.5 8.5 36 167 C12-205 C18-62-B 52.7 39.1 8.2 8.5 86 227.4

Example 12

(616) A low-dose response was performed on C12-200 and/or C12-205. The knockdown and body weight loss results are shown in FIG. 18a and FIG. 18b. The results show that efficient knockdown is achieved and extremely low doses of siRNA. In comparison to ND98, our previous best compound from the original lipidoid library, comparable knockdown can be achieved with 100-fold lower doses of siRNA. The formulation parameters and characterization data are shown in Table 6.

(617) TABLE-US-00007 TABLE 6 Formulation parameters and characterization data for C12-200 and/or C12-205 and ND98 formulations Formulation Parameters Total Entrapment Size Formulation Lipid Chol PEG Lipid:siRNA (%) (nm) C12-200 and/or 48.2 42.8 8.9 8.5 45 154.1 C12-205 ND98 42.0 48.0 10.0 8.5 99 83.9

Example 13

(618) To further improve delivery efficacy, the percent composition of the C12-200 and/or C12-205 formulation was modified incrementally. These formulations were screened at a dose of 0.01 mg/kg to identify formulations which may perform better than the previous compositions. The results of these experiments are shown in FIG. 19 along with the formulation parameters and characteristics in Table 7. As expected, more efficacious delivery can be achieved by tuning the composition of the formulation. This optimization work is currently ongoing, along with synthesis of longer and shorter tailed versions of the C12-200 and/or C12-205 structure.

(619) TABLE-US-00008 TABLE 7 Formulation parameters and characterization data for C12-200 and/or C12-205formulations Formulation Parameters Total Entrapment Size Formulation Lipid Chol PEG Lipid:siRNA (%) (nm) 1 65.0 25.0 10.0 8.5 0 129 2 60.0 30.0 10.0 8.5 1 128 3 55.0 35.0 10.0 8.5 16 156 4 50.0 40.0 10.0 8.5 30 136 5 45.0 45.0 10.0 8.5 46 140 6 40.0 50.0 10.0 8.5 44 168 7 35.0 55.0 10.0 8.5 40 154 8 50.0 45.0 5.0 8.5 29 157 9 45.0 50.0 5.0 8.5 34 154 10 40.0 55.0 5.0 8.5 27 159 11 35.0 60.0 5.0 8.5 34 155

Example 14

(620) Part 1: Lipidoids Based on Amine 111

(621) Amine 111 (tetraethylenepentamine, or TEPA) is represented as the linear polyamine of the following structure:

(622) ##STR00339##

(623) The expected products of the reaction between amine 111 and the 12 carbon terminal epoxide C12 are illustrated as follows.

(624) ##STR00340##

(625) This reaction was performed, and the crude reaction mixture was separated based on the assumption that the order of product elution from polar silica gel would be: a) 7 tail (max substitution on 111 amine); b) 6 tail isomers (the isomers corresponding to 6 epoxides having reacted with the 111 amine); c) 5 tail isomers, and so on. It was expected that the MALDI-MS spectra of the crude reaction mixture would reveal peaks corresponding to the m/z ratios of these compounds (calculated [M+H.sup.+] for the expected 7 tail, 6 tail, and 5 tail products: 1481, 1295, and 1111, respectively). Material was isolated from the crude reaction mixture that, based on TLC analysis, was assumed to be the mixture of 6 tail isomers. This “purified” material performed quite well in the in vivo anti-Factor VII transfection assay.

(626) MALDI-MS spectra (see FIG. 20a) of the crude reaction mixture and of the purified “6 tail” material suggested compounds (see FIG. 20b). Technical grade tetraethlenepentamine (TEPA) is a mixture of compounds with similar boiling points; some of these compounds are of the following formulae:

(627) ##STR00341##

(628) Reaction of the C12 epoxide with these compounds accounts for most of the intense peaks in the MALDI mass spectra of the crude reaction mixture (FIG. 20a). The m/z ratios observed for the “purified” material (FIG. 20b) are consistent with amine 200 or 205 reacting with 5 equivalents of epoxide (calculated m/z for [M+H.sup.+] 1137, found 1137) and with amine 210 or 220 reacting with 4 equivalents of epoxide (calculated m/z for [M+H.sup.+] 910, found 910). The structures of these compounds are illustrated as follows:

