Multimotif dendrons and their supramolecular structures and uses thereof

Abstract

The present application includes dendrons of Formula I, compositions containing these dendrons, and their uses, particularly for the delivery of agents such as nucleic acids and drugs to cells and subjects. [Formula 1] JPEG2024543272000161.jpg23159 wherein each repeating group is the same or different.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 398,936, filed August 18, 2022, and U.S. Provisional Patent Application No. 63 / 283,588, filed November 29, 2021, the entire contents of both of which are incorporated herein by reference.

[0002] This application relates generally to the technical field of dendrons. In particular, the present invention relates to dendron nanoparticles and compositions thereof. More particularly, the present invention relates to dendron nanoparticle compositions for delivery of agents such as nucleic acids and drugs. [Background technology]

[0003] Nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), including messenger ribonucleic acid (mRNA), small interfering RNA (siRNA), microRNA (miRNA), and long noncoding RNA (lncRNA), hold immense potential for the development of new therapeutics and vaccines. Delivery of nucleic acids into cells is a direct way to affect gene expression at the cellular level. For example, mRNA has shown therapeutic potential with a wide range of applications including viral vaccines and genome editing [Pardi, N., Hogan, MJ, Porter, FW & Weissman, D., mRNA vaccines-a new era in accinology, Nat. Rev. Drug Discov. 17, 261-279, 2018; Barbier, AJ, Jiang, AY, Zhang, P., Wooster, R. & Anderson, DG, The clinical progress of mRNA vaccines and immunotherapies, Nat. Biotechnol. 1-15, 2022]. In 2021, the first mRNA vaccines, Comirnaty (BNT162b) and Spikevax (mRNA-1273), were approved to protect against COVID-19 [Corbett, KSet al., Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates, N. Engl. J. Med. 383, 1544-1555, 2020; Chaudhary, N., Weissman, D. & Whitehead, KA, mRNA vaccines for infectious diseases: principles, delivery and clinical translation, Nat. Rev. Drug Discov. 20, 817-838, 2021]. However, current materials used to sequester and deliver nucleic acids have drawbacks in terms of both manufacturing and biological effectiveness. For example, high molecular weight materials are difficult to synthesize, and delivery materials are often toxic to the body and / or trigger an inflammatory immune response.Furthermore, the co-isolation of different types of nucleic acids in a form suitable for delivery is currently not well developed. Collectively, these current limitations of delivery materials prevent widespread use of nucleic acids.

[0004] Currently, lipid nanoparticles (LNPs), proteins, modified dendrimers, and cationic nanoemulsions are used to deliver nucleic acids. For example, LNPs have been successfully used to deliver nucleic acids [AJ Geall, A. Verma, GR Otten, et al., Proceedings of the National Academy of Sciences of the United States of America 2012, 109, 14604-14609; A. Hekele, S. Bertholet, J. Archer, et al., Emerging Microbes and Infections 2013, 2]. A typical LNP contains four components: ionizable lipids, phospholipids, cholesterol, and lipid-conjugated polyethylene glycol (PEG). Ionizable lipids bind to nucleic acids during LNP formulation and act as the main driver of nucleic acid expression and immunogenicity [Hassett,KJ et al.,Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines,Mol.Ther. Nucleic Acids 15,1-11,2019;Han,X.et al.,An ionizable lipid toolbox for RNA delivery,Nat.Commun.12,1-6,2021]. These lipids are bifunctional. One end contains a protonated tertiary amine that electrostatically associates with anionic nucleic acids, and the other end has a lipid tail for nanoparticle self-assembly. However, these materials typically require a series of helper lipids such as DSPC to add charge to improve nucleic acid sequestration, resulting in complex formulations that can cause processing errors.Ionizable LNPs have a neutral charge at physiological pH, mitigating the toxicity arising from permanently cationic particles, and after cellular uptake, ionizable lipids acquire a positive charge in acidic endosomes, facilitating the release of nucleic acids into the cytosol [Heyes, J., Palmer, L., Bremner, K. & MacLachlan, I., Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids, J. Controlled Release 107, 276-287, 2005; Semple, SC et al., Rational design of cationic lipids for siRNA delivery, Nat. Biotechnol. 28, 172-176, 2010; Heyes, J., Palmer, L., Bremner, K. & MacLachlan, I., Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids, J. Controlled Release 107, 276-287, 2005].

[0005] Ionizable lipids in the literature share a series of structural features: 1) one or more tertiary amines that bind RNA at low pH; 2) multiple alkyl tails for hydrophobic self-assembly, and 3) a common "cone" shape for disrupting endosomal membranes [Cullis, PR, Hope, MJ & Tilcock, CPS, Lipid polymorphism and the roles of lipids in membranes, Chemistry and Physics of Lipids 40, 127-144, 1986; Hafez, IM, Maurer, N. & Cullis, PR, On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids, Gene Ther. 8, 1188-1196, 2001; Semple, SC et al., Rational design of cationic lipids for siRNA delivery, Nat. Biotechnol. 28, 172-176, 2010]. Careful and deliberate design of ionizable lipid structures is desirable, as small modifications in molecular structure can dramatically alter the efficacy of RNA delivery.

[0006] Crosslinked protein particles have been used to encapsulate and deliver nucleic acids. However, to enhance delivery and transfection, degradable crosslinks along with cationic lipids are required [J. Xu, JC Luft, X. Yi, et al., Molecular Pharmaceutics 2013, 10, 3366-3374.], which also leads to overly complicated formulations and multiple error-prone processing steps.

[0007] Modified dendrimers are larger molecular weight polymers that branch symmetrically from a central point in a starburst pattern [J.S. Chahal,O.F. Khan,C.L. Cooper,et al.,Proc Natl Acad Sci USA 2016,113,E4133-4142.;O.F. Khan,E.W. Zaia,S. Jhunjhunwala,et al.,Nano Lett 2015,15,3008-3016;O.F. Khan,E.W. Zaia,H. Yin,et al.,Angewandte Chemie 2014,53,14397-14401]. Because they are branched, they contain more amines for protonation and improved nucleic acid binding. Nonpolar alkanes are incorporated around these symmetrically branched polymers for self-assembly of nanoparticles. Although additional helper lipids may not be required to increase the charge content, there is significant steric hindrance from the exterior alkane groups, preventing intimate electrostatic binding with nucleic acids and incorporation of the dendrimer into the self-assembly of nanoparticles. This steric hindrance can lead to inefficient packing of the nucleic acid within the nanoparticle, leaving large nucleic acids near or on the surface of the nanoparticle, exposed and unprotected. If two or more amines are located on the same side of the core, it may be possible to grow the dendrimer asymmetrically. However, the presence of multiple amines on one side of the core rapidly leads to crowding, increasing steric hindrance and preventing the formation of higher generation dendrimers. In addition, steric hindrance also prevents the incorporation of terminal alkanes into asymmetric dendrimers. Furthermore, modified dendrimers are difficult to synthesize as single molecular species because the reaction results in multiple degrees of substitution, requiring lengthy and expensive purification. These multiple degrees of substitution produce numerous isomers that can affect performance, safety, and purity. Isomers include stereoisomers, positional isomers, and chirality, all of which are difficult to predict, isolate, and control. Additionally, modified dendrimers have a high molecular weight, which can affect their ability to be cleared from the body.When dendrimers are incorporated with degradable groups, the degradation products from these high molecular weight molecules also become large and remain difficult to remove from the body. Furthermore, the large number of amines in dendrimers adversely affects the ability to purify these materials using normal phase chromatographic separation processes, as many amines interact with the stationary phase of the chromatography column, resulting in delayed elution and multiple isomers that detrimentally co-elute without separation. This separation problem creates great difficulties when trying to test the efficacy and safety of individual isomers. Dendrimers also contain a significant amount of void space between the branches. Thus, when dendrimers are used to form nanoparticles, the nanoparticles contain a large amount of void space that cannot be filled with the nucleic acid payload. This wasted space reduces the overall efficiency of nucleic acid delivery. Furthermore, dendrimers are known to be toxic. Although terminal modifications can reduce toxicity, toxicity caused by the large amount of charge per molecule remains a drawback (see, e.g., Labieniec-Watala, M. and Watala, CJ Pharm. Sci. 2015, 104(1):2-14, and Mendes, LP et al. Molecules, 2017, 22(9):1401).

[0008] Cationic nanoemulsions are nanoparticles formed with MF59 adjuvant, with nucleic acid adsorbed on the surface [LA Brito, M. Chan, CA Shaw, et al., Molecular Therapy 2014, 22, 2118-2129; WM Bogers, H. Oostermeijer, P. Mooij, et al., Journal of Infectious Diseases 2015, 211, 947-955]. However, the mass ratio of (delivery material):(nucleic acid) is an order of magnitude higher than in lipid nanoparticle systems, which may result in a dose-limiting substance-induced effect.

[0009] Nanoparticle compositions for nucleic acid delivery comprising modified dendrimers are disclosed, for example, in PCT Patent Application Publication No. WO2020 / 132196.

[0010] Thus, there is an unmet need for new delivery materials that simultaneously have low molecular weight, the ability to load large amounts of nucleic acids and other drugs, the ability to reduce wasted internal voids, and the ability to contain multiple types of nucleic acids and / or other drugs. Summary of the Invention

[0011] Given the aforementioned limitations, considerations for an optimal delivery system include the following engineering design criteria: (1) multiple ionized charges z per delivery molecule; (2) maximizing the electrostatic force F by minimizing the distance L between the delivery charge and the nucleic acid according to the relationship F∝1 / L^2; (3) minimizing the (delivery molecule):(nucleic acid) mass ratio by maximizing the binding affinity KD; (4) optimizing the molecular packing of the delivery material through formulation conditions; and (5) incorporating molecular groups that modulate cellular responses.

[0012] Based on these design criteria, a new class of dendron-inspired branched molecules (generally shown in Figure 1) was prepared that is an ideal delivery material. These delivery materials allow separation of charged and self-assembling groups, thereby reducing steric hindrance and increasing accessibility to nucleic acids. Flexible chemical structures facilitate optimization of charge density, self-assembly, and solubility. Importantly, the molecular architecture can be tailored to modulate cellular response, degradation, clearance, and other fundamental properties.

[0013] Thus, the present application includes a dendron of formula I, or a salt and / or solvate thereof: [ka] During the ceremony, Each repeat group may be the same or different; [ka] and; n is 1, 2, 3, 4, or 5, and each L nand each X n are the same or different and are as defined below depending on the value of n; When n is 1, the dendron is a first generation dendron and the compound of formula I has the structure: [ka] In the formula, each X 1 are the same or different and are either H or a terminal group, provided that at least one X 1 are terminal groups, and each L 1 are the same or different and are linking groups; When n is 2, the dendron is a second generation dendron and the compound of formula I has the structure: [ka] In the formula, each X 2 are the same or different and are either H or a terminal group, provided that at least one X 2 are terminal groups, and each L 1 and each L 2 are the same or different and are linking groups; When n is 3, the dendron is a third generation dendron and the compound of formula I has the structure: [ka] In the formula, each X 3 are the same or different and are either H or a terminal group, provided that at least one X 3 are terminal groups, and each L 1 , each L 2 , and each L 3 are the same or different and are linking groups; When n is 4, the dendron is a fourth generation dendron and the compound of formula I has the structure: [ka] In the formula, each X4 are the same or different and are either H or a terminal group, provided that at least one X 4 are terminal groups, and each L 1 , each L 2 , each L 3 , and each L 4 are the same or different and are linking groups; When n is 5, the dendron is a fifth generation dendron and the compound of formula I has the structure: [ka] In the formula, each X 5 are the same or different and are either H or a terminal group, provided that at least one X 5 are terminal groups, and each L 1 , each L 2 , each L 3 , each L 4 , and each L 5 are the same or different and are linking groups; R 1 is C 1-20 Alkyl, C 2-20 Alkenyl, C 2 - 20 Alkynyl, C 1-20 Alkylene aryl, C 1-20 Alkylene Heteroaryl, C 1-20 Alkylene C 3-8 Heterocycloalkyl, C 1-20 Alkylene C 3-8 Cycloalkyl and C 1-6 Alkylene-SSC 1-6 alkyl, each of which is unsubstituted or selected from halo, OH, OC 1-20 Alkyl, C(O)OC 1-20 Alkyl and NR 2 R 2’ and the aryl, heteroaryl, heterocycloalkyl, and cycloalkyl groups are further optionally substituted with one or more C 1-10 is substituted with alkyl; R 2 and R 2’ are independently H and C 1-10 alkyl; The linker group is C 1-20 Alkylene or C 2-20 alkenylene, each of which is optionally S, SS, O, NH, N(C 1-4 Alkyl), C(O), C(O)O, OC(O), C(O)NH, NHC(O), NHC(NH), NHC(NC 1-4 alkyl), C(NH)NH, and C(NC 1-4 alkyl)NH, each alkyl, alkylene, and alkenylene being unsubstituted or substituted with one or more of halo and OH; The terminal group is C 1-40 Alkyl, C 2-40 Alkenyl, C 1-40 Alkylenearyl, and C 1-40 alkenylenearyl, each of alkyl, alkenyl, alkylene, and alkenylene is optionally selected from S-S, C(O), OC(O), C(O)O, OC(O)O, NR 3 C(O)O, OC(O)NR 3 , C(O)S, SC(O), NR 3 C(O), C(O)NR 3 , N.R. 3 C(O)NR 4 , and C(NC 1 - 20 alkyl), wherein alkyl, alkenyl, alkylenearyl, and alkenylenearyl are optionally interrupted by one or more groups selected from halo, NR 5 R 5’ and OH; R 3 , R 4 , R 5 , and R 5’ are independently hydrogen or C 1 - 10 is alkyl; wherein all available hydrogen atoms attached to carbon are optionally replaced with fluorine atoms.

[0014] The present application also includes nanoparticles comprising one or more dendrons of the present application, colloids comprising one or more dendrons of the present application, or supramolecular structures comprising one or more dendrons of the present application. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0015] The present application also includes compositions comprising one or more dendrons of the present application, as well as one or more nanoparticles, one or more colloids, and / or one or more supramolecular structures, each of which comprises one or more dendrons of the present application.

[0016] In some embodiments, the compositions of the present application further comprise one or more agents to be delivered to a cell or a subject.

[0017] In some embodiments, the compositions of the present application comprise one or more dendrons of the present application, one or more PEG lipids, one or more phospholipids, one or more steroids, and one or more agents to be delivered.

[0018] Also included in the application are methods of delivering one or more agents to a cell or a subject, comprising contacting the cell or subject with one or more compositions of the present application, wherein the cell or subject is contacted under conditions that cause uptake of the agents into the cell or subject.

[0019] The present application also includes kits that include one or more of the dendrons of the present application, or one or more of the compositions of the present application.

[0020] Certain embodiments of the present application will now be described in more detail with reference to the accompanying drawings. [Brief description of the drawings]

[0021] [Figure 1]1 shows a general schematic of an exemplary dendron of the present application, where each L1 is a linker group which may be the same or different, each Xn is H, a terminal group, or additional repeating LnN(Xn)2 groups which may be the same or different, where n is a generation number (e.g., 1, 2, 3, 4, or 5). The top shaded box represents the self-assembly and degradable regions of the dendron, the next shaded box represents the ionizable charge regions of the dendron for binding agents such as nucleic acids, and the bottom shaded oval is a focal group. [Diagram 2] FIG. 1 is a schematic diagram showing the contents of LNPs and their ratios, and screening of LNPs for intramuscular delivery of firefly luciferase mRNA in C57BL / 6 mice. [Diagram 3] a-e show a comparison of alkylation methods; a) Chemical structures of exemplary ionizable lipids alkylated via reductive amination (I-18) or epoxide ring-opening (I-1); b) Total luminescence flux (mean ± s.d., n = 4 hind limbs, **p < 0.01, independent and combined) in hind limb muscles of C57BL / 6 mice 6, 24, 48, and 96 hours after injection of SM-102 (●, top line), I-1 (▼, middle line), and I-18 (▲, bottom line). two-tailed t-test); c) 6-(p-toluidino)-2-naphthalenesulfonic acid (TNS) assay results for I-1 (▼), SM-102 (●), and I-18 (▲); d) nanoparticle size distribution by intensity for I-1 (▼), SM-102 (●), and I-18 (▲); and e) LNP characterization summary for SM-102, I-1, and I-18 showing the results of size distribution, polydispersity index (PDI), encapsulation efficiency (EE), and pKa. [Figure 4]a-e show a comparison of alkyl tail lengths (group X1); a) chemical structures of exemplary ionizable lipids alkylated via epoxide ring opening at the 10 (I-26) or 15 (I-25) carbon tail; b) total luminescence flux in hindlimb muscle of C57BL / 6 mice 6 h after injection of (from left to right) I-1, I-26, and I-25 (mean ± sd., n = 4 or 5, hindlimb); c) TNS assay results of I-25 (▲), I-1 (●), and I-26 (▼); d) nanoparticle size distribution of I-25 (▲), I-1 (●), and I-26 (▼); and e) summary of LNP characterization of I-1, I-26, and I-25 showing size distribution, PDI, EE, and pKa results. [Diagram 5] a-e show a comparison of linker lengths (group L1); a) chemical structure of an exemplary ionizable lipid with a 3-carbon linker (I-27); b) total luminescence flux in hindlimb muscle of C57BL / 6 mice 6 hours after I-26 (●) and I-27 (▲) injection (mean ± sd, n = 4 or 6, hindlimb, **p < 0.01, unpaired two-tailed t-test), c) TNS assay results of I-26 (●) and I-27 (▲); d) nanoparticle size distribution of I-27 (▲) and I-26 (●); and e) summary of LNP characterization of I-26 and I-27 showing size distribution, PDI, EE, and pKa results. [Figure 6]a-f show the effect of adding a hydroxyl group to the core (group R1); a) Chemical structures of exemplary ionizable lipids with an ethanolamine (I-28) or propanolamine (I-29) core; b) Total luminescence flux in hindlimb muscles of C57BL / 6 mice injected with unmodified mRNA using (from left to right) comparative compound SM-102 and exemplary I-27, I-28, and I-29 (mean ± sd, n = 8 or 10, hindlimb, **p < 0.01, *p < 0.05, ns = not significant, unpaired two-tailed t-test); c) comparative compound SM-102 (left) and exemplary I-28 (right) in hindlimb muscle of C57BL / 6 mice injected with modified mRNA (mean ± sd, n = 4, ns = not significant, unpaired two-tailed t-test); d) TNS assay results for SM-102 (●), I-27 (▲), I-28 (▼), and I-29 (◆); e) nanoparticle size distribution for SM-102 (●), I-27 (▲), I-28 (▼), and I-29; and f) LNP characterization summary for SM-102, I-27, I-28, and I-29 showing size distribution, PDI, EE, and pKa results. [Figure 7] a-f show the morphology and mRNA copies per particle of SM-102 and exemplary I-28-LNPs; a) CryoTEM image of SM-102 LNPs, scale bar = 200 nm; b) CryoTEM image of exemplary I-28 LNPs, scale bar = 200 nm; c) nanoparticle size distribution of comparative compound SM-102 (●) and exemplary I-28 (▲); d) total lipid to RNA mass ratio of comparative compound SM-102 (left column) and exemplary I-28 (right column); e) predicted number of mRNA copies per particle (average) for comparative compound SM-102 (left column) and exemplary I-28 (right column); and f) measured number of mRNA copies per particle for comparative compound SM-102 (left column) and exemplary I-28 (right column). [Figure 8]a-c show intramuscular gene editing in transgenic mice; a) Schematic showing the method demonstrating the removal of the triple-stop cassette by the CRISPR-cas9 system to enable expression of tdTomato; b) tdTomato fluorescence in hindlimb muscles of Ai9 mice captured with IVIS spectra (535 nm / 600 nm), the left leg treated with saline and the right leg treated with exemplary I-28 LNPs; c) tdTomato fluorescence (535 nm / 600 nm) in hindlimb muscles treated with saline (left) and exemplary I-28 LNPs (right) (mean ± s.d., n=4, hindlimb, **p<0.01, unpaired two-tailed t-test); d) Histological and confocal microscopy analysis of Ai9 mouse hindlimb muscles after treatment with CRISPR-cas9 I-28 LNPs (bottom row). Phosphate-buffered saline (PBS) was used as a negative control (top row). The tdTomato column (left) shows tdTomato signal (white) upon treatment with I-28, confirming successful gene editing. Nuclear stain (DAPI, middle column) shows nuclei in white. tdTomato and DAPI signals are merged in the right column. [Figure 9] IL-6 responses to comparative compound SM-102 (left bar) and exemplary I-28-LNP (middle bar) compared to saline (right bar) are shown. IL-6 concentrations (pg / mg total protein) in draining lymph nodes of C57BL / 6 mice 6 hours post-injection (mean ± sd, n = 3, ns = not significant, unpaired two-tailed t-test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] definition As will be appreciated by those of skill in the art, unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the application described herein where they are suitable.

