Membrane fusion compounds for delivery of biologically active molecules

Abstract

The present application relates to membrane fusion compounds for the delivery of biologically active molecules. The invention includes membrane fusion compounds, compositions thereof, and methods of use. The membrane fusion compounds can be used to manufacture nanoparticle compositions for use in biopharmaceuticals and therapeutics. More particularly, the present invention relates to compounds, compositions, and methods for providing nanoparticles incorporating or encapsulating active agents for the delivery and distribution of the active agents to cells, tissues, organs, and subjects.

Description

[0001] This application is a divisional application of the Chinese Invention Patent Application No. 201880071515.7 (PCT Application No. PCT / US2018 / 059504) with the title “Membrane Fusion Compounds for Delivery of Biologically Active Molecules” and a filing date of November 6, 2018. TECHNICAL FIELD

[0002] The present invention relates to the field of biopharmaceuticals and therapeutic agents. More particularly, the present invention relates to compounds, compositions, and methods comprising membrane fusion molecules for providing nanoparticles for delivery and distribution of active agents or pharmaceutical compounds to cells, tissues, organs, and subjects. BACKGROUND

[0003] Therapeutic agents such as pharmaceutical compounds, nucleic acid molecules, and other active agents act by being taken up into cells, tissues, and organs of a subject. Transfection of agents and molecules into cells is often a limiting step in therapeutic behavior.

[0004] When active agent molecules are sensitive to attack or degradation in serum or other biological environments, the molecules must be protected in order to achieve their medicinal effect.

[0005] For example, one way of performing nucleic acid transfection is to incorporate or encapsulate the active molecules into nanoparticles. One drawback of such methods is low cell penetration rates.

[0006] There is a long-felt need for molecules with membrane fusion properties for providing nanoparticles with advantageous transfection properties to improve cell penetration rates and deliver active agents to cells.

[0007] What is needed are compositions and compounds to form nanoparticles for active agents. There is a continuing need for molecules and compositions for effectively transfecting and distributing nucleic acid molecules and other agents into cells and subjects. SUMMARY

[0008] The present invention relates to molecules and compositions thereof for biopharmaceuticals and therapeutic agents. More particularly, the present invention relates to compounds, compositions, and methods for providing nanoparticles for delivery and distribution of active agents or pharmaceutical compounds to cells, tissues, organs, and subjects.

[0009] The present invention provides a series of membrane fusion compounds. The membrane fusion compounds of the present invention can be used to form nanoparticles for delivery and distribution of active agents.

[0010] Examples of active agents in the present disclosure include biologically active molecules, nucleic acids, DNA, RNA, mRNA, siRNA, and microRNA, among others.

[0011] Embodiments of the invention include the following:

[0012] membrane fusion compounds of formula I,

[0013]

[0014] wherein each amphiphile independently comprises one to two lipophilic chains, wherein the lipophilic chains each independently comprise 8 to 22 carbon atoms;

[0015] wherein each AA is independently an amino acid comprising a side chain having an amino group, wherein the amino acid is linked at each of its amino groups to an amphiphile and at its C-terminus to a linker;

[0016] wherein the linker has the structure:

[0017]

[0018] or

[0019]

[0020] wherein Q 1 is branched or unbranched C(2-8)alkanediyl, branched or unbranched C(2-8)alkenediyl, branched or unbranched C(2-8)alkynediyl, or

[0021]

[0022] or

[0023]

[0024] wherein Q 2 is

[0025]

[0026] wherein Q 3 is

[0027]

[0028] wherein X is -O-, -S-, or -NH-;

[0029] n, p, q, and t are each independently present, 1 to 3;

[0030] m is independently 1 to 10;

[0031] r and s are each independently present, 1 to 5.

[0032] the above membrane fusion compounds, wherein AA is selected from the following structures, and any stereoisomer thereof:

[0033]

[0034]

[0035] The above membrane fusion compound, wherein one or both amphiphiles are missing and replaced by an alkyl group, or a pharmaceutically acceptable organic chemical group having 1-400 atoms selected from the group consisting of carbon, oxygen, nitrogen, sulfur, fluorine, and hydrogen.

[0036] The above membrane fusion compound, wherein the pharmaceutically acceptable organic chemical group is an alkyl group, an alkenyl group, an alkynyl group, an acetyl group, Boc, Fmoc, TFA, or CBZ, preferably an alkyl group, an acetyl group, more preferably an acetyl group.

[0037] The above membrane fusion compound, wherein the compound is selected from the group consisting of:

[0038]

[0039]

[0040] The above membrane fusion compound, wherein one or more of the amphiphiles has the structure of Formula II:

[0041]

[0042] wherein R 1 and R 2 are:

[0043] R 1 = CH2(CH2) n O(C=O)R 4 , CH2(CH2) n NH(C=O)R 4 , CH2(CH2) n (C=O)OR 4 , CH2(CH2) n (C=O)NHR 4

[0044] R 2 = CH2(CH2) m O(C=O)R 5 , CH2(CH2) m NH(C=O)R 5 , CH2(CH2) m (C=O)OR 5 , CH2(CH2) m (C=O)NHR 5

[0045] wherein n and m are each independently 1 to 2; R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl;

[0046] wherein R 3 is selected from branched or unbranched C(1-8)alkanediyl, substituted or unsubstituted C(2-8)alkenediyl, substituted or unsubstituted C(2-8)alkynediyl, substituted or unsubstituted C(3-8)cycloalkanediyl, substituted or unsubstituted aralkanediyl, substituted or unsubstituted C(4-8)heteroaralkanediyl, and substituted or unsubstituted heterocycloalkanediyl, and combinations thereof; wherein R 3 is optionally interrupted by one or more -0-, -S-, -SO-, -SO2-, -NH-, -NR 6 -, -NH(C=0)-, -0(C=0)-, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0047] R 3 Preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkenediyl, substituted or unsubstituted C(2-8)alkynediyl, C(3-8)cycloalkanediyl, substituted or unsubstituted C(4-8)aralkanediyl, even more preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0048] As used herein, the term "and combinations thereof" in relation to a chemical formula indicates other structural variations on the basis of the combination of listed groups. For example, the combination of C(1-8)alkanediyl and C(4-8)heteroaralkanediyl refers to C(1-8)alkanediyl-C(4-8)heteroaralkanediyl, as well as C(1-8)alkanediyl-C(4-8)heteroaralkanediyl-C(1-8)alkanediyl.

[0049] The above-mentioned membrane fusion compound, wherein R 3 is selected from:

[0050] branched or unbranched C(2-8)alkanediyl,

[0051] substituted or unsubstituted C(2-8)alkenediyl,

[0052] substituted or unsubstituted C(2-8)alkynediyl,

[0053] substituted or unsubstituted C(3-8)cycloalkanediyl,

[0054] substituted or unsubstituted C(4-8)aralkanediyl,

[0055]

[0056] preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0057] The above membrane fusion compound, wherein one or more of the amphiphilic molecules has a structure according to Formula III:

[0058]

[0059] wherein R 1 and R 2 are:

[0060] R 1 = CH2(CH2) n O(C=O)R 4 , CH2(CH2) n NH(C=O)R 4 ,

[0061] CH2(CH2) n (C=O)OR 4 , CH2(CH2) n (C=O)NHR 4

[0062] R 2 = CH2(CH2) m O(C=O)R 5 , CH2(CH2) m NH(C=O)R 5 ,

[0063] CH2(CH2) m (C=O)OR 5 , CH2(CH2) m (C=O)NHR 5

[0064] wherein n and m are each independently 1 to 2; R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl;

[0065] wherein R 3 is selected from:

[0066] -alkyl-(C=O)-, which is attached to AA;

[0067] -alkyl-O(C=O)-, which is attached to AA;

[0068] -alkyl-NH(C=O)-, which is attached to AA;

[0069] - alkyl-(C=0)-alkyl-(C=0)-, which is attached to AA;

[0070] - alkyl-0(C=0)-alkyl-(C=0)-, which is attached to AA;

[0071] - alkyl-NH(C=0)-alkyl-(C=0)-, which is attached to AA;

[0072] - alkenyl-(C=0)-, which is attached to AA;

[0073] - alkenyl-0(C=0)-, which is attached to AA;

[0074] - alkenyl-NH(C=0)-, which is attached to AA;

[0075] - alkenyl-(C=0)-alkenyl-(C=0)-, which is attached to AA;

[0076] - alkenyl-0(C=0)-alkenyl-(C=0)-, which is attached to AA;

[0077] - alkenyl-NH(C=0)-alkenyl-(C=0)-, which is attached to AA;

[0078] - alkynyl-(C=0)-, which is attached to AA;

[0079] - alkynyl-0(C=0)-, which is attached to AA;

[0080] - alkynyl-NH(C=0)-, which is attached to AA;

[0081] - alkynyl-(C=0)-alkynyl-(C=0)-, which is attached to AA;

[0082] - alkynyl-0(C=0)-alkynyl-(C=0)-, which is attached to AA;

[0083] - alkynyl-NH(C=0)-alkynyl-(C=0)-, which is attached to AA;

[0084] wherein R 3 any alkyl group of R 3 any alkenyl group of R 3 any alkynyl group of R

[0085]

[0086] and positional isomers thereof;

[0087]

[0088]

[0089] in,

[0090] Each R 6 Independently selected from H, alkyl, alkoxy, and alkoxyalkoxy, provided that it is an R 6 -(C=O)- or -alkyl-(C=O)- linked to AA;

[0091] Each R 8 Independently selected from H and alkyl groups, provided that one of them is R. 8 -(C=O)- or -alkyl-(C=O)- linked to AA;

[0092] q is between 0 and 4;

[0093] Q can be O or N.

[0094] The above-mentioned membrane fusion compound, wherein R 6 and R 8 Each alkyl group is independently a branched or unbranched C(1-6) alkyl group, R 6 The alkoxy group is a C(1-6) alkoxy group, R 6 The alkoxy group is C(1-6)alkoxy C(1-6)alkoxy.

[0095] The above-mentioned membrane fusion compound, wherein R 4 and R 5 Each exists independently and is a C(14-18)alkyl or C(14-18)alkenyl, preferably a C(14-18)alkenyl having 2 to 4 double bonds.

[0096] The above-mentioned membrane fusion compounds, wherein the compounds are selected from the following:

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109] The above membrane fusion compound, wherein one or more of the amphiphilic molecules has a structure according to Formula III:

[0110]

[0111] wherein R 1 and R 2 are:

[0112] R 1 = CH2(CH2) n O(C=O)R 4 , CH2(CH2) n NH(C=O)R 4 , CH2(CH2) n (C=O)OR 4 , CH2(CH2) n (C=O)NHR 4

[0113] R 2 = CH2(CH2) m O(C=O)R 5 , CH2(CH2) m NH(C=O)R 5 , CH2(CH2) m (C=O)OR 5 , CH2(CH2) m (C=O)NHR 5

[0114] wherein

[0115] n and m are each independently 1 to 2;

[0116] R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl;

[0117] R 3 is C(1-12)alkyl or C(4-12)alkenyl substituted with -(C=O)- or -alkyl-(C=O)- attached to AA.

[0118] The above membrane fusion compound, wherein R4 and R 5 each independently is present, is C(14-18)alkyl or C(14-18)alkenyl, preferably C(14-18)alkenyl having 2 to 4 double bonds.

[0119] The membrane fusion compound described above, wherein one or more of the amphiphilic molecules has the structure of Formula IV:

[0120]

[0121] wherein R 1 and R 2 are:

[0122] R 1 = (C=O)OR 4 , (C=O)NHR 4 , O(C=O)R 4 , (C=O)R 4

[0123] R 2 = (C=O)OR 5 , (C=O)NHR 5 , O(C=O)R 5 , NH(C=O)R 5

[0124] wherein R 4 and R 5 each independently is present, is C(12-20)alkyl or C(12-20)alkenyl;

[0125] Z is O or NH; p is 0 to 5;

[0126] wherein R 3 is selected from the group consisting of branched or unbranched C(1-8)alkanediyl-(C=O)- attached to AA, substituted or unsubstituted C(2-8)alkenediyl-(C=O)- attached to AA, substituted or unsubstituted C(2-8)alkyndiyl-(C=O)- attached to AA, substituted or unsubstituted C(3-8)cycloalkanediyl-(C=O)- attached to AA, substituted or unsubstituted arlyidene-(C=O)- attached to AA, substituted or unsubstituted C(4-8)heteroarylidene-(C=O)- attached to AA, and substituted or unsubstituted heterocycloalkanediyl-(C=O)- attached to AA; wherein R 3 is optionally interrupted by one or more -O-, -S-, -SO-, -SO2-, -NH-, -NR 6 -, -NH(C=O)-, -O(C=O)-, wherein R 6C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0127] R 3 Preferably, branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkenediyl, substituted or unsubstituted C(2-8)alkynediyl, C(3-8)cycloalkanediyl, substituted or unsubstituted C(4-8)arylenyl, even more preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0128] The above membrane fusion compound, wherein one or more of the amphiphiles has the structure of Formula IV:

[0129]

[0130] wherein R 1 and R 2 are:

[0131] R 1 = (C=O)OR 4 , (C=O)NHR 4 , O(C=O)R 4 , (C=O)R 4

[0132] R 2 = (C=O)OR 5 , (C=O)NHR 5 , O(C=O)R 5 , NH(C=O)R 5

[0133] wherein R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl;

[0134] Z is O or NH; p is 0 to 5;

[0135] wherein R 3 is selected from:

[0136] C(1-12)alkyl or C(2-12)alkenyl substituted with -(C=O)- attached to AA;

[0137]

[0138] and positional isomers thereof;

[0139]

[0140] wherein,

[0141] each R 6 is independently selected from H, alkyl, alkoxy, and alkoxyalkoxy, provided that one R 6 is -(C=0)- or -alkyl-(C=0)- attached to AA;

[0142] each R 8 is independently selected from H, alkyl, provided that one R 8 is -(C=0)- or -alkyl-(C=0)- attached to AA;

[0143] q is 0 to 4;

[0144] Q is O or N.

[0145] The above membrane fusion compound, wherein R 4 and R 5 each independently present, is C(14-18)alkyl or C(14-18)alkenyl, preferably C(14-18)alkenyl having 2 to 4 double bonds.

[0146] The above membrane fusion compound, wherein the compound is compound T10:

[0147]

[0148] The above membrane fusion compound, wherein one or more amphiphilic molecules has the structure of Formula IV:

[0149]

[0150] wherein R 1 and R 2 are:

[0151] R 1 is C(12-20)alkyl, or C(12-20)alkenyl;

[0152] R 2 is (CH2) n XR 4 wherein n is 0 to 3, and X is O, S, SO, S02, NH;

[0153] wherein R 4 is C(12-20)alkyl, or C(12-20)alkenyl;

[0154] wherein Z is O or NH;

[0155] wherein p is 1;

[0156] wherein R 3selected from C(1-12)alkyl or C(2-12)alkenyl substituted with -(C=O)- attached to AA.

[0157] The above membrane fusion compound, wherein one or more of the amphiphiles has the structure of Formula V:

[0158]

[0159] wherein R 1 and R 2 are

[0160] R 1 = (C=O)OR 4 , (C=O)NHR 4 , O(C=O)R 4 , NH(C=O)R 4

[0161] R 2 = (C=O)OR 5 , (C=O)NHR 5 , O(C=O)R 5 , NH(C=O)R 5

[0162] wherein R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl;

[0163] wherein R 3 is selected from branched or unbranched -O(C=O)-C(1-8)alkanediyl- (C=O)- attached to AA, substituted or unsubstituted -O(C=O)-C(2-8)alkenediyl- (C=O)- attached to AA, substituted or unsubstituted -O(C=O)-C(2-8)alkynediyl- (C=O)- attached to AA, substituted or unsubstituted -O(C=O)-C(3-8)cycloalkanediyl- (C=O)- attached to AA, substituted or unsubstituted -O(C=O)-arylene-(C=O)- attached to AA, substituted or unsubstituted -O(C=O)-C(4-8)heteroarylene-(C=O)- attached to AA, and substituted or unsubstituted -O(C=O)-heterocycloalkanediyl- (C=O)- attached to AA; wherein R 3 is optionally interrupted by one or more -O-, -S-, -SO-, -SO2-, -NH-, -NR 6 -, -NH(C=O)-, -O(C=O)-, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0164] R 3 Preferred are branched or unbranched C(2-8) alkyl diesters, substituted or unsubstituted C(2-8) alkenyl diesters, substituted or unsubstituted C(2-8) alkynyl diesters, C(3-8) cycloalkyl diesters, substituted or unsubstituted C(4-8) arylene dies, and even more preferred are branched or unbranched C(2-8) alkyl diesters, substituted or unsubstituted C(3-8) cycloalkyl diesters.

[0165] The above-mentioned membrane fusion compound, wherein R 4 and R 5 Each exists independently and is a C(14-18)alkyl or C(14-18)alkenyl, preferably a C(14-18)alkenyl having 2 to 4 double bonds.