(629) ##STR00342##

(630) To determine if this result was reproducible, an epoxide ring opening reaction was performed using the C12 epoxide and two different batches of amine 111. MALDI-MS was performed on each crude reaction mixture. In the reaction between the C12 epoxide and an older batch of 111 amine, an array of compounds were observed that were also observed in the original crude reaction mixture (see FIG. 20a). The MALDI spectrum of the crude reaction mixture using the C12 epoxide and a newer batch of amine 111 (see FIG. 21a) contains predominant peaks with m/z ratios of 1481 (linear amine 111 and 7 epoxide tails) and 1137 (consistent with amine 200 or 205 with 5 epoxide tails). The peak at m/z 910 in this spectrum was small. This might be a result of batch to batch differences in the 111 amine. Purification of the second reaction allowed isolation of a highly pure sample of the m/z 1137 material; we have designated this material “C12-200 and/or C12-205”. The .sup.1H NMR spectrum of C12-200 and/or C12-205 is consistent with the proposed structure (see FIG. 22).

(631) We are developing a library of materials based on the reaction of amines 200, 205, 210, and 220 and related amine structures with epoxides of varying length as depicted below:

(632) ##STR00343##

(633) These amines are being prepared in pure form using techniques familiar to one skilled in the art. We also propose a library of materials derived from free-based commercially available 111 amine pentahydrochloride according to the following scheme:

(634) ##STR00344##
the actual linear pentamine having the following structure:

(635) ##STR00345##

Example 15

(636) Part 2: Lipidoids Based on Amine 96

(637) This Example describes the synthesis of a library of structures that are variations of the amino alcohols lipidoid derived from the reaction of amine 96 with a C16 epoxide as follows:

(638) ##STR00346##
based upon the core 96 amine. First, variations at the position of the methyl group are achieved according to the following scheme:

(639) ##STR00347##
by reacting the terminal epoxides with an assortment of commercially available amines as depicted below.

(640) ##STR00348## ##STR00349##
Based on this strategy, the resulting library would contain approximately 800 possible amino alcohols of varying structure.

(641) Similar amine starting materials are available wherein the length of the carbon chain between the two amines is longer or shorter than amine 96 as depicted below.

(642) ##STR00350## ##STR00351##
A library of compounds resulting from the reaction of these amines with the various terminal epoxides would provide an additional ˜700 amino alcohol lipidoids.

(643) A protection/deprotection synthetic strategy could provide multiple variations, where the two core amines are functionalized with different alkyl epoxides according to the following scheme:

(644) ##STR00352##
This strategy could allows for substitution at one amine position with a functional group other than an epoxide (e.g., alkyl halide, isothiocyanate, chloroformate, acid halide) generating two different functional groups on the same amine core as follows:

(645) ##STR00353##
The following is an exemplary scheme illustrates general synthetic procedures to generate various compounds having two different functional groups on the same amine core as follows:

(646) ##STR00354##
wherein, Y is an aryl, heteroaryl, alkyl group (unreactive with epoxides, isocyanates, isothiocyanates and/or alkyl halides); and Z represents fragment from isocyanate, isothiocyante, alkyl halide, having the following exemplary structures:

(647) ##STR00355##

(648) Various multi-step sequences could be used to introduce additional hydroxyl groups near the amine core at positions different from those generated through epoxide ring opening as follows:

(649) ##STR00356##
Similar routes could provide the means to generate both hydroxyl groups and additional unsaturation as follows:

(650) ##STR00357##
Reductive amination as a first step after differential protection of the amine core provides access to a multitude of commercially available aldehydes and perhaps a way to introduce multiple hydroxyl groups through reductive amination using simple carbohydrates (a known procedure) as follows:

(651) ##STR00358##

Example 16

Synthesis of 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200 and/or C12-205)