[0023] All features disclosed in this specification, including any accompanying claims, abstract, and drawings, and / or all steps of any method or process disclosed may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. Each feature disclosed in this specification, including the claims, abstract, and drawings, may be replaced by an alternative feature serving the same, equivalent, or similar purpose, unless expressly stated otherwise.

[0024] As used in this application and the claim(s), the terms "comprising" (and any form of including, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), or "containing" (and any form of including, such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0025] As used herein, the term "consisting of" and its derivatives are intended to be closed language specifying the presence of stated features, elements, components, groups, integers and / or steps and excluding the presence of other unstated features, elements, components, groups, integers and / or steps.

[0026] As used herein, the term "consisting essentially of" and its derivatives are intended to specify the presence of the specified features, elements, components, groups, integers and / or steps, as well as those that do not materially affect the basic and novel characteristic(s) of those features, elements, components, groups, integers and / or steps.

[0027] As used herein, the terms "about," "substantially," and "approximately" refer to a reasonable amount of deviation from the modified term such that the end result is not materially different. These degrees of deviation should be construed to include at least ±5% deviation from the modified term unless it negates the meaning of the word it modifies or the context would suggest otherwise to one of ordinary skill in the art.

[0028] As used in this application, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. For example, an embodiment including "a compound" should be understood to provide a particular aspect having one compound, or two or more additional compounds.

[0029] In embodiments that include an "additional" or "secondary" component or effect, e.g., an additional or second compound, a second compound, as used herein, is different from the other compound or the first compound. A "third" compound is different from the other first and second compounds, and further recited or "additional" compounds are similarly different.

[0030] When "one or more" molecules or materials are referred to (such as one or more dendrons), it is understood that this refers to a "type" or "identity" of molecules or materials. Thus, a "second" molecule or material is different from the one or first molecule or material. Similarly, a "third" molecule or material is different from the one, first, and second molecule or material, and further recited or "additional" molecules or materials are similarly different.

[0031] As used herein, the term "and / or" means that the listed items are present or used individually or in combination. In practice, the term means that "at least one" or "one or more" of the listed items are used or present. For example, the term "and / or" with respect to salts and / or solvates thereof means that the compounds of the present application are present as individual salts and hydrates, as well as combinations of salts, for example, solvates of the compounds of the present application.

[0032] As used herein, the terms "dendron of the application" or "dendron of the application" and the like refer to the dendron of Formula I and salts and / or solvates thereof.

[0033] As used herein, the terms "the composition of the application" or "composition of the application" and the like refer to a composition comprising one or more of the dendrons of the application.

[0034] As used herein, the term "suitable" means that the selection of a particular compound or conditions will depend on the particular synthetic operation being performed, the identity of the molecule(s) being converted, and / or the particular use of the compound, but that such selection is well within the skill of one of ordinary skill in the art.

[0035] This description refers to numerous chemical terms and abbreviations used by those of ordinary skill in the art. Nonetheless, definitions of selected terms are provided for clarity and consistency.

[0036] As used herein, terms such as "protecting group" or "PG" refer to a chemical moiety that protects or masks a reactive portion of a molecule to prevent side reactions at the reactive portion of the molecule while allowing a different portion of the molecule to be manipulated or reacted. After the manipulation or reaction is complete, the protecting group is removed under conditions that do not degrade or decompose the remaining portion of the molecule. Selection of a suitable protecting group can be made by one of ordinary skill in the art. Many conventional protecting groups are known in the art and are described, for example, in Greene, TW and Wuts, PGM, "Protective Groups in Organic Synthesis", John Wiley & Sons, 3 rd Edition, 1999 and Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas), in “Protective Groups in Organic Chemistry” McOmie, J. F. W. Ed., Plenum Press, 1973.

[0037] As used herein, the term "inert organic solvent" refers to a solvent that is generally considered to be non-reactive with the functional groups present in the compounds that are combined together in any reaction, and thus will not interfere with or inhibit the required synthetic transformations. Organic solvents are typically non-polar and will dissolve compounds that are not soluble in aqueous solutions.

[0038] As used herein, the term "alkyl" whether used alone or as part of another group, means a straight or branched chain saturated alkyl group. The number of carbon atoms that may be contained in the referred alkyl group is specified by the prefix "C n1-n2 For example, C 1-10 The term alkyl refers to alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Unless otherwise indicated, all alkyl groups are optionally fluoro substituted.

[0039] The term "alkylene", whether used alone or as part of another group, means a straight or branched saturated alkylene group, i.e., a saturated carbon chain containing substituents at two of its termini. The number of carbon atoms that may be contained in the referred alkylene group is indicated by the prefix "C". n1-n2 For example, C 1~6 The term alkylene refers to alkylene groups having 1, 2, 3, 4, 5, or 6 carbon atoms. Unless otherwise indicated, all alkylene groups are optionally fluoro-substituted.

[0040] As used herein, the term "alkenyl" whether used alone or as part of another group, means a straight or branched chain unsaturated alkyl group containing at least one double bond. The number of carbon atoms that may be contained in the referred alkylene group is specified by the prefix "C n1-n2 For example, C 2-6 The term alkenyl refers to an alkenyl group having 2, 3, 4, 5, or 6 carbon atoms and at least one double bond. Unless otherwise indicated, all alkenyl groups are optionally fluoro-substituted.

[0041] As used herein, the term "alkynyl" whether used alone or as part of another group, means a straight or branched chain unsaturated alkynyl group containing at least one triple bond. The number of carbon atoms that may be contained in the referred alkyl group is specified by the prefix "C n1-n2 For example, C 2-6 The term alkynyl refers to alkynyl groups having 2, 3, 4, 5 or 6 carbon atoms.

[0042] As used herein, the term "aryl", whether used alone or as part of another group, refers to a monovalent unsaturated aromatic group whose ring atoms are all carbon. An aryl group can contain 6 or more carbon atoms. Unless otherwise indicated, all aryl groups are optionally fluoro-substituted.

[0043] As used herein, the term "cycloalkyl" whether used alone or as part of another group, means a saturated carbocyclic group containing one or more rings. The number of carbon atoms that may be contained in the referenced cycloalkyl group is indicated by the numerical prefix "C". n1-n2 For example, C 3-10 The term cycloalkyl means a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Unless otherwise indicated, all cycloalkyl groups are optionally fluoro substituted.

[0044] As used herein, the term "heteroaryl," whether used alone or as part of another group, refers to a cyclic group that contains at least one heteroaromatic ring in which one or more of the atoms is a heteroatom selected from O, S, and N. A heteroaryl group may be denoted by the prefix C. n1-n2 When the prefix includes the radical "aryl", it indicates the number of carbon atoms in the corresponding carbocyclic group in which one or more, suitably one to five, of the ring atoms is replaced with a heteroatom as defined above. Unless otherwise indicated, all heteroaryl groups are optionally fluoro-substituted.

[0045] As used herein, the term "heterocycloalkyl," whether used alone or as part of another group, refers to a cyclic group containing at least one non-aromatic ring in which one or more of the atoms is a heteroatom selected from O, S, and N. Heterocycloalkyl groups are either saturated or unsaturated (i.e., containing one or more double bonds). Heterocycloalkyl groups are denoted by the prefix C.n1-n2 When the prefix includes the radical "-", it indicates the number of carbon atoms in the corresponding carbocyclic group in which one or more, suitably one to five, of the ring atoms is replaced with a heteroatom as defined above. Unless otherwise indicated, all heterocycloalkyl groups are optionally fluoro substituted.

[0046] The term "fluoro-substituted" refers to one or more (including all) available hydrogens in the referenced group being replaced with fluoro.

[0047] As used herein, the terms "halo" or "halogen," whether used alone or as part of another group, refer to a halogen atom and include fluoro, chloro, bromo, and iodo.

[0048] Symbols drawn perpendicular to the bond TIFF2024543272000009.tif11164 shows the attachment points of the groups.

[0049] The term "available", such as "available hydrogen atom" or "available atom", refers to an atom known to those of skill in the art that can be substituted with a substituent, such as a fluorine atom.

[0050] It is understood that all available hydrogen atoms in the compounds of the present application and all embodiments thereof are optionally substituted with fluorine atoms, unless otherwise indicated.

[0051] As used herein, the term "cell" refers to a single cell or to multiple cells, including cells either in cell culture or in a subject.

[0052] As used herein, the term "subject" refers to any target for delivery of one or more agents using the compositions of the present application.Subjects can be living objects, including all members of the animal and plant kingdoms, or inanimate objects.Thus, the methods and uses of the present application are applicable to human treatment, veterinary treatment, plant applications, and material processing.

[0053] The term "pharmaceutical acceptable" means compatible with the treatment of a subject, e.g., a human.

[0054] The term "pharmaceutical acceptable carrier" means a non-toxic solvent, dispersant, excipient, adjuvant, or other substance with which an active ingredient is mixed to enable the formation of a pharmaceutical composition, i.e., a dosage form that can be administered to a subject.

[0055] The term "pharmacologically acceptable salt" means either an acid addition salt or a base addition salt suitable or compatible with the treatment of a subject.

[0056] As used herein, the term "solvate" refers to a compound, or a salt and / or prodrug of a compound, wherein molecules of a suitable solvent are incorporated into the crystal lattice. A suitable solvent is one that is physiologically acceptable at the dosage administered.

[0057] As used herein, and as is well understood in the art, the term "treating" or "treatment" refers to an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, reduction or amelioration of one or more symptoms or conditions, whether detectable or undetectable, reduction in the extent of the disease, stabilization (i.e., not worsening) of the disease state, prevention of disease spread, delay or slowing of disease progression, remission or palliation of the disease state, reduction in recurrence of disease, and remission (whether partial or complete remission). "Treating" and "treatment" can also mean extended survival compared to expected survival in the absence of treatment. As used herein, "treating" and "treatment" also include prophylactic treatment. For example, a subject with early stage cancer can be treated to prevent progression, or a subject in remission can be treated with a compound or composition of the present application to prevent recurrence. A method of treatment includes administering to a subject a therapeutically effective amount of one or more compounds or compositions of the present application, optionally consisting of a single dose or including a series of doses.

[0058] "Alleviating" a disease or disorder means that the severity and / or undesirable clinical symptoms of the disorder or disease state are reduced and / or the time course of progression is slowed or prolonged compared to if the disorder was not treated.

[0059] As used herein, the terms "prevention" or "prophylaxis" or synonyms thereof refer to the reduction of the risk or probability that a patient will suffer from a disease, disorder, or condition.

[0060] As used herein, the term "effective amount" or "therapeutically effective amount" means an amount of a compound of the present application or one or more compounds of the present application, or a composition of the present application or one or more compositions of the present application, that is effective at dosages and for periods of time necessary to achieve a desired result.

[0061] By "inhibition, blocking, and / or interference" is meant detectable inhibition, blocking, and / or interference in the presence of a compound or composition compared to otherwise identical conditions, except for the absence of the compound or composition.

[0062] As used herein, the term "administered" means administering a therapeutically effective amount of a compound or one or more compounds of the present application, or a composition or one or more compositions of the present application to a cell, tissue, or organ in vivo, in vitro, or ex vitro.

[0063] The term "encapsulation efficiency" refers to the percentage of drug encapsulated within a particle, such as a nanoparticle, that is trapped within the nanoparticle.

[0064] As used herein, the terms "nanoparticle," "nanoparticles," or variations thereof, are intended to mean particles whose size is measured on the nanometer scale.

[0065] As used herein, the term "colloid" refers to a mixture of microscopically dispersed insoluble particles suspended in another substance, usually a liquid.

[0066] As used herein, the term "supramolecular structure" refers to a complex of molecules held together by non-covalent bonds.

[0067] Dendrons and compositions of the present application Multi-amine structures, such as poly(amidoamine) (PAMAM) dendrons, are biocompatible materials that contain a core amine group that branches unidirectionally into successive generations containing repeating amine and amide units. Multi-amine structures, such as poly(amidoamine) PAMAM dendrons, may be chemically modified to alter or improve their functionality.

[0068] A variety of structural modifications to the basic multi-amine structure, such as poly(amidoamine) PAMAM dendrons, are possible. These modifications are aimed at improving the efficacy and reducing toxicity of multi-amine structures, such as poly(amidoamine) PAMAM dendrons. Nitrogen-containing modified dendrons are multifunctional, with distinct structural motifs playing different roles to effectively deliver nucleic acids and other drugs with the appropriate overall negative charge. For example, nitrogen-containing tertiary amines in the structure become protonated and cationic at low pH. These ionizable cations can interact with the negative charges, for example along the sugar phosphate backbone of nucleic acids, allowing the delivery material to bind to its cargo and self-assemble into nanoparticles. Amides enhance biodegradability and reduce toxicity of the delivery material. Hydrophobic tails, when conjugated with nucleic acids, promote self-assembly of the delivery material. Disulfides, esters, thioesters, carbamates, ureas, imines, enamines, ketones or other hydrolyzable groups can be optionally included to control the release of the nucleic acid cargo and improve biodegradability. The inclusion of unsaturated, fluorinated, sterically hindered, or inflexible groups alters the self-assembly and packing within each nanoparticle.

[0069] Thus, the present application includes a dendron of formula I, or a salt and / or solvate thereof: [ka] During the ceremony, Each repeat group may be the same or different; [ka] and; n is 1, 2, 3, 4, or 5, and each L n and each X n are the same or different and are as defined below depending on the value of n; When n is 1, the dendron is a first generation dendron and the compound of formula I has the structure: [ka] In the formula, each X 1 are the same or different and are either H or a terminal group, provided that at least one X 1 are terminal groups, and each L 1 are the same or different and are linking groups; When n is 2, the dendron is a second generation dendron and the compound of formula I has the structure: [ka] In the formula, each X 2 are the same or different and are either H or a terminal group, provided that at least one X 2 are terminal groups, and each L 1 and each L 2 are the same or different and are linking groups; When n is 3, the dendron is a third generation dendron and the compound of formula I has the structure: [ka] In the formula, each X 3 are the same or different and are either H or a terminal group, provided that at least one X 3 are terminal groups, and each L 1 , each L 2 , and each L 3 are the same or different and are linking groups; When n is 4, the dendron is a fourth generation dendron and the compound of formula I has the structure: [ka] In the formula, each X 4 are the same or different and are either H or a terminal group, provided that at least one X 4 are terminal groups, and each L 1 , each L 2 , each L 3 , and each L 4are the same or different and are linking groups; When n is 5, the dendron is a fifth generation dendron and the compound of formula I has the structure: [ka] In the formula, each X 5 are the same or different and are either H or a terminal group, provided that at least one X 5 are terminal groups, and each L 1 , each L 2 , each L 3 , each L 4 , and each L 5 are the same or different and are linking groups; R 1 is C 1-20 Alkyl, C 2-20 Alkenyl, C 2 - 20 Alkynyl, C 1-20 Alkylene aryl, C 1-20 Alkylene Heteroaryl, C 1-20 Alkylene C 3-8 Heterocycloalkyl, C 1-20 Alkylene C 3-8 Cycloalkyl and C 1-6 Alkylene-SSC 1-6 alkyl, each of which is unsubstituted or selected from halo, OH, OC 1-20 Alkyl, C(O)OC 1-20 Alkyl and NR 2 R 2’ and the aryl, heteroaryl, heterocycloalkyl, and cycloalkyl groups are further optionally substituted with one or more C 1-10 is substituted with alkyl; R 2 and R 2’ are independently H and C 1-10 alkyl; The linker group is C 1-20 Alkylene or C 2-20alkenylene, each of which is optionally S, SS, O, NH, N(C 1-4 Alkyl), C(O), C(O)O, OC(O), C(O)NH, NHC(O), NHC(NH), NHC(NC 1-4 alkyl), C(NH)NH, and C(NC 1-4 alkyl)NH, each alkyl, alkylene, and alkenylene being unsubstituted or substituted with one or more of halo and OH; The terminal group is C 1-40 Alkyl, C 2-40 Alkenyl, C 1-40 Alkylenearyl, and C 1-40 alkenylenearyl, each of alkyl, alkenyl, alkylene, and alkenylene is optionally selected from S-S, C(O), OC(O), C(O)O, OC(O)O, NR 3 C(O)O, OC(O)NR 3 , C(O)S, SC(O), NR 3 C(O), C(O)NR 3 , N.R. 3 C(O)NR 4 , and C(NC 1 - 20 alkyl), wherein alkyl, alkenyl, alkylenearyl, and alkenylenearyl are optionally interrupted by one or more groups selected from halo, NR 5 R 5’ and OH; R 3 , R 4 , R 5 , and R 5’ are independently hydrogen or C 1 - 10 is alkyl; wherein all available hydrogen atoms attached to carbon are optionally replaced with fluorine atoms.