[0166] The above-mentioned membrane fusion compound, wherein the compound is compound T12:

[0167]

[0168] The above-mentioned membrane fusion compounds, wherein one or more amphiphilic molecules have the structure shown in chemical formula V:

[0169]

[0170] Where R 1 and R 2 for:

[0171] R 1 =(C=O)OR 4 (C=O)NHR 4 O(C=O)R 4 NH(C=O)R 4

[0172] R 2 =(C=O)OR 5 (C=O)NHR 5 O(C=O)R 5 NH(C=O)R 5

[0173] Where R 4 and R 5 Each exists independently and is a C(12-20) alkyl or C(12-20) alkenyl; wherein R 3 Selected from:

[0174] -(C=O)- or -alkyl-(C=O)-, which is connected to AA;

[0175] -O(C=O)- or -alkyl-O(C=O)-, which is connected to AA;

[0176] -0(C=0)-alkanediyl-(C=0)-, which is attached to AA;

[0177] -0(C=0)-alkenediyl-(C=0)-, which is attached to AA;

[0178] -0(C=0)-alkynediyl-(C=0)-, which is attached to AA;

[0179] -NH(C=0)- or -alkyl-NH(C=0)-, which is attached to AA;

[0180] -alkyl-(C=0)-alkyl-(C=0)-, which is attached to AA;

[0181] -alkyl-0(C=0)-alkyl-(C=0)-, which is attached to AA;

[0182] -alkyl-NH(C=0)-alkyl-(C=0)-, which is attached to AA;

[0183] -alkenyl-(C=0)-, which is attached to AA;

[0184] -alkenyl-0(C=0)-, which is attached to AA;

[0185] -alkenyl-NH(C=0)-, which is attached to AA;

[0186] -alkenyl-(C=0)-alkenediyl-(C=0)-, which is attached to AA;

[0187] -alkenyl-0(C=0)-alkenediyl-(C=0)-, which is attached to AA;

[0188] -alkenyl-NH(C=0)-alkenediyl-(C=0)-, which is attached to AA;

[0189] -alkynyl-(C=0)-, which is attached to AA;

[0190] -alkynyl-0(C=0)-, which is attached to AA;

[0191] -alkynyl-NH(C=0)-, which is attached to AA;

[0192] -alkynyl-(C=0)-alkynediyl-(C=0)-, which is attached to AA;

[0193] -alkynyl-0(C=0)-alkynediyl-(C=0)-, which is attached to AA;

[0194] -alkynyl-NH(C=0)-alkynediyl-(C=0)-, which is attached to AA;

[0195]

[0196] and positional isomers thereof;

[0197] wherein R 3 Any alkyl group of R 3 Any alkenyl group of R 3 Any alkynyl group of R

[0198] The above membrane fusion compound, wherein one or more amphiphiles have the structure of Formula VI:

[0199]

[0200] wherein R 1 and R 2 are:

[0201] R 1 = (C=O)OR 4 , (C=O)NHR 4 , O(C=O)R 4 , NH(C=O)R 4

[0202] R 2 = (C=O)OR 5 , (C=O)NHR 5 , O(C=O)R 5 , NH(C=O)R 5

[0203] wherein,

[0204] R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl;

[0205] wherein R 3 is selected from the group consisting of branched or unbranched C(1-8)alkanediyl-(C=O)- attached to AA, substituted or unsubstituted C(2-8)alkenediyl-(C=O)- attached to AA, substituted or unsubstituted C(2-8)alkynediyl-(C=O)- attached to AA, substituted or unsubstituted C(3-8)cycloalkanediyl-(C=O)- attached to AA, substituted or unsubstituted aralkanediyl-(C=O)- attached to AA, substituted or unsubstituted C(4-8)heteroaralkanediyl-(C=O)- attached to AA, and substituted or unsubstituted heterocycloalkanediyl-(C=O)- attached to AA; wherein R 3 is optionally substituted with one or more -O-, -S-, -SO-, -SO2-, -NH-, -NR6 -, -NH(C=O)-, -O(C=O)-interruptions, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0206] R 3 Preferred are branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkenediyl, substituted or unsubstituted C(2-8)alkynediyl, C(3-8)cycloalkanediyl, substituted or unsubstituted C(4-8)arylen, even more preferred are branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0207] The above membrane fusion compound, wherein one or more of the amphiphilic molecules has the structure of Formula VI:

[0208]

[0209] wherein R 1 and R 2 are:

[0210] R 1 = (C=O)OR 4 , (C=O)NHR 4 , O(C=O)R 4 , NH(C=O)R 4

[0211] R 2 = (C=O)OR 5 , (C=O)NHR 5 , O(C=O)R 5 , NH(C=O)R 5

[0212] wherein,

[0213] R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl;

[0214] R 3 is selected from:

[0215] -alkyl-(C=O)-, which is attached to AA;

[0216] -alkyl-O(C=O)-, which is attached to AA;

[0217] -alkyl-NH(C=O)-, which is attached to AA;

[0218] - alkyl-(C=0)-alkyl-(C=0)-, which is attached to AA;

[0219] - alkyl-O(C=0)-alkyl-(C=0)-, which is attached to AA;

[0220] - alkyl-NH(C=0)-alkyl-(C=0)-, which is attached to AA;

[0221] - alkenyl-(C=0)-, which is attached to AA;

[0222] - alkenyl-O(C=0)-, which is attached to AA;

[0223] - alkenyl-NH(C=0)-, which is attached to AA;

[0224] - alkenyl-(C=0)-alkenyl-(C=0)-, which is attached to AA;

[0225] - alkenyl-O(C=0)-alkenyl-(C=0)-, which is attached to AA;

[0226] - alkenyl-NH(C=0)-alkenyl-(C=0)-, which is attached to AA;

[0227] - alkynyl-(C=0)-, which is attached to AA;

[0228] - alkynyl-O(C=0)-, which is attached to AA;

[0229] - alkynyl-NH(C=0)-, which is attached to AA;

[0230] - alkynyl-(C=0)-alkynyl-(C=0)-, which is attached to AA;

[0231] - alkynyl-O(C=0)-alkynyl-(C=0)-, which is attached to AA;

[0232] - alkynyl-NH(C=0)-alkynyl-(C=0)-, which is attached to AA;

[0233]

[0234] and positional isomers thereof;

[0235] wherein R 3 any alkyl group of R 3 any alkenyl group of R 3 any alkynyl group of R

[0236] the above-mentioned membrane fusion compound, wherein R 4 and R5 Each exists independently and is a C(14-18)alkyl or C(14-18)alkenyl, preferably a C(14-18)alkenyl having 2 to 4 double bonds.

[0237] The above-mentioned membrane fusion compound, wherein the compound is compound T11:

[0238]

[0239] Membrane fusion compounds having chemical formula VII,

[0240]

[0241] Each amphiphilic molecule independently contains one or two lipophilic chains, each of which independently contains 8 to 22 carbon atoms;

[0242] Each AA a Independently, it refers to an amino acid containing a side chain with an acyl group, wherein the amino acid is attached to an amphiphilic molecule with each acyl group and to a linker with its N-terminus;

[0243] The connector has the following structure:

[0244]

[0245] or

[0246]

[0247] Q 1 It is a branched or unbranched C(2-8) chain alkyl diel, a branched or unbranched C(2-8) alkenyl diel, a branched or unbranched C(2-8) ynyl diel, or

[0248]

[0249] or

[0250]

[0251] Q 2 yes

[0252]

[0253] Q 3 yes

[0254]

[0255] Where X is -O-, -S-, or -NH-;

[0256] n and p exist independently and are 1 to 3;

[0257] m is independently 1 to 10;

[0258] r and s are each independently present, 1 to 5.

[0259] The above membrane fusion compound, wherein AA a is selected from the following structures, and any stereoisomer thereof:

[0260]

[0261] The above membrane fusion compound, wherein one or both amphiphiles are missing and replaced with an alkyl group, or a pharmaceutically acceptable organic chemical group having 1-400 atoms selected from the group consisting of carbon, oxygen, nitrogen, sulfur, fluorine, and hydrogen.

[0262] The above membrane fusion compound, wherein the pharmaceutically acceptable organic chemical group is selected from the group consisting of alkyl, alkenyl, alkynyl, alkyl ether, aryl ether, alkoxy, and alkoxyalkoxy.

[0263] The above membrane fusion compound, wherein the pharmaceutically acceptable organic chemical group is selected from the group consisting of methoxy, ethoxy, t-butyl ether, and benzyloxy.

[0264] The above membrane fusion compound, wherein one or more of the amphiphiles has the structure of Formula VIII:

[0265]

[0266] wherein R 1 and R 2 are:

[0267] R 1 = CH2(CH2) n O(C=O)R 4 , CH2(CH2) n NH(C=O)R 4 , CH2(CH2) n (C=O)OR 4 , CH2(CH2) n (C=O)NHR 4

[0268] R 2 = CH2(CH2) m O(C=O)R 5 , CH2(CH2) m NH(C=O)R 5 , CH2(CH2) m (C=O)OR 5 , CH2(CH2) m(C=O)NHR 5

[0269] wherein,

[0270] n and m are each independently 1 to 2;

[0271] R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl.

[0272] The above membrane fusion compound, wherein one or more amphiphiles has the structure of Formula IX:

[0273]

[0274] wherein R 1 and R 2 are:

[0275] R 1 =CH2(CH2) n O(C=O)R 4 , CH2(CH2) n NH(C=O)R 4 , CH2(CH2) n (C=O)OR 4 , CH2(CH2) n (C=O)NHR 4

[0276] R 2 =CH2(CH2) m O(C=O)R 5 , CH2(CH2) m NH(C=O)R 5 , CH2(CH2) m (C=O)OR 5 , CH2(CH2) m (C=O)NHR 5

[0277] wherein,

[0278] n and m are each independently 1 to 2;

[0279] R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl.

[0280] wherein R 3selected from branched or unbranched C(1-8)alkanediyl, substituted or unsubstituted C(2-8)alkenediyl, substituted or unsubstituted C(2-8)alkynediyl, substituted or unsubstituted C(3-8)cycloalkanediyl, substituted or unsubstituted aralkanediyl, substituted or unsubstituted C(4-8)heteroaralkanediyl, and substituted or unsubstituted heterocycloalkanediyl; wherein R 3 is optionally interrupted by one or more -0-, -S-, -SO-, -SO2-, -NH-, -NR 6 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0281] R 3 Preferably, R is branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkenediyl, substituted or unsubstituted C(2-8)alkynediyl, C(3-8)cycloalkanediyl, substituted or unsubstituted C(4-8)aralkanediyl, and even more preferably, R is branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0282] The above membrane fusion compound, wherein R 4 and R 5 are each independently present, C(14-18)alkyl, or C(14-18)alkenyl, preferably C(14-18)alkenyl having 2 to 4 double bonds.

[0283] The above membrane fusion compound, wherein the compound is compound T9:

[0284]

[0285] The above membrane fusion compound, wherein one or more of the amphiphilic molecules has the structure shown in formula X:

[0286]

[0287] wherein,

[0288] R 1 and R 2 are:

[0289] R 1 = CH2(CH2) n O(C=O)R 4 , CH2(CH2) n NH(C=O)R 4 ,

[0290] CH2(CH2) n (C=O)OR​4 CH2(CH2) n (C=O)NHR 4

[0291] R 2 =CH2(CH2) m O(C=O)R 5 CH2(CH2) m NH(C=O)R 5 ,

[0292] CH2(CH2) m (C=O)OR 5 CH2(CH2) m (C=O)NHR 5

[0293] R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl;

[0294] wherein R 3 is absent or selected from branched or unbranched *-NH-C(1-8)alkanediyl- (C=O)-, substituted or unsubstituted *-NH-C(2-8)alkenediyl-(C=O)-, substituted or unsubstituted *-NH-C(2-8)alkynediyl-(C=O)-, substituted or unsubstituted *-NH-C(3-8)cycloalkanediyl-(C=O)-, substituted or unsubstituted *-NH-arylene-(C=O)-, substituted or unsubstituted *-NH-C(4-8)heteroarylene-(C=O)-, and substituted or unsubstituted *-NH-heterocycloalkanediyl-(C=O)-, wherein * indicates the terminal end of attachment to AA a ; wherein R 3 is optionally interrupted by one or more -O-, -S-, -SO-, -SO2-, -NH-, -NR 6 -, -NH(C=O)-, -O(C=O)-, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0295] R 3 Preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkenediyl, substituted or unsubstituted C(2-8)alkynediyl, C(3-8)cycloalkanediyl, substituted or unsubstituted C(4-8)arylene, even more preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0296] The above membrane fusion compound, wherein one or more of the amphiphilic molecules has a structure according to Formula XI:

[0297]

[0298] wherein,

[0299] R 1 and R 2 are:

[0300] R 1 = (C=O)OR 4 , (C=O)NHR 4 , O(C=O)R 4 , NH(C=O)R 4

[0301] R 2 = (C=O)OR 5 , (C=O)NHR 5 , O(C=O)R 5 , NH(C=O)R 5

[0302] R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl;

[0303] wherein R 3 is absent or selected from the group consisting of branched or unbranched *-NH-C(1-8)alkanediyl-(C=O)-, substituted or unsubstituted *-NH-C(2-8)alkenediyl-(C=O)-, substituted or unsubstituted *-NH-C(2-8)alkynediyl-(C=O)-, substituted or unsubstituted *-NH-C(3-8)cycloalkanediyl-(C=O)-, substituted or unsubstituted *-NH-arylen-(C=O)-, substituted or unsubstituted *-NH-C(4-8)heteroarylen-(C=O)-, and substituted or unsubstituted *-NH-heterocycloalkanediyl-(C=O)-, wherein * indicates the terminal end of attachment to AA a ; wherein R 3 is optionally interrupted by one or more -O-, -S-, -SO-, -SO2-, -NH-, -NR 6 , -NH(C=O)-, -O(C=O)-, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0304] R 3Preferred are branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkenediyl, substituted or unsubstituted C(2-8)alkynediyl, C(3-8)cycloalkanediyl, substituted or unsubstituted C(4-8)arylen, even more preferred are branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0305] The above membrane fusion compound, wherein one or more of the amphiphilic molecules has a structure according to Formula III:

[0306]

[0307] wherein R 1 and R 2 are:

[0308] R 1 = CH2(CH2) n O(C=O)R 4 , CH2(CH2) n NH(C=O)R 4 ,

[0309] CH2(CH2) n (C=O)OR 4 , CH2(CH2) n (C=O)NHR 4

[0310] R 2 = CH2(CH2) m O(C=O)R 5 , CH2(CH2) m NH(C=O)R 5 ,

[0311] CH2(CH2) m (C=O)OR 5 , CH2(CH2) m (C=O)NHR 5

[0312] wherein n and m are each independently 1 to 2; R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl;

[0313] wherein

[0314] R 3 is selected from:

[0315] -(C=O)-alkyl-NH-, which is attached to AA a ;

[0316] -(C=O)-alkenyl-NH-, which is related to AA a connect;

[0317] -(C=O)-alkynyl-NH-, which reacts with AA a connect;

[0318] Where R 3 All alkyl groups are branched or unbranched C(1-6) alkyl groups, R 3 All alkenyl groups are either branched or unbranched C(2-6) alkenyl groups, R 3 All ynyl groups are either branched or unbranched C(2-6) ynyl groups;

[0319]

[0320] and its positional isomers;

[0321]

[0322]

[0323] in,

[0324] Each R 6 Independently selected from H, alkyl, alkoxy, and alkoxyalkoxy, provided that it is an R 6 It is -(C=O)-alkyl-NH-, where NH and AA a connect;

[0325] Each R 8 Independently selected from H and alkyl groups, provided that one of them is R. 8 It is -(C=O)-alkyl-NH-, where NH and AA a connect;

[0326] q is between 0 and 4;

[0327] Q can be O or N.

[0328] The above-mentioned membrane fusion compounds, wherein one or more amphiphilic molecules have the structure shown in chemical formula V:

[0329]

[0330] Where R 1 and R 2 for:

[0331] R 1 =(C=O)OR 4 (C=O)NHR 4 O(C=O)R 4 NH(C=O)R 4

[0332] R 2 = (C=0)OR 5 , (C=0)NHR 5 , 0 (C=0)R 5 , NH (C=0)R 5

[0333] wherein R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl;

[0334] wherein,

[0335] wherein R 3 is absent or selected from branched or unbranched *-NH-C(1-8)alkanediyl- (C=0)-, substituted or unsubstituted *-NH-C(2-8)alkenediyl-(C=0)-, substituted or unsubstituted *-NH-C(2-8)alkynediyl-(C=0)-, substituted or unsubstituted *-NH-C(3-8)cycloalkanediyl-(C=0)-, substituted or unsubstituted *-NH-arylene-(C=0)-, substituted or unsubstituted *-NH-C(4-8)heteroarylene-(C=0)-, and substituted or unsubstituted *-NH-heterocycloalkanediyl-(C=0)-, wherein * indicates the terminal end of attachment to AA a ; wherein R 3 is optionally interrupted by one or more -0-, -S-, -SO-, -S02-, -NH-, -NR 6 -, -NH(C=0)-, -0(C=0)-, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0336] R 3 is preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkenediyl, substituted or unsubstituted C(2-8)alkynediyl, C(3-8)cycloalkanediyl, substituted or unsubstituted C(4-8)arylene, even more preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0337] Embodiments of the present application also relate to compositions comprising the above-mentioned membrane fusion compounds and a pharmaceutically acceptable carrier. The compositions can comprise nanoparticles or liposomes.

[0338] The pharmaceutical compositions of the present application can comprise a membrane fusion compound, an active agent, and a pharmaceutically acceptable carrier. In the composition, the membrane fusion compound can be from 0.01 mol% to 20 mol% of the lipids of the composition. The composition can comprise nanoparticles or liposomes.

[0339] The membrane fusion molecules and formulations of the present application can be used for delivery of active agents.

[0340] In some embodiments, the active agent is one or more nucleic acids.

[0341] In certain embodiments, the active agent is one or more DNA, RNA, mRNA, siRNA, or microRNA. The active agent can be one or more RNA molecules.

[0342] The active agent can be one or more RNAi molecules, one or more mRNA molecules, and modified versions thereof.

[0343] Embodiments of the present application include compositions for use in the distribution of active agents in a subject to treat a condition or disease. The compositions can contain an active agent, a membrane fusion compound, an ionizable lipid, a structural lipid, a stabilizer lipid, and a lipid for reducing the immunogenicity of the composition.

[0344] The present application includes methods for preventing, alleviating, or treating a disease or condition in a subject in need thereof, comprising administering to the subject a composition described above. The compositions of the present application can be used to treat the human or animal body.