(652) ##STR00359##

(653) A 250 mL glass pressure vessel was charged with 2-decyloxirane (20.0 grams, 109 mmoles), tetraethylenepentamine (Sigma-Aldrich technical grade, 2.93 grams, 15.5 mmoles) and a magnetic stir bar. The vessel was sealed and immersed in a silicone oil bath at 90° C. The reaction mixture was stirred vigorously for ˜72 hours at 90° C. The pressure vessel was then removed from the oil bath, allowed to cool to room temperature, then opened with caution. ˜9 grams of the resulting viscous, slightly yellow oil were purified via chromatography on silica gel (gradient elution from dichloromethane to 83.5:16.3:1.5 dichloromethane/methanol/aqueous ammonium hydroxide). Fractions containing the desired compound were pooled and concentrated by rotary evaporation. The resulting yellow oil was dissolved in ˜15 mL of ethyl acetate; decolorizing charcoal was added to this mixture. The solution was warmed to 68° C. and then filtered through Celite; the filtrate was concentrated by rotary evaporation; residual solvent was removed under reduced pressure overnight affording ˜1.3 grams of a pale yellow viscous oil. The starting material may contain inseparable isomer N1-(2-aminoethyl)-N2-(2-(piperazin-1-yl)ethyl)ethane-1,2-diamine; and the product may contain an inseparable isomer 1,1′-(2-((2-hydroxydodecyl)(2-((2-hydroxydodecyl)(2-(4-(2-hydroxydodecyl)piperazin-1-yl)ethyl)amino)ethyl)-amino)-ethylazanediyl)-didodecan-2-ol.

Example 17

(654) Amino Alcohol Lipidoids Prepared from Chiral Epoxides

(655) Antimicrobial lipidoids (e.g., C12-200, C16-96) can be prepared by reacting lipophilic, racemic terminal epoxides with low molecular weight polyamines. This approach is illustrated directly below with amine 200 and a generic terminal epoxide. There are two problems with this approach that complicate the isolation of pure products: the use of racemic epoxides, and addition of amines to the second carbon atom (C2) in the epoxide chain. In the following Examples, we report: a) the issues that may arise from the use of racemic epoxides can be avoided through the use of stereochemically pure terminal epoxides; and b) side products that may arise from additions at C2 of the epoxide can be avoided through an alternate synthetic route involving reductive amination.

(656) ##STR00360##
Reactions of Racemic Epoxides

(657) The epoxides used in the initial library synthesis were purchased from commercial sources as racemic mixtures: each epoxide contained an equal proportion of the R and S enantiomer. Achiral amines are equally likely to react with either stereoisomer. The effect of using racemic epoxides can be illustrated by considering the simple case of the reaction between an amine with one reactive site (e.g., piperidine) and a racemic epoxide (illustrated directly below). In this case, two aminoalcohol lipidoid products are generated: the R and S enantiomers. In theory, these products are separable through chromatography on a chiral stationary phase; in practice, developing and scaling up a method to perform this separation is difficult and expensive.

(658) ##STR00361##

(659) The situation becomes more complex when the starting amine has multiple reactive sites. For N reactive sites of an amine starting material, 2.sup.N stereoisomers are generated. For example, amine 200 (five reactive sites) reacts with a racemic epoxide generating 32 stereoisomers. In our experience, these products are inseparable. This issue can be resolved by performing the reaction with epoxides that are stereochemically pure (e.g., a single enantiomer of an epoxide). This is illustrated directly below.

(660) ##STR00362##

(661) A few terminal epoxides are commercially available as single enantiomers, but the cost of these compounds is prohibitive. Racemic epoxides can be resolved (separated into constituent enantiomers) by several means, including chromatography on a chiral stationary phase. We resolved the epoxides using a chemical method known as hydrolytic kinetic resolution (HKR). Efficient HKR of racemic epoxides can be achieved using a procedure described by Jacobsen (Schaus, et al., J. Am. Chem. Soc. 2002, 124, 1307-1315; which is incorporated herein by reference). The process is illustrated directly below. A chiral catalyst and water are added to a solution containing the racemic epoxide. In the presence of the chiral catalyst, the rate of hydrolysis of one epoxide enantiomer is much greater than the rate of hydrolysis for the other enantiomer. This allows selective hydrolysis of the unwanted epoxide enantiomer (to a 1,2-diol). The 1,2-diol can be separated from the epoxide by removing the epoxide through distillation under reduced pressure.