[0070] In some embodiments, R 1 is C 1-10 Alkyl, C 2-10 Alkenyl, C 2- 10 Alkynyl, C 1-10 Alkylene Ph, C 1-10 Alkylene Heteroaryl, C 1-10 Alkylene C 5-6 Heterocycloalkyl, C 1-10 Alkylene C 5-6 Cycloalkyl and C 1-4 Alkylene-SSC 1-4 alkyl, each of which is unsubstituted or selected from OH, OC 1-15 Alkyl, C(O)OC 1-15 Alkyl and NR 2 R 2’ and / or one or more of fluoro, phenyl, heteroaryl, heterocycloalkyl, and cycloalkyl groups are optionally further substituted with C 1-4 Alkyl and C 1-4 fluoroalkyl, R 2 and R 2’ are independently H and C 1-4 is selected from alkyl.

[0071] In some embodiments, R 1 is C 1-6 Alkyl, C 2-6 Alkenyl, C 2 - 6 Alkynyl, C 1-6 Alkylene Ph, C 1-6 Alkylene C 6 Heterocycloalkyl, and C 1-4 Alkylene-SSC 1-4 alkyl, each of which is unsubstituted or selected from OH, OC 1-12 Alkyl, C(O)OC 1-15 Alkyl and NR 2 R 2’ and / or one or more of the fluoro, phenyl, and heterocycloalkyl groups are further optionally substituted with C 1-4 Alkyl and C 1-4 fluoroalkyl, R 2 and R2’ are independently H and C 1-4 In some embodiments, R 1 is C 1-6 Alkyl, C 2-6 Alkenyl, and C 1-6 fluoroalkyl.

[0072] In some embodiments, R 1 teeth, [ka] is selected from.

[0073] In some embodiments, n is 1 or 2.

[0074] In some embodiments, L 1 , L 2 , L 3 , L 4 , and L 5 is independently 1-10 Alkylene or C 2-10 alkenylene-containing linker groups, each of which is optionally S, SS, O, NH, N(C 1-2 Alkyl), C(O), C(O)O, OC(O), C(O)NH, NHC(O), NHC(NH), NHC(NC 1-2 alkyl), C(NH)NH, C(NC 1-2 In some embodiments, L is interrupted by one or two moieties independently selected from: 1 , L 2 , L 3 , L 4 , and L 5 is independently 1-10 Alkylene or C 2-10A linker group comprising alkenylene, each of which is optionally interrupted by one or two moieties independently selected from S, SS, O, C(O)O, OC(O), C(O)NH, NHC(O), and each alkylene and alkenylene is unsubstituted or substituted with one or two OH and / or one or more fluoro.

[0075] In some embodiments, L 1 , L 2 , L 3 , L 4 , and L 5 is independently 1-6 Alkylene or C 2-6 A linker group comprising alkenylene, each of which is optionally interrupted by one or two moieties independently selected from SS, C(O)NH, and NHC(O), and each alkylene and alkenylene is unsubstituted or substituted with one or two OH and / or one or more fluoro.

[0076] In some embodiments, L 1 , L 2 , L 3 , L 4 , and L 5 is, independently, [ka] where d, e, f, g, u, v, w, x, and y are independently selected from 1, 2, 3, 4, 5, and 6. In some embodiments, d, e, f, g, u, v, w, x, and y are independently selected from 1 and 2.

[0077] In some embodiments, L 1 , L 2 , L 3 , L 4 , and L 5 is, independently, [ka] wherein u, v, w, x, and y are independently selected from 1, 2, 3, 4, 5, and 6. In some embodiments, u, v, w, x, and y are independently selected from 1 and 2.

[0078] In some embodiments, each L in the first generation dendron 1 In some embodiments, each L in the second generation dendron is the same. 2 In some embodiments, each L in the third generation dendron is the same. 3 In some embodiments, each L in the fourth generation dendron is the same. 4 In some embodiments, each L in the fifth generation dendron is the same. 5 is the same.

[0079] In some embodiments, the terminal group is C 1-20 Alkyl, C 2-20 Alkenyl, C 1-20 Alkylenearyl, and C 1-20 alkenylenearyl, each of alkyl, alkenyl, alkylene, and alkenylene is optionally selected from S-S, C(O), OC(O), C(O)O, OC(O)O, NR 3 C(O)O, OC(O)NR 3 , C(O)S, SC(O), NR 3 C(O), C(O)NR 3 , N.R. 3 C(O)NR 4 , and C(NC 1 - 10 alkyl), wherein alkyl, alkenyl, alkylenearyl, and alkenylenearyl are optionally interrupted by one or more groups selected from OH and NR 5 R 5’ substituted with 1 to 4 of the following: R 3 , R 4 , R 5 , and R 5’ are independently hydrogen or C 1 - 10 is alkyl; wherein all available hydrogen atoms attached to carbon are optionally replaced with fluorine atoms.

[0080] In some embodiments, the end group is [ka] is selected from Each of these may optionally contain one or two OH, one or two NH, provided that the total number of carbon atoms in the group is 20 or less. 2 and / or substituted with one or more fluorines. In some embodiments, the terminal group is [ka] is selected from In the formula, i is an integer from 1 to 10.

[0081] In some embodiments, each X in the first generation dendron 1 are terminal groups and are the same or different. In some embodiments, each X in the first generation dendron 1 are terminal groups and are the same.

[0082] In some embodiments, at least two X 2 are terminal groups and are the same or different. In some embodiments, at least three X in the second generation dendron 2 are terminal groups and may be the same or different. In some embodiments, all X 2 are terminal groups and are the same or different. In some embodiments, at least one X on each nitrogen of the second generation dendron 2 is an end group, each end group being the same or different. In some embodiments, at least one X on each nitrogen of the second generation dendron 2 are terminal groups, and each terminal group is the same. In some embodiments, all X2 are terminal groups and are the same.

[0083] In some embodiments, at least two X 3 are terminal groups and are the same or different. In some embodiments, at least three X in the third generation dendron 3 are terminal groups and are the same or different. In some embodiments, at least four X in the third generation dendron 3 are terminal groups and are the same or different. In some embodiments, at least five X in the third generation dendron 3 are terminal groups and are the same or different. In some embodiments, at least six X in the third generation dendron 3 are terminal groups and are the same or different. In some embodiments, at least seven X in the third generation dendron 3 are terminal groups and may be the same or different. In some embodiments, all X 3 are terminal groups and are the same or different. In some embodiments, at least one X on each nitrogen of the third generation dendron 3 is an end group, each end group being the same or different. In some embodiments, at least one X on each nitrogen of the third generation dendron 3 are terminal groups, and each terminal group is the same. In some embodiments, all X 3 are terminal groups and are the same.

[0084] In some embodiments, at least two X 4 are terminal groups and are the same or different. In some embodiments, at least three X in the fourth generation dendron 4 are terminal groups and are the same or different. In some embodiments, at least four X 4 are terminal groups and are the same or different. In some embodiments, at least five X 4are terminal groups and are the same or different. In some embodiments, at least six X in the fourth generation dendron 4 are terminal groups and are the same or different. In some embodiments, at least seven X in the fourth generation dendron 4 are terminal groups and are the same or different. In some embodiments, at least eight X in the fourth generation dendron 4 are terminal groups and are the same or different. In some embodiments, at least nine X in the fourth generation dendron 4 are terminal groups and are the same or different. In some embodiments, at least 10 X in the fourth generation dendron 4 are terminal groups and are the same or different. In some embodiments, at least 11 X in the fourth generation dendron 4 are terminal groups and are the same or different. In some embodiments, at least 12 X in the fourth generation dendron 4 are terminal groups and are the same or different. In some embodiments, at least 13 X in the fourth generation dendron 4 are terminal groups and are the same or different. In some embodiments, at least 14 X in the fourth generation dendron 4 are terminal groups and are the same or different. In some embodiments, at least 15 X 4 are terminal groups and may be the same or different. In some embodiments, all X 4 are terminal groups and are the same or different. In some embodiments, at least one X on each nitrogen of the fourth generation dendron 4 are terminal groups, each of which may be the same or different. In some embodiments, at least one X on each nitrogen of the fourth generation dendron 4 are terminal groups, and each terminal group is the same. In some embodiments, all X 4 are terminal groups and are the same.

[0085] In some embodiments, at least two X 5 are terminal groups and are the same or different. In some embodiments, at least three X in the fifth generation dendron 5 are terminal groups and are the same or different. In some embodiments, at least four X in the fifth generation dendron 4 are terminal groups and are the same or different. In some embodiments, at least five X 5 are terminal groups and are the same or different. In some embodiments, at least six X in the fifth generation dendron 5 are terminal groups and are the same or different. In some embodiments, at least seven X in the fifth generation dendron 5 are terminal groups and are the same or different. In some embodiments, at least eight X in the fifth generation dendron 5 are terminal groups and are the same or different. In some embodiments, at least nine X in the fifth generation dendron 5 are terminal groups and are the same or different. In some embodiments, at least 10 X 5 are terminal groups and are the same or different. In some embodiments, at least 11 X in the fifth generation dendron 5 are terminal groups and are the same or different. In some embodiments, at least 12 X in the fifth generation dendron 5 are terminal groups and are the same or different. In some embodiments, at least 13 X in the fifth generation dendron 5 are terminal groups and are the same or different. In some embodiments, at least 14 X in the fifth generation dendron 5 are terminal groups and are the same or different. In some embodiments, at least 15 X 5 are terminal groups and are the same or different. In some embodiments, at least 16 X in the fifth generation dendron 5 are terminal groups and are the same or different. In some embodiments, at least 17 X in the fifth generation dendron5 are terminal groups and are the same or different. In some embodiments, at least 18 X in the fifth generation dendron 5 are terminal groups and are the same or different. In some embodiments, at least 19 X in the fifth generation dendron 5 are terminal groups and are the same or different. In some embodiments, at least 20 X 5 are terminal groups and are the same or different. In some embodiments, at least 21 X in the fifth generation dendron 5 are terminal groups and are the same or different. In some embodiments, at least 22 X 5 are terminal groups and are the same or different. In some embodiments, at least 23 X 5 are terminal groups and are the same or different. In some embodiments, at least 24 X 5 are terminal groups and are the same or different. In some embodiments, at least 25 X 5 are terminal groups and are the same or different. In some embodiments, at least 26 X 5 are terminal groups and are the same or different. In some embodiments, at least 27 X 5 are terminal groups and are the same or different. In some embodiments, at least 28 X 5 are terminal groups and are the same or different. In some embodiments, at least 29 X 5 are terminal groups and are the same or different. In some embodiments, at least 30 X 5 are terminal groups and are the same or different. In some embodiments, at least 31 X 5 are terminal groups and are the same or different. In some embodiments, at least 32 X 5are terminal groups and are the same or different. In some embodiments, at least 33 X 5 are terminal groups and are the same or different. In some embodiments, at least 34 X 5 are terminal groups and are the same or different. In some embodiments, at least 35 X 5 are terminal groups and are the same or different. In some embodiments, all X 5 are terminal groups and are the same or different. In some embodiments, at least one X on each nitrogen of the fifth generation dendron 5 is an end group, each end group being the same or different. In some embodiments, at least one X on each nitrogen of the fifth generation dendron 5 are terminal groups, and each terminal group is the same. In some embodiments, all X 5 are terminal groups and are the same.

[0086] In some embodiments, the dendron of formula I is [ka] I-1(G1-nPr-C14E); [ka] I-2(G1-nPr-C10O1E); [ka] I-3 (G1-nPr-C7RfE); [ka] I-4(G2-nPr-C14E); [ka] I-5(G2-nPr-C10O1E); [ka] I-6(G2-nPr-C16E); [ka] I-7(G1-nPr-S8-C14E); [ka] I-8 (G1-benzyl-S-1); [ka] I-9 (G2-Azine-S-1); [ka] I-10 (G2-c1-amide-t1); [ka] I-11 (G2-c2-amide-t2); [ka] I-12 (G1-c3-amide-t3); [ka] I-13 (G1-nPr-amide-t4); [ka] I-14 (G1-nPr-amide-t5); [ka] I-15(G1-nPr-C14K); [ka] I-16 (G2-c4-amide-t7); [ka] I-17 (G1-c5-amide-t8); [ka] I-18(G1-nPr-amide-t9); [ka] I-19 (G1-nPr-amide-t10); [ka] I-20 (G1-nPr-amide-t11); [ka] I-21(G0-nPr-amide-t12); [ka] I-22(G1-nPr-C14); [ka] I-23(G1-nPr-C16E); [ka] I-24(G2-nPr-S14-C14E); [ka] I-25(G1-C3-K2-E15); [ka] I-26(G1-C3-K2-E10); [ka] I-27(G1-C3-K3-E10); [ka] I-28(G1-OC2-K3-E10); [ka] I-29(G1-OC3-K3-E10), [ka] I-30 (DPA-GABA-E10), [ka] I-31 (TPA-GABA-E10), [ka] I-32 (BU2-S3-E10); and [ka] I-33(BU3-S3-E10), or a salt and / or solvate thereof.

[0087] The present application also includes compositions comprising one or more dendrons of the present application. In some embodiments, the dendrons of the present application are incorporated into nanoparticles in the composition. Thus, the present application also includes nanoparticles comprising one or more dendrons of the present application. The present application also includes compositions comprising these nanoparticles. In some embodiments, the nanoparticles are formed by self-assembly of one or more dendrons. In some embodiments, the nanoparticles comprise two or more dendrons of the present application. In some embodiments, the nanoparticles are lipid nanoparticles (LNPs).

[0088] In some embodiments, the LNPs comprise one or more ionizable lipids (one or more dendrons of the present application), one or more bilayer-forming lipids, one or more structural lipids, and one or more lipid-conjugated polyethylene glycol (PEG lipids).

[0089] In some embodiments, the nanoparticles have a maximum longest linear dimension (e.g., diameter) of 200 nm or more. In some embodiments, the nanoparticles have a maximum longest linear dimension (e.g., diameter) of 200 nm or less. In some embodiments, the nanoparticles have an average diameter of about 150 nm, about 125 nm, about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm or less. In some embodiments, the nanoparticles have an average diameter of 50 nm or less. In some embodiments, the nanoparticles have an average diameter of 40 nm or less, 30 nm or less, 20 nm or less, 10 nm or less, or 5 nm or less. Thus, in some embodiments, the nanoparticles have an average diameter of about 1 nm to about 50 nm, about 5 nm to about 30 nm, about 10 nm to about 25 nm, about 10 nm to about 20 nm, or about 15 nm to about 20 nm. In some embodiments, the nanoparticles disclosed herein have an average diameter of about 150 nm, about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, about 15 nm, about 10 nm, or about 5 nm. In some embodiments, the average diameter is determined using dynamic light scattering intensity.

[0090] In some embodiments, one or more dendrons of the present application form colloids or supramolecular structures. Thus, in some embodiments, the present application also includes colloids comprising one or more dendrons of the present application, or supramolecular structures comprising one or more dendrons of the present application, as well as compositions comprising these colloids or supramolecular structures.

[0091] The present application also includes compositions comprising one or more dendrons of the present application and one or more agents to be delivered to a cell or subject. In some embodiments, the one or more agents to be delivered to a cell or subject are selected from unmodified or modified nucleic acids, mitochondria, plasmids, PolyIC and related adjuvants, ribonucleoproteins, proteins, peptides, cells, stains, dyes, small molecule drugs, and other organic and inorganic moieties. In some embodiments, the one or more agents to be delivered to a cell or subject are selected from polynucleotides, chemically modified polynucleotides, small molecules, biologics, and other organic or inorganic molecules. In some embodiments, the one or more agents to be delivered to a cell or subject are selected from dyes and radiolabeled compounds. In some embodiments, the one or more agents to be delivered to a cell or subject are vaccines. In some embodiments, the one or more agents to be delivered to a cell or subject are drugs. In some embodiments, the one or more agents to be delivered to a cell or subject are referred to as payloads.

[0092] In some embodiments, one or more of the agents to be delivered to a cell or subject has an overall negative charge.

[0093] In some embodiments, one or more agents to be delivered to a cell or subject are located within a nanoparticle, colloid, or supramolecular structure comprising one or more dendrons of the present application. By "located within," it is understood that the one or more agents are encapsulated within the nanoparticle, colloid, or supramolecular structure and / or are non-covalently associated with any portion of the one or more dendrons that make up the nanoparticle, colloid, or supramolecular structure.

[0094] In some embodiments, the proteins and peptides are selected from endonucleases, meganucleases, proteases, and kinases.

[0095] In some embodiments, the one or more agents delivered to a cell or subject are one or more nucleic acids. In some embodiments, the one or more nucleic acids are selected from short interfering RNA (e.g., small interfering RNA or siRNA), circular RNA, cyclic RNA, long non-coding RNA, microRNA (miRNA), pri-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), transactivating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), exDNA, proform RNA, single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA). In some embodiments, the one or more nucleic acids are selected from siRNA, tRNA, and nucleic acids used in CRISPR process. In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid used in the CRISPR process is a clustered regularly interspaced short palindromic repeats (CRISPR) associated nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), and / or a transactivating crRNA (tracrRNA). In some embodiments, the nucleic acid is a miRNA. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid is a tRNA. In some embodiments, the nucleic acid is a guide RNA. In some embodiments, a guide RNA is used in the CRISPR process. In some embodiments, the nucleic acid is pDNA.

[0096] In some embodiments, the compositions of the present application are for use in gene editing. In some embodiments, these compositions comprise cas9 mRNA and one or more guide RNAs (gRNAs) designed to target specific genes. In some embodiments, these compositions further comprise DNA or are formulated to be co-delivered with DNA for homology-directed repair.

[0097] In some embodiments, the compositions of the present application are for use in gene silencing. In some embodiments, these compositions comprise siRNA.

[0098] In some embodiments, the compositions of the present application are used for gene regulation. In some embodiments, these compositions comprise non-coding RNA (ncRNA).

[0099] In some embodiments, the compositions of the present application are used for upregulating gene expression or downregulating gene expression. In some embodiments, these compositions comprise unmodified or chemically modified messenger RNA (mRNA). In some embodiments, chemically modified mRNA refers to partial or complete replacement of nucleotides with chemically modified nucleotides. In one embodiment, uridine is completely replaced by N1-methylpseudouridine.

[0100] In some embodiments, the compositions of the present application are used for antiviral treatment.In some embodiments, these compositions include an mRNA encoding Ca9 protein and one or more sgRNAs.In some embodiments, the one or more sgRNAs identify the viral gene for deletion.

[0101] In some embodiments, the dendron(s) and the one or more agents to be delivered to a cell or subject are present in a weight ratio of about 100:1 to about 1:5. In some embodiments, the weight ratio of dendron(s) to one or more agents to be delivered to a cell or subject is about 50:1 to about 2:1. In some embodiments, the weight ratio of dendron(s) to one or more agents to be delivered to a cell or subject is 25:1.

[0102] In some embodiments, the composition further comprises one or more lipids, hi some embodiments, the one or more lipids are selected from steroids, steroid derivatives, PEG lipids, and phospholipids.