[0345] Embodiments of the present application also include the following:

[0346] a compound of Formula (A),

[0347]

[0348] wherein the linker is a divalent radical comprising a PEG moiety,

[0349] X1and X2are independently C1-C5alkanediyl,

[0350] R1, R2, R3, and R4are independently

[0351]

[0352] X3is a single bond, C1-C5alkanediyl, or C2-C5alkenediyl, X4and X5are independently C2-5alkanediyl,

[0353] Z1, Z2, and Z3are independently -O-, -S-, or -NH-, and R5and R6are independently C11-23alkyl or C11-23alkenyl. Embodiments of the present application also include the following:

[0354] the compound of formula (B),

[0355]

[0356] wherein the linker is a divalent group comprising a PEG moiety,

[0357] X6and X7are independently C1-C5alkanediyl,

[0358] X8and X9are independently C1-C5alkanediyl,

[0359] Z4and Z5are independently -O-, -S-, or -NH-,

[0360] R7, R8, R9, and R 10 are independently

[0361]

[0362] X4and X5are independently C2-5alkanediyl,

[0363] Z2and Z3are independently -O-, -S-, or -NH-, and

[0364] R5and R6are independently C11-23alkyl or C11-23alkenyl.

[0365] the above compound, wherein the linker is

[0366]

[0367] wherein m is an integer from 1 to 12,

[0368] Y1is -O-, -NH-, or -NHCH2-,

[0369] Y2is -O-, -NH-, or -CH2NH-,

[0370] n and q are independently integers from 1 to 5,

[0371] p is an integer from 0 to 5,

[0372] Y3and Y5are independently -O-, -NH-, or -NHCH2-, and

[0373] Y4and Y6are independently -O-, -NH-, or -CH2NH-.

[0374] the above compound, wherein X1and X2are independently C1-C5straight chain alkanediyl, preferably C2-C4straight chain alkanediyl, more preferably C4straight chain alkanediyl.

[0375] The above compound, wherein R1, R2, R3, and R4 are the same group.

[0376] The above compound, wherein X3 is a single bond or a C1-C5 straight-chain alkanediyl group, preferably X3 is a C2-C4 straight-chain alkanediyl group, more preferably ethylene, i.e., an ethanediyl group.

[0377] The above compound, wherein X4 and X5 are independently a C2-5 straight-chain alkanediyl group, preferably X4 and X5 are a C2-4 straight-chain alkanediyl group, more preferably ethylene, i.e., an ethanediyl group.

[0378] The above compound, wherein Z1 is -NH-.

[0379] The above compound, wherein Z2 and Z3 are -O-.

[0380] The above compound, wherein R5 and R6 are independently a C11-23 straight-chain alkenyl group containing 1-6 double bonds, preferably 1-3, more preferably 2-3, still more preferably 2.

[0381] The above compound, wherein R5 and R6 are independently a C11-23 straight-chain alkenyl group containing 1-6 double bonds, preferably 1-3, more preferably 2-3, still more preferably 2.

[0382] The above compound, wherein R5 and R6 are independently a C11-23 straight-chain alkenyl group containing 1-6 double bonds, preferably 1-3, more preferably 2-3, still more preferably 2.

[0383] The above compound, wherein R5 and R6 are independently a C11-23 straight-chain alkenyl group containing 1-6 double bonds, preferably 1-3, more preferably 2-3, still more preferably 2.

[0384] The above compound, wherein R5 and R6 are independently a C11-23 straight-chain alkenyl group containing 1-6 double bonds, preferably 1-3, more preferably 2-3, still more preferably 2.

[0385] The above compound, wherein R5 and R6 are independently a C11-23 straight-chain alkenyl group containing 1-6 double bonds, preferably 1-3, more preferably 2-3, still more preferably 2.

[0386] The above compound, wherein R5 and R6 are independently a C11-23 straight-chain alkenyl group containing 1-6 double bonds, preferably 1-3, more preferably 2-3, still more preferably 2.

[0387] The above composition, further comprising a nucleic acid.

[0388] The above composition, wherein the nucleic acid is an siRNA, an mRNA, or a microRNA.

[0389] The above composition, wherein the composition is a pharmaceutical composition.

[0390] The present application relates to the following:

[0391] Item 1, a membrane fusion compound of formula I,

[0392]

[0393] wherein each amphiphilic molecule independently comprises one to two lipophilic chains, wherein the lipophilic chains each independently comprise 8 to 22 carbon atoms;

[0394] wherein each AA is independently an amino acid comprising a side chain having an amino group, wherein the amino acid is linked at each of its amino groups to an amphiphilic molecule and at its C-terminus to a linker;

[0395] wherein the linker has the structure:

[0396]

[0397] or

[0398]

[0399] wherein Q 1 is branched or unbranched C(2-8)alkanediyl, branched or unbranched C(2-8)alkenediyl, branched or unbranched C(2-8)alkynediyl, or

[0400]

[0401] or

[0402]

[0403] wherein Q 2 is

[0404]

[0405] wherein Q 3 is

[0406]

[0407] wherein X is -0-, -S-, or -NH-;

[0408] n, p, q, and t are each independently present, 1 to 3;

[0409] m is independently 1 to 10;

[0410] r and s are each independently present, 1 to 5.

[0411] Item 2. The membrane fusion compound of item 1, wherein AA is selected from the following structures, and any stereoisomer thereof:

[0412]

[0413]

[0414] Item 3. The membrane fusion compound of item 1, wherein one or both amphiphiles are missing and replaced with an alkyl group, or a pharmaceutically acceptable organic chemical group having 1-400 atoms selected from carbon, oxygen, nitrogen, sulfur, fluorine, and hydrogen.

[0415] Item 4. The membrane fusion compound of item 3, wherein the pharmaceutically acceptable organic chemical group is an alkyl group, an alkenyl group, an alkynyl group, an acetyl group, Boc, Fmoc, TFA, or CBZ.

[0416] Item 5. The membrane fusion compound of item 3, wherein the compound is selected from the following: Compound T13, Compound T14.

[0417] Item 6. The membrane fusion compound of item 1, wherein one or more of the amphiphiles has the structure of Formula II:

[0418]

[0419] wherein R 1 and R 2 are:

[0420] R 1 = CH2(CH2) n O(C=O)R 4 , CH2(CH2) n NH(C=O)R 4 ,

[0421] CH2(CH2) n (C=O)OR 4 , CH2(CH2) n (C=O)NHR 4

[0422] R 2 = CH2(CH2) m O(C=O)R 5 , CH2(CH2) m NH(C=O)R 5 ,

[0423] CH2(CH2) m (C=O)OR 5 , CH2(CH2) m (C=O)NHR 5

[0424] wherein n and m are each independently 1 to 2; R 4 and R 5each independently present, is C(12-20)alkyl or C(12-20)alkenyl;

[0425] wherein R 3 is selected from branched or unbranched C(1-8)alkanediyl, substituted or unsubstituted C(2-8)alkenediyl, substituted or unsubstituted C(2-8)alkynediyl, substituted or unsubstituted C(3-8)cycloalkanediyl, substituted or unsubstituted aralkanediyl, substituted or unsubstituted C(4-8)heteroaralkanediyl, and substituted or unsubstituted heterocycloalkanediyl; wherein R 3 is optionally interrupted by one or more -0-, -S-, -SO-, -S02-, -NH-, -NR 6 -, -NH(C=0)-, -0(C=0)-, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0426] Item 7. The membrane fusion compound of item 6, wherein R 3 is selected from:

[0427] branched or unbranched C(2-8)alkanediyl,

[0428] substituted or unsubstituted C(2-8)alkenediyl,

[0429] substituted or unsubstituted C(2-8)alkynediyl,

[0430] substituted or unsubstituted C(3-8)cycloalkanediyl,

[0431] substituted or unsubstituted C(4-8)heteroaralkanediyl,

[0432]

[0433] Item 8. The membrane fusion compound of item 1, wherein one or more amphiphilic molecules has a structure according to Formula III:

[0434]

[0435] wherein R 1 and R 2 are:

[0436] R 1 = CH2(CH2) n O(C=0)R 4 , CH2(CH2) n NH(C=0)R 4 ,

[0437] CH2(CH2) n (C=0)OR4 , CH2(CH2) n (C=O)NHR 4

[0438] R 2 =CH2(CH2) m O(C=O)R 5 , CH2(CH2) m NH(C=O)R 5 ,

[0439] CH2(CH2) m (C=O)OR 5 , CH2(CH2) m (C=O)NHR 5

[0440] wherein n and m are each independently 1 to 2; R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl;

[0441] wherein R 3 is selected from:

[0442] -alkyl-(C=O)-, which is attached to AA;

[0443] -alkyl-O(C=O)-, which is attached to AA;

[0444] -alkyl-NH(C=O)-, which is attached to AA;

[0445] -alkyl-(C=O)-alkyl-(C=O)-, which is attached to AA;

[0446] -alkyl-O(C=O)-alkyl-(C=O)-, which is attached to AA;

[0447] -alkyl-NH(C=O)-alkyl-(C=O)-, which is attached to AA;

[0448] -alkenyl-(C=O)-, which is attached to AA;

[0449] -alkenyl-O(C=O)-, which is attached to AA;

[0450] -alkenyl-NH(C=O)-, which is attached to AA;

[0451] -alkenyl-(C=O)-alkenyl-(C=O)-, which is attached to AA;

[0452] -alkenyl-O(C=O)-alkenyl-(C=O)-, which is attached to AA;

[0453] -Alkenyl-NH(C=O)-Alkenyl-(C=O)-, which is linked to AA;

[0454] -Alynyl group-(C=O)-, which is linked to AA;

[0455] -Alynyl group-O(C=O)-, which is linked to AA;

[0456] -Alynyl group -NH(C=O)-, which is linked to AA;

[0457] -Alynyl-(C=O)-Alynyl-(C=O)-, which is linked to AA;

[0458] -Alynyl-O(C=O)-Alynyl-(C=O)-, which is linked to AA;

[0459] -Alynyl-NH(C=O)-Alynyl-(C=O)-, which is linked to AA;

[0460] Where R 3 All alkyl groups are branched or unbranched C(1-6) alkyl groups, R 3 All alkenyl groups are either branched or unbranched C(2-6) alkenyl groups, R 3 All ynyl groups are either branched or unbranched C(2-6) ynyl groups;

[0461]

[0462] and its positional isomers;

[0463]

[0464] in,

[0465] Each R 6 Independently selected from H, alkyl, alkoxy, and alkoxyalkoxy, provided that it is an R 6 -(C=O)- or -alkyl-(C=O)- linked to AA;

[0466] Each R 8 Independently selected from H and alkyl groups, provided that one of them is R. 8 -(C=O)- or -alkyl-(C=O)- linked to AA;

[0467] q is between 0 and 4;

[0468] Q can be O or N.

[0469] Item 9, the membrane fusion compound as described in Item 8, wherein R 6 and R 8 Each alkyl group is independently a branched or unbranched C(1-6) alkyl group, R 6The alkoxy group is a C(1-6) alkoxy group, R 6 The alkoxy group is C(1-6)alkoxy C(1-6)alkoxy.

[0470] Item 10, the membrane fusion compound as described in Item 8, wherein R 4 and R 5 They exist independently and are C(14-18)alkyl or C(14-18)alkenyl.

[0471] Item 11. The membrane fusion compound as described in Item 8, wherein the compound is selected from the following: compound R4, compound S6, compound S7, compound S8, compound T1, compound T2, compound T4, compound T5, compound T6, compound T7, compound T8, and compound T3.

[0472] Item 12. A membrane fusion compound as described in Item 1, wherein one or more amphiphilic molecules have the structure shown in Formula III:

[0473]

[0474] Where R 1 and R 2 for:

[0475] R 1 =CH2(CH2) n O(C=O)R 4 CH2(CH2) n NH(C=O)R 4 ,

[0476] CH2(CH2) n (C=O)OR 4 CH2(CH2) n (C=O)NHR 4

[0477] R 2 =CH2(CH2) m O(C=O)R 5 CH2(CH2) m NH(C=O)R 5 ,

[0478] CH2(CH2) m (C=O)OR 5 CH2(CH2) m (C=O)NHR 5

[0479] in,

[0480] n and m are each independently 1 to 2;

[0481] R 4 and R 5 each independently is present, is C(12-20)alkyl or C(12-20)alkenyl;

[0482] R 3 is C(1-12)alkyl or C(4-12)alkenyl substituted with -(C=O)- or -alkyl-(C=O)- attached to AA.

[0483] Item 13. The membrane fusion compound of item 12, wherein R 4 and R 5 each independently is present, is C(14-18)alkyl or C(14-18)alkenyl.

[0484] Item 14. The membrane fusion compound of item 1, wherein one or more amphiphiles has the structure of Formula IV:

[0485]

[0486] wherein R 1 and R 2 are:

[0487] R 1 = (C=O)OR 4 , (C=O)NHR 4 , O(C=O)R 4 , (C=O)R 4

[0488] R 2 = (C=O)OR 5 , (C=O)NHR 5 , O(C=O)R 5 , NH(C=O)R 5

[0489] wherein R 4 and R 5 each independently is present, is C(12-20)alkyl or C(12-20)alkenyl;

[0490] Z is O or NH;

[0491] p is 1 to 4;

[0492] wherein R 3selected from the group consisting of branched or unbranched C(1-8)alkanediyl-(C=0)- linked to AA, substituted or unsubstituted C(2-8)alkenediyl-(C=0)-linked to AA, substituted or unsubstituted C(2-8)alkynediyl-(C=0)-linked to AA, substituted or unsubstituted C(3-8)cycloalkanediyl-(C=0)-linked to AA, substituted or unsubstituted aralkanediyl-(C=0)-linked to AA, substituted or unsubstituted C(4-8)heteroaralkanediyl-(C=0)-linked to AA, and substituted or unsubstituted heterocycloalkanediyl-(C=0)-linked to AA; wherein R 3 is optionally interrupted by one or more -0-, -S-, -SO-, -S02-, -NH-, -NR 6 -, -NH(C=0)-, -0(C=0)-, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0493] Item 15. The membrane fusion compound of item 1, wherein one or more amphiphiles has the structure of Formula IV:

[0494]

[0495] wherein R 1 and R 2 are:

[0496] R 1 = (C=0)OR 4 , (C=0)NHR 4 , 0(C=0)R 4 , (C=0)R 4

[0497] R 2 = (C=0)OR 5 , (C=0)NHR 5 , 0(C=0)R 5 , NH(C=0)R 5

[0498] wherein R 4 and R 5 are each independently present, C(12-20)alkyl, or C(12-20)alkenyl; Z is O or NH;

[0499] p is 1 to 4;

[0500] wherein R 3 is selected from the group consisting of:

[0501] C(1-12)alkyl or C(2-12)alkenyl substituted with -(C=0)- attached to AA;

[0502]

[0503] and positional isomers thereof;

[0504]

[0505] wherein,

[0506] each R 6 is independently selected from the group consisting of H, alkyl, alkoxy, and alkoxyalkoxy, provided that one R 6 is -(C=0)- or -alkyl-(C=0)- attached to AA;

[0507] each R 8 is independently selected from the group consisting of H, alkyl, provided that one R 8 is -(C=0)- or -alkyl-(C=0)- attached to AA;

[0508] q is 0 to 4;

[0509] Q is O or N.

[0510] Clause 16, the membrane fusion compound of Clause 15, wherein R 4 and R 5 are each independently present, C(14-18)alkyl or C(14-18)alkenyl.

[0511] Clause 17, the membrane fusion compound of Clause 15, wherein the compound is Compound T10.

[0512] Clause 18, the membrane fusion compound of Clause 1, wherein one or more amphiphilic molecules has the structure of Formula IV:

[0513]

[0514] wherein R 1 and R 2 are:

[0515] R 1 is C(12-20)alkyl, or C(12-20)alkenyl;

[0516] R 2 is (CH2) n XR 4 wherein n is 0 to 3, and X is O, S, SO, S02, NH;

[0517] wherein R 4C(12-20)alkyl, or C(12-20)alkenyl;

[0518] wherein Z is O or NH;

[0519] wherein p is 1 to 4;

[0520] wherein R 3 is selected from C(1-12)alkyl or C(2-12)alkenyl substituted with -(C=O)- attached to AA.

[0521] Clause 19. The membrane fusion compound of clause 1, wherein one or more amphiphiles has the structure of Formula V:

[0522]

[0523] wherein R 1 and R 2 are:

[0524] R 1 = (C=O)OR 4 , (C=O)NHR 4 , O(C=O)R 4 , NH(C=O)R 4

[0525] R 2 = (C=O)OR 5 , (C=O)NHR 5 , O(C=O)R 5 , NH(C=O)R 5

[0526] wherein R 4 and R 5 are each independently present, C(12-20)alkyl, or C(12-20)alkenyl;

[0527] wherein R 3 is selected from branched or unbranched -O(C=O)-C(1-8)alkanediyl- (C=O)- attached to AA, substituted or unsubstituted -O(C=O)-C(2-8)alkenediyl- (C=O)- attached to AA, substituted or unsubstituted -O(C=O)-C(2-8)alkynediyl- (C=O)- attached to AA, substituted or unsubstituted -O(C=O)-C(3-8)cycloalkanediyl- (C=O)- attached to AA, substituted or unsubstituted -O(C=O)-arylene-(C=O)- attached to AA, substituted or unsubstituted -O(C=O)-C(4-8)heteroarylene-(C=O)- attached to AA, and substituted or unsubstituted -O(C=O)-heterocycloalkanediyl- (C=O)- attached to AA; wherein R 3optionally interrupted by one or more -0-, -S-, -SO-, -SO2-, -NH-, -NR 6 -, -NH(C=0)-, -0(C=0)-, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0528] Clause 20, the membrane fusion compound of Clause 19, wherein R 4 and R 5 are each independently present, C(14-18)alkyl, or C(14-18)alkenyl.

[0529] Clause 21, the membrane fusion compound of Clause 19, wherein the compound is compound T12.