(662) ##STR00363##

Step 1. The Resolution of Epoxydodecane by HKR PGP-332C3

(663) ##STR00364##

(R)-(+)-1,2-epoxydodecane

(664) An oven-dried round bottom flask containing a magnetic stir bar was charged with the (R,R)-HKR catalyst (Schaus, et al., J. Am. Chem. Soc. 2002, 124, 1307-1315); CAS Number 176763-62-5, 1.31 g, 2.17 mmol. Dichloromethane (34 mL) and then glacial acetic acid (1.30 mL) were added to the flask. The resulting solution was stirred vigorously for 1.5 h; during this time the color of the mixture changed from dark red to brown. The solvent was removed by rotary evaporation until the material appeared dry. 1,2-epoxydodecane (40.0 g, 217 mmol) then isopropyl alcohol (reagent grade, 47 mL) were added to the flask containing the oxidized catalyst and a magnetic stir bar. The flask was immersed in an ice bath. H.sub.2O (2.15 mL, 119 mmol, 0.55 equiv relative to epoxide) was added dropwise to the stirred mixture. The flask was sealed with a rubber septum and the solution was allowed to warm to room temperature. After stirring for 2 days, the reaction mixture was diluted with ˜200 mL of hexanes. The resulting solution was filtered through paper to remove the white precipitate (1,2-diol). The filtrate was concentrated by rotary evaporation. The resulting dark red oily liquid was dissolved in ˜150 mL of hexanes and filtered in order to remove a substantial amount of white crystalline precipitate (diol). The filtrate was transferred into a 250 mL round bottom flask and concentrated by rotary evaporation. The desired product was isolated by distillation under vacuum (literature: 124° C./15 mm Hg). The desired product (14.3 grams, 71.5% of theoretical yield) was collected as a clear oil. The product was determined to be 100% ee by chiral chromatography of the 2-napthylenethiol derivative.

Step 2. The Synthesis of (R)-C12-200

(665) ##STR00365##

(666) (R)-C12-200.

(667) Amine 200 (640 mg, 2.97 mmol) and (R)-1,2-epoxydodecane (2.27 g, 12.1 mmol) were added to a vial containing a magnetic stir bar. The vial was sealed and warmed on a 80° C. reaction block for 5 days. The reaction mixture was allowed to cool to room temperature, and the desired product was isolated by chromatography on silica gel (gradient elution from CH.sub.2Cl.sub.2 to 175:22:3 CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH (aq.)). Fractions were pooled and concentrated affording (R)-C12-200 (665 mg) as a pale yellow oil. .sup.1H NMR (600 MHz, CDCl.sub.3): δ 4.37 (br s, —OH, 4H), 3.63 (app. br s, 3H), 3.56 (app. br s, 2H), 2.84-2.21 (m, 30H), 1.43-1.26 (m, 90H), 0.88 (t, J=7.0 Hz, 15H); MALDI-TOF-MS m/z: calcd for C.sub.70H.sub.146N.sub.5O.sub.5 [M+H.sup.+] 1137.1, found 1137.6.

Example 18

In Vivo Transfection with Chiral Amino Alcohol Lipidoids

(668) Preliminary in vivo transfections using anti-Factor VII siRNA formulated with were performed using (R)-C12-200 and (S)-C12-200 in mice. At 0.01 mg/kg siRNA dosing, approximately 50% reduction of systemic Factor VII was achieved using either the R or S forms of C12-200; there difference between these results and those obtained using C12-200 (the lipidoid prepared using amine 200 and racemic C12 epoxide) were insignificant.

Example 19

Synthesis of Amino Alcohol Lipidoids by the Reductive Amination Approach

(669) The first carbon atom in a terminal epoxide is the preferred site of attack during nucleophilic addition. 2D-NMR analysis of amino alcohol lipidoids shows that the majority of addition occurs at C1 of the epoxide, as illustrated directly below. Nevertheless, a trace amount of addition at C2 does occur. 2D-NMR analysis of compounds (S)-C12-205 and (R)-C12-200 suggest that roughly 10% of the lipid “tails” are the result of amine attack at C2 of the epoxide. These regioisomeric tails are likely distributed randomly throughout the entire population of lipid tails in the material. Efforts to limit this side reaction with the epoxides have not been successful. To avoid this side reaction, we proposed and executed an alternate synthetic strategy.