[0103] In some embodiments, the PEG lipid comprises one or more C linked to a linker group having a PEG chain. 6-24 Alkyl group or C 6-24 Alkenyl group, or C 6-24 A compound that contains a fatty acid group. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-ceramide conjugate, PEG-modified dialkylamine, PEG-modified 1,2-diacyloxypropan-3-amine, PEG-modified diacylglycerol, and / or dialkylglycerol.

[0104] In some embodiments, the PEG lipid is PEG-modified 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, PEG-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, PEG-modified 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, PEG-modified 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, PEG-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, PEG-modified 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol, and / or PEG-modified distearoyl-rac-glycerol.

[0105] In some embodiments, PEG is measured by the molecular weight of the PEG component of the lipid. In some embodiments, PEG has a molecular weight (g / mol or Daltons) of about 100 to about 15,000. In some embodiments, the molecular weight is about 200 to about 10,000, about 400 to about 8,000, about 1,000 to about 6,000, or about 2,000 to about 5,000. In some embodiments, the molecular weight of PEG is about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. Some non-limiting examples of lipids that can be used in the present invention are taught by U.S. Patent No. 5,820,873, WO2010 / 141069, or U.S. Patent No. 8,450,298, which are incorporated herein by reference.

[0106] In some embodiments, the PEG lipid is a PEG phospholipid conjugate, such as a PEGylated phosphoethanolamine lipid of formula II: [ka] During the ceremony, R ’ C has one or two double bonds 6-24 Alkyl or C 6-24 alkenyl; m is an integer from 20 to 200; Y is any suitable counter cation.

[0107] In some embodiments, R ’ C has one double bond 12-20 Alkyl, or C 12-20 In some embodiments, R is alkenyl. ’ is C 14 Alkyl, C 16 Alkyl or C 18 alkyl or R ’ is C14 Alkenyl, C 16 Alkenyl or C 18 alkenyl, each alkenyl group having one double bond.

[0108] In some embodiments, m is an integer from 40 to 120. In some embodiments, m is 45 (PEG2000) or 113 (PEG5000).

[0109] In some embodiments, Y is ammonium, or any other suitable counter cation.

[0110] In some embodiments, the composition comprises a molar ratio of PEG lipid to dendron of about 1:1 to about 1:400 or 1:1 to about 1:250. In some embodiments, the molar ratio is about 1:1, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:125, 1:150, 1:175, 1:200, 1:225 to about 1:250, or any range derivable therein. In some embodiments, the PEG lipid is present at 0-50 mol% of the total lipid, or any range derivable therein. In some embodiments, the PEG lipid is present at 0 mol%, 1.5 mol%, 3 mol%, 10 mol%, 15 mol%, 20 mol%, or 40 mol%, or any range derivable therein.

[0111] In some embodiments, the structured lipid is a steroid or steroid derivative. In some embodiments, the steroid or steroid derivative is unmodified or modified cholesterol, phytosterol, cholecalciferol, dexamethasone, or any combination thereof. In some embodiments, the modified cholesterol is oxidized on the beta ring or on the hydrocarbon tail structure. In another embodiment, the phytosterol includes, but is not limited to, β-sitosterol, stigmasterol, β-sitostanol, campesterol, brassicasterol, salts and esters thereof. In some embodiments, the structured lipid and dendron are present in a molar ratio of 2:1 to 1:20, or any range derivable therein. In some embodiments, the structured lipid is present in 0-50 mol% of the total lipid, or any range derivable therein.

[0112] In some embodiments, the bilayer-forming lipids are composed of naturally occurring lipids or are of synthetic origin including phospholipids, sphingolipids, and glycolipids. Phospholipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the sphingolipid is sphingomyelin. In some embodiments, the bilayer-forming lipids and dendrons are present in a molar ratio of 2:1 to 1:20, or any range derivable therein. In some embodiments, the structured lipids are present at 0-50 mol % of the total lipids, or up to 50 mol %, or any range derivable therein.

[0113] In some embodiments, the present application includes compositions comprising one or more dendrons of the present application, one or more PEG lipids, one or more phospholipids, and one or more steroids. In some embodiments, the one or more dendrons are present in an amount of about 45 mol% to about 55 mol%, the one or more phospholipids are present in an amount of about 33.5 mol% to about 43.5 mol%, the one or more PEG lipids are present in an amount of about 0.5 mol% to about 2.5 mol%, and the one or more steroids are present in an amount of about 5 mol% to about 15 mol%. In some embodiments, the one or more dendrons are present in an amount of about 50 mol%, the one or more phospholipids are present in an amount of about 38.5 mol%, the one or more PEG lipids are present in an amount of about 1.5 mol%, and the one or more steroids are present in an amount of about 10 mol%.

[0114] In some embodiments, the present application includes compositions comprising one or more dendrons of the present application, one or more PEG-lipids, one or more phospholipids, one or more steroids, and one or more agents to be delivered. In some embodiments, the steroid is cholesterol. In some embodiments, the steroid is β-sitosterol.

[0115] In some embodiments, when lipids are present in a composition comprising one or more agents to be delivered, the one or more agents to be delivered to a cell or subject are present in a weight ratio of one or more dendrons and lipid to agent(s) of about 100:1 to about 1:5, about 50:1 to about 2:1, or about 25:1. In some embodiments, the mol % ratio of one or more dendrons and lipid:agent(s) is within the range of about 95 mol %:5 mol % to about 80 mol %:20 mol %.

[0116] In some embodiments, when one or more of the agents to be delivered is RNA, the composition comprises a lipid nitrogen to RNA phosphate ratio (N / P) of about 5.

[0117] The present application also includes a pharmaceutical composition comprising a composition or dendron of the present application and a pharma- ceutically acceptable carrier.

[0118] In some embodiments, the pharma- ceutically acceptable carrier is a solvent or solution. In some embodiments, the pharmaceutical composition is formulated for administration orally, intraadiposely, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrathoracically, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesically, intravitreally, liposomally, topically, mucosally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, topically, buccally, transdermally, intravaginally, in creams, in lipid compositions, via catheter, via irrigation, via continuous infusion, via drip, via inhalation, via injection, local delivery, or via local perfusion. In some embodiments, the pharmaceutical composition is formulated for intravenous or intraarterial injection. In some embodiments, the pharmaceutical composition is formulated as a unit dose. In some embodiments, the pharmaceutical composition is formulated for intramuscular administration.

[0119] In some embodiments, the compositions and pharmaceutical compositions of the present application have a pH of about 2 to about 8.5. In some embodiments, the compositions are formulated in 10 to 100 mM citrate buffer at pH 3, 4, 5, or 6. It should be understood that the pH of the compositions of the present application can be any suitable pH during preparation and storage. However, to deliver the compositions of the present application to a cell or subject, the pH is adjusted to be physiologically acceptable, for example, to a pH of about 7. In some embodiments, the compositions and pharmaceutical compositions of the present application have a polydispersity index (PDI) of less than 0.2. In some embodiments, the compositions and pharmaceutical compositions of the present application have a pKa of about 4 to about 8. In some embodiments, the compositions and pharmaceutical compositions of the present application have a pKa of about 5 to about 7. In some embodiments, the compositions and pharmaceutical compositions of the present application have a pKa of about 5.5.

[0120] In some embodiments, the compositions and pharmaceutical compositions of the present application have an encapsulation efficiency of greater than about 90%. In some embodiments, the encapsulation efficiency is greater than about 93%. In some embodiments, the encapsulation efficiency is greater than about 95%.

[0121] This application also includes kits. Any of the components disclosed herein can be combined in the form of a kit. In some embodiments, the kit includes the dendron or composition described above.

[0122] The kit typically includes at least one vial, test tube, flask, bottle, syringe, or other container into which the components are placed and optionally appropriately aliquoted. If there are multiple components in the kit, the kit typically also includes a second, third, or other additional container into which the additional components are separately placed. However, various combinations of components may be included in the container. In some embodiments, all of the delivery components are combined in a single container. In other embodiments, some or all of the dendron delivery components are provided in separate containers.

[0123] The kits of the present application typically also include packaging to house the various containers in a closed state for commercial sale. Such packaging may include cardboard, injection molded or blow molded plastic packaging in which the desired containers are held. In some embodiments, the kits also include instructions for using the components of the kit. The instructions may include implementable variations. In some embodiments, the instructions are for delivery of one or more dendrons or one or more compositions to a subject or cell. Methods and Uses of the Present Application

[0124] The present application includes methods for delivering one or more agents to a cell or a subject using the dendrons and / or compositions of the present application. Thus, in some embodiments, the present application includes methods for delivering one or more agents to a cell comprising contacting the cell with one or more dendrons or compositions or pharmaceutical compositions of the present application under conditions that cause uptake of the agent into the cell. In some embodiments, the present application includes methods for delivering one or more agents to a subject comprising contacting the subject with one or more dendrons or compositions or pharmaceutical compositions of the present application. In some embodiments, the cell is contacted in vitro. In some embodiments, the cell is contacted in vivo. In some embodiments, the cell is contacted ex vivo. In some embodiments, the contacting is for treating a disease, disorder, or condition. In some embodiments, the contacting is for diagnosing a disease, disorder, or condition. In some embodiments, the contacting is by administering an effective amount of one or more dendrons or compositions or pharmaceutical compositions of the present application to a cell or subject in need thereof.

[0125] In some embodiments, the application also includes the use of one or more dendrons or compositions or pharmaceutical compositions of the application for diagnostic, prophylactic, or therapeutic applications. In some embodiments, the use is associated with a payload delivered by the composition. In some embodiments, the payload includes a polynucleotide, a chemically modified polynucleotide, a small molecule, a biologic, or other organic or inorganic moiety. Diagnostic applications include the use of one or more dendrons or compositions or pharmaceutical compositions of the application as a carrier of a distinguishable label, such as a dye or radioactive label, that can be detected after administration and indicates a disease, disorder, or condition. Preventive applications include use as a vaccine, including a vaccine against infectious disease or another expected or potential condition of a subject. Therapeutic applications include use to treat any disease, disorder, or condition, including, but not limited to, infectious disease, autoimmune disease, cancer, genetic disease, chronic disease, trauma, wound healing, traumatic brain injury, muscular disease, neuromuscular disease, and / or gastrointestinal disease. Therapeutic applications may be in vivo or may be ex vivo therapeutic treatments where cells, tissues, or organs are treated ex vivo and transplanted or grafted into a subject.

[0126] In some embodiments, the present application also includes the use of one or more dendrons or compositions or pharmaceutical compositions of the present application to deliver one or more agents to cells, either in vitro, in vivo, or ex vivo.

[0127] In some embodiments, the one or more agents delivered to the cell are one or more nucleic acids. Thus, the present application also includes a method of modulating expression of a gene, comprising delivering one or more nucleic acids to a cell, the method comprising contacting the cell with one or more dendrons or compositions or pharmaceutical compositions of the present application under conditions that cause uptake of the one or more nucleic acids into the cell. In some embodiments, the cell is contacted in vitro. In some embodiments, the cell is contacted in vivo. In some embodiments, the cell is contacted ex vivo. In some embodiments, the modulation of gene expression is sufficient to treat a disease, disorder, or condition.

[0128] In some embodiments, the disease, disorder, or condition is, for example, but not limited to, an infectious disease, an autoimmune disease, a cancer, a genetic disease, a chronic disease, trauma, wound healing, traumatic brain injury, a neuromuscular disease, and / or a gastrointestinal disease.

[0129] In some embodiments, the present application also includes the use of one or more dendrons or compositions or pharmaceutical compositions of the present application to modulate expression of a gene. In some embodiments, expression of a gene is modulated by delivering one or more nucleic acids to a cell, either in vitro, in vivo, or ex vivo.

[0130] In some embodiments, when the compositions and pharmaceutical compositions of the present application include siRNA, miRNA, and mRNA as one or more agents delivered to a cell or subject, the formulated delivery composition is designed to target bone marrow endothelial cells and suppress, regulate, or induce genes that cause events that lead to chronic inflammation. Currently, autoimmune diseases are the leading cause of chronic inflammation and affect approximately 2 million Canadians. Autoimmune diseases are not fully understood due to their complex nature, which creates challenges when discussing treatment options. Current treatments include immunosuppressants, corticosteroids, and pain management. These current treatment options do not provide long-term or permanent results and need to be optimized. Chronic and severe inflammation is caused by the overexpression and migration of monocytes and monocyte-derived macrophages to target areas in the body. The majority of monocytes and monocyte-derived macrophages are generated in the bone marrow and can then cross the endothelial barrier and migrate through the bloodstream to target areas. Because the bone marrow is the primary source of monocytes, the process of monocyte proliferation and egress into the bloodstream can be altered by directly targeting areas of mass production. The silencing effect of the siRNA contained in the nanoparticles can not only inhibit the proliferation of monocytes, but also inhibit the ability of monocytes to leave the bone marrow. By reducing their ability to move freely, as well as their total number in the body, it can effectively reduce the inflammatory response that has harmful effects on people suffering from chronic inflammation. Thus, in some embodiments, the present application includes a method of treating chronic inflammation, comprising administering to a cell or subject in need thereof an effective amount of a composition comprising one or more dendrons, siRNA, miRNA, and mRNA of the present application. Also included is the use of a composition comprising one or more dendrons, siRNA, miRNA, and mRNA of the present application to treat chronic inflammation. In some embodiments, the siRNA is an siRNA against a bone marrow endothelial gene required for monocyte adhesion and migration into the blood circulation.

[0131] In some embodiments, the compositions of the present application are for use in gene editing. Thus, in some embodiments, the present application includes a method of editing the genome of a cell, comprising contacting the cell with one or more compositions of the present application. The use of one or more compositions of the present application for gene editing is also included. In some embodiments, these compositions include cas9 mRNA and one or more guide RNAs (gRNAs) designed to target a specific gene, and the one or more compositions are contacted with the cell under conditions that cause uptake of the cas9 mRNA and one or more gRNAs into the cell. Once uptaken into the cell, the cas9 mRNA is translated into the Cas9 protein, which incorporates the gRNA to target the sequence of interest in the nucleus. DNA can also be co-delivered for homologous recombination repair.

[0132] In some embodiments, the compositions of the present application are for use in gene silencing.Thus, in some embodiments, the present application includes a method for silencing a gene in a cell, comprising contacting the cell with one or more compositions of the present application.Also included is the use of one or more compositions of the present application for gene silencing.In some embodiments, these compositions include siRNA, and when delivered to a cell, silence the gene corresponding to the siRNA.Thus, the one or more compositions are contacted with the cell under conditions that cause the siRNA to be taken up into the cell.

[0133] In some embodiments, the compositions of the present application are used for gene regulation.In some embodiments, these compositions comprise non-coding RNA (ncRNA), and one or more compositions are contacted with cells under conditions that cause the incorporation of the ncRNA into the cell, and upon incorporation into the cell, regulate the gene associated with the ncRNA.

[0134] In some embodiments, the compositions of the present application are used for upregulating gene expression or downregulating gene expression. Thus, in some embodiments, the present application includes a method for regulating the expression of a gene in a cell, comprising contacting the cell with one or more compositions of the present application. Also included is the use of one or more compositions of the present application for regulating the expression of a gene in a cell. In some embodiments, these compositions include messenger RNA (mRNA), and the one or more compositions are contacted with the cell under conditions that cause the mRNA to be taken up into the cell, and upon taking up into the cell, express the protein.

[0135] In some embodiments, the compositions of the present application may be used to treat or prevent an infectious disease, an autoimmune disease, a cancer, a genetic disease, a chronic disease, trauma, wound healing, traumatic brain injury, a neuromuscular disease, and / or a gastrointestinal disease.

[0136] In some embodiments, the compositions of the present application are used for antiviral treatment. Thus, in some embodiments, the present application includes a method for treating a viral infection, comprising administering one or more compositions of the present application to a cell or subject in need thereof. Also included is the use of one or more compositions of the present application for treating a viral infection. In some embodiments, these compositions include an mRNA encoding a Ca9 protein and one or more sgRNAs. In some embodiments, the one or more sgRNAs identify a viral gene for deletion. In some embodiments, the virus is a DNA virus or an RNA virus.

[0137] In some embodiments, the compositions of the present application are used in a method of delivering one or more proteins to a cell, a method of delivering one or more small molecule drugs to a cell, or a method of delivering one or more DNA molecules to a cell.

[0138] In some embodiments, the compositions of the present application are used in cosmetic and / or personal care products, and the one or more agents delivered to a cell or subject are any such agents. In some embodiments, the one or more agents for cosmetic and / or personal care products include, but are not limited to, hair moisturizers, hair growth agents, hair anti-frizz agents, skin moisturizers, anti-aging agents, and temporary bioluminescent proteins.

[0139] In some embodiments, the compositions of the present application are used in methods to prevent counterfeiting, for example, to verify that a product or packaging has not been tampered with or is in its original form. In these embodiments, one or more agents include a unique DNA or RNA sequence (barcode) that is placed into the product's packaging / label that can be read to verify that the product is authentic or has not been tampered with, and delivered onto or into a subject in which the compositions of the present application are the packaging.

[0140] In some embodiments, the composition in the methods and uses is a pharmaceutical composition as defined above.

[0141] In some embodiments, the cells in the methods and uses of the present application are in vitro. In some embodiments, the cells in the methods and uses of the present application are in vivo. In some embodiments, the cells in the methods and uses of the present application are ex vivo.

[0142] In some embodiments, the cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a plant cell.

[0143] Preparation of the dendrons and compositions of the present application The dendrons of the present application can be prepared by a variety of synthetic processes. The selection of certain structural features and / or substituents may influence the selection of one process over another. The selection of a particular process for preparing a given dendron of formula (I) is within the knowledge of one of ordinary skill in the art. Some starting materials for preparing the dendrons of the present application are available from commercial chemical sources. Other starting materials are readily prepared from available precursors using simple transformations well known in the art.

[0144] In some embodiments, dendrons of formula I are assembled by linking different parts of the molecule in a specific order depending on the functional groups present. Standard chemistry known in the art can be used to assemble dendrons of formula I, including but not limited to nucleophilic substitution, cross-coupling, Michael reactions, and / or activating group strategies. Many of the moieties of the dendrons are known or based on known compounds, so compounds that can be used to link the parts of the molecule are readily available from commercial sources or using synthetic methods known in the art. For example, many of such compounds have at least one functional group in their structure that can form a covalent bond with a linker group. Such examples include amine, thiol, halo, hydroxyl, alkoxy, carboxyl, ester, amide, and / or oxo groups.