[0530] Clause 22, the membrane fusion compound of Clause 1, wherein one or more amphiphilic molecules has the structure of Formula V:

[0531]

[0532] wherein R 1 and R 2 are:

[0533] R 1 = (C=0)OR 4 , (C=0)NHR 4 , 0(C=0)R 4 , NH(C=0)R 4

[0534] R 2 = (C=0)OR 5 , (C=0)NHR 5 , 0(C=0)R 5 , NH(C=0)R 5

[0535] wherein R 4 and R 5 are each independently present, C(12-20)alkyl, or C(12-20)alkenyl;

[0536] wherein R 3 is selected from:

[0537] -(C=0)- or -alkyl-(C=0)-, which is attached to AA;

[0538] -0(C=0)- or -alkyl-0(C=0)-, which is attached to AA;

[0539] -O(C=O)-alkanediyl-(C=O)-, which is attached to AA;

[0540] -O(C=O)-alkanediyl-(C=O)-, which is attached to AA;

[0541] -O(C=O)-alkanediyl-(C=O)-, which is attached to AA;

[0542] -NH(C=O)- or -alkyl-NH(C=O)-, which is attached to AA;

[0543] -alkyl-(C=O)-alkyl-(C=O)-, which is attached to AA;

[0544] -alkyl-O(C=O)-alkyl-(C=O)-, which is attached to AA;

[0545] -alkyl-NH(C=O)-alkyl-(C=O)-, which is attached to AA;

[0546] -alkenyl-(C=O)-, which is attached to AA;

[0547] -alkenyl-O(C=O)-, which is attached to AA;

[0548] -alkenyl-NH(C=O)-, which is attached to AA;

[0549] -alkenyl-(C=O)-alkanediyl-(C=O)-, which is attached to AA;

[0550] -alkenyl-O(C=O)-alkanediyl-(C=O)-, which is attached to AA;

[0551] -alkenyl-NH(C=O)-alkanediyl-(C=O)-, which is attached to AA;

[0552] -alkynyl-(C=O)-, which is attached to AA;

[0553] -alkynyl-O(C=O)-, which is attached to AA;

[0554] -alkynyl-NH(C=O)-, which is attached to AA;

[0555] -alkynyl-(C=O)-alkanediyl-(C=O)-, which is attached to AA;

[0556] -alkynyl-O(C=O)-alkanediyl-(C=O)-, which is attached to AA;

[0557] -alkynyl-NH(C=O)-alkanediyl-(C=O)-, which is attached to AA;

[0558]

[0559] and positional isomers thereof;

[0560] wherein R 3 Any alkyl group of R 3 Any alkenyl group of R 3 Any alkynyl group of R

[0561] Item 23. The membrane fusion compound of item 1, wherein one or more amphiphiles has the structure of Formula VI:

[0562]

[0563] wherein R 1 and R 2 are:

[0564] R 1 = (C=0)OR 4 , (C=0)NHR 4 , 0(C=0)R 4 , NH(C=0)R 4

[0565] R 2 = (C=0)OR 5 , (C=0)NHR 5 , 0(C=0)R 5 , NH(C=0)R 5

[0566] wherein,

[0567] R 4 and R 5 are each independently present, C(12-20)alkyl, or C(12-20)alkenyl;

[0568] wherein R 3 is selected from the group consisting of branched or unbranched C(1-8)alkanediyl-(C=0)- attached to AA, substituted or unsubstituted C(2-8)alkenediyl-(C=0)- attached to AA, substituted or unsubstituted C(2-8)alkynediyl-(C=0)- attached to AA, substituted or unsubstituted C(3-8)cycloalkanediyl-(C=0)- attached to AA, substituted or unsubstituted arlyenediyl-(C=0)- attached to AA, substituted or unsubstituted C(4-8)heteroarylenediyl-(C=0)- attached to AA, and substituted or unsubstituted heterocycloalkanediyl-(C=0)- attached to AA; wherein R 3 is optionally substituted with one or more -0-, -S-, -SO-, -S02-, -NH-, -NR 6- NH(C=0)-, -0(C=0)-, interrupted, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0569] Clause 24. The membrane fusion compound of clause 1, wherein one or more amphiphiles has the structure of Formula VI:

[0570]

[0571] wherein R 1 and R 2 are:

[0572] R 1 = (C=0)OR 4 , (C=0)NHR 4 , 0(C=0)R 4 , NH(C=0)R 4

[0573] R 2 = (C=0)OR 5 , (C=0)NHR 5 , 0(C=0)R 5 , NH(C=0)R 5

[0574] wherein,

[0575] R 4 and R 5 are each independently present, C(12-20)alkyl, or C(12-20)alkenyl;

[0576] R 3 is selected from:

[0577] - alkyl-(C=0)-, which is attached to AA;

[0578] - alkyl-0(C=0)-, which is attached to AA;

[0579] - alkyl-NH(C=0)-, which is attached to AA;

[0580] - alkyl-(C=0)-alkyl-(C=0)-, which is attached to AA;

[0581] - alkyl-0(C=0)-alkyl-(C=0)-, which is attached to AA;

[0582] - alkyl-NH(C=0)-alkyl-(C=0)-, which is attached to AA;

[0583] - alkenyl-(C=0)-, which is attached to AA;

[0584] -alkenyl-O(C=O)-, which is attached to AA;

[0585] -alkenyl-NH(C=O)-, which is attached to AA;

[0586] -alkenyl-(C=O)-alkenyl-(C=O)-, which is attached to AA;

[0587] -alkenyl-O(C=O)-alkenyl-(C=O)-, which is attached to AA;

[0588] -alkenyl-NH(C=O)-alkenyl-(C=O)-, which is attached to AA;

[0589] -alkynyl-(C=O)-, which is attached to AA;

[0590] -alkynyl-O(C=O)-, which is attached to AA;

[0591] -alkynyl-NH(C=O)-, which is attached to AA;

[0592] -alkynyl-(C=O)-alkynyl-(C=O)-, which is attached to AA;

[0593] -alkynyl-O(C=O)-alkynyl-(C=O)-, which is attached to AA;

[0594] -alkynyl-NH(C=O)-alkynyl-(C=O)-, which is attached to AA;

[0595]

[0596] and positional isomers thereof;

[0597] wherein R 3 each alkyl group of R 3 each alkenyl group of R 3 each alkynyl group of R

[0598] Clause 25, the membrane fusion compound of Clause 24, wherein R 4 and R 5 are each independently present, C(14-18)alkyl or C(14-18)alkenyl.

[0599] Clause 26, the membrane fusion compound of Clause 24, wherein the compound is compound T11.

[0600] Clause 27, the membrane fusion compound of Formula VII,

[0601]

[0602] wherein each amphiphilic molecule independently comprises one to two lipophilic chains, wherein the lipophilic chains each independently comprise 8 to 22 carbon atoms;

[0603] wherein each AA a is independently an amino acid comprising a side chain having a carboxyl group, wherein the amino acid is linked at each of its carboxyl groups to an amphiphilic molecule and at its N-terminus to a linker;

[0604] wherein the linker has the structure:

[0605]

[0606] or

[0607]

[0608] wherein Q 1 is branched or unbranched C(2-8)alkanediyl, branched or unbranched C(2-8)alkenediyl, branched or unbranched C(2-8)alkynediyl, or

[0609]

[0610] or

[0611]

[0612] wherein Q 2 is

[0613]

[0614] wherein Q 3 is

[0615]

[0616] wherein X is -0-, -S-, or -NH-;

[0617] n and p are each independently present, 1 to 3;

[0618] m is independently 1 to 10;

[0619] r and s are each independently present, 1 to 5.

[0620] Item 28, the membrane fusion compound of item 27, wherein AA a is selected from the following structures, and any stereoisomer thereof:

[0621]

[0622] Item 29. The membrane fusion compound of item 27, wherein one or both amphiphiles are missing and replaced with an alkyl group, or a pharmaceutically acceptable organic chemical group having 1-400 atoms selected from the group consisting of carbon, oxygen, nitrogen, sulfur, fluorine, and hydrogen.

[0623] Item 30. The membrane fusion compound of item 29, wherein the pharmaceutically acceptable organic chemical group is selected from the group consisting of alkyl, alkenyl, alkynyl, alkyl ether, aryl ether, alkoxy, and alkoxyalkoxy.

[0624] Item 31. The membrane fusion compound of item 29, wherein the pharmaceutically acceptable organic chemical group is selected from the group consisting of methoxy, ethoxy, t-butyl ether, and benzyloxy.

[0625] Item 32. The membrane fusion compound of item 27, wherein one or more of the amphiphiles has the structure of Formula VIII:

[0626]

[0627] wherein R 1 and R 2 are:

[0628] R 1 = CH2(CH2) n O(C=O)R 4 , CH2(CH2) n NH(C=O)R 4 ,

[0629] CH2(CH2) n (C=O)OR 4 , CH2(CH2) n (C=O)NHR 4

[0630] R 2 = CH2(CH2) m O(C=O)R 5 , CH2(CH2) m NH(C=O)R 5 ,

[0631] CH2(CH2) m (C=O)OR 5 , CH2(CH2) m (C=O)NHR 5

[0632] wherein,

[0633] n and m are each independently 1 to 2;

[0634] R 4and R 5 each independently is present, is C(12-20)alkyl or C(12-20)alkenyl.

[0635] Clause 33. The membrane fusion compound of Clause 27, wherein one or more amphiphiles has the structure of Formula IX:

[0636]

[0637] wherein R 1 and R 2 are:

[0638] R 1 =CH2(CH2) n O(C=O)R 4 , CH2(CH2) n NH(C=O)R 4 ,

[0639] CH2(CH2) n (C=O)OR 4 , CH2(CH2) n (C=O)NHR 4

[0640] R 2 =CH2(CH2) m O(C=O)R 5 , CH2(CH2) m NH(C=O)R 5 ,

[0641] CH2(CH2) m (C=O)OR 5 , CH2(CH2) m (C=O)NHR 5

[0642] wherein,

[0643] n and m are each independently 1 to 2;

[0644] R 4 and R 5 each independently is present, is C(12-20)alkyl or C(12-20)alkenyl.

[0645] wherein R 3 is selected from the group consisting of branched or unbranched C(1-8)alkanediyl, substituted or unsubstituted C(2-8)alkenediyl, substituted or unsubstituted C(2-8)alkynediyl, substituted or unsubstituted C(3-8)cycloalkanediyl, substituted or unsubstituted aralkanediyl, substituted or unsubstituted C(4-8)heteroaralkanediyl, and substituted or unsubstituted heterocycloalkanediyl; wherein R3 optionally interrupted by one or more -0-, -S-, -SO-, -S02-, -NH-, -NR 6 -, -NH(C=0)-, -0(C=0)-, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0646] Clause 34, the membrane fusion compound of Clause 33, wherein R 4 and R 5 are each independently present, C(14-18)alkyl, or C(14-18)alkenyl.

[0647] Clause 35, the membrane fusion compound of Clause 33, wherein the compound is Compound T9.

[0648] Clause 36, the membrane fusion compound of Clause 27, wherein one or more amphiphilic molecules has the structure shown in Chemical Formula X:

[0649]

[0650] wherein,

[0651] R 1 and R 2 are:

[0652] R 1 =CH2(CH2) n O(C=0)R 4 , CH2(CH2) n NH(C=0)R 4 ,

[0653] CH2(CH2) n (C=0)OR 4 , CH2(CH2) n (C=0)NHR 4

[0654] R 2 =CH2(CH2) m O(C=0)R 5 , CH2(CH2) m NH(C=0)R 5 ,

[0655] CH2(CH2) m (C=0)OR 5 , CH2(CH2) m (C=0)NHR 5

[0656] R4 and R 5 each independently is present, is C(12-20)alkyl or C(12-20)alkenyl;

[0657] wherein R 3 is absent or selected from branched or unbranched *-NH-C(1-8)alkanediyl- (C=0)-, substituted or unsubstituted *-NH-C(2-8)alkenediyl-(C=0)-, substituted or unsubstituted *-NH-C(2-8)alkynediyl-(C=0)-, substituted or unsubstituted *-NH-C(3-8)cycloalkanediyl-(C=0)-, substituted or unsubstituted *-NH- arylene-(C=0)-, substituted or unsubstituted *-NH-C(4-8)heteroarylene-(C=0)-, and substituted or unsubstituted *-NH-heterocycloalkanediyl-(C=0)-, wherein * indicates the terminal end of attachment to AA a wherein R 3 is optionally interrupted by one or more -0-, -S-, -SO-, -S02-, -NH-, -NR 6 -, -NH(C=0)-, -0(C=0)-, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0658] Item 37. The membrane fusion compound of item 27, wherein one or more amphiphiles has the structure of Formula XI:

[0659]

[0660] wherein,

[0661] R 1 and R 2 are:

[0662] R 1 = (C=0)OR 4 , (C=0)NHR 4 , 0(C=0)R 4 , (C=0)R 4

[0663] R 2 = (C=0)OR 5 , (C=0)NHR 5 , 0(C=0)R 5 , NH(C=0)R 5

[0664] R 4 and R 5each independently present, is C(12-20)alkyl or C(12-20)alkenyl;

[0665] wherein R 3 is absent or selected from branched or unbranched *-NH-C(1-8)alkanediyl- (C=0)-, substituted or unsubstituted *-NH-C(2-8)alkenediyl-(C=0)-, substituted or unsubstituted *-NH-C(2-8)alkynediyl-(C=0)-, substituted or unsubstituted *-NH-C(3-8)cycloalkanediyl-(C=0)-, substituted or unsubstituted *-NH- arylene-(C=0)-, substituted or unsubstituted *-NH-C(4-8)heteroarylene-(C=0)-, and substituted or unsubstituted *-NH-heterocycloalkanediyl-(C=0)-, wherein * indicates the terminal attachment to AA a wherein R 3 is optionally interrupted by one or more -0-, -S-, -SO-, -S02-, -NH-, -NR 6 -, -NH(C=0)-, -0(C=0)-, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0666] Item 38. The membrane fusion compound of item 27, wherein one or more amphiphiles has a structure according to Formula III:

[0667]

[0668] wherein R 1 and R 2 are:

[0669] R 1 = CH2(CH2) n O(C=0)R 4 , CH2(CH2) n NH(C=0)R 4 ,

[0670] CH2(CH2) n (C=0)OR 4 , CH2(CH2) n (C=0)NHR 4

[0671] R 2 = CH2(CH2) m O(C=0)R 5 , CH2(CH2) m NH(C=0)R 5 ,

[0672] CH2(CH2) m (C=O)OR 5 , CH2(CH2) m (C=O)NHR 5

[0673] wherein n and m are each independently 1 to 2; R 4 and R 5 are each independently present, C(12-20)alkyl or C(12-20)alkenyl;

[0674] wherein,

[0675] R 3 is selected from:

[0676] -(C=O)-alkyl-NH- which is attached to AA a ;

[0677] -(C=O)-alkenyl-NH- which is attached to AA a ;

[0678] -(C=O)-alkynyl-NH- which is attached to AA a ;

[0679] wherein any alkyl of R 3 is branched or unbranched C(1-6)alkyl, any alkenyl of R 3 is branched or unbranched C(2-6)alkenyl, and any alkynyl of R 3 is branched or unbranched C(2-6)alkynyl;

[0680]

[0681]

[0682] and positional isomers thereof;

[0683]

[0684] wherein,

[0685] each R 6 is independently selected from H, alkyl, alkoxy, and alkoxyalkoxy, provided that one R 6 is -(C=O)-alkyl-NH- wherein NH is attached to AA a ;

[0686] each R 8 is independently selected from H, alkyl, provided that one R 8 is -(C=O)-alkyl-NH- wherein NH is attached to AA a ;

[0687] q is 0 to 4;

[0688] Q is O or N.

[0689] Clause 39. The membrane fusion compound of Clause 27, wherein one or more amphiphiles has a structure according to Formula V:

[0690]

[0691] wherein R 1 and R 2 are:

[0692] R 1 = (C=O)OR 4 , (C=O)NHR 4 , O(C=O)R 4 , NH(C=O)R 4

[0693] R 2 = (C=O)OR 5 , (C=O)NHR 5 , O(C=O)R 5 , NH(C=O)R 5

[0694] wherein R 4 and R 5 are each independently present, C(12-20)alkyl, or C(12-20)alkenyl;

[0695] wherein R 3 is absent or selected from branched or unbranched *-NH-C(1-8)alkanediyl- (C=O)-, substituted or unsubstituted *-NH-C(2-8)alkenediyl-(C=O)-, substituted or unsubstituted *-NH-C(2-8)alkynediyl-(C=O)-, substituted or unsubstituted *-NH-C(3-8)cycloalkanediyl-(C=O)-, substituted or unsubstituted *-NH-arylene-(C=O)-, substituted or unsubstituted *-NH-C(4-8)heteroarylene-(C=O)-, and substituted or unsubstituted *-NH-heterocycloalkanediyl-(C=O)-, wherein * indicates the terminal end of attachment to AA a ; wherein R 3 is optionally interrupted by one or more -O-, -S-, -SO-, -SO2-, -NH-, -NR 6 -, -NH(C=O)-, -O(C=O)-, wherein R 6 is C(1-6)alkyl-, C(1-6)alkoxy-, or C(1-6)alkoxy-C(1-6)alkoxy-.

[0696] Item 40. A composition comprising the membrane fusion compound of any one of items 1-39 and a pharmaceutically acceptable carrier.

[0697] Item 41. The composition of item 40, wherein the composition comprises a nanoparticle or a liposome.

[0698] Item 42. A pharmaceutical composition comprising the membrane fusion compound of any one of items 1-39, an active agent, and a pharmaceutically acceptable carrier.

[0699] Item 43. The composition of item 42, wherein the membrane fusion compound is 0.01 mol% to 20 mol% of the lipids of the composition.

[0700] Item 44. The composition of item 42, wherein the composition comprises a nanoparticle or a liposome.

[0701] Item 45. The composition of item 42, wherein the active agent is one or more nucleic acids.

[0702] Item 46. The composition of item 42, wherein the active agent is one or more DNA, RNA, mRNA, siRNA, or microRNA.

[0703] Item 47. The composition of item 42, wherein the active agent is one or more RNA molecules.

[0704] Item 48. The composition of item 42, wherein the active agent is selected from one or more RNAi molecules, one or more mRNA molecules, and modified versions thereof.

[0705] Item 49. A composition for use in delivering an active agent to treat a disorder or disease in a subject, the composition comprising the active agent, the membrane fusion compound of any one of items 1-39, an ionizable lipid, a structural lipid, a stabilizer lipid, and a lipid for reducing immunogenicity of the composition.

[0706] Item 50. The composition of item 49, wherein the active agent is one or more nucleic acids.

[0707] Item 51. A method for preventing, mitigating, or treating a disease or disorder in a subject in need thereof, comprising administering to the subject the composition of item 42.