(670) ##STR00366##

(671) A retrosynthetic analysis of this strategy is presented directly below. The desired product is C (as illustrated directly below), from addition of amine A to C1 of epoxide B. D is the undesired constitutional isomer formed when amine A attacks C2 of epoxide B. Reductive amination of aldehyde fragment E with amine A and a reducing agent (giving F), followed by removal of the protecting group on the secondary alcohol, should generate product C. This route does not generate undesired structure D. This approach has two advantages: it does not generate the side product from reaction at C2 of the epoxide (e.g., D, directly below), and avoids the generation of a mixture of stereoisomers. To demonstrate that this strategy is feasible, we prepared a substrate analogous to E and reacted this component with an amine, ultimately generating the desired product (analogous to C).

(672) ##STR00367##

Synthesis of (S)-C12-205 by Reductive Amination Approach

(673) ##STR00368##

Step 1. Synthesis of Fragment 404 for the Reductive Amination Approach

(674) ##STR00369##

(S)-2-((trityloxy)methyl)oxirane (401)

(675) Trityl protected glycidol derivative 401 was prepared as described previously (Schweizer, et al., Synthesis 2007, 3807-3814; which is incorporated herein by reference.) A solution of (R)-glycidol (5.0 g, 61 mmol) in CH.sub.2Cl.sub.2 (30 mL) was added by syringe to a stirred solution of trityl chloride (18.6 g, 66.8 mmol) and triethylamine (16.9 mL, 122 mmol) in CH.sub.2Cl.sub.2 (67 mL) in an ice bath under argon. DMAP (742 mg, 6.08 mmol) was added to the reaction mixture following addition of the glycidol. The reaction was allowed to warm to room temperature. After 14 hours, the reaction mixture was diluted with 300 mL saturated aqueous NH.sub.4Cl. The mixture was further diluted to ˜1 L with water to dissolve precipitated salts. The product was extracted from the quenching solution with Et.sub.2O (3×); combined ethereal layers were washed with brine, dried over MgSO.sub.4, filtered through paper, and concentrated by rotary evaporation to a white solid. The crude product was purified by recrystallization from boiling MeOH (200 mL) affording the desired product 401 (14.1 g, 73%) as white crystals. NMR analysis of this material was consistent with that reported in the literature. (Schweizer, et al., Synthesis 2007, 3807-3814.)

(676) ##STR00370##

(S)-2-(benzyloxy)dodecan-1-ol (403)

(677) A 60 wt. % suspension of NaH in mineral oil (2.01 g, 50.3 mmol) was added to an oven-dried round bottom flask containing a magnetic stir bar. THF (120 mL) was added to the flask by syringe under Ar, and the flask was submerged in an ice bath. Crude 402 (14.9 g, 33.5 mmol) was dissolved in anhydrous THF (50 mL) and was added slowly to the stirred suspension of NaH. The reaction mixture was allowed to warm to room temperature. Benzyl chloride (5.8 mL, 50 mmol) was added to the reaction mixture. The flask was fitted with a reflux condenser, and the mixture was warmed to reflux under Ar overnight. After the reaction mixture had cooled, NH.sub.4Cl (sat. aq., ˜300 mL) was added slowly to quench residual NaH. The suspension was transferred into a separatory funnel using H.sub.2O (300 mL) and Et.sub.2O (200 mL). The organic layer was extracted with additional Et.sub.2O; ethereal layers were dried over MgSO.sub.4, filtered through paper, and concentrated to a yellow oil. This material was purified by chromatography on silica (gradient elution from hexanes to EtOAc); desired fractions were pooled and concentrated affording 15 g of a slightly yellow oil. This oil was dissolved in 1:1 MeOH/THF (100 mL). p-TsOH.H.sub.2O (572 mg) was added to the mixture; the solution was stirred for 6 hours. The reaction mixture was concentrated onto Celite by rotary evaporation and purified by chromatography on silica gel (gradient elution from hexanes to ethyl acetate). Fractions containing the desired product were pooled and concentrated affording 403 (5.44 g, 66%) as a clear oil. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.39-7.29 (m, 5H), 4.64 (d, J=11.6 Hz, 1H), 4.55 (d, J=11.6 Hz, 1H), 3.74-3.67 (m, 1H), 3.60-3.49 (m, 2H), 1.93-1.90 (m, 1H), 1.68-1.60 (m, 1H), 1.53-1.45 (m, 1H), 1.40-1.40 (m, 16H), 0.89 (t, J=6.9 Hz, 3H).