[0145] In an exemplary embodiment, the synthesis of first generation (G1) and second generation (G2) polyamidoamine (PAMAM) dendron polymers can be performed by following the steps shown in Scheme 1. For example, n-propylamine (or a compound of formula R 1 -NH 2or any other suitable amine) to form intermediate A, which is then condensed, for example, with ethylenediamine to form intermediate B. Intermediate B can then undergo alkylation, for example, via epoxide ring opening, to form a first generation dendron in which R is a terminal group, or a second set of addition and condensation reactions to form intermediates C and D, respectively. Alkylation of intermediate D, for example, via epoxide ring opening, forms a second generation dendron in which each R is a terminal group. [ka]

[0146] In a further exemplary embodiment shown in Scheme 2, to synthesize a bioreducible first generation modified dendron, for example, n-propylamine (or a compound of formula R 1 -NH 2 or any other suitable amine) with, for example, methyl acrylate (or any other suitable linker group) to form intermediate A. Further condensation reaction with cystamine forms intermediate E, which can then undergo alkylation, for example via epoxide ring opening, to form a bioreducible, R-terminal first generation dendron. [ka]

[0147] In a further exemplary embodiment shown in Scheme 3, to synthesize a bioreducible second generation modified dendron, intermediate C is formed as in Scheme 1. Further condensation reaction with cystamine forms intermediate F, which can then undergo alkylation, for example via epoxide ring opening, to form a bioreducible, second generation dendron in which R is a terminal group. [ka]

[0148] In a further exemplary embodiment shown in Scheme 4, the alkylation of intermediates B or D of Scheme 1 is alternatively carried out by reductive amination of an aldehyde or ketone in the presence of a reducing agent such as sodium triacetoxyborohydride. [ka]

[0149] It should be understood that throughout the process, suitable protecting groups are added to and subsequently removed from the various reactants and intermediates, as necessary, in a manner readily understood by one of ordinary skill in the art. Conventional procedures for the use of such protecting groups and examples of suitable protecting groups are described, for example, in "Protective Groups in Organic Synthesis", TW Green, PG M Huts, Wiley-Interscience, New York, (1999). It should also be understood that the conversion of a group or substituent to another group or substituent by chemical manipulation can be performed on any intermediate or final product on the synthetic route toward the final product, and the types of transformations possible are limited only by the inherent incompatibility of other functional groups carried by the molecule at that stage with the conditions or reagents used in the transformation. Such inherent incompatibilities, and how to overcome them by carrying out the appropriate transformations and synthetic steps in a suitable order, will be readily understood by one of ordinary skill in the art. Examples of transformations are provided herein, and it should be understood that the transformations described are not limited only to the general groups or substituents for which the transformations are exemplified. References and descriptions of other suitable transformations are provided in "Comprehensive Organic Transformations-A Guide to Functional Group Preparations" RC Larock, VHC Publishers, Inc. (1989). References and descriptions of other suitable reactions can be found in organic chemistry textbooks, such as "Advanced Organic Chemistry", March, 4th ed. McGraw Hill (1992), or "Organic Synthesis", Smith, McGraw Hill, (1994).

[0150] Techniques for purification of intermediates and final products include, for example, normal and reverse phase chromatography on columns or rotating plates, recrystallization, distillation, and liquid-liquid or solid-liquid extraction and will be readily apparent to those skilled in the art.

[0151] Salts of the dendrons of the present application are generally formed by dissolving the neutral compound in an inert organic solvent, adding the desired acid or base, and isolating the resulting salt by filtration or other known means.

[0152] The formation of solvates of the dendrons of the present application will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in a suitable solvent and isolating the solvate by cooling or by use of an antisolvent. The solvate is typically dried under ambient conditions or azeotroped. Selection of appropriate conditions for forming a particular solvate can be made by one of skill in the art. Examples of suitable solvents are ethanol, water, etc. When water is the solvent, the molecule is called a "hydrate". The following non-limiting examples are illustrative of the present application.

[0153] The compositions of the present application are prepared by combining the dendrons of the present application with optional excipients under conditions to form nanoparticles, colloids, and / or supramolecular structures, suitably including one or more agents to be delivered. In some embodiments, the conditions to form nanoparticles, colloids, and / or supramolecular structures include first combining one or more dendrons with one or more optional excipients, including one or more PEG lipids, one or more phospholipids, and / or one or more steroids, in a suitable solvent, such as ethanol. In some embodiments, sonication and / or warming are used to remove precipitates. One or more agents to be delivered are prepared separately in a suitable solvent, such as an aqueous buffer, such as an aqueous citrate buffer. The two solutions are then combined under conditions to form nanoparticles, colloids, and / or supramolecular structures. In some embodiments, the two solutions are combined under continuous flow conditions, for example using microfluidics. In some embodiments, the resulting nanoparticles, colloids, and / or supramolecular structures are processed using dialysis to adjust the pH. In some embodiments, the particles can be stored at room temperature or at -20°C for at least 48 hours without noticeable change. EXAMPLES

[0154] Common methods Several modified first (G1) and second (G2) generation polyamidoamine (PAMAM) dendron polymers are synthesized. These polymers are combined with lipid-polyethylene glycol (PEG lipid) excipients in ethanol (EtOH) phase to create delivery materials for sequestering nucleic acids. RNA nanoparticles are then formed by combining the delivery materials with various nucleic acids in citrate buffers at different pH levels.

[0155] Example 1: Synthesis of multi-motif PAMAM dendron polymers For PAMAM polymers, unless otherwise indicated, measurements were performed in deuterated chloroform as the solvent, using tetramethylsilane (TMS) or the residual solvent signal as the internal reference. 1 H, 13 C, and 19 The F NMR spectrum is 1 500MHz for H NMR; 13 126MHz, 400MHz, or 500MHz for C NMR, and 19 F NMR was recorded on a machine operating at 400 MHz. All chemical shifts reported are expressed in ppm on the delta scale and fine divisions of signals appearing in the recordings are generally indicated, e.g.: br = broad, s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, m, multiplet, coupling constant, in Hz. 1 H and 13 C NMR was recorded on either an Agilent DD2 500 MHz equipped with a direct cryoprobe (126 MHz) or an Agilent VnmrS 400 MHz, yielding a peak at 7.26 ppm ( 1 H) and 77.16 ppm ( 13 C) CDCl 3Matrix-assisted laser desorption / ionization time-of-flight mass spectra (MALDI-TOF MS) were recorded on a Bruker AutoFlex Speed ​​using a dithranol matrix, and direct analysis in real time mass spectra (DART MS) were recorded on a JEOL AccuTOF Plus 4G. Electrospray ionization mass spectra (ESI MS) were recorded on an Agilent 6538 UHD.

[0156] [ka] Preparation 1: A solution of n-propylamine (2.5 g, 42.3 mmol, 1 equiv.) in methanol was added dropwise to a solution of methyl acrylate (35.31 g, 422.9 mmol, 10 equiv.) in methanol. The mixture was stirred at room temperature for 72 h. The solvent and excess methyl acrylate were removed under reduced pressure. Further purification (if necessary) was performed by flash chromatography using a gradient from 100% dichloromethane (DCM) to 7% MeOH / DCM. Intermediate A was isolated as a pale yellow clear oil (8.47 g, 86%).

[0157] 1 H NMR (400 MHz, CDCl 3 )δ 3.61(s,6H),2.71(t,J=7.2 Hz,4H),2.39(t,J=7.2 Hz,4H),2.32(t,2H,J=7.3Hz),1.38(sextet,J=7.3 Hz,2H),0.80(t,J=7.3 Hz,3H). 13 C NMR (400 MHz, CDCl 3 )δ 173.04,77.40,77.08,76.76,55.72,51.43,49.31,32.59,20.32,11.65.

[0158] Preparation 2: Methyl acrylate (7.28 g, 84.6 mmol, 5 equiv.) was added along with n-propylamine (1.0 g, 16.9 mmol, 1 equiv.) in methanol (10 mL). The mixture was purged with nitrogen, covered with foil, and stirred at room temperature for 72 h. Consumption of n-propylamine was monitored by thin layer chromatography (TLC) (10% MeOH / dichloromethane (DCM)). Solvent and excess methyl acrylate were removed under reduced pressure. Intermediate A was isolated as a clear, colorless oil containing a small amount of methyl acrylate impurity (4.08 g, 17.6 mmol, 104%).

[0159] [ka] Preparation 1: A solution of ethylenediamine (20.79 g, 345.9 mmol, 40 equiv.) in methanol was added dropwise to a solution of intermediate A (2.00 g, 8.65 mmol, 1 equiv.) in methanol. The mixture was stirred at room temperature for 1 week. The solvent and excess ethylenediamine were removed under reduced pressure to give intermediate B (2.88 g, 116%) as a yellow viscous oil containing residual ethylenediamine.

[0160] 1 H NMR (500 MHz, CDCl 3 )δ 7.49(t,J=5.7 Hz,2H),3.27(q,J=5.7 Hz,4H),2.81(t,J=6.0Hz,4H),2.71(t,4H),2.41-2.30(m,6H),1.45(sextet,J=7.4Hz,2H),0.85(t,J=7.4 Hz,3H). 13 C NMR (500 MHz, CDCl 3 )δ 172.86,55.67,50.23,42.04,41.53,34.13,19.99,12.02.

[0161] Preparation 2: Ethylenediamine (15.6 g, 259 mmol, 20 equiv.) and intermediate A (3.0 g, 13 mmol, 1 equiv.) were added together in methanol (10 mL). The mixture was purged with nitrogen, covered with foil, and stirred at room temperature for 5 days. Consumption of intermediate A was monitored by TLC (15% MeOH / DCM). Solvent and excess ethylenediamine were removed under reduced pressure. Residual ethylenediamine was removed by successive washes with diethyl ether (5×30 mL). Residual ether was removed under vacuum to give intermediate B as a clear, colorless, viscous oil (1.604 g, 5.58 mmol, 43%).

[0162] 1 H NMR (400 MHz, CDCl 3 )δ 7.71(t,J=5.7 Hz,2H),2.98(q,J=5.9 Hz,4H),2.52(t,J=6.0 Hz,4H),2.46(t,J=6.3 Hz,4H),2.17-2.03(m,6H),1.35(s,8H),1.19(sextet,J=7.4 Hz,1H),0.59(t,J=7.3 Hz,3H). 13 C NMR (400 MHz, CDCl 3 )δ 172.44,55.01,49.55,48.88,41.80,41.12,33.33,19.46,11.46.

[0163] [ka] Preparation 1: A solution of 1,2-epoxytetradecane (1.03 g, 4.4 equiv.) in EtOH was added to a solution of intermediate B (0.3 g, 1 equiv.) in EtOH in a sealed pressure vessel. The mixture was stirred at 80° C. for 72 h. The solvent was removed under reduced pressure. The crude oil was purified by flash chromatography using a gradient of 20% to 100% ULTRA solvent in DCM. The desired fractions were combined and concentrated under reduced pressure to give a pale yellow viscous oil (0.76 g, 64%). 1 H NMR (500 MHz, CDCl 3)δ 7.78(br s,<2H),3.28(br s,<1H),3.0-2.2(m,20H),1.6-1.1(m,105H),0.87(m,15H). 13 C NMR (500 MHz, CDCl 3 )δ 172.69,70.68,68.10,64.74,61.08,50.02,31.92,29.66,25.75,22.68,14.10,11.82. MS(MALDI-TOF pos)m / z:[M+H] + C 69 H 141 N 5 O 6 Theoretical value: 1137.09; measured value: 1137.16.

[0164] Preparation 2: A mixture of 1,2-epoxytetradecane (1.17 g, 5.51 mmol, 4.4 equiv.) and Intermediate B (0.36 g, 1.25 mmol, 1 equiv.) was added together to an 8-dram screw-cap vial. The vial was covered with foil and stirred at 90° C. overnight. Consumption of Intermediate B was monitored by TLC (35% ULTRA / DCM). The crude oil was purified by flash chromatography using a 50 g silica cartridge and a solvent gradient from 10% ULTRA / DCM to 20% ULTRA / DCM. The desired fractions were combined and concentrated under vacuum to give a pale yellow, clear, viscous oil (0.39 g, 0.34 mmol, 28%). 1 H NMR (500 MHz, CDCl 3 )δ 7.96-7.86(m,2H),3.57-3.45(m,5H),3.19(q,J=4.7 Hz,1H),2.71-2.63(m,4H),2.62-2.53(m,3H),2.53-2.45(m,2H),2.41-2.20(m,10H),1.53-1.04(m,105H),0.91-0.66(m,15H). 13 C NMR (500 MHz, CDCl 3)δ 70.46,70.36,70.27,68.09,67.92,64.87,64.74,64.63,61.50,61.43,56.77,55.71,55.19,49.77,49.72,49.68,38.51,37. 35,35.26,35.16,35.01,34.94,33.68,33.54,31.89,29.84,29.68,29.64,29.34,25.78,25.74,22.64,19.58,14.06,11.81. MS(ESI+)m / z:[M+H] + C 69 H 141 N 5 O 6 Theoretical value: 1137.92; measured value: 1137.09.

[0165] [ka] A solution of 1,2-epoxyhexadecane (1.3 g, 4 eq.) in EtOH was added to a solution of intermediate B (0.3 g, 1 eq.) in EtOH in a sealed pressure vessel. The mixture was stirred at 80° C. for 72 h. The solvent was removed under reduced pressure. The crude oil was purified by flash chromatography using a gradient of 20% to 100% ULTRA solvent in DCM. The desired fractions were combined and concentrated under reduced pressure to give a pale yellow viscous oil (0.42 g, 32%). 1 H NMR (500 MHz, CDCl 3 )δ 7.95-7.80(m,1H),3.25(br s,1H),2.80-2.20(m,20H),1.6-1.1(m,125H),0.84(t,J=6.9 Hz,15H). 13 C NMR (500 MHz, CDCl 3 )δ 172.98,70.55,68.08,64.73,61.25,58.02,53.37,31.90,29.69,22.65,14.07. MS(DART pos)m / z:[M-OH] + C 69 H 140 N 5 O 5 Theoretical value: 1232.21; measured value: 1232.66.

[0166] [ka] A solution of 1,2-epoxy-9-decene (0.49 g, 4 eq.) in EtOH was added to a solution of intermediate B (0.23 g, 1 eq.) in EtOH in a sealed pressure vessel. The mixture was stirred at 80° C. for 72 h. The solvent was removed under reduced pressure. The crude oil was purified by flash chromatography using a gradient of 20% to 100% ULTRA solvent in DCM. The desired fractions were combined and concentrated under reduced pressure to give a pale yellow viscous oil (0.21 g, 33%). 1 H NMR (500 MHz, CDCl 3 ) δ 8.30-7.3 (m, mostly impurities. br s containing 2H), 5.80-5.70 (m, 4H), 4.90 (dd, 8H), 3.23 (br s, 2H), 2.80-2.20 (m, 22H), 1.98 (q, J = 6.7Hz, 8H), 1.50-1.20 (m, 46H), 0.81 (t, J = 7.3Hz, 3H). 13 C NMR (500 MHz, CDCl 3 )δ 172.74,139.00,133.91,131.53,128.44,128.42,127.23,127.20,114.15,70.2 4,67.97,64.54,61.38,57.95,33.70,29.60,29.01,28.80,25.63,25.61,18.25. MS(DART pos)m / z:[M+H] + C 53 H 101 N 5 O 6 Theoretical value: 904.78; measured value: 904.81.

[0167] [ka] A solution of 2,2,3,3,4,4,5,5,5-nonafluoropentyloxirane (15 g, 4 eq.) in EtOH was added to a solution of Intermediate B (0.4 g, 1 eq.) in EtOH in a sealed pressure vessel. The mixture was stirred at 80° C. for 72 h. The solvent was removed under reduced pressure. The crude oil was purified by flash chromatography using a gradient of 20% to 100% ULTRA solvent in DCM. The desired fractions were combined and concentrated under reduced pressure to give a pale yellow viscous oil (0.25 g, 13%). 1 H NMR (500 MHz, CDCl 3 ) δ 7.90-7.35 (m, mostly br s with impurity 2H), 4.35 (p, J=5.5Hz, <1H), 3.34-3.26 (m, 2H), 2.85-2.00 (m, 55H), 1.55-1.40 (m, 2H), 0.88-0.81 (m, 3H). 19 F NMR (400 MHz, CDCl 3 )δ-81.49(3F),-112.87(1.4F),-113.26(0.4F),-124.87(2F),126.26(2F). MS(MALDI-TOF pos)m / z:[M+H] + C 41 H 49 F 36 N 5 O 6 Theoretical value: 1392.31; measured value: 1392.3.

[0168] [ka] A solution of intermediate B (2.74 g, 9.53 mmol, 1 eq.) in methanol was added dropwise to a solution of methyl acrylate (16.4 g, 190.7 mmol, 20 eq.) in methanol. The mixture was stirred at room temperature for 72 h. The solvent and excess methyl acrylate were removed under reduced pressure. Further purification was carried out by flash chromatography using a gradient of 10% to 50% ULTRA in DCM. Intermediate C was isolated as a pale yellow clear oil (1.46 g, 24%). 1 H NMR (500 MHz, CDCl 3)δ 7.13(t,J=5.5Hz,2H),3.15(q,J=5.6 Hz,4H),2.63(t,J=6.8 Hz,12H),2.41(t,J=6.4Hz,4H),2.30(t,J=6.8 Hz,10H),2.23(t,J=6.8Hz,4H),1.33(sextet,J=7.4 Hz,2H),0.73(t,J=7.3 Hz,3H). 13 C NMR (500 MHz, CDCl 3 )δ 172.86,172.18,55.02,52.86,51.46,49.60,49.12,36.93,33.32,32.56,19.83,11.75. MS(DART pos)m / z:[M+H] + C 29 H 53 N 5 O 10 Theoretical value: 632.38; measured value: 632.28.

[0169] [ka] A solution of ethylenediamine (10.6 g, 110.8 mmol, 80 equiv.) in methanol was added dropwise to a solution of intermediate C (1.4 g, 2.22 mmol, 1 equiv.) in methanol. The mixture was stirred at room temperature for 1 week. The solvent and excess ethylenediamine were removed under reduced pressure to give intermediate D (1.88 g, 114%) as a yellow viscous oil containing residual ethylenediamine. 1 H NMR (500 MHz, CDCl 3 )δ 8.01(t,J=5.7 Hz,1H),7.65(t,J=5.7 Hz,2H),3.25(q,J=5.8 Hz,4H),3.20(q,J=6.1 Hz,2H),2.79(t,J=5.7 Hz,3H),2.74-2.65(m)2.50(t,J=6.2 Hz,2H),2.40-2.28(m,14H)1.42(h,J=7.3 Hz,1H),0.83(t,J=7.3 Hz,1H). 13 C NMR (500 MHz, CDCl 3)δ 173.07,172.88,55.22,52.86,50.60,50.22,49.73,45.78,44.77,42.07,41.42,37.88,34.37,33.60,19.63,11.91.