[0708] Item 52. The composition of item 42 for use in the treatment of the human or animal body. BRIEF DESCRIPTION OF DRAWINGS

[0709] Figure 1 A scheme showing the preparation of compound R1 is shown.

[0710] Figure 2A preparation scheme for compound R2 is shown.

[0711] Figure 3A A preparation scheme for compound R3 is shown.

[0712] Figure 3B A structure for compound R3 is shown.

[0713] Figure 4A A preparation scheme for compound R4 is shown.

[0714] Figure 4B A structure for compound R4 is shown.

[0715] Figure 5 A preparation scheme for compound R5 is shown.

[0716] Figure 6 A preparation scheme for compound R6 is shown.

[0717] Figure 7 An alternative preparation scheme for compound R6 is shown.

[0718] Figure 8 A preparation scheme for compound S2 is shown.

[0719] Figure 9 A preparation scheme for compound S3 is shown.

[0720] Figure 10A A preparation scheme for compound S4 is shown.

[0721] Figure 10B A structure for compound S4 is shown.

[0722] Figure 11 A preparation scheme for compound S5 is shown.

[0723] Figure 12A A preparation scheme for compound S6 is shown.

[0724] Figure 12B A structure for compound S6 is shown.

[0725] Figure 13A A preparation scheme for compound S7 is shown.

[0726] Figure 13B A structure for compound S7 is shown.

[0727] Figure 14A A preparation scheme for compound S8 is shown.

[0728] Figure 14B A structure for compound S8 is shown.

[0729] Figure 15AA preparation scheme for compound T1 is shown.

[0730] Figure 15B A structure for compound T1 is shown.

[0731] Figure 16A A preparation scheme for compound T2 is shown.

[0732] Figure 16B A structure for compound T2 is shown.

[0733] Figure 17A A preparation scheme for compound T4 is shown.

[0734] Figure 17B A structure for compound T4 is shown.

[0735] Figure 18A A preparation scheme for compound T5 is shown.

[0736] Figure 18B A structure for compound T5 is shown.

[0737] Figure 19A A preparation scheme for compound T6 is shown.

[0738] Figure 19B A structure for compound T6 is shown.

[0739] Figure 20A A preparation scheme for compound T7 is shown.

[0740] Figure 20B A structure for compound T7 is shown.

[0741] Figure 21A A preparation scheme for compound T8 is shown.

[0742] Figure 21B A structure for compound T8 is shown.

[0743] Figure 22A A preparation scheme for compound T9 is shown.

[0744] Figure 22B A structure for compound T9 is shown.

[0745] Figure 23A A preparation scheme for compound T3 is shown.

[0746] Figure 23B A structure for compound T3 is shown.

[0747] Figure 24 Results showing the in vitro delivery of biologically active molecules using the membrane fusion molecules of the application are shown. As Figure 24Liposomal delivery formulation #5 containing 2% (of total lipids) of the membrane fusion compound R4 of the present application provided unexpectedly increased gene expression knockdown activity of an example siRNA targeting HSP47 in stellate cells compared to control formulation #1 which did not contain a membrane fusion compound of the present application.

[0748] Figure 25 Results of in vitro delivery of biologically active molecules using the membrane fusion molecules of the present application are shown. The HSP47 gene expression knockdown activity of several siRNA liposomal delivery formulations was measured. The formulations provided high gene expression knockdown activity in stellate cells. The formulations contained 2% (of total lipids) of a membrane fusion compound of the present application, providing high activity siRNA targeting HSP47.

[0749] Figure 26 Results of in vivo delivery of biologically active molecules using the membrane fusion molecules of the present application are shown. Liposomal delivery formulations of siRNA targeting HSP47 are shown to provide gene expression knockdown activity in vivo (mice). Active formulations #2-#8 contained 2% (of total lipids) of a specified membrane fusion compound of the present application.

[0750] Figure 27 The left half of the structure of compound T10 is shown.

[0751] Figure 28 The right half of the structure of compound T10 is shown.

[0752] Figure 29 A preparation scheme for compound T10 is shown.

[0753] Figure 30 A preparation scheme for compound T10 is shown.

[0754] Figure 31 The left half of the structure of compound T11 is shown.

[0755] Figure 32 The right half of the structure of compound T11 is shown.

[0756] Figure 33 A preparation scheme for compound T11 is shown.

[0757] Figure 34 A preparation scheme for compound T11 is shown.

[0758] Figure 35 The left half of the structure of compound T12 is shown.

[0759] Figure 36 The right half of the structure of compound T12 is shown.

[0760] Figure 37 The preparation method of compound T12 is shown.

[0761] Figure 38 The preparation method for compound T12 is shown.

[0762] Figure 39 The left half of the structure of compound T13 is shown.

[0763] Figure 40 The right half of the structure of compound T13 is shown.

[0764] Figure 41 The preparation method of compound T13 is shown.

[0765] Figure 42 The preparation method of compound T13 is shown.

[0766] Figure 43 The left and right halves of the structure of compound T14 are shown.

[0767] Figure 44 The preparation method of compound T14 is shown.

[0768] Figure 45 The preparation method of compound T14 is shown.

[0769] Figure 46 The in vitro activity of knockdown of gene expression of example siRNA in rat astrocytes for liposome formulations containing the membrane fusion compound of the present invention is shown. (1) Results of a liposome formulation containing lipids HEDC and S104, which does not contain the membrane fusion compound of the present invention. (2) Results of a liposome formulation similar to (1), except that it contains 2% (of total lipids) of the membrane fusion compound R4. (3) Results of a liposome formulation similar to (1), except that it contains 10% (of total lipids) of the membrane fusion compound R4. The presence of the membrane fusion compound in the formulation greatly increases the delivery activity of the example siRNA, and the increased activity is directly attributed to the presence of the membrane fusion compound R4.

[0770] Figure 47The in vitro activity of knockdown of gene expression of example siRNA in rat astrocytes for liposome formulations containing the membrane fusion compound of the present invention is shown. (1) Results of a liposome formulation containing lipids HEDC and S104, which does not contain the membrane fusion compound of the present invention. (2) Results of a liposome formulation similar to (1), except containing 2% (of total lipids) of membrane fusion compound R4. (3) Results of a liposome formulation similar to (1), except containing 2% (of total lipids) of membrane fusion compound T3. The presence of membrane fusion compounds R4 and T3 in the formulations respectively greatly increased the delivery activity of the example siRNA, and the increased activity was directly attributed to the presence of membrane fusion compounds R4 and T3.

[0771] Figure 48 The results show the delivery and transfection of GFP mRNA into A549 cells in vitro at 5 nM (top panel) and 2 nM (bottom panel) using the LNP nanoparticles of the present invention, comprising the composition HEDC:S104:CH:DOPE:DMPE-PEG2000:compound T3. The fluorescence image of GFP expression (left panel) was obtained using fluorescence microscopy 48 hours post-transfection. The overlay image of fluorescence and bright-field (BF) images (right panel) confirms that nearly 100% of the cells were transfected and expressed GFP.

[0772] Figure 49 Results of in vivo delivery of GFP mRNA using the membrane-fused lipid-like molecule of the present invention are shown. GFP mRNA was transfected into Balb / c mice using LNP nanoparticles of the present invention having the composition HEDC:S104:CH:DOPE:DMPE-PEG2000:compound T3. Figure 49 As shown, the delivery of mRNA to various tissues and cells was determined using the MAXDISCOVER GFP ELISA. Surprisingly, GFP mRNA was selectively transfected and / or translated in the lungs, while transfection and / or translation were lower in muscle, liver, heart, and kidneys.

[0773] Figure 50 Results of in vivo delivery of luciferase mRNA using the membrane-fused lipid-like molecules of the present invention are shown. Luciferase mRNA was transfected into Balb / c mice using LNP nanoparticles of the present invention having the composition HEDC:S104:CH:DOP E:DMPE-PEG2000:compound T3. Figure 50As shown, the relative delivery, transfection, and / or translation of mRNA in various tissues and cells were determined using the Promega E4510 assay kit. Surprisingly, Fluc mRNA was selectively delivered, transfected, and / or translated in the lungs and spleen, while delivery, transfection, and / or translation were lower in the liver, heart, kidneys, and muscle.

[0774] Figure 51 Results of in vivo delivery of luciferase mRNA using the membrane-fused lipid-like molecules of the present invention are shown. Luciferase mRNA was transfected into Balb / c mice by injection of 2 mpk using LNP nanoparticles of the present invention having the composition: (-01) HE2DC:S104:CH:DOPE:DMPE-PEG2000:compound T3, or (-02) HEDC:S104:CH:DOPE:DMPE-PEG2000:compound T3. Figure 51 As shown, the relative delivery, transfection, and / or translation of mRNA in various tissues and cells were determined using the Promega E4510 assay kit. Fluc mRNA was selectively delivered, transfected, and / or translated in the lungs and spleen, while delivery, transfection, and / or translation were lower in the pancreas, kidneys, liver, testes, and small intestine.

[0775] Figure 52 The results show the autofluorescence of luciferase mRNA delivered in vivo in mice using the membrane-fused lipid-like molecule of the present invention. Figure 52 As shown, autofluorescence imaging was used to determine the relative delivery, transfection, and / or translation of mRNA in various tissues 7 hours after injection.

[0776] Figure 53 The results of delivering luciferase mRNA in mice using the membrane-fused lipid-like molecule of the present invention are shown. Figure 53 As shown, the relative delivery of mRNA in the formulation containing the membrane fusion molecule of the present invention (2035-03-03) is much greater than that in the same formulation without the membrane fusion molecule (2035-13-01). Invention Details

[0777] This invention provides a series of membrane fusion molecules. The membrane fusion compounds of this invention can be used to deliver therapeutic agents to cells, tissues or organs, organisms, and objects.

[0778] In some aspects, the present invention provides platform compounds for forming membrane fusion molecules. Membrane fusion molecules can be formed by linking one or more neutral molecules to a platform structure, such as hydrocarbon molecules, aliphatic molecules, saturated fatty acid molecules, unsaturated fatty acid molecules, monounsaturated fatty acid molecules, or polyunsaturated fatty acid molecules.

[0779] Examples of the membrane fusion compounds of the present invention include, but are not limited to, those mentioned above. Figure 4B Compound R4 is shown; Figure 12B Compound S6 is shown; Figure 13B Compound S7 is shown; Figure 14B Compound S8 is shown; Figure 15B Compound T1 is shown; Figure 16B Compound T2 is shown; Figure 17B Compound T4 is shown; Figure 18B Compound T5 is shown; Figure 19B Compound T6 is shown; Figure 20B Compound T7 is shown; Figure 21B Compound T8 is shown; Figure 22B Compound T9 is shown; Figure 23B Compound T3 is shown; Figure 27 and 28 Compound T10 is shown; Figure 31 and 32 Compound T11 is shown; Figure 35 and 36 Compound T12 is shown; Figure 39 and 40 Compound T13 is shown; Figure 43 The compound shown is T14.

[0780] For the purpose of illustrating the method of synthesizing the compound, the following is shown Figure 1 , 2 Compounds shown in 3A, 3B and 6-11.

[0781] In some aspects, the present invention provides a series of membrane fusion molecules that can be used in formulations for forming and utilizing lipid nanoparticles for delivering active agents to cells and objects.

[0782] The membrane fusion compound of the present invention may have one or two amphiphilic molecules linked to an amino acid group, the amino acid group being linked to a separate amino acid group carrying one or two other amphiphilic molecules via a linker.

[0783] The lipophilic chains of amphiphilic molecules can each contain 8 to 22 carbon atoms independently.

[0784] The amphiphilic molecular group can be a lipid-like group, having one or two lipophilic chains linked to an organic chemical group. The organic chemical group can have up to 400 atoms, or 20-400 atoms, or 10-400 atoms, or 4-400 atoms, or 3-400 atoms, or 2-400 atoms, or 1-400 atoms, wherein the atoms are selected from carbon, oxygen, nitrogen, sulfur, fluorine, and hydrogen, and can have any structure suitable for linking one or two lipophilic chains and being linked to an amino acid group. The organic chemical group can be neutral, zwitterionic, or can provide hydrophilic properties. In some embodiments, the organic chemical group can be ionizable. Examples of organic chemical groups include alkyl, alkenyl, alkynyl, and acetyl groups, as well as protecting groups such as Boc, Fmoc, TFA, and CBZ (benzyloxycarbonyl).

[0785] Unwilling to be bound by any particular theory, amphiphilic molecules can possess lipid-like structures, allowing them to enter the lipid bilayer in a manner similar to that of lipid molecules in a bilayer, while maintaining connections with larger membrane fusion compounds. The membrane fusion compounds of this invention can disrupt the dynamic structure of the bilayer to enhance membrane fusion with cells.

[0786] The amino acid group of the membrane fusion compound (named AA or AA) a It can be modified with substituents. The amino acid group of the membrane fusion compound can be any compound with the chemical formula -NR. N -CR 1 R 2 -(C=O)- D or L type amino acid groups, where R 1 It is a substituted or unsubstituted side chain of certain natural amino acids. R 2 and R N Each of them can be an organic group consisting of hydrogen, or composed of carbon, oxygen, nitrogen, sulfur and hydrogen atoms, and having 1 to 20 carbon atoms, or can be C(1-6)alkyl, cycloalkyl, cycloalkylalkyl, C(2-6)alkenyl, C(2-6)alkynyl, C(1-6)alkanoyl, C(1-6)alkanoyloxy, C(1-6)alkoxy, C(1-6)alkoxy-C(1-6)alkyl, C(1-6)alkoxy-C(1-6)alkoxy.

[0787] As used herein, the term "attached to AA" is used to indicate the point of attachment of a group to AA. For example, the term "-alkyl-(C=O)-, attached to AA" refers to the attachment of a group to AA via a (C=O)- group, thereby forming -alkyl-(C=O)-AA. Unless otherwise indicated, it is intended to indicate that the last group appearing in the phrase "attached to AA" is the group attached to AA.

[0788] The present invention can provide compositions for dispensing active agents in cells, tissues or organs, organisms and objects, wherein the compositions comprise one or more membrane fusion molecules of the present invention.

[0789] The membrane fusion compounds of the present invention can advantageously provide compositions and formulations for delivering therapeutic agents to cells, tissues and objects without significant aggregation of the composition components.

[0790] The membrane fusion compounds of the present invention can advantageously provide liposome formulations for delivering therapeutic agents to cells, tissues and subjects without significant aggregation of the liposomes in the composition.

[0791] The compositions of the present invention may comprise one or more membrane fusion molecules, structural lipids, and one or more lipids for reducing the immunogenicity of the composition.

[0792] In some aspects, the present invention provides novel membrane-fused lipids that facilitate the delivery of bioactive molecules to cells. Membrane-fused lipids can be incorporated into formulations, such as nanoparticles or liposomes, to deliver therapeutic molecules, including nucleic acids or oligonucleotides, to cells, including tumors. In some embodiments, nanoparticles or liposomes containing membrane-fused lipids can fuse with the cell membrane or intracellular membrane of cells, promoting the release of therapeutic molecules and increasing transfection efficiency.

[0793] In other embodiments, a range of novel membrane-fusion lipids can be synthesized and incorporated into nanoparticles or liposomes. The nanoparticles or liposomes can incorporate or encapsulate therapeutic molecules, including nucleic acid-based molecules such as siRNA, miRNA, or mRNA, as well as small molecule drugs and any active therapeutic agent that can be delivered using nanoparticles or liposomes.

[0794] The particle size of nanoparticles or liposomes can range from 50 to 200 nm, and their polydispersity is less than 0.2. Compared with nanoparticles or liposomes lacking one or more novel membrane-fused lipids, the transfection efficiency of nanoparticles or liposomes in a variety of cell lines can be enhanced.

[0795] In other embodiments, cellular uptake of the nanoparticles or liposomes of the present invention can be enhanced via endocytosis or micropinocytosis mechanisms.

[0796] The nanoparticles or liposomes of the present invention can also reduce lysosomal degradation of therapeutic molecules during delivery.

[0797] In some embodiments, the present invention includes a composition comprising one or more membrane fusion molecules and other lipid molecules for forming nanoparticles. In some embodiments, the membrane fusion molecule may comprise 0.1 to 40 mol% of the lipids in the composition. In other embodiments, the membrane fusion compound may comprise 1 to 20 mol% of the lipids in the composition. In other embodiments, the membrane fusion compound may comprise 1 to 10 mol% or 2 to 10 mol% of the lipids in the composition. In other embodiments, the membrane fusion molecule may comprise 2 mol% of the lipids in the composition.

[0798] In some embodiments, the membrane fusion compound may comprise a fourth or fifth component of the lipids of the composition, or the membrane fusion molecule may replace one of the lipid components of the composition.

[0799] The membrane fusion molecule of the present invention may include a platform structure and be connected to the platform structure by one to four amphiphilic molecular groups by suitable chemical bonds.

[0800] The compositions of this invention may include the membrane fusion molecules disclosed herein. The membrane fusion molecules may be 1-10 mol% or more of the composition. The compositions may form nanoparticles or liposomes.

[0801] In some embodiments, the compositions of the present invention may comprise cationic lipids, ionizable lipids, and membrane-fused lipid molecules, which can be combined to form lipid nanoparticles. In some embodiments, the lipid nanoparticles may have a bilayer of lipid molecules.

[0802] In some embodiments, one or both amphiphilic molecules may be missing; when missing, they can be protected by the protective group R. P Replacement.

[0803] In other embodiments, one or two amphiphilic molecules may be missing, and when missing, they may be replaced by alkyl, alkenyl, or alkynyl groups, or organic chemical groups having up to 400 atoms, or 20-400 atoms, or 10-400 atoms, or 4-400 atoms, or 3-400 atoms, or 2-400 atoms, or 1-400 atoms, wherein the atoms are selected from carbon, oxygen, nitrogen, sulfur, fluorine, and hydrogen.

[0804] Methods for preparing various organic groups and protecting groups are known in the art, and their use and modification generally fall within the capabilities of those skilled in the art. See, for example, Stanley R. Sandler and Wolf Karo, Organic Functional Group Preparations (1989); Greg T. Hermanson, Bioconjugate Techniques (1996); Leroy G. Wade, Compendium of Organic Synthetic Methods (1980); some examples of protecting groups can be found in TW Greene and PGM Watts, Protective Groups in Organic Synthesis (3rd ed. 1991). See, for example, Helmut Vorbrüggen, Handbook of Nucleoside Synthesis (2001).