(678) ##STR00371##

(S)-2-(benzyloxy)dodecanal (404)

(679) CH.sub.2Cl.sub.2 (10 mL) and oxalyl chloride (1.72 mL, 20.3 mmol) were added to an oven-dried 2-neck round bottom flask containing a magnetic stir bar under Ar. The flask was immersed in a dry ice/acetone bath. A solution of DMSO (2.88 mL, 40.6 mmol) in CH.sub.2Cl.sub.2 (10 mL) was added to the stirred solution of oxalyl chloride slowly. 403 (5.4 g, 18.5 mmol) was dissolved in CH.sub.2Cl.sub.2 and added dropwise, over a period of 15 minutes, to the cold, stirred reaction mixture. After stirring for 2 hours, Et.sub.3N (12.9 mL, 18.46 mmol) was added to the reaction mixture, which was then allowed to warm to room temperature. The mixture was diluted with Et.sub.2O (˜300 mL) and water. The ether layer was washed with sat. aq. NaHCO.sub.3, 1M aq. HCl, and brine. The Et.sub.2O layer was then dried over MgSO.sub.4, filtered through paper, and concentrated by rotary evaporation. The crude product was purified by chromatography on silica (gradient elution from hexanes to 1:1 EtOAc/hexanes); fractions containing the desired product were pooled and concentrated affording 404 (3.58 g, 67%) as a clear, slightly viscous oil. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 9.66 (d, J=1.9 Hz, 1H), 7.37-7.31 (m, 5H), 4.69 (d, J=11.7 Hz, 1H), 4.55 (d, J=11.7 Hz, 1H), 3.78-3.75 (m, 1H), 1.68 (dd, J=14.3, 7.2 Hz, 2H), 1.49-1.35 (m, 2H), 1.25 (br s, 14H), 0.89 (t, J=6.7, 3H)

Step 2. Reductive Amination Pure Form of (S)-C12-205

(680) ##STR00372##

(681) 1-(2-aminoethyl)piperazine (205, 39 μL, 0.3 mmol) was added to a vial containing a magnetic stir bar. MeOH (10 mL) and the aldehyde 404 (971 mg, 3.34 mmol) were added to the vial. NaCNBH.sub.3 (188 mg, 3 mmol) was then added to the mixture. Glacial AcOH was added dropwise to the stirred solution until the pH (as measured using indicator strips) was approximately 5.5. The mixture bubbled during the addition of the AcOH. The mixture was stirred for 4 days, whereupon it was diluted with 1M NaOH (aq.) and CH.sub.2Cl.sub.2. The aqueous layer was extracted an additional time with CH.sub.2Cl.sub.2. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtered through paper and concentrated. The desired intermediate was purified by chromatography on silica (CH.sub.2Cl.sub.2 to 10% MeOH/CH.sub.2Cl.sub.2) affording a yellow oil (83 mg). This oil was dissolved in 25 mL 7:2:1 MeOH/H.sub.2O/AcOH. A portion of 10 wt. % Pd/C was added to the solution. The reaction mixture was stirred under H.sub.2 (slightly above atmospheric pressure) for 8 hours. The reaction mixture was filtered through Celite to remove the Pd/C and then concentrated to a film. Mass spectral analysis of this material indicated that it was the pure, desired product (S)-C12-205. MALDI-TOF-MS m/z: calcd for C.sub.42H.sub.88N.sub.3O.sub.3 [M+H.sup.+], 682.7; found 682.9.

(682) 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 ordinal 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.

(683) 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.