[0170] [ka] A solution of 1,2-epoxytetradecane (1.14 g, 5.38 mmol, 8 equiv) in EtOH was added to a solution of intermediate D (0.5 g, 0.672 mmol, 1 equiv) in EtOH in a sealed pressure vessel. The mixture was stirred at 80° C. for 72 h. The solvent was removed under reduced pressure. The crude oil was purified by flash chromatography using a gradient of 20% to 50% ULTRA in DCM. The desired fractions were combined, dried over anhydrous sodium, and concentrated under reduced pressure to give an off-white amorphous solid (0.19 g, 12%). 1 H NMR (500 MHz, CDCl 3 )δ 8.05-7.80(m,2H),3.30-3.00(m,3H),2.90-2.10(m,36H),1.50-1.00(m,198H),0.82(t,J=7.0 Hz,27H). 13 C NMR (126 MHz, cdc 3 )δ 174.94,173.15,70.35,70.32,69.33,67.73,64.62,64.23,61.63,57.81,56.17,55.11,55.06,47.85,31.88,29.88,29 .84,29.80,29.75,29.73,29.68,29.67,29.64,29.63,29.57,29.33,29.31,25.79,25.76,25.73,22.64,18.28,14.05. MS(ESI pos)m / z:[M+H] + C 145 H 293 N 13 O 14 Theoretical value: 2442.26; measured value: 2442.26.

[0171] [ka] A solution of 1,2-epoxyhexadecane (2.71 g, 11.3 mmol, 10 equiv.) in EtOH was added to a solution of intermediate D (0.84 g, 1.13 mmol, 1 equiv.) in EtOH in a sealed pressure vessel. The mixture was stirred at 80° C. for 72 h and then extracted with dichloromethane and water. The organic fraction was collected and the solvent was removed under reduced pressure to give the crude product (1.52 g, 50%). 1 H NMR (500 MHz, CDCl 3 )δ 7.98(br s,3.7H),3.42(br s,3H),3.35-3.20(m,4.2H),3.00-2.00(m,53H),1.60-1.00(m,209H),0.87(t,J=7.0Hz,27H). 13 C NMR(500 MHz,CDCl3)δ 172.90,70.38,67.73,64.21,61.99,52.63,52.62,31.91,29.71,29.35,25.78,22.67,14.09. MS(ESI pos)m / z:[M+H] + C 161 H 325 N 14 O 14 Theoretical value: 2666.51; measured value: 2666.51.

[0172] [ka] A solution of 1,2-epoxy-9-decene (1.2 g, 12 eq.) in EtOH was added to a solution of intermediate D (0.5 g, 1 eq.) in EtOH in a sealed pressure vessel. The mixture was stirred at 80° C. for 72 h. The solvent was removed under reduced pressure. The crude oil was purified by flash chromatography using a gradient of 20% to 50% ULTRA in DCM. The desired fractions were combined, dried over anhydrous salt, and concentrated under reduced pressure to give a yellow viscous oil (0.75 g, 56%). 1 H NMR (500 MHz, CDCl 3)δ 8.10-7.75(m,6H),5.80-5.70(m,8H),4.98-4.86(m,16H),3.56(br s,9H),3.30-3.15(m,8H),2.80-2.20(m,53H),1.99(q,J=6.7 Hz,16H),1.50-1.15(m,90H),0.83(t,J=7.3 Hz,3H). 13 C NMR (126 MHz, CDCl 3 )δ 173.23,173.17,139.03,114.17,114.16,70.16,70.10,67.96,67.90,64.55,61.84,56.88,54.89,52.55,50.31,50.30,50.20,49.65, 38.67,37.49,35.13,34.88,34.85,33.73,29.66,29.64,29.61,29.05,29.04,29.02,28.83,28.81,25.67,25.65,25.63,19.25,11.89. MS(ESI pos)m / z:[M+H] + C 133 H 213 N 13 O 14 Theoretical value: 1977.64; measured value: 1977.64.

[0173] [ka] A solution of free base cystamine (20.79 g, 136 mmol, 15 equiv) in methanol was added dropwise to a solution of intermediate A (2.00 g, 8.65 mmol, 1 equiv) in methanol. The mixture was stirred at room temperature for 1 week. The solvent was removed under reduced pressure to give a crude oil. The crude oil was purified by tetrahydrofuran (TFA) with a 70:26:4 ratio of DCM:MeOH:NH 4 The desired fractions were collected and purified by flash chromatography using an eluent of 0.5 mL of anhydrous NaOH. 2 SO 4 It was dried over water and concentrated to give a pale yellow viscous oil (0.55 g, 13%). 1 H NMR (500 MHz, CDCl 3)δ 7.56(t,J=5.9 Hz,2H),3.52(q,J=6.1 Hz,4H),2.99(t,J=6.2 Hz,4H),2.82-2.73(m,8H),2.71(t,J=6.5 Hz,4H),2.42-2.29(m,6H),1.87(s,6H),1.45(h,J=7.3 Hz,2H),0.85(t,J=7.4 Hz,3H). 13 C NMR (126 MHz, CDCl 3 )δ 172.54,57.55,55.04,49.58,42.17,41.98,40.41,40.39,37.96,37.75,33.28,19.69,18.42,11.92. MS(DART pos)m / z:[M+H] + C 17 H 37 N 5 O 2 S 4 Theoretical value: 472.18; measured value: 472.19.

[0174] [ka] A solution of 1,2-epoxytetradecane (0.67 g, 3.14 mmol, 4 equiv) in EtOH was added to a solution of intermediate E (0.37 g, 0.78 mmol, 1 equiv) in EtOH in a sealed pressure vessel. The mixture was stirred at 70° C. for 72 h. The solvent was removed under reduced pressure. The crude oil was purified by flash chromatography using a gradient of 20% to 40% ULTRA solvent in DCM. The desired fractions were combined and concentrated under reduced pressure to give a viscous yellow oil (0.15 g, 14.6%). 1 H NMR (500 MHz, CDCl 3 )δ 7.64-7.54(m,2H),3.53(q,J=6.2Hz,4H),3.00-2.25(m,30H),1.52-1.15(m,90H),0.86(t,J=0.69Hz,15H). MS(MALDI-TOF pos)m / z:[M+H] + C 73 H 149 N 5 O 6 S 4Theoretical value: 1321.04; measured value: 1321.2.

[0175] [ka] A solution of free base cystamine (13.2 g, 87 mmol, 39 equiv) in methanol was added dropwise to a solution of intermediate C (1.41 g, 2.23 mmol, 1 equiv) in methanol. The mixture was stirred at room temperature for 1 week. The solvent was removed under reduced pressure to give a crude oil. The crude oil was purified by precipitation in MeOH / diethyl ether to give a viscous yellow oil (0.86 g, 0.35%). 1 H NMR (500 MHz, CDCl 3 )δ 7.77(m,2H),7.65(t,J=5.8 Hz,4H),3.53(q,J=6.5 Hz,8H),3.24(m,4H)3.05-2.95(m,16H),2.85-2.65(m,30H),2.60-2.25(m,15H),1.65(br s,38H),1.45-1.40(m,2H),0.86(t,J=7.3 Hz,3H).MS(ESI pos)m / z:[M+H] + C 41 H 85 N 5 O 6 S 8 Theoretical value: 1112.45; measured value: 1112.46.

[0176] [ka] A solution of 1,2-epoxytetradecane (1.97 g, 9.27 mmol, 12 equiv) in EtOH was added to a solution of intermediate F (0.86 g, 0.77 mmol, 1 equiv) in EtOH in a sealed pressure vessel. The mixture was stirred at 50° C. for 72 h. The solvent was removed under reduced pressure. The crude oil was purified by flash chromatography using a gradient of 30% to 50% ULTRA solvent in DCM. The desired fractions were combined and concentrated under reduced pressure to give a viscous yellow oil (0.36 g, 17%). 1 H NMR (400 MHz, CDCl 3)δ 7.83(br s,2H),7.71(br s,4H)3.63(m,8H),3.51(m,8H),3.24(m,4H),3.10-2.20(m,70H),1.60-1.00(m,187H),0.87(t,J=7.1 Hz,1H).

[0177] [ka] Any compound produced using the ring-opening reaction of an epoxide can be used to reduce the -OH group to a ketone by oxidation. An example of an oxidizing agent is Dess-Martin periodinane.

[0178] [ka] Dess-Martin periodinane (2.545 g, 1.5 equiv.) was added to a solution of 1-tetradecanol (0.858 g, 1 equiv.) in DCM (30 mL). The mixture was stirred at room temperature under nitrogen for 16 h. A 1:1 mixture of saturated aqueous sodium bicarbonate and sodium thiosulfate (100 mL) was added to quench the reaction. The organic phase was then extracted three times with diethyl ether (3×40 mL). The combined organic extracts were washed with water (3×30 mL) and brine (90 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude was further purified by flash chromatography using a gradient of 0% to 10% ethyl acetate in hexanes. The desired fractions were combined and concentrated under reduced pressure to give the tetradecanal as a white waxy solid (0.48 g, 57%).

[0179] A solution of intermediate B (0.108 g, 1 eq.) in dichloroethane (DCE) (5 mL) was added to tetradecanal (0.48 g, 6 eq.) on ice. A solution of sodium triacetoxyborohydride (0.48 g, 6 eq.) and glacial acetic acid (0.136 g, 6 eq.) in DCE (5 mL) was added to the mixture and the reaction was allowed to warm to room temperature. The mixture was stirred at room temperature under nitrogen for 16 hours. The reaction was quenched by slow addition of saturated aqueous sodium bicarbonate (50 mL). The organic phase was extracted four times with diethyl ether (4×40 mL). The combined extracts were washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was further purified by flash chromatography using a gradient of 0% to 20% ULTRA solvent in DCM. The desired fractions were combined and concentrated under reduced pressure to afford I-22 (G1-nPr-C14) as a colorless oil (0.17 g, 45%). 1 H NMR(500 MHz,CDCL3)δ:6.98(t,J=5.2 Hz,2H),3.24(q,J=6.0 Hz,3H),2.71(t,J=6.6 Hz,3H),2.50(t,J=6.3 Hz,3H),2.42-2.33(m,9H),2.29(t,J=6.6 Hz,4H),1.47-1.33(m,10H),1.23(s,92H),0.84(td,J=7.1,5.5 Hz,15H).MS(ESI pos)m / z:[M+H] + C 69 H 141 N 5 O 2 Theoretical value 1073.11; measured value 1073.11.

[0180] [ka] Dess-Martin periodinane (2.545 g, 1.5 equiv.) was added to a solution of 1-tetradecanol (0.858 g, 1 equiv.) in DCM (30 mL). The mixture was stirred at room temperature under nitrogen for 16 h. A 1:1 mixture of saturated aqueous sodium bicarbonate and sodium thiosulfate (100 mL) was added to quench the reaction. The organic phase was then extracted with diethyl ether (3×40 mL). The combined organic extracts were washed with water (3×30 mL) and brine (90 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude was further purified by flash chromatography using a gradient of 0% to 10% ethyl acetate / hexanes. The desired fractions were combined and concentrated under reduced pressure to give the tetradecanal as a white waxy solid (0.48 g, 57%).

[0181] Dichloroethane was dried over molecular sieves (Sigma-Aldrich, 3 Å, 8-12 mesh) for 1 week before use. A solution of intermediate B (0.108 g, 0.376 mmol, 1 equiv.) in dichloroethane (5 mL) was added to tetradecanal (0.48 g, 2.3 mmol, 6 equiv.) at 0 °C. A solution of sodium triacetoxyborohydride (STAB) (0.48 g, 2.3 mmol, 6 equiv.) and glacial acetic acid (0.136 g, 2.26 mmol, 6 equiv.) in dichloroethane (5 mL) was added to the mixture and the reaction was allowed to warm to room temperature. The mixture was purged with nitrogen, covered with foil, and stirred for 16 h. The reaction was quenched by slow addition of saturated aqueous sodium bicarbonate (50 mL). The organic phase was extracted with diethyl ether (4 × 40 mL). The combined extracts were washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was further purified by flash chromatography using a gradient of 0% to 20% ULTRA / DCM. The desired fractions were combined and concentrated under reduced pressure to give a clear, colorless, viscous oil (0.17 g, 45%). 1 H NMR (500 MHz, CDCl 3)δ 6.98(t,J=5.2 Hz,2H),3.24(q,J=6.0 Hz,4H),2.71(t,J=6.6 Hz,4H),2.50(t,J=6.3 Hz,4H),2.42-2.33(m,9H),2.29(t,J=6.6 Hz,4H),1.47-1.33(m,10H),1.23(s,92H),0.84(t,J=7.1Hz,15H).MS(MALDI-TOF+)m / z:[M+H] + C 69 H 141 N 5 O 2 Theoretical value 1073.11; measured value 1073.11.

[0182] [ka] 1-Pentadecane (0.5 g, 2.37 mmol, 1 equiv.) was dissolved in dichloromethane (10 mL) and added dropwise to a flask containing m-chloroperoxybenzoic acid (1.17 g, 4.75 mmol, 2 equiv.) dissolved in dichloromethane (10 mL). The reaction was stirred for 18 h and then quenched with saturated sodium sulfite (40 mL) with stirring. The mixture was transferred to a separatory funnel and extracted with dichloromethane (3×30 mL). The organic fraction was collected, washed with 1 M sodium hydroxide (3×30 mL), brine (1×50 mL), and then dried over anhydrous salt. The organic fraction was concentrated under vacuum to give 1,2-epoxypentane (0.49 g, 2.16 mmol, 91%) as a clear, colorless oil containing a small amount of benzoic acid impurity. The epoxide was used without further purification. 1 H NMR (500 MHz, CDCl 3 )δ 2.86(tdd,J=5.5,4.0,2.7 Hz,1H),2.70(dd,J=5.1,3.9 Hz,1H),2.42(dd,J=5.1,2.7 Hz,1H),1.52-1.17(m,26H),0.85(t,J=7.0 Hz,3H). 13 C NMR (500 MHz, CDCl 3)δ 52.37,47.07,32.57,32.00,29.76,29.74,29.73,29.71,29.63,29.52,29.43,26.04,22.75,14.14.

[0183] I-25 (G1-C3-K2-E15) was synthesized similarly to I-1 (G1-nPr-C14E), using 1,2-epoxypentadecane instead of 1,2-epoxytetradecane. The purified compound was a pale yellow, clear, viscous oil (73.9 mg, 0.062 mmol, 11%). 1 H NMR (500 MHz, CDCl 3 )δ 7.99-7.90(m,1H),7.87(t,J=5.6 Hz,0.5H),3.63-3.47(m,4H),3.24(q,J=5.5 Hz,1H),2.71(q,J=6.7 Hz,5H),2.66-2.58(m,2H),2.58-2.50(m,2H),2.45-2.23(m,9H),1.52-1.09(m,104H),0.82(dt,J=11.9,7.0 Hz,15H). 13 C NMR (500 MHz, CDCl 3 )δ 172.91,172.87,172.77,172.50,70.49,68.09,64.77,61.43,55.48,53.36,49.83,35.15,35.02,34.9 1,33.86,31.88,29.89,29.84,29.81,29.80,29.68,29.63,29.33,25.81,25.72,22.64,14.06,11.82. MS(MALDI-TOF+)m / z:[M+H] + C 73 H 149 N 5 O 6 Theoretical value: 1193.15; measured value: 1193.2.

[0184] [ka] I-26 (G1-C3-K2-E10) was synthesized similarly to I-1 (G1-nPr-C14E), using 1,2-epoxydecane instead of 1,2-epoxytetradecane. The purified compound was a pale yellow, clear, viscous oil (0.5961 g, 0.6533 mmol, 38%). 1 H NMR (400 MHz, CDCl 3 )δ 7.99-7.75(m,2H),3.69-3.51(m,4H),3.28(q,J=5.6 Hz,2H),2.82-2.73(m,4H),2.73-2.55(m,5H),2.53-2.26(m,13H),1.57-1.11(m,60H),0.86(q,J=6.9 Hz,15H). 13 C NMR (400 MHz, CDCl 3 )δ 70.81,68.27,64.87,50.16,35.33,35.15,32.02,29.96,29.73,29.44,25.88,22.80,14.23,11.97. MS(MALDI-TOF+)m / z:[M]+ C 53 H 109 N 5 O 6 Theoretical value: 912.5; measured value: 912.8.

[0185] [ka] 1,3-Diaminopropane (9.62 g, 129.7 mmol, 15 equiv.) was mixed with Intermediate A (2.0 g, 8.6 mmol, 1 equiv.) in methanol (10 mL). The mixture was purged with nitrogen, covered with foil, and stirred at room temperature for 5 days. Consumption of Intermediate A was monitored by TLC (15% MeOH / DCM). The solvent was removed under reduced pressure. Residual propanediamine was removed by successive washes with 5% methanol in diethyl ether (5×30 mL). Residual solvent was removed under vacuum to give Intermediate B′ (0.558 g, 20%) as a clear, colorless, viscous oil. 1 H NMR (400 MHz, CDCl 3)δ 7.72(t,J=5.8 Hz,2H),3.09(q,J=6.4 Hz,4H),2.54(td,J=6.6,2.5 Hz,8H),2.39(s,7H),2.24-2.18(m,1H),2.14(t,J=6.5 Hz,3H),1.44(p,J=6.7 Hz,4H),1.28(sextet,J=7.3 Hz,1H),0.69(t,J=7.4 Hz,3H). 13 C NMR (400 MHz, CDCl 3 )δ 172.38,54.89,49.50,49.24,39.08,36.41,33.15,32.38,19.51,11.57. MS(DART+)m / z:[M+H] + C 15 H 33 N 5 O 2 Theoretical value: 316.26; measured value: 316.27.

[0186] [ka] A mixture of 1,2-epoxydecane (0.300 g, 1.92 mmol, 5 equiv.) and Intermediate B' (0.121 g, 0.38 mmol, 1 equiv.) was added together to an 8-dram screw-cap vial. The mixture was covered with foil and stirred at 90° C. overnight. Consumption of Intermediate B was monitored by TLC (35% ULTRA / DCM). The crude oil was purified by flash chromatography using a 50 g silica cartridge and a solvent gradient from 20% ULTRA / DCM to 40% ULTRA / DCM. The desired fractions were combined and concentrated under vacuum to give a pale yellow, clear, viscous oil (0.12 g, 34%). 1 H NMR (400 MHz, CDCl 3 )δ 7.96-7.84(m,2H),3.71-3.58(m,4H),3.30(q,J=5.2 Hz,2H),3.22-3.10(m,1H),2.77(q,J=6.8 Hz,3H),2.68-2.58(m,3H),2.57-2.16(m,13H),1.63(p,J=6.3 Hz,4H),1.54-1.11(m,58H),0.85(td,J=7.0,3.1 Hz,15H).13 C NMR (400 MHz, CDCl 3 )δ 172.34,70.24,68.00,63.19,60.76,49.94,35.43,35.24,33.82,31.94,29.92,29.66,29.36,25.82,25.74,22.72,14.15,11.88. MS(ESI+)m / z:[M+H] + C 55 H 113 N 5 O 6 Theoretical value: 940.87; measured value: 940.87.