[0805] Protective group R P Examples include Fmoc (fluorenylmethoxycarbonyl).

[0806] Protective group R P Examples include Boc (tert-butyloxycarbonyl).

[0807] Protective group R P Examples include OTrt (o-triphenylmethyl).

[0808] Amino protective group R P Examples include Ac(acetamide (C=O)CH3).

[0809] Examples of amino-protecting groups include Fmoc, Boc, Trt, Dde, and Alloc.

[0810] Examples of protective alkoxy groups include OTrt, OClt, OMmt, OMtt, ODpm, and OtBu.

[0811] Examples of protecting groups include tert-butyl ethers.

[0812] Examples of carboxylic acid protecting groups include benzyl esters.

[0813] Cationic lipids and ionizable lipids

[0814] Examples of cationic and ionizable lipids in this disclosure are given in US20130022665A and US20130330401A.

[0815] The structure of HEDC is described in section

[0146] of US2013 / 0022665A.

[0816] The structure of S104 is described in section

[0046] of US2013 / 0115274A1.

[0817] Compositions containing three or more components

[0818] As used herein, a component of a formulation, such as “lipid,” can be a single compound or a combination of one or more suitable lipid compounds. For example, “stabilizing lipid” can refer to a single stabilizing lipid or a combination of one or more suitable stabilizing lipids. Those skilled in the art will readily understand that certain combinations of the compounds described herein can be used without excessive experimentation, and that various combinations of compounds are covered in the description of the formulation components.

[0819] The ionizable compound in the composition of the present invention may be 20 mol% to 80 mol% of the lipid component of the composition. In some embodiments, the ionizable molecule of the composition may be 55 mol% to 65 mol% of the lipid component of the composition. In other embodiments, the ionizable molecule of the composition may be about 60 mol% of the lipid component of the composition.

[0820] The structural lipids in the compositions of the present invention may be 20 mol% to 50 mol% of the lipid components of the composition. In some embodiments, the structural lipids in the compositions may be 35 mol% to 45 mol% of the lipid components of the compositions.

[0821] One or more lipids used to reduce the immunogenicity of the composition may be from 1 mol% to 8 mol% of the total lipid components of the composition. In some embodiments, one or more lipids used to reduce the immunogenicity of the composition may be from 1 mol% to 5 mol% of the total lipid components of the composition.

[0822] In other aspects, the compositions of the present invention may further comprise cationic lipids, which may be 5 mol% to 25 mol% of the lipid component of the composition. In some embodiments, the compositions of the present invention may further comprise cationic lipids, which may be 5 mol% to 15 mol% of the lipid component of the composition. In these aspects, the molar ratio of the concentration of cationic lipids to ionizable molecules in the compositions of the present invention may be from 5:80 to 25:50.

[0823] In the compositions of the present invention, all lipid components may comprise one or more ionizable compound molecular components, structural lipids, and one or more lipids for reducing the immunogenicity of the composition.

[0824] In addition to the components described above, the compositions of the present invention may also include the membrane fusion molecules disclosed herein. The membrane fusion molecules may be 1-10 mol% of the composition.

[0825] Compositions containing four or more components

[0826] The ionizable molecules of the compositions of the present invention may be 15 mol% to 40 mol% of the lipid component of the composition. In some embodiments, the ionizable molecules of the compositions may be 20 mol% to 35 mol% of the lipid component of the composition. In other embodiments, the ionizable molecules of the compositions may be 25 mol% to 30 mol% of the lipid component of the composition.

[0827] The structural lipids in the compositions of the present invention may be 25 mol% to 40 mol% of the lipid components of the composition. In some embodiments, the structural lipids in the compositions may be 30 mol% to 35 mol% of the lipid components of the compositions.

[0828] The total amount of stabilizer lipids in the compositions of the present invention may be 25 mol% to 40 mol% of the lipid components of the composition. In some embodiments, the total amount of stabilizer lipids in the compositions may be 30 mol% to 40 mol% of the lipid components of the compositions.

[0829] In some embodiments, the compositions of the present invention may comprise two or more stabilizer lipids, wherein each stabilizer lipid may individually comprise 5 mol% to 35 mol% of the lipid component of the composition. In some embodiments, the compositions of the present invention may comprise two or more stabilizer lipids, wherein each stabilizer lipid may individually comprise 10 mol% to 30 mol% of the lipid component of the composition.

[0830] In some embodiments, the total amount of one or more stabilizer lipids may be 25 mol% to 40 mol% of the lipids in the composition, wherein each stabilizer lipid may individually be 5 mol% to 35 mol%.

[0831] In some embodiments, the total amount of one or more stabilizer lipids may be 30 mol% to 40 mol% of the lipids in the composition, wherein each stabilizer lipid may individually be 10 mol% to 30 mol%.

[0832] One or more lipids used to reduce the immunogenicity of the composition may be from 1 mol% to 8 mol% of the total lipid components of the composition. In some embodiments, one or more lipids used to reduce the immunogenicity of the composition may be from 1 mol% to 5 mol% of the total lipid components of the composition.

[0833] In other aspects, the compositions of the present invention may further comprise cationic lipids, which may be 5 mol% to 25 mol% of the lipid component of the composition. In some embodiments, the compositions of the present invention may further comprise cationic lipids, which may be 5 mol% to 15 mol% of the lipid component of the composition. In these aspects, the molar ratio of the concentration of cationic lipids to ionizable molecules in the compositions of the present invention may be 5:35 to 25:15.

[0834] In some embodiments, the entire lipid component of the composition may comprise one or more ionizable compound molecules, structural lipids, one or more lipids for reducing the immunogenicity of the composition, and one or more stabilizer lipids.

[0835] In addition to the components described above, the compositions of the present invention may also include the membrane fusion molecules disclosed herein. The membrane fusion molecules may be 1-10 mol% of the composition.

[0836] Examples of lipid compositions

[0837] In some embodiments, the composition may contain one or more ionizable molecules, structural lipids, one or more lipids for reducing the immunogenicity of the composition, and the membrane fusion molecule of the present invention, which will represent 100% of the lipid component of the composition. In some embodiments, cationic lipids may be included.

[0838] Examples of the compositions of the present invention are shown in Table 1.

[0839] Table 1: Composition of lipid components (mol% of total lipids)

[0840]

[0841]

[0842] Examples of the compositions of the present invention are shown in Table 2.

[0843] Table 2: Composition of lipid components (mol% of total lipids)

[0844]

[0845] Structural lipids

[0846] Examples of structural lipids include cholesterol, sterols, and steroids.

[0847] Examples of structural lipids include cholestanes, cholestane, ergosterane, campestanes, poriferastanes, stigmastanes, gorgostanes, lanosterane, gonanes, estrostanes, androstanes, pregnanes, and cyclopinene.

[0848] Examples of structural lipids include sterols and animal sterols, such as cholesterol, lanosterol, zymosterol, zymostenol, desmosterol, stigmasterol, dihydrolanosterol, and 7-dehydrocholesterol.

[0849] Examples of structural lipids include PEGylated cholesterol and cholesterol-3-keto-(C1-22) acyl compounds, such as cholesterol acetate, cholesterol arachidonic acid ester, cholesterol butyrate, cholesterol hexanoate, cholesterol myristate, cholesterol palmitate, cholesterol behenate, cholesterol stearate, cholesterol caprylate, cholesterol decanoate, cholesterol dodecanoate, cholesterol nervate, cholesterol nonanoate, cholesterol valerate, cholesterol oleate, cholesterol trans oleate, cholesterol erucic acid, cholesterol heptanate, cholesterol trans linoleic acid, and cholesterol linoleic acid.

[0850] Examples of structural lipids include sterols such as phytosterol, β-sitosterol, campesterol, ergosterol, brassicasterol, δ-7-stigmasterol, and δ-7-amaresterol.

[0851] Stabilizer lipids

[0852] Examples of stabilizer lipids include zwitterionic lipids.

[0853] Examples of stabilizer lipids include compounds such as phospholipids.

[0854] Examples of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoyl lecithin, dioleoyl phosphatidylcholine, and dilinoleoyl phosphatidylcholine.

[0855] Examples of stabilizer lipids include phosphatidylethanolamine compounds and phosphatidylcholine compounds.

[0856] Examples of stabilizer lipids include 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC).

[0857] Examples of stabilizer lipids include diphyranoylphosphatidylethanolamine (DPhPE) and 1,2-diphyranoyl-sn-glycerol-3-phosphatidylcholine (DPhPC).

[0858] Examples of stabilizer lipids include 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), and 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE).

[0859] Examples of stabilizer lipids include 1,2-dilauroyl-sn-glycerol (DLG); 1,2-dimyristoyl-sn-glycerol (DMG); 1,2-dipalmitoyl-sn-glycerol (DPG); 1,2-distearatel-sn-glycerol (DSG); 1,2-diaarachidonicoyl-sn-glycerol-3-phosphocholine (DAPC); 1,2-dilauroyl-sn-glycerol-3-phosphocholine (DLPC); 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC); and 1,2-dipalmitoyl-sn-glycerol-O-ethyl-3-phosphocholine (DPePC). ; 1,2-Dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE); 1,2-Dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE); 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE); 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate choline; 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC); 1-palmitoyl-2-hemolysin-sn-glycerol-3-phosphate choline (P-Lyso-PC); and 1-stearoyl-2-hemolysin-sn-glycerol-3-phosphate choline (S-Lyso-PC).

[0860] Lipids for reducing immunogenicity

[0861] Examples of lipids used to reduce immunogenicity include polymers and polymer-lipid conjugates.

[0862] Examples of lipids used to reduce immunogenicity include PEGylated lipids having a polyethylene glycol (PEG) region. The PEG region can be of any molecular weight. In some embodiments, the PEG region may have a molecular weight of 200, 300, 350, 400, 500, 550, 750, 1000, 1500, 2000, 3000, 3500, 4000, or 5000 Da.

[0863] Examples of lipids used to reduce immunogenicity include compounds with methoxy polyethylene glycol regions.

[0864] Examples of lipids used to reduce immunogenicity include compounds with carbonyl-methoxy polyethylene glycol regions.

[0865] Examples of lipids used to reduce immunogenicity include compounds with multi-branched PEG regions.

[0866] Examples of lipids used to reduce immunogenicity include compounds with polyglycerol regions.

[0867] Examples of lipids used to reduce immunogenicity include polymeric lipids such as DSPE-mPEG, DMPE-mPEG, DPPE-mPEG, and DOPE-mPEG.

[0868] Examples of lipids used to reduce immunogenicity include PEG-phospholipids and PEG-ceramides.

[0869] Cationic lipids

[0870] Examples of cationic lipids include cationic HEDC (2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethan-aminium bromide), as described in US 2013 / 0330401 A1. Some examples of cationic lipids are given in US 2013 / 0115274 A1.

[0871] Lipid compositions

[0872] In some embodiments, the composition may contain a membrane fusion molecule, an ionizable compound, cholesterol, lipids DOPC and DOPE, and DPPE-mPEG. In some embodiments, the membrane fusion molecule may be 1-20 mol% of the composition, the ionizable molecule may be 15-25 mol% of the composition; the cholesterol, DOPC, and DOPE combined may be 75-85 mol% of the composition; and the DPPE-mPEG may be 2-5 mol% of the composition.

[0873] In one embodiment, the membrane fusion molecule may be 2 mol% of the composition, the ionizable molecule may be 24 mol% of the composition, cholesterol may be 29 mol% of the composition, DOPC may be 20 mol% of the composition, DOPE may be 20 mol% of the composition, and DPPE-mPEG(2000) may be 5 mol% of the composition.

[0874] Nanoparticles

[0875] Embodiments of the present invention can provide liposome nanoparticle compositions. The membrane fusion molecules of the present invention can be used to form liposome compositions having a bilayer structure of one or more lipid-like molecules.

[0876] The nanoparticle composition may contain one or more membrane fusion molecules of the present invention in a liposome structure, bilayer structure, microcapsule, layered structure, or mixture thereof.

[0877] In some embodiments, the composition may comprise one or more liquid carrier components. The liquid vehicle suitable for delivering the active agent of the present invention may be a pharmaceutically acceptable liquid carrier. The liquid carrier may comprise an organic solvent, or a combination of water and an organic solvent.

[0878] Embodiments of the present invention can provide lipid nanoparticles having a size of 10 to 1000 nm. In some embodiments, the liposome nanoparticles may have a size of 10 to 150 nm.

[0879] Pharmaceutical compositions

[0880] The present invention also relates to a method for dispensing an active agent to an organ of a subject to treat fibrosis, wherein the composition of the present invention is administered to the subject. Treatable organs include the lungs, liver, pancreas, kidneys, colon, heart, bone marrow, skin, intestines, brain, and eyes.

[0881] In some embodiments, the present invention provides a method for treating pulmonary fibrosis, wherein the composition of the present invention is applied to a subject.

[0882] Examples of fibrotic diseases include idiopathic pulmonary fibrosis and cirrhosis of the liver.

[0883] In other respects, the present invention provides a series of pharmaceutical formulations.

[0884] The pharmaceutical formulations described herein may include active agents, pharmaceutical carriers, or lipids of the present invention, and pharmaceutically acceptable carriers or diluents.

[0885] Generally, the active agents described in this specification include siRNA, active agents for fibrosis, and any small molecule drugs. The active agent can be a nucleic acid, siRNA, mRNA, or microRNA.

[0886] The pharmaceutical formulations described herein may contain one or more of the following: surfactants, diluents, excipients, preservatives, stabilizers, dyes, and suspending agents.

[0887] Some of the drug carriers, diluents and components used in pharmaceutical formulations, as well as methods for formulating and administering the compounds and compositions of the present invention, are described in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Penn. (1990).

[0888] Examples of preservatives include sodium benzoate, ascorbic acid, and esters of p-hydroxybenzoic acid.

[0889] Examples of surfactants include alcohols, esters, and sulfated fatty alcohols.

[0890] Examples of excipients include sucrose, glucose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium aluminum silicate, synthetic aluminum silicate, calcium carbonate, sodium acid carbonate, calcium hydrogen phosphate, and calcium carboxymethyl cellulose.

[0891] Examples of suspending agents include coconut oil, olive oil, sesame oil, peanut oil, soybean oil, cellulose acetate, methyl acetate-methacrylate copolymer, and phthalates.

[0892] Structure of the molecular tail

[0893] The compounds of the present invention may have one or more lipophilic tails containing one or more alkyl or alkenyl groups. Examples of lipophilic tails containing alkenyl groups include C(14:1(5))alkenyl, C(14:1(9))alkenyl, C(16:1(7))alkenyl, C(16:1(9))alkenyl, C(18:1(3))alkenyl, C(18:1(5))alkenyl, C(18:1(7))alkenyl, C(18:1(9 ... 11)) alkenyl, C(18:1(12)) alkenyl, C(18:2(9,12)) alkenyl, C(18:2(9,11)) alkenyl, C(18:3(9,12,15)) alkenyl, C(18:3(6,9,12)) alkenyl, C(18:3(9,11,13)) alkenyl, C(18:4(6,9,12,15)) alkenyl, C(18:4(9,11,13,15))alkenyl, C(20:1(9))alkenyl, C(20:1(11))alkenyl, C(20:2(8,11))alkenyl, C(20:2(5,8))alkenyl, C(20:2(11,14))alkenyl, C(20:3(5,8,11))alkenyl, C(20:4(5 The following are examples of tail structures: C(20:4(7,10,13,16)), C(20:5(5,8,11,14,17)), C(20:6(4,7,10,13,16,19)), C(22:1(9)), C(22:1(13)), and C(24:1(9)). Some examples of tail structures can be found in Donald Voet and Judith Voet, Biochemistry, 3rd Edition (2005), p. 383.

[0894] Some examples of lipophilic tails include the following structures:

[0895]

[0896] Any of these example structures with lipophilic tails may have one or more other chemical branches.

[0897] Other embodiments

[0898] Embodiments of the present invention further include:

[0899] Compounds with chemical formula (A),

[0900]

[0901] The connector is a divalent group containing the PEG moiety.

[0902] X1 and X2 are independently C1-C5 alkyl dimethyl groups.

[0903] R1, R2, R3, and R4 are independent

[0904]

[0905] X3 is a single bond, a C1-C5 alkyl diester, or a C2-C5 olefin diester; X4 and X5 are independently C2-5 alkyl diesters.

[0906] Z1, Z2, and Z3 are independently -O-, -S-, or -NH-, and R5 and R6 are independently C11-23 alkyl or C11-23 alkenyl. Compounds of formula (B),

[0907]

[0908] The connector is a divalent group containing the PEG moiety.

[0909] X6 and X7 are independently C1-C5 alkyl dimethyl groups.

[0910] X8 and X9 are independently C1-C5 alkyl dimethyl groups.

[0911] Z4 and Z5 are independently -O-, -S-, or -NH-.

[0912] R7, R8, R9 and R 10 Independently

[0913]

[0914] X4 and X5 are independently C2-5 alkyl dimethyl groups.

[0915] Z2 and Z3 are independently -O-, -S-, or -NH-, and

[0916] R5 and R6 are independently C11-23 alkyl or C11-23 alkenyl.

[0917] The above-mentioned compound, wherein the connector is

[0918]

[0919] Where m is an integer from 1 to 12,

[0920] Y1 is -O-, -NH-, or -NHCH2-.

[0921] Y2 is -O-, -NH-, or -CH2NH-.

[0922] n and q are independent integers from 1 to 5.

[0923] p is an integer between 0 and 5.

[0924] Y3 and Y5 are independently -O-, -NH-, or -NHCH2-, and

[0925] Y4 and Y6 are independently -O-, -NH-, or -CH2NH-.

[0926] The above compounds, wherein X1 and X2 are independently C1-C5 straight-chain alkyldiyl, preferably C2-C4 straight-chain alkyldiyl, and more preferably C4 straight-chain alkyldiyl.