[0187] [ka] 2-Amino-1-ethanol (0.916, 15 mmol, 1 equiv.) was added to a mixture of imidazole (2.042 g, 30 mmol, 2 equiv.) and tert-butyldimethylsilyl chloride (2.487 g, 16.5 mmol, 1.1 equiv.) in dichloromethane (50 mL) and stirred at room temperature for 3 h. The mixture was then diluted with water (60 mL) and extracted with dichloromethane (3×30 mL). The organic fraction was washed with brine (1×50 mL) and then dried over anhydrous salt. The organic fraction was concentrated under vacuum to yield 2-((tert-butyldimethylsilyl)oxy)ethan-1-amine (2.96 g, 112%), which was used without further purification. 1 H NMR (400 MHz, CDCl 3 )δ 3.58(t,J=5.3 Hz,2H),2.73(t,2H),0.86(s,9H),0.02(s,6H).

[0188] [ka] 2-Amino-1-ethanol (0.916, 15 mmol, 1 equiv.) was added to a mixture of imidazole (2.042 g, 30 mmol, 2 equiv.) and tert-butyldimethylsilyl chloride (2.487 g, 16.5 mmol, 1.1 equiv.) in dichloromethane (50 mL) and stirred at room temperature for 3 h. The mixture was then diluted with water (60 mL) and extracted with dichloromethane (3×30 mL). The organic fraction was washed with brine (1×50 mL) and then dried over anhydrous salt. The organic fraction was concentrated under vacuum to yield 2-((tert-butyldimethylsilyl)oxy)ethan-1-amine (2.96 g, 112%), which was used without further purification. 1 H NMR (400 MHz, CDCl 3 )δ 3.58(t,J=5.3 Hz,2H),2.73(t,2H),0.86(s,9H),0.02(s,6H).

[0189] 2-((tert-Butyldimethylsilyl)oxy)ethan-1-amine (2.96 g, 16.9 mmol, 1 equiv.) was mixed with methyl acrylate (3.87 g, 45 mmol, 3 equiv.) in methanol (10 mL). The mixture was purged with nitrogen, covered with foil, and stirred at room temperature for 3 days. Consumption of TBS-A' was monitored by TLC (20% MeOH / DCM). The solvent and excess methyl acrylate were removed under vacuum to yield TBS-A' as a clear, colorless oil (3.72 g, 71%). 1 H NMR (400 MHz, CDCl 3 )δ 3.59(s,7H),2.76(t,J=7.2 Hz,4H),2.53(t,J=6.4 Hz,2H),2.38(d,J=14.3 Hz,2H),0.82(s,9H),-0.02(s,6H).

[0190] [ka] 1,3-Diaminopropane (6.35 g, 85.6 mmol, 8 equiv.) was added to a flask containing TBS-A' (3.72 g, 10.7 mmol, 1 equiv.) dissolved in methanol (10 mL). The flask was purged with nitrogen, covered with foil, and stirred for 5 days. Consumption of TBS-A' was monitored by TLC (15% MeOH / DCM). The solvent was removed under reduced pressure, and the majority of the 1,3-diaminopropane was removed by successive washes with 15% ethyl acetate in hexanes (5 x 20 mL). Residual solvent was removed under vacuum to give TBS-B''' as a clear, colorless, viscous oil containing residual 1,3-diaminopropane impurity (1.59 g, 3.68 mmol, 34%). 1 H NMR (500 MHz, CDCl 3 )δ 7.62(t,J=5.7 Hz,2H),3.42(t,J=6.0 Hz,2H),3.02(q,J=6.6 Hz,4H),2.59-2.44(m,18H,1,3-diaminopropane impurity),2.37(t,J=6.1 Hz,2H),2.12-2.02(m,6H),1.75(br s,16H,water),1.41-1.30(m,9H),0.63(s,7H),0.62(s,2H),-0.20(s,4H),-0.20(s,2H). 13 C NMR (500 MHz, CDCl 3 )δ 172.02,60.67,59.88,54.93,50.08,48.86,39.37,39.02,36.52,36.26,33.33,32.44,25.45,20.55,17.79,13.71,-5.79.

[0191] [ka] A mixture of 1,2-epoxydecane (2.3 g, 14.7 mmol, 12 equiv.) and intermediate TBS-B''' (0.53 g, 1.23 mmol, 1 equiv.) was added together in an 8-dram screw-cap vial. A higher equivalent of 1,2-epoxydecane was required to alkylate the residual 1,3-diaminopropane, which could be separated during flash chromatography. The mixture was covered with foil and stirred at 90° C. overnight. The crude oil was purified by flash chromatography using a 50 g silica cartridge and a solvent gradient from 20% ULTRA / DCM to 40% ULTRA / DCM. The desired fractions were combined and concentrated under vacuum to give TBS-I-28 as a pale yellow, clear, viscous oil (0.83 g, 0.79 mmol, 64%). 1 H NMR (500 MHz, CDCl 3 )δ 7.76-7.67(m,2H),),3.75-3.57(m,6H),3.33-3.25(m,2H),3.23-3.11(m,2H),2.82-2.74(m,4H),2.66-2.58(m,4H),2.51(dd,J=13.4,3.1 Hz,2H),2.45-2.37(m,6H),2.31(t,J=6.5 Hz,4H),2.23(dd,J=13.0,2.3 Hz,2H),1.69-1.57(m,4H),1.48-1.18(m,66H),0.86(t,J=6.9 Hz,24H),0.04(s,6H). 13 C NMR (500 MHz, CDCl 3 )δ 172.60,172.56,70.28,68.00,63.17,60.86,60.83,60.78,55.96,55.16,55.09,53.75,53.47,50.65,50.61,50.59,50.56,38.05,37.89 ,37.85,35.40,35.20,34.35,34.32,34.16,31.95,29.92,29.66,29.37,27.17,26.58,25.99,25.83,25.77,22.73,18.33,14.16,-5.24. MS(DART+)m / z:[M+H]+C 60 H 125 N 5 O 7Theoretical value of Si is 1056.93; measured value is 1056.95.

[0192] [ka] TBS-I-28 (0.830 g, 0.785 mmol, 1 equiv.) was then dissolved in diethyl ether (50 mL), cooled to 0° C., and a solution of tetra-n-butylammonium fluoride (TBAF) (3.14 mL, 3.14 mmol, 4 equiv.) was added. The reaction mixture was allowed to warm to room temperature, covered with foil, and stirred for 18 h. Consumption of the TBS-protected material was monitored by TLC (35% ULTRA / DCM). The organic fraction was washed with 20% 1M sodium hydroxide in saturated ammonium chloride (5×15 mL) and brine (1×50 mL), then dried over anhydrous salt. The organic fraction was concentrated to a crude oil and purified by flash chromatography using a 50 g silica cartridge and a solvent gradient from 20% ULTRA / DCM to 50% ULTRA / DCM. The desired fractions were collected and concentrated in vacuo to give 16 (0.20 g, 0.21 mmol, 27%) as a clear, colorless, viscous oil. 1 H NMR (500 MHz, CDCl 3 )δ 7.97-7.85(m,2H),4.46(s,6H),3.69-3.50(m,9H),3.35-3.24(m,3H),3.08-2.93 (m,1H),2.74-2.13(m,20H),1.71-1.54(m,4H),1.45-1.17(m,73H),0.85(t,J=6.8 Hz,15H). 13 C NMR (500 MHz, CDCl 3)δ 172.96,172.76,172.72,70.58,70.47,68.13,68.06,63.18,60.66,58.51,56.22,55.98,55.61,55.37,53.62,49.62,49.49,49.44,39.2 4,38.95,38.53,38.26,35.39,35.22,34.57,34.35,31.91,29.86,29 .61,29.32,26.94,26.86,26.08,25.77,25.70,22.69,20.71,14.14. MS(ESI+)m / z:[M+H] + C 54 H 111 N 5 O 7 Theoretical value: 942.85; measured value: 942.85.

[0193] [ka] Intermediate TBS-A was synthesized similarly to intermediate TBS-A', using 3-amino-1-propanol instead of 2-amino-1-ethanol. The compound was a clear, colorless oil (2.38 g, 6.58 mmol, 80%). 1 H NMR (400 MHz, CDCl 3 )δ 3.61(s,6H),2.71(t,J=7.2 Hz,5H),2.45(t,J=7.3 Hz,2H),2.40(t,J=7.2 Hz,4H),1.58(p,J=6.3 Hz,2H),0.84(s,9H),-0.01(s,6H). 13 C NMR (400 MHz, CDCl 3 )δ 173.11,61.02,51.53,50.28,49.37,32.58,30.33,25.98,18.32,-3.52,-5.30.

[0194] [ka] Intermediate TBS-B was synthesized similarly to intermediate TBS-A'. This compound was a clear, colorless, viscous oil containing residual 1,3-diaminopropane impurity (0.52 g, 1.16 mmol, 38%).1 H NMR (400 MHz, CDCl 3 )δ 7.36(t,J=5.7 Hz,2H),3.58(t,J=5.9 Hz,2H),3.26(q,J=6.4 Hz,4H),2.72(q,J=6.8 Hz,8H),2.66(t,J=6.4 Hz,4H),2.48(t,J=6.9 Hz,2H),2.27(t,J=6.3 Hz,4H),1.57(p,J=6.7 Hz,6H),0.85(s,9H),0.01(s,6H). 13 C NMR (400 MHz, CDCl 3 )δ 172.46,60.71,50.12,49.46,39.94,39.80,37.31,37.15,34.06,32.79,29.79,25.92,18.22,-5.22.

[0195]

change

[0196] [ka] Intermediate TBS-I-29 was deprotected in the same manner as intermediate TBS-I-29 to give I-29. The purified compound was a clear, colorless, viscous oil (0.080 g, 0.084 mmol, 35%). 1 H NMR (500 MHz, CDCl 3 )δ 7.94-7.74(m,2H),4.49(br s,7H),3.72-3.62(m,7H),3.57-3.45(m,1H),3.38-3.24(m,2H),3.16-3.04(m,1H),2.79-2.66(m,4H),2.62(t,J=6.0 Hz,2H),2.58-2.42(m,6H),2.41-2.30(m,4H),2.25(dt,J=13.0,2.6 Hz,2H),1.74-1.60(m,8H),1.47-1.18(m,75H),0.86(t,J=6.9 Hz,12H). 13 C NMR (500 MHz, CDCl 3 )δ 172.28,70.22,70.09,67.96,62.99,60.70,55.82,55.69,54.52,53.58,50.55,38.69,38.60,38.19,38.08,35.47,35 .25,34.51,34.40,34.26,31.95,29.90,29.65,29.37,28.05,26.70,26.20,25.78,25.72,22.73,20.38,14.17,13.74. MS(DART+)m / z:[M+H]+ C 55 H 113 N 5 O 7 Theoretical value: 956.86; measured value: 956.88.

[0197] Example 2: Synthesis of multiplexed RNA nanoparticles Prior to formulating the RNA nanoparticles, the surfaces were sterilized. Glassware and stir bars were endotoxin baked at 250 °C for 2-24 h, followed by a NaOH rinse with 10 N NaOH. Glassware was then covered in foil to ensure sterility before use. An appropriate amount of material could be prepared in 25% excess to account for material loss prior to formulation. Dialysis cassettes were presoaked in 1x phosphate-buffered saline (PBS) prior to adding the material.

[0198] Materials such as PEG lipids were vortexed as solutions in EtOH prior to use. According to the formula sheet, appropriate amounts of EtOH, PAMAM dendron polymer, PEG lipids, and other additional excipients were added in this order to a 1.5 mL Eppendorf tube. The delivery materials were subjected to brief sonication and slightly warmed to remove precipitates if necessary. Similarly, in a biosafety cabinet (BSC), appropriate amounts of citrate buffer, ultrapure water, and selected RNA were added in this order to a 1.5 mL Eppendorf tube.

[0199] Sterile airtight glass syringes with Zeus tubing with a 10 cm inlet and 10 cm outlet were rinsed with 70% ethanol prior to loading. The syringe designated to hold the RNA material received an additional citrate buffer rinse. The delivery material and RNA substance were loaded into their respective syringes. After ensuring there were no air bubbles and the tubing was primed, the two syringes were loaded into the syringe pump and all tubing was attached to the microfluidic chip.

[0200] Further, the outlet tubing was attached to the dialysis machine. The syringe pump was set appropriately and started at the same time. Once stopped, the tubing was removed from the dialysis machine and the mixed material was dialyzed in 1x PBS buffer to bring the nanoparticles back to a neutral pH of 7.4. After the appropriate time, the nanoparticles were removed from the dialysis machine using a syringe and needle, added to an Eppendorf tube, and stored at ambient temperature. The particles can be safely stored at room temperature or at -20°C for at least 48 hours without any noticeable changes.

[0201] Formulations were completed in 10 mM to 95 mM citrate buffer, pH 3.0, 5.0, or 6.0. Total volumetric flow rates for the nucleic acid and delivery material streams ranged from approximately 1 mL / min to 7.5 mL / min flowing at aqueous to organic phase volume ratios of 1:1, 2.5:1, and 5:1. Final RNA nanoparticles were formulated to have dendron to RNA mass ratios ranging from 5:1 to 25:1.

[0202] These nanoparticles were characterized using dynamic light scattering (DLS) to demonstrate successful formulation.

[0203] The nanoparticles in Table 1 below were made in a similar manner. ID indicates the identification number of the nanoparticle formulation. siRNA is small interfering RNA. LNC RNA is long non-coding RNA. Fip-mKate2 plasmid is a DNA plasmid. Rluc IVT RNA is an mRNA lacking a 5' cap and polyA tail. 14:0PEG2000PE is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 14:0 can be replaced by 18:0 and 2000 can be replaced by 5000. 18:1PEG2000PE is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 2000 can be replaced by 5000. DMG-PEG2000 is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. DSG-PEG2000 is distearoyl-rac-glycerol-PEG2K, and DSPC is 1,2-distearoyl-sn-glycero-3-phosphocholine. Cas9 IVT is mRNA lacking the 5' cap and polyA tail. Rluc and Fluc are Renilla luciferase mRNA and Firefly luciferase mRNA, respectively. [Table 1] TIFF2024543272000092.tif199159TIFF2024543272000093.tif202159TIFF2024543272000094.tif215159TIFF2024543272000095.tif21115 9TIFF2024543272000096.tif221159TIFF2024543272000097.tif215159TIFF2024543272000098.tif219159TIFF2024543272000099.tif34159

[0204] Example 3: RNA encapsulation efficiency assay Three different nanoparticles were formulated at pH 3.0 using I-1 (G1-nPr-C14E), 25 bp siRNA, and PEG lipid excipient. The PEG lipid excipient was one of 14:0PEG2000PE, 18:0PEG2000PE, or 18:1PEG2000PE. The mass ratio of nanomaterial to RNA was 5:1, and the molar ratio of nanomaterial to PEG was 96.5:3.5. The assay was performed in triplicate in a 96-well plate. Briefly, particles were diluted (1:25) in TE buffer or water and treated with 1% TritonX for 10 min at 37 °C to disrupt self-assembly. An siRNA standard curve was generated to interpolate concentrations. Immediately after addition of RiboGreen reagent, fluorescence intensity was measured on a Tecan Infinite M200 plate reader (excitation = 480 nm, emission = 525 nm, integration time = 40 μs).

[0205] The RNA concentration and RNA encapsulation efficiency of the I-1(G1-nPr-C14E) nanoparticles of the present application were quantified using a modified Quant-iT RiboGreen assay (Walsh C et al., Microfluidic-based manufacture of siRNA-lipid nanoparticles for therapeutic applications. Methods Mol Biol. 2014; 1141: 109-20).

[0206] Example 4: Functional delivery of siRNA to cells in vitro I-1(G1-nPr-C14E) nanoparticles delivering GFP siRNA to reduce GFP expression in HEK293-GFP cells Nanoparticles were formulated in 10 mM citrate buffer (pH 3) containing I-1 (G1-nPr-C14E), PEG lipid, and siRNA against green fluorescent protein (GFP). The weight ratio of nanomaterial to RNA was 5:1. The molar ratio of nanomaterial to PEG was 96.5:3.5. In vitro delivery of siRNA to cells was evaluated by treating green fluorescent protein (GFP)-positive human embryonic kidney cells (HEK293-GFP&RFP, GenTarget Inc.).

[0207] Cells were seeded into black 96-well plates at a density of approximately 14,000 cells per well and incubated at 37°C, 5% CO 2 The cells were incubated overnight at 4°C for 12 h. I-1 (G1-nPr-C14E) formulated with 14:0PEG2000PE or 18:0PEG2000PE with or without GFP siRNA (Invitrogen) was then added to the cells along with a PBS control. After 48 hours, Hoechst stain was added to the treated cells in the 96-well plate for 15 minutes. The 96-well plate was then briefly centrifuged. The Hoechst-containing medium was removed and the cells were resuspended in PBS. The fluorescence intensity of GFP and Hoechst was quantified using a plate reader.

[0208] I-1(G1-nPr-C14E) and 14:0PEG2000PE nanoparticles carrying GFP siRNA and I-1(G1-nPr-C14E) and 18:0PEG2000PE nanoparticles carrying GFP siRNA both reduced GFP levels after 48 hours compared to their corresponding nanoparticles without siRNA and PBS treatment. Mean fluorescence intensity (MFI) is normalized to Hoechst and PBS treated controls (n=3). [Table 2]

[0209] Example 5: Functional delivery of mRNA to cells in vitro LNPs were formulated using I-1(G1-nPr-C14E), cholesterol, DSPC, DSG-PEG2000 (50 / 38.5 / 10 / 1.5 mol%), and Renilla luciferase (RLuc) mRNA. Briefly, 300,000 human embryonic kidney cells (HEK293) were transfected with 1 μg of mRNA in a 24-well plate. 24 hours after transfection, cells were lysed and processed according to the Renilla luciferase assay system (Promega). Relative luminescence units (RLU) were measured using a TECAN Infinite M200 plate reader (n=3).

[0210] I-1(G1-nPr-C14E) / cholesterol / DSPC / DSG-PEG2000 nanoparticles significantly increased RLuc expression in HEK293 cells compared with the commercially available transfection reagent Lipofectamine Messenger Max (Invitrogen). [Table 3]

[0211] In another experiment, cells were treated in a similar manner with nanoparticles formulated with I-1(G1-nPr-C14E), 14:0PEG2000PE (96.5 / 3.5 mol%), and RLuc mRNA.