[0927] In the above compounds, R1, R2, R3, and R4 are the same functional groups.

[0928] In the above compounds, X3 is a single bond or a C1-C5 straight-chain alkyl diester, preferably a C2-C4 straight-chain alkyl diester, and more preferably an ethylene, i.e. an ethane diester.

[0929] In the above compounds, X4 and X5 are independently C2-5 straight-chain alkyldiyl groups, preferably C2-4 straight-chain alkyldiyl groups, and more preferably ethylene groups, i.e. ethanediyl groups.

[0930] The above compounds, wherein Z1 is -NH-.

[0931] The above compounds, wherein Z2 and Z3 are -O-.

[0932] The above compounds, wherein R5 and R6 are independently C11-23 straight-chain alkenyl groups.

[0933] The above compounds, wherein R5 and R6 are independently C11-23 straight-chain alkenyl groups containing 1-6 double bonds, wherein the number of double bonds is preferably 1-3, more preferably 2-3, and even more preferably 2.

[0934] The above compounds, wherein R5 and R6 are independently C11-23 straight-chain alkenyl groups containing two double bonds.

[0935] In the above compounds, R5 and R6 are independently C13-17 straight-chain alkenyl groups, preferably C15-17 straight-chain alkenyl groups, and more preferably C17 straight-chain alkenyl groups.

[0936] The above compounds, wherein R5 and R6 are independently C17 straight-chain alkenyl groups.

[0937] In the above compounds, R5 and R6 are heptadec-8,11-dienyl.

[0938] A composition comprising cationic lipids, ionizable lipids, and lipids of the aforementioned compounds in lipid nanoparticles containing bilayer lipid molecules.

[0939] The above composition also contains nucleic acids.

[0940] The above composition contains nucleic acids that are siRNA, mRNA, or microRNA.

[0941] The above-mentioned composition, wherein the composition is a pharmaceutical composition.

[0942] Chemical definitions

[0943] As used herein, the term "alkyl" refers to a hydrocarbon group of a saturated aliphatic group, which can be of any length unless otherwise specified. An alkyl group can be a branched or unbranched, substituted or unsubstituted aliphatic group containing 1 to 22 carbon atoms. This definition also applies to the alkyl portion of other groups, such as cycloalkyl, alkoxy, alkanoyl, and aralkyl groups.

[0944] As used herein, for example, the term “C(1-5)alkyl” includes C(1)alkyl, C(2)alkyl, C(3)alkyl, C(4)alkyl, and C(5)alkyl. Similarly, for example, the term “C(3-22)alkyl” includes C(1)alkyl, C(2)alkyl, C(3)alkyl, C(4)alkyl, C(5)alkyl, C(6)alkyl, C(7)alkyl, C(8)alkyl, C(9)alkyl, C(10)alkyl, C(11)alkyl, C(12)alkyl, C(13)alkyl, C(14)alkyl, C(15)alkyl, C(16)alkyl, C(17)alkyl, C(18)alkyl, C(19)alkyl, C(20)alkyl, C(21)alkyl, and C(22)alkyl.

[0945] As used herein, alkyl groups can be named using terms such as Me (methyl, -CH3), Et (ethyl, -CH2CH3), Pr (arbitrary propyl). n Pr(n-Pr, n-propyl), i Pr (i-Pr, isopropyl), Bu (any butyl), n Bu(n-Bu, n-butyl), i Bu(i-Bu, isobutyl), s Bu (s-Bu, sec-butyl) and t Bu(t-Bu, tert-butyl).

[0946] As used herein, the term "alkenyl" refers to a hydrocarbon group having at least one carbon-carbon double bond. An alkenyl group can be a branched or unbranched, substituted or unsubstituted hydrocarbon group having 2 to 22 carbon atoms and at least one carbon-carbon double bond. An "alkenyl" group has one or more carbon-carbon double bonds.

[0947] As used herein, the term "substituted" refers to atoms having one or more substitutions or substituents, which may be the same or different, and may include hydrogen substituents. Thus, for example, the terms alkyl, cycloalkyl, alkenyl, alkoxy, alkanoyl, and aryl refer to groups that may include variations in substitution. Variations in substitution include linear, branched, and cyclic variations, and groups having substituents that replace one or more hydrogen atoms bonded to any carbon atom of the group.

[0948] Generally, a compound may contain one or more chiral centers. Compounds containing one or more chiral centers may include those described as “isomers,” “stereoisomers,” “diastereomers,” “enantiomers,” “optical isomers,” or “racemic mixtures.” Conventions for stereochemical nomenclature (e.g., the stereoisomer nomenclature rules of Cahn, Ingold, and Prelog) and methods for determining stereochemistry and isolating stereoisomers are known in the art. See, for example, Michael B. Smith and Jerry March, March's Advanced Organic Chemistry, 5th edition, 2001. The compounds and structures (including chemical figures) of this disclosure are intended to cover all possible isomers, chemically plausible positional isomers, stereoisomers, diastereomers, enantiomers, and / or optical isomers, including any of their compounds, racemates, or others, that are understood to exist for a particular compound or structure.

[0949] This invention covers any and all tautomers, solvated or desolvated, hydrated or dehydrated forms, and any atomic isotopic forms of the compounds and compositions disclosed herein.

[0950] This invention covers any and all crystal polymorphisms or different crystal forms of the compounds and compositions disclosed herein.

[0951] Abbreviations used:

[0952] DMAP–4-N,N-dimethylaminopyridine

[0953] DCM – dichloromethane

[0954] TEA – Triethylamine

[0955] EDC–1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0956] Na2SO4 – Sodium sulfate

[0957] EtOAc – Ethyl acetate

[0958] DMF–N,N-dimethylformamide

[0959] ELSD – Evaporative Light Scattering Detector

[0960] NaCl – Sodium chloride

[0961] K2CO3 – Potassium carbonate

[0962] MeOH – Methanol

[0963] TFA – Trifluoroacetic acid

[0964] DIEA–N,N-Diisopropylethylamine

[0965] PEG – Polyethylene glycol, also known as polyethylene oxide

[0966] MgSO4 – Magnesium sulfate

[0967] LCMS – Liquid Chromatography-Mass Spectrometry

[0968] NaHCO3 – Sodium bicarbonate

[0969] H2O – water

[0970] HCl – hydrochloric acid

[0971] KI – Potassium Iodide

[0972] DMSO – dimethyl sulfoxide

[0973] TBAF – Tetra-n-Butylammonium Fluoride

[0974] NaBH4 – Sodium borohydride

[0975] THF – Tetrahydrofuran

[0976] TBDMS – tert-butyldimethylsilyl

[0977] LiOH – Lithium hydroxide

[0978] MeI – Iodomethane

[0979] BOC – tert-Butyloxycarbonyl

[0980] Fmoc–fluorenemethyloxycarbonyl Example

[0981] Example 1: The membrane fusion molecule of the present invention can be used to deliver one or more bioactive agents into cells. This example demonstrates that the membrane fusion molecule of the present invention provides remarkable active delivery for gene knockdown of an example siRNA targeting HSP47. The example siRNA is delivered in a liposome formulation containing the membrane fusion molecule. The presence of the membrane fusion molecule of the present invention in the delivery formulation surprisingly provides high activity for the formulation used to achieve gene knockdown via the example siRNA.

[0982] The following experimental procedure was followed to measure the in vitro activity of gene expression knockdown using siRNA in DMEM medium using rat astrocytes: One day before transfection, 100 μl of medium containing 10% FBS was used at a concentration of 3 × 10⁶ cells per well. 3 Seed cells into 96-well plates and incubate at 37°C in a humidified atmosphere with 5% CO2. Replace the culture medium with 90 μl of antibiotic-free medium before transfection. Prepare an appropriate dilution of the solution collected from the tubes in PBS solution, such that 10 μl is added to each well to achieve the desired concentration. 48 hours after transfection, wash cells once with ice-cold PBS. Lyse cells at room temperature with 50 μl of Cell-to-Ct lysis buffer for 5–30 minutes. Add 5 μl of stop solution and incubate at room temperature for 2 minutes. Measure mRNA levels immediately using TAQMAN via qPCR. Alternatively, samples can be frozen at -80°C and measured later. For qRT-PCR assays: Thaw all reagents on ice. Mix the combined reagents in 0.2 ml PCR tubes. Dispense the prepared mixture into 384-well plates, 10 μl / well × 3. Seal the plate with a film and twist the mixture down to the bottom of the well. Perform qRT-PCR assays.

[0983] In this embodiment, HSP47 siRNA was used to knock down the HSP47 gene. For TaqMan gene expression detection, an HSP47 gene-specific TaqMan probe was used.

[0984] The results of in vitro gene silencing activity in rat stellate cells are shown in Tables 3 and 4.

[0985] Table 3: In vitro activity in rat stellate cells

[0986]

[0987] Table 4: In vitro activity in rat stellate cells

[0988]

[0989] These data demonstrate that siRNA formulations containing the membrane fusion molecule of the present invention are remarkably effective in delivering active siRNA reagents into cells. This indicates that the membrane fusion molecule of the present invention provides remarkable active delivery for gene knockdown of example siRNAs targeting HSP47.

[0990] Example 2: A series of membrane fusion molecules of the present invention have demonstrated their suitability for delivering active agents into cells. This example shows that a series of membrane fusion molecules of the present invention provide remarkable active delivery for gene knockdown of an example siRNA targeting HSP47. The example siRNA was delivered in a liposomal formulation containing the membrane fusion molecules.

[0991] The in vitro gene expression knockdown activity measured in astrocytes is shown in Table 5, using example siRNAs in liposome formulations containing various membrane fusion molecules of the present invention.

[0992] Table 5: HSP47 expression % in stellate cells

[0993]

[0994] As shown in Table 5, the presence of the membrane fusion molecule of the present invention in the liposome delivery formulation surprisingly provides high activity for the formulation used to achieve gene expression knockdown via example siRNA.

[0995] Example 3: The membrane fusion molecule of the present invention is remarkably effective in increasing the intracellular delivery activity of active agents. The activity of reagents delivered in liposome formulations containing the membrane fusion molecule of the present invention is greatly increased compared to the activity of liposome formulations that do not contain the membrane fusion molecule of the present invention.

[0996] In this embodiment, the use of a liposome delivery formulation containing the compound R4 of the present invention surprisingly increased the gene expression knockdown activity of an example siRNA targeting HSP47.

[0997] The liposome delivery formulation was prepared according to the following procedure: HEDC (2-(bis(2-(tetradecyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethylammonium bromide) and S104 (((2-((2-(2-(dimethylamino)ethyl)thio)acetyl)ureido)bis(ethane-2,1-diyl)bistetradecanoate) were dissolved in anhydrous EtOH (200 proof) at a molar ratio of 1:1. HSP47 siRNA was dissolved in 50 mM citrate buffer, and the temperature was adjusted to 35-40 °C. Then, an ethanol / lipid mixture was added to the siRNA-containing buffer, and the mixture was stirred to allow spontaneous formation of siRNA-loaded liposomes. The lipid-siRNA ratio was adjusted to a final total lipid to siRNA ratio of 5:1 to 15:1 (wt:wt). The siRNA-loaded liposomes were dialyzed against 10 × volume PBS (pH 7.2) to remove ethanol and replace the buffer. The final product is filtered through a 0.22μm sterile-grade PES filter to reduce bioburden.

[0998] In this embodiment, the in vitro activity of gene expression knockdown using the example siRNA in rat stellate cells was performed in the same manner as in Example 1. The results are shown in... Figure 46 The liposome formulation contains lipids HEDC and S104, as well as membrane fusion compounds.

[0999] exist Figure 46 Among them, the results of the liposome formulation containing lipids HEDC and S104 but not containing membrane fusion compounds were indicated as (1). The HSP47 gene expression knockdown of the control formulation was only sufficient at the highest concentration of 300 nm.

[1000] exist Figure 46 In this study, the results of a liposome formulation containing lipids HEDC and S104 and containing the membrane fusion compound R4 were indicated as (2). The amount of membrane fusion compound R4 in this formulation was 2% of the total lipids. Compared with the control formulation that did not contain membrane fusion compound R4, the HSP47 gene expression knockdown of this formulation was significantly and dramatically increased at all siRNA concentrations. Therefore, the presence of membrane fusion compound R4 in the formulation greatly increased the delivery activity of the example siRNA, and the increased activity was directly attributed to the presence of membrane fusion compound R4.

[1001] exist Figure 46 Among them, the results of the liposome formulation containing lipids HEDC and S104 and containing membrane fusion compound R4 are indicated as (3). The amount of membrane fusion compound R4 in this formulation is 10% of the total lipids. Compared with the control formulation that does not contain membrane fusion compound R4, the HSP47 gene expression knockdown of this formulation is significantly and dramatically increased at all siRNA concentrations. Therefore, the presence of membrane fusion compound R4 in the formulation greatly increases the delivery activity of the example siRNA, and the increased activity is directly attributed to the presence of membrane fusion compound R4.

[1002] like Figure 46 As shown, for liposome delivery formulations containing lipids HEDC and S104, the gene expression knockdown activity of an example siRNA targeting HSP47 in stellate cells was surprisingly increased in formulations containing 2–10% (total lipids) of the membrane fusion compound R4.

[1003] Example 4: The membrane fusion molecule of the present invention results in a remarkable increase in the intracellular activity of active nucleic acid reagents. The activity of nucleic acid reagents delivered in liposome formulations containing the membrane fusion molecule of the present invention is significantly increased compared to the activity of liposome formulations that do not contain the membrane fusion molecule of the present invention.

[1004] In this embodiment, the in vitro activity of gene expression knockdown using the example siRNA in rat stellate cells was performed in a manner similar to that in Example 1. The results are shown in... Figure 47 The liposome formulation contains lipids HEDC and S104, as well as membrane fusion compounds.

[1005] exist Figure 47 In the study, the results of a liposomal formulation containing lipids HEDC and S104 but not any membrane fusion compounds of the present invention were indicated as (1). HSP47 gene expression knockdown in this control formulation was only sufficient at the highest concentration of 300 nm siRNA.

[1006] exist Figure 47 In this study, the results of a liposome formulation containing lipids HEDC and S104 and containing the membrane fusion compound R4 were indicated as (2). The amount of membrane fusion compound R4 in this formulation was 2% of the total lipids. Compared with the control formulation that did not contain membrane fusion compound R4, the HSP47 gene expression knockdown of this formulation was significantly and dramatically increased at all siRNA concentrations. Therefore, the presence of membrane fusion compound R4 in the formulation greatly increased the delivery activity of the example siRNA, and the increased activity was directly attributed to the presence of membrane fusion compound R4.

[1007] exist Figure 47 Among them, the results of the liposome formulation containing lipids HEDC and S104 and containing the membrane fusion compound T3 are indicated as (3). The amount of membrane fusion compound T3 in this formulation is 2% of the total lipids. Compared with the control formulation that does not contain membrane fusion compound T3, the HSP47 gene expression knockdown of this formulation was significantly and dramatically increased at all siRNA concentrations. Therefore, the presence of membrane fusion compound T3 in the formulation greatly increased the delivery activity of the example siRNA, and the increased activity is directly attributed to the presence of membrane fusion compound T3.

[1008] Example 5: The membrane fusion molecule of the present invention is effective for the in vitro delivery of one or more bioactive molecules. For example, due to the presence of the membrane fusion compound of the present invention in the liposome delivery formulation, the activity of gene expression knockdown using siRNA (HSP47 siRNA, see Example 1) is surprisingly increased.

[1009] In this embodiment, liposome delivery formulations of the comparative compound and membrane fusion compound R4 were prepared. The liposome delivery formulations were prepared in the same manner as in Example 1, and the compositions are shown in Table 6 (CH refers to cholesterol).

[1010] Table 6: Membrane-fused liposome formulations

[1011]

[1012] like Figure 24 As shown, for the liposome delivery formulations in Table 6, compared to the control formulations without the membrane fusion compound of the present invention, the gene knockdown activity of the example siRNA targeting HSP47 in stellate cells was remarkably increased in the formulation containing 2% (of total lipids) of the membrane fusion compound R4 of the present invention. The structure of the membrane fusion compound R4 of the present invention provides remarkable delivery activity for gene knockdown of the example siRNA in stellate cells.

[1013] Example 6: The membrane-fused lipid molecules of the present invention are effective for delivering one or more bioactive molecules into cells. In this example, a liposome delivery formulation containing an example siRNA (HSP47 siRNA, see Example 1) and the membrane-fused compound of the present invention provides activity for gene expression knockdown. In this example, as shown in Table 7, liposome delivery formulations containing siRNAs of various compounds T3 to T9 (No. 1 to No. 7) were prepared, each containing 2% (of total lipids) of the membrane-fused compound of the present invention. The liposome delivery formulations were prepared in the same manner as in Example 1.

[1014] Table 7: Membrane-fused liposome formulations

[1015] Numbering Formulations 1 HEDC:S104:DOPE:CH:DMPE-PEG:T4 2 HEDC:S104:DOPE:CH:DMPE-PEG:T5 3 HEDC:S104:DOPE:CH:DMPE-PEG:T6 4 HEDC:S104:DOPE:CH:DMPE-PEG:T7 5 HEDC:S104:DOPE:CH:DMPE-PEG:T8 6 HEDC:S104:DOPE:CH:DMPE-PEG:T9 7 HEDC:S104:DOPE:CH:DMPE-PEG:T3

[1016] like Figure 25 As shown, in the activity measurements of the liposome delivery formulations of the siRNAs in Table 7, the formulations provided high activity for gene expression knockdown in astrocytes. Therefore, the formulation containing 2% (of total lipids) of the membrane fusion compound of the present invention provides high activity of siRNA targeting HSP47.

[1017] Example 7: In vivo activity of the membrane fusion formulation. The membrane fusion molecules of the present invention can be used for in vivo delivery of active agents. For example, due to the presence of the membrane fusion compounds of the present invention, liposome delivery formulations for gene expression knockdown using example siRNAs targeting HSP47 are effective.

[1018] like Figure 26 As shown, using siRNA targeting HSP47, the liposome-delivered formulation exhibited gene expression knockdown activity in vivo (mice). The formulation contains 2% (of total lipids) of the membrane fusion compound specified in this invention. The formulation was delivered via rapid infusion bolus. Delivery parameters are shown in Table 8.

[1019] Table 8: Membrane-fused liposome formulations in vivo

[1020]

[1021] The following is an example of the procedure used for the above results.

[1022] 1.1. Animals

[1023] Eighty male Sprague-Dawley rats, aged 49 days, were purchased from Charles River Laboratories and transported to the laboratory. At arrival, the animals weighed an average of approximately 200-210 grams. The animals were housed in a standard cage system, two rats per cage, on an alternating 12-hour day / night cycle. The room temperature was maintained at 64-79°F (18-26°C) and humidity at 30-70%, with at least 10 air changes per hour using 100% fresh air, without repeated recirculation. Fresh, radiation-sterilized, qualified standard rat food and tap water were provided for the animals to consume freely.

[1024] From day 0 to 5, 72 animals were treated with DMN and then randomly divided into 9 groups of 8 rats each based on their body weight, ensuring no significant difference in body weight between groups before siRNA treatment. Validation was performed using one-sided ANOVA analysis. As expected, the DMN-treated animals exhibited significantly lower body weights than the untreated animals.

[1025] 1.2. DMN treatment

[1026] DMN was obtained from Wako (batch number DSP2369) and prepared as a formulation for intraperitoneal (IP) injection by dissolving the compound in phosphate-buffered saline (PBS) at a concentration of 5 mg / mL. From day 0 to day 2, 10 mg / kg DMN was administered daily to 72 rats at a dose volume of 2 mL / kg. Then, from day 3 to day 5, 5 mg / kg DMN was administered daily at a dose volume of 1 mL / kg. Eight animals not treated with DMN served as blank controls for this procedure. Animals were weighed daily, and the DMN dose was adjusted accordingly.

[1027] 1.3. siRNA treatment

[1028] On day 5 of the experiment, animals were assigned to different treatment groups and administered drugs according to appropriate dosing regimens. The experimental substance was administered to treatment groups 2 through 8 (#1 through #8); while animals in group 1 (#1) received a single intravenous injection of 3 mL / kg of the carrier (physiological saline) via the caudal vein. Animals in group 9 (the original group, #9) received no treatment.

[1029] 1.4. Euthanasia and necropsy

[1030] On day 6 of the experiment, 24 hours after treatment, the animals were euthanized by excessive carbon dioxide inhalation. The liver was immediately flushed via the portal vein with PBS (40 mL, 20 mL / min) at pH 7.4 to remove residual blood and blood-related preparations. A 2 mm thick transverse section of the liver was collected from the left lateral lobe and immediately immersed in 2 mL of RNAlater in a microcentrifuge tube. The sample was stored at 4°C until further processing for RNA isolation.

[1031] 1.6. RNA analysis

[1032] HSP47 mRNA abundance in liver samples was assessed. Total RNA was extracted using an RNeasy column (Qiagen) according to the manufacturer's instructions. RNA concentration for each sample was quantified using a Nanodrop spectrophotometer and then diluted to 10 ng / μl with nuclease-free water. 20 ng of total RNA was used for each PCR reaction. In summary, total RNA was extracted from left lobe sections of liver tissue using an RNeasy column (Qiagen) according to the manufacturer's instructions. RNA quantification was performed using a Nanodrop spectrophotometer. RNA was adjusted to 10 ng / μl with nuclease-free water. Real-time PCR was performed on a ViiA7 system in 96-well configuration. Measurements were repeated three times for each sample using TaqMan Gene Expression Master Mix. The cycling program was set to 48 °C for 15 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 1 min. Raw cycle threshold data and calculated ΔCt values ​​were normalized using the mean cycle threshold of the housekeeping gene MRP119. The ΔCt of each target gene (GOI) was calculated by subtracting the average ΔCt of the GOI in the control samples from the ΔCt of each target gene (GOI) in the target samples. Data for each animal were presented both as a percentage of the mean load-treated group and as a fold change relative to the naive group. Differences between the siRNA-treated and load-treated groups were analyzed using one-sided ANOVA followed by Dunnett's post-hoc multiple comparisons. For all analyses, a p-value less than 0.05 was considered significant.

[1033] Example 8: Preparation of mRNA nanoparticles. Cationic lipids such as HEDC or HE2DC (2-(bis(2-(palmitoyloxy)ethyl)amino)-N,N-bis(2-hydroxyethyl)-N-methyl-2-oxoethane-1-ammonium bromide), ionizable lipids such as S104 or TU104, Dlin ((9Z,9′Z,12Z,12′Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)ureidyl)bis(ethane-2,1-diyl)bis(octadec-9,12-dienoate)), and auxiliary lipids cholesterol and DOPE were dissolved in ethanol. mRNA was dissolved in 50 mM citrate buffer (pH 3.5). An appropriate amount of the ethanol solution of lipids was injected into the citrate buffer containing mRNA at a flow rate of 25 mL / min at 37 °C, thereby preparing lipid nanoparticles (LNPs). The molar percentages of the LNP composition are 20% HEDC, 20% S104, 30% DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine) (Avanti Polar Lipid), 25% cholesterol (ultrapure grade) (Wilshire Technologies), 5% DMPE-Peg (1,2-dimyristicoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) (ammonium salt), and 2% of the membrane fusion molecule of the present invention (e.g., compound T3).

[1034] High-speed injection resulted in highly encapsulated mRNA and a controlled particle size distribution. The mixed LNP solution was then diluted with 20 mM HEPES buffer and 9% sucrose (w / v) at a 1:1 ratio, reducing the ethanol content from 35% to 17.5%. The diluted LNP solution was transferred to a tangential flow filtration (TFF) step for ultrafiltration and dialyzed against 20 mM HEPES buffer in 9% sucrose (w / v). To remove ethanol from the LNP solution, a total of 10 dialysate volumes of HEPES buffer in 9% sucrose were used. The TFF-concentrated LNP solution was aliquoted into EMD Millipore flasks for further centrifugation; however, this step is only necessary for small-scale batches. After final concentration, the LNP was filtered through a 0.2 μm filter. Encapsulation efficiency and mRNA yield were calculated using the RiboGreen method.

[1035] Example 9: In vitro transfection and delivery of mRNA to cells. In vitro transfection of cells with mRNA was performed in three different cell lines: Hek-293, A549, and lung fibroblasts. JET MESSENGER (Polyplus Transfection Company) was used as a positive transfection control. GFP mRNA (TriLink), encapsulated in the LNP nanoparticles of this invention or mixed with the JET MESSENGER control, was used to transfect cells at different concentrations. Cells were observed under a confocal microscope 24, 48, and 72 hours after transfection, and the fluorescence produced by GFP expressed in the cells was detected and captured. When using the LNP nanoparticles of this invention, GFP mRNA was well transfected into all three cell lines, and the mRNA was translated.

[1036] Example 10: In vivo transfection and delivery of mRNA to cells. mRNA was transfected into tissues and cells in vivo using the LNP nanoparticles of this invention. Two different mRNAs of different sizes, GFP mRNA, and luciferase mRNA (TriLink) were delivered and transfected into Balb / c mice. In some luciferase mRNA delivery studies, Viromer's in vivo mRNA transfection reagent was also used as a positive control for mRNA delivery. Animals were given a single intravenous injection of mRNA encapsulated in the LNP nanoparticles or positive control particles of this invention at doses of 1.0, 2.0, or 4.0 mg / kg. Six to eight hours after mRNA injection, mice were anesthetized, and whole-body fluorescence was detected and analyzed using an IVIS system to conduct luciferase mRNA delivery studies. Animals were then immediately sacrificed, different organs were harvested, and stored at -80°C until further analysis. In GFP mRNA delivery studies, the MAXDISCOVER GFP ELISA kit was used to determine mRNA delivery to various tissues and cells to analyze GFP protein levels in tissues. For the luciferase mRNA delivery study, tissues were homogenized in CCLR lysis buffer and centrifuged. The resulting supernatant was used for luciferase activity assays using the Promega E4510 assay kit. Surprisingly, both GFP mRNA and luciferase mRNA were primarily transfected and / or translated in the lungs and spleen, with much lower transfection and / or translation rates in other tissues.

[1037] Example 11: In vitro transfection and delivery of mRNA to cells.

[1038] According to the methods described in Examples 9 and 10 above, GFP mRNA (CleanCap EGF P mRNA, 5 molU) of the present invention, comprising the composition HEDC:S104:CH:DOPE:DMPE-PEG2000:compound T3, was transfected into A549 cells in vitro. Results 48 hours after transfection are shown in... Figure 48 Cells were observed under a confocal microscope, and the fluorescence produced by GFP expressed in the cells was then detected. Results showed that GFP mRNA was transfected into A549 cells and translated therein.

[1039] Example 12: In vivo transfection and delivery of mRNA to cells.

[1040] According to the methods described in Examples 9 and 10 above, GFP mRNA (CleanCap EGFP mRNA, 5 molU) was transfected into Balb / c mice using LNP nanoparticles of the present invention having the composition HEDC:S104:CH:DOPE:DMPE-PEG2000:compound T3, as shown in Table 9:

[1041] Table 9: In vivo transfection in Balb / c mice

[1042]

[1043] like Figure 49 As shown, the delivery of mRNA in various tissues and cells was determined using the MAXDISCOVER GFP ELISA. Surprisingly, GFP mRNA was selectively transfected and / or translated in the lungs, while transfection and / or translation were lower in muscle, liver, heart, and kidneys.

[1044] Example 13: In vivo transfection and delivery of mRNA to cells.

[1045] According to the method described in Examples 9 and 10 above, luciferase mRNA (Fluc mRNA(5meC)) was transfected into Balb / c mice using LNP nanoparticles of the present invention having the composition HEDC:S104:CH:DOPE:DMPE-PEG2000:compound T3, as shown in Table 10.

[1046] Table 10: In vivo transfection in Balb / c mice

[1047]

[1048] like Figure 50As shown, the relative delivery, transfection, and / or translation of mRNA in various tissues and cells were determined using the Promega E4510 assay kit. Surprisingly, Fluc mRNA was selectively delivered, transfected, and / or translated in the lungs and spleen, while delivery, transfection, and / or translation were lower in the liver, heart, kidneys, and muscle.

[1049] Example 14: In vivo transfection and delivery of mRNA to cells.

[1050] According to the methods described in Examples 9 and 10 above, luciferase mRNA (Fluc mRNA (5meC)) was transfected into Balb / c mice using LNP nanoparticles of the present invention having the following composition:

[1051] (-01)HE2DC:S104:CH:DOPE:DMPE-PEG2000:compound T3, or

[1052] (-02)HEDC:S104:CH:DOPE:DMPE-PEG2000:Compound T3,

[1053] Inject 2 mpk and image using autofluorescence 7 hours after injection.

[1054] like Figure 51 As shown, the relative delivery, transfection, and / or translation of mRNA in various tissues and cells were determined using a Promega E4510 assay. Surprisingly, Fluc mRNA was selectively delivered, transfected, and / or translated in the lungs and spleen, while delivery, transfection, and / or translation were lower in the pancreas, kidneys, liver, testes, and small intestine.

[1055] like Figure 52 As shown, autofluorescence imaging was used to determine the relative delivery, transfection, and / or translation of mRNA in various tissues 7 hours after injection. Figure 52 In the image, the top column shows Balb / c mice transfected with (-01) LNP nanoparticles. The bottom column shows Balb / c mice transfected with (-02) LNP nanoparticles.

[1056] Example 15: In vivo delivery of mRNA to cells using membrane fusion compounds. The membrane fusion compounds of the present invention greatly enhance the delivery of active agents to cells, organs, and tissues in vivo.

[1057] In this embodiment, the formulation for in vivo delivery of mRNA was prepared using the membrane fusion compound T3 and compared with the same formulation without the membrane fusion compound, as shown in Table 11.

[1058] Table 11: Formulations delivered in mice

[1059]

[1060] Figure 53 The results of delivering luciferase mRNA in mice using the membrane-fused lipid-like molecule of the present invention are shown. Figure 53 As shown, in the formulation containing the membrane fusion molecule of the present invention (2035-03-03), the relative delivery of mRNA was significantly higher than in the same formulation without the membrane fusion molecule (2035-13-01). In all organs observed (including the pancreas, spleen, liver, kidney, lung, testis, and intestine), the delivery of the formulation containing the membrane fusion compound T3 was advantageously and surprisingly higher.

[1061] The embodiments described herein are not limiting, and those skilled in the art will readily understand that the specific combinations of modifications described herein can be tested without excessive experimentation to identify nucleic acid molecules with improved RNAi activity.

[1062] All publications, patents and documents specifically mentioned in this article are incorporated herein by reference for all purposes.

[1063] It is understood that the present invention is not limited to the specific methods, procedures, materials, and reagents described herein, all of which are subject to change. It is also understood that the terminology used herein is merely for describing particular embodiments and is not intended to limit the scope of the invention. It will be apparent to those skilled in the art that substitutions and modifications can be made to the description disclosed herein without departing from the scope and spirit of the specification, and such embodiments also fall within the scope of this specification and the appended claims.

[1064] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “this” include the plural reference unless otherwise expressly indicated by superscript or superscript. Similarly, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein. It should also be noted that the terms “comprising,” “including,” “containing,” “comprise,” and “having” are used interchangeably and should be interpreted broadly without limitation.

[1065] Unless otherwise stated herein, references to numerical ranges herein are merely intended as a convenient way to individually indicate each independent value falling within the range, and each independent value is integrated into the specification as if it were mentioned separately herein. For Markush groups, those skilled in the art will recognize that this specification includes individual members of Markush groups, as well as subgroups of members.

[1066] The compounds, molecules, or compositions of the present invention may have ionic forms that do not exhibit corresponding balance ions. Those skilled in the art will readily understand that balance ions are present where necessary. Examples of balance ions include alkali metals, Cl... - And pharmaceutically acceptable balanced ions.

[1067] For example, when a list of embodiments or components applicable to the present invention is given, such as a list of compounds, molecules or compositions, it will be apparent to those skilled in the art that mixtures of the listed compounds, molecules or compositions may also be suitable.

[1068] Without further elaboration, it is believed that those skilled in the art can utilize the present invention to the fullest extent based on the foregoing description. Therefore, the following specific embodiments are considered merely examples and are in no way intended to limit the remainder of the disclosure herein.

[1069] All features disclosed in this specification can be combined in any way. Each feature disclosed in this specification can be replaced by alternative features that serve the same, equivalent, or similar purpose.

[1070] The images in the appended claims are adapted to fit the page size, and the appearance of the molecules in the images does not necessarily reflect any obvious shape or properties of the compound.

Claims

1. A membrane fusion compound having the chemical formula VII, Each AA a Independently, it is an amino acid selected from the following structures. The amino acid is attached to an amphiphilic molecule by each of its carboxyl groups and to a linker by its N-terminus; The connector has the following structure: or Q 1 It is a branched or unbranched C(2-8) chain alkyl diester; or Q 2 yes Q 3 yes Where X is -O-, -S-, or -NH-; n and p exist independently and are 1 to 3; m is independently 1 to 10; r and s exist independently and are 1 to 5; Each amphiphilic molecule is independently selected from chemical formulas (XI) and (V), as shown below: in R 1 and R 2 for R 1 =(C=O)R 4 R 2 =(C=O)OR 5 R 4 and R 5 Each exists independently and is a C(12-20)alkyl or C(12-20)alkenyl; Where R 3 It is a missing, branched, or unbranched *-NH-C(1-8) alkyldiyl-(C=O)-, where * indicates a relationship with AA. a The end of the connection; Where R 1 and R 2 for R 1 =(C=O)OR 4 R 2 =NH(C=O)R 5 Where R 4 and R 5 Each exists independently and is a C(12-20)alkyl or C(12-20)alkenyl; in R 3 It is -O- or branched or unbranched *-NH-C(1-8) alkyl diel-(C=O)-, where * indicates that it is related to AA a The end of the connection; One or both of the amphiphilic molecules may be optionally omitted and replaced by a pharmaceutically acceptable organic chemical group having 1-400 atoms selected from alkyl, alkenyl, alkynyl, acetyl, Boc, Fmoc, TFA and CBZ, said atoms being selected from carbon, oxygen, nitrogen, sulfur, fluorine and hydrogen.

2. The membrane fusion compound of claim 1, wherein one or both of the amphiphilic molecules are missing and replaced by the pharmaceutically acceptable organic chemical group.

3. The membrane fusion compound of claim 2, wherein the pharmaceutically acceptable organic chemical group is selected from alkyl, alkenyl, alkynyl, alkyl ether, aryl ether, alkoxy, and alkoxyalkoxy.

4. The membrane fusion compound of claim 2, wherein the pharmaceutically acceptable organic chemical group is selected from methoxy, ethoxy, tert-butyl ether, and benzyloxy.

5. A composition comprising the membrane fusion compound of any one of claims 1-4 and a pharmaceutically acceptable carrier.

6. The composition of claim 5, wherein the composition comprises nanoparticles or liposomes.

7. A pharmaceutical composition comprising the membrane fusion compound of any one of claims 1-4, an active agent, and a pharmaceutically acceptable carrier.

8. The composition of claim 7, wherein the membrane fusion compound is 0.01 mol% to 20 mol% of the lipids in the composition.

9. The composition of claim 7, wherein the composition comprises nanoparticles or liposomes.

10. The composition of claim 7, wherein the active agent is one or more nucleic acids.

11. The composition of claim 7, wherein the active agent is one or more DNA, RNA, mRNA, siRNA, or microRNA.

12. The composition of claim 7, wherein the active agent is one or more RNA molecules.

13. The composition of claim 7, wherein the active agent is selected from one or more RNAi molecules, one or more mRNA molecules, and modified forms thereof.

14. The composition of claim 7, used for treating human or animal bodies.

15. A composition for dispensing an active agent in a subject to treat a condition or disease, said composition comprising an active agent, a membrane fusion compound according to any one of claims 1-4, an ionizable lipid, a structural lipid, a stabilizing lipid, and a lipid for reducing the immunogenicity of the composition.

16. The composition of claim 15, wherein the active agent is one or more nucleic acids.

Images