[0212] I-1(G1-nPr-C14E) / 14:0PEG2000PE nanoparticles significantly increased the relative luminescence units (RLU) compared to PBS-treated cells. This suggests that I-1(G1-nPr-C14E) / 14:0PEG2000PE particles can transfect cells without the need for helper lipids. Table 4 below shows RLuc expression 24 hours after transfection. [Table 4]

[0213] Example 6: Effect of formulation pH on RNA release The formulation pH of nanoparticles containing I-1(G1-nP4-C14E) and 14:0PEG2000PE influences the stability of the particles in basic conditions. The Ribogreen Assay (ThermoFisher) is a common method used to determine the encapsulation efficiency of LNPs. The assay is typically performed in TE buffer (pH 8.0), where the addition of 1% TritonX destroys the particles and determines the capture of RNA. The encapsulation efficiency of particles formulated in 10 mM citrate buffer (pH 5.0) cannot be determined in TE buffer because the particles are destroyed without the presence of TritonX. It was discovered that water can be used instead of TE buffer to accurately determine the encapsulation efficiency, since water does not destroy the particles. Interestingly, nanoparticles formulated in 10 mM citrate buffer (pH 3.0) can be assayed in both TE buffer and water. [Table 5]

[0214] Example 7: In vivo expression of firefly luciferase mRNA LNPs were formulated using I-1(G1-nPr-C14E), cholesterol, DSPC, DMG-PEG2000 (50 / 38.5 / 10 / 1.5 mol%), and Firefly Luciferase (FLuc) mRNA from Trilink Biotechnologies (L-7202).

[0215] In one experiment, 9-10 week old C57BL / 6 mice (n = 3) were intravenously injected with 0.4 mg / kg LNPs. Total flux is expressed as photons per second (p / s) and the negative control was phosphate buffered saline (PBS). [Table 6]

[0216] In a separate experiment, 10-week-old C57BL / 6 mice (n = 1) were injected intramuscularly with the same LNPs at 0.01 mg / kg. Total flux is measured in photons per second (p / s), with phosphate-buffered saline (PBS) as the negative control. [Table 7]

[0217] Six hours after injection, all mice were injected with D-luciferin (150 mg / kg, i.p.). Firefly luciferase emits bioluminescence in the presence of a substrate. Organs were harvested within 10–15 min, and luminescence was detected using an IVIS Lumina system (Perkin Elmer).

[0218] Mice injected intravenously expressed luciferase in the liver, spleen, lungs, kidneys, heart, and bone, while mice injected intramuscularly expressed luciferase at the injection site and in the draining lymph nodes.

[0219] Example 8: Ionizable lipid structures (LNPs) Materials and Methods General lipid synthesis All chemicals and solvents were commercially available and used as received unless otherwise stated. Purification was performed by flash chromatography (Buchi Pure C-815 flash chromatography system, ELSD detector) on normal phase silica gel (silica gel, 230-400 mesh, grade 60, Fisher Chemical) columns manually packed into reusable cartridges (Biotage Sfar DLV). Flash chromatography used ULTRA as polar solvent (DCM:MeOH:NH in a volume ratio of 75:22:3). 4 OH(H 2 The reaction was analyzed by thin layer chromatography (Supelco TLC silica gel 60 F 254 ) and visualized using iodine staining on silica.

[0220] All ionizable lipids were synthesized by the method shown in Scheme 1. Detailed synthesis methods and molecular characterization are described in Example 1.

[0221] Firefly luciferase mRNA synthesis The firefly luciferase DNA template was ordered from Integrated DNA Technologies as a custom gene (Table 8) containing a T7 promoter, a minimal 5′ untranslated region, and a 3′ untranslated region derived from the mouse alpha globin sequence [Trepotec, Z. et al., Maximizing the Translational Yield of mRNA Therapeutics by Minimizing 5′-UTRs, Tissue Eng. Part A 25, 69-79, 2019]. In vitro transcription of the DNA template was performed using the HiScribe T7 kit (NEB). Modified mRNA was prepared by replacing uridines with N 1 The mRNA was generated by complete substitution with 2'-O-methyl-pseudouridine-5'-triphosphate (Trilink Biotechnologies). The RNA was capped with the cap-1 structure using the vaccinia system and 2'-O-methyltransferase (NEB), and a poly(A) tail of approximately 100 nucleotides (nt) was enzymatically added (NEB). The resulting mRNA was purified by silica column chromatography. The concentration was measured using a Nanodrop, and the purity was determined by gel electrophoresis. [Table 8] TIFF2024543272000107.tif98159

[0222] LNP formulation SM-102, used as a reference LNP, was purchased from Cayman Chemicals. Cholesterol and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from VWR. 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG) was purchased from Avanti Polar Lipids. Staggered herringbone microfluidic mixers were fabricated by soft lithography as previously reported [Chen, D. et al., Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation, J. Am. Chem. Soc. 134, 6948-6951, 2012]. The lipid components (50% ionizable lipids, 38.5% cholesterol, 10% DSPC, 1.5% PEG-DMG) were dissolved in ethanol and RNA was dissolved in 75 mM sodium citrate (pH 3.0). LNPs were prepared by mixing the organic and aqueous phases in the microfluidic chip at a total flow rate of 7 mL / min (aqueous:organic flow ratio of 2.5:1). The formulated LNPs were collected in Slide-A-Lyzer™ dialysis cassettes 10K MWCO (ThermoScientific, 87729) and dialyzed 1:1000 against 1× phosphate buffered saline (PBS) (pH 7.4) for 2 h twice. The LNPs were removed from the dialysis machine, sterile filtered using a 0.2 μm polyethersulfone syringe filter (Whatman, Uniflo 13), and stored at 4 °C for up to 1 week before use.

[0223] LNP size distribution LNPs were equilibrated at room temperature and diluted 1:100 in 1 mL of RNAse-free distilled water. Size distribution measurements were performed using a Zetasizer Nano (Malvern, Ltd. Malvern, UK) equipped with a 633 nm He-Ne ion laser.

[0224] mRNA concentration and encapsulation efficiency The RNA concentration and encapsulation efficiency of the LNPs were determined using a modified Quant-iT RiboGreen assay (ThermoFisher). The assay was performed in triplicate in black opaque 96-well plates. Briefly, LNPs were diluted 1:50 in RNAse-free distilled water or 2% TritonX. A standard curve of the corresponding RNA was prepared in 2% TritonX. The plates were incubated at 37 °C for 10 min to destroy the LNPs. RiboGreen reagent was then added to each well, and the fluorescence intensity was measured with a Tecan Infinite M200 plate reader (480 nm / 525 nm). Raw fluorescence values ​​were subtracted with background fluorescence, and the total RNA concentration in the LNP formulations was determined by linear interpolation of the standard curve. Encapsulation efficiency was calculated from the ratio of untreated wells to treated wells as previously reported [Walsh, C. et al., Microfluidic-Based Manufacture of siRNA-Lipid Nanoparticles for Therapeutic Applications, Drug Delivery System (ed. Jain, KK) vol. 1141 109-120 (Springer New York, 2014)].

[0225] Apparent pK a Apparent pK of LNP aDetermination of the Ionization Constants, SAR, and the Impact of Lipid p K was performed using a protocol previously described [Heyes, J., Palmer, L., Bremner, K. & MacLachlan, I., Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids, J. Controlled Release 107, 276-287, 2005; Zhang, J., Fan, H., Levorse, DA & Crocker, LS, Ionization Behavior of Amino Lipids for siRNA Delivery: Determination of Ionization Constants, SAR, and the Impact of Lipid p K a on Cationic Lipid-Biomembrane Interactions, Langmuir 27, 1907-1914, 2011]. Briefly, a series of buffer solutions with pH values ​​varying from 3.0 to 8.5 in increments of 0.5 were prepared by titrating a solution containing 10 mM citrate, 10 mM phosphate, 10 mM borate, and 150 mM NaCl with 1.0 M HCl and aliquoted into a black 96-well plate. LNP and 6-(p-toluidino)-2-naphthalenesulfonic acid (TNS, Sigma Aldrich) were diluted into these buffers to final concentrations of 25 and 5.45 μM, respectively. The plate was equilibrated at room temperature for 20 min. Fluorescence intensity was determined using a Tecan Infinite M200 plate reader (325 nm / 435 nm). The raw fluorescence values ​​were normalized between 0 and 1 and fitted using graphing software to obtain a pK value corresponding to the pH value at 50% ionization. a It was decided.

[0226] Cryo-Transmission Electron Microscopy (CryoTEM) CryoTEM samples were prepared using a Vitrobot Mark IV system (ThermoScientific). To prepare cryoTEM grids, 4 μL of sample was applied to a Quantifoil R2 / 2 300 mesh grid (Electron Microscopy Sciences, Q325CR2). The following parameters were used: temperature = 4 °C, humidity = 100%, wait time = 5 s, blot time = 2 s, blot force = 2, drain time = 0 s, number of blots = 1. Images were taken on a Talos L120 C at an accelerating voltage of 120 kV and a magnification of 57,000 times.

[0227] Intramuscular firefly luciferase mRNA expression C57BL / 6 mice (female, 8-9 weeks old, 19-21 g, Charles River) were injected intramuscularly with LNPs (50 μL / quadriceps) containing 500 ng of firefly luciferase mRNA. Six hours later, mice were injected intraperitoneally (200 μL) with 3 mg of D-luciferin (ThermoScientific), legs were harvested and imaged for bioluminescence within 10 min using an IVIS Spectrum (PerkinElmer) (open filter, height = 1 cm). Total luminescence flux (photons / sec) within the muscle was determined using the automated region of interest tool.

[0228] In vivo gene editing The modified Cas9 mRNA replaces the uridine with N 1 Cas9 mRNA was generated by IVT of a fully substituted Cas9 DNA template (Sigma-Aldrich, CAS9P) with 5'-methyl-pseudouridine-5'-triphosphate (Table 9). Modified sgRNA with phosphorothioate linkages was purchased from Synthego (Table 13). LNPs were formulated with Cas9 mRNA and sgTOM (3 / 1, wt / wt) at a lipid nitrogen to RNA phosphate ratio (N / P) of 10. Female B6.Cg-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze / J(Ai9) mice (The Jackson Laboratory, 8–9 weeks, n = 2) were intramuscularly injected (50 μL) with 2 mg / kg total RNA into one quadriceps on days 0 and 2. The other quadriceps was simultaneously injected with 50 μL of 1× PBS. On day 5, hindlimb muscles were harvested and fluorescence (535 nm / 600 nm) was detected with an IVIS Spectrum (PerkinElmer).

[0229] [Table 9] TIFF2024543272000109.tif212159TIFF2024543272000110.tif153159

[0230] Chemical Parameter Prediction Predicted LogD and pK of ionizable lipids a was determined using MarvinSketch 21.18. The molecular volumes of SM-102 and I-28, used to predict the number of mRNA copies per particle, were approximated using the volume() function of InstantJChem 21.20.0, which calculates van der Waals volumes.

[0231] mRNA copy number per particle The expected number of mRNA copies per particle was calculated based on a previously reported method [Carrasco, MJ et al., Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration, Commun. Biol. 4, 1-15, 2021]. The predicted value is an overestimate because it utilizes the van der Waals volume of ionizable lipids instead of the molecular volume. The actual number of mRNA copies per particle was calculated by dividing the concentration of encapsulated mRNA copies (obtained from measuring mRNA concentration and encapsulation efficiency) by the concentration of nanoparticles. Nanoparticle concentration was measured by nanoparticle tracking analysis (NanoSight NS300) diluted 1:100 in water.

[0232] Detection of IL-6 in draining lymph nodes I-28 LNPs and SM-102 LNPs were formulated with modified firefly luciferase mRNA. C57BL / 6 mice (female, 8-9 weeks old, 19-21 g, Charles River) were injected intramuscularly with 7.5 μg mRNA LNPs (50 μL / quadriceps) or saline. Six hours after injection, draining lymph nodes were harvested. Popliteal and inguinal lymph nodes were combined in Powerbead Tubes (Qiagen) containing T-Per™ lysis buffer (ThermoScientific) in the presence of Halt™ protease inhibitor cocktail (ThermoScientific). Samples were flash frozen in a mixture of isopropanol and dry ice and stored at -80°C. Frozen samples were thawed on ice and homogenized using a bead mill homogenizer. Lysates were removed by centrifugation, transferred to new tubes, and stored at -80°C until use. Undiluted samples were used for quantification of total protein content using the Pierce™ BCA Protein Assay Kit (ThermoScientific). IL-6 concentrations were determined by enzyme-linked immunosorbent assay (RnD Systems). Each sample was assayed undiluted or diluted 1:1 with lysis buffer. Absorbance measurements were performed on a Biotek Synergy H1 plate reader. IL-6 concentrations were normalized for each sample by dividing the IL-6 concentration by the total protein content. Normalized IL-6 concentrations from undiluted and diluted samples were averaged before plotting.

[0233] statistics Statistical analysis was performed using GraphPad Prism 8. An unpaired two-tailed t-test was used to compare means of two independent samples with each other. A two-tailed paired t-test was used to compare mean differences between pairs of measurements. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 were considered statistically significant.

[0234] result Design and synthesis of modular ionizable lipid structures The general synthetic strategy consists of three molecular regions that can be easily modified using commercially available reagents: the amine core (R 1 ), linker length (L 1 ), and alkyl tail (X 1 The present invention was designed to generate a poly(amidoamine) ionizable lipid of Formula I having the formula:

[0235] Iterative screening strategy of LNPs for intramuscular mRNA delivery Ionizable lipids were formulated into LNPs based on the Spikevax composition, which consists of ionizable lipid, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG) in the ratio of 50:38.5:10:1.5 mol%, respectively (Figure 2). All LNPs had a fixed N / P ratio of 5, consistent with the Spikevax formulation (Spikevax Full Prescribing Information, US FDA). The ultimate goal was to construct a new chemically distinct ionizable lipid that was non-inferior to SM-102 in mRNA expression.

[0236] Design parameters with specific target criteria were used to guide the optimization process (Table 10). The average size of the LNPs should be less than 200 nm to allow for sterile filtration, and the polydispersity index (PDI) should be less than 0.2 to create a reproducible and stable formulation. Encapsulation efficiency refers to the percentage of encapsulated mRNA in the formulation. An encapsulation efficiency >90% maximizes mRNA delivery to cells and reduces immune activation induced by free transcripts [Kariko, K., Buckstein, M., Ni, H. & Weissman, D., Suppression of RNA Recognition by Toll-like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA, Immunity 23, 165-175, 2005]. Currently, the apparent pKa of LNPs is considered a relevant parameter for determining therapeutic efficacy. The apparent pKa should be between 6-7 so that LNPs are nearly neutral in physiological conditions but become protonated and cationic during formulation in acidic endosomes and at low pH. For systemic delivery, a pKa range of 6.2-6.5 is optimal for liver expression [Jayaraman,M.et al.,Maximizing the Potency of siRNA Lipid Nanoparticles for Hepatic Gene Silencing In Vivo,Angew. Chem.124,8657-8661,2012]. Meanwhile, for intramuscular delivery, a slightly higher range of 6.6-6.9 has been reported in one study [Hassett,KJet al.,Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines,Mol.Ther. Nucleic Acids 15,1-11,2019]. [Table 10]

[0237] Rather than synthesizing a large combinatorial library of ionizable lipids, we opted for an iterative approach to optimize lipid structures based on the design criteria in Table 10. Briefly, LNPs were formulated with unmodified firefly luciferase mRNA, characterized, and injected into both quadriceps of C57BL / 6 mice. Hind limbs were harvested and mRNA expression was determined by measuring the total luminescence flux within the muscle (Figure 2). We decided to start by optimizing the alkyl tail, based on the assumption that it would have the greatest impact on the physical parameters. Linker length and amine core were then explored. Alkylation of ionizable lipids Two commonly used methods for alkylating amines from primary to tertiary were compared: epoxide ring-opening and reductive amination. Lipids with the same core and linker length were alkylated with a symmetrical 14-carbon tail using either method (epoxide ring-opening is shown in Scheme 1). The epoxide ring-opening reaction introduces a hydroxyl group at the beta position next to the tertiary amine (I-1), whereas reductive amination produces a fully saturated tail (I-18). The LNP size distribution by intensity showed some differences (Figure 3d). The apparent pK of I-1-LNPs a The pK of I-1 was 0.7 points lower than that of I-18-LNP (Figures 3c and 3e). The hydroxyl group of I-1 pulls electron density away from the neighboring amines due to an inductive effect, resulting in a pK a This reduces the amount of erythrocyte lysate that is carried over into the formulated LNP.

[0238] Prior to screening these formulations in mice, I-18-LNPs demonstrated higher encapsulation efficiency and an apparent pK within 6–7. aBased on the results, I-1-LNP was expected to have higher mRNA expression than I-18-LNP (Fig. 3e). Conversely, I-1-LNP was found to have higher luminescence flux than I-18-LNP at all time points (Fig. 3b). As the only structural difference between I-1 and I-18 lipids is the presence of a hydroxyl group, it follows that mRNA delivery is improved by the ability of ionizable lipids to form hydrogen bonds with nucleobases [Cornebise, M. et al., Discovery of a Novel Amino Lipid That Improves Lipid Nanoparticle Performance through Specific Interactions with mRNA, Adv. Funct. Mater.32,2106727,2022]. These results highlight the important role of hydrogen bonds in mRNA binding and delivery. Going forward, epoxide ring-opening was selected as the alkylation method of choice.

[0239] SM-102 was included in this first set of experiments to serve as a benchmark and to measure encapsulation efficiency, apparent pK a and the need to improve mRNA delivery. Furthermore, for all LNPs, the highest luminescence flux was detected 6 h after injection, which then steadily decreased over the next 96 h (Figure 3b). Therefore, in the following experiments, the 6 h time point was chosen to quantify mRNA expression. Reducing the length of the alkyl tail reduces the pK a and the encapsulation efficiency of mRNA increased.

[0240] Epoxide ring opening is the same C 3 Alkyl core (group R 1 ) and C 2 Alkyl linker (group L 1 ), while maintaining the shorter 10-carbon tail and the longer 15-carbon tail (group X 1) was used to alkylate lipids (Fig. 4a). Tail lengths longer than 15 carbons could not be successfully formulated due to solubility issues. Initially, it was expected that ionizable lipids with longer tails would interact more favorably with cell membranes due to their higher lipophilicity, closer to the predicted LogD of SM-102, but no significant difference in mRNA expression was found (Fig. 4b). a was highly dependent on the length of the tail and inversely proportional (Figure 4c). Without being limited by theory, it is possible that the 10 carbon tail reduces the overall hydrophobic character of the LNP compared to longer tails. This allows for greater partitioning of protons from the surrounding aqueous phase into the lipid phase of the particle, resulting in increased ionization of tertiary amines at higher pH values ​​[Carrasco, MJ et al., Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration, Commun. Biol.4,1-15,2021]. I-26 has the highest pK a (6.3) and produced LNPs with encapsulation efficiency (93%), both of which met the design criteria ( Figure 4 e). Thus, a 10-carbon tail was selected for the next structural iteration.

[0241] Increasing the linker length increases the apparent pK a and mRNA expression was increased Apparent pK a To further optimize the compound, lipids with three-carbon linkers (group L 1 ) was synthesized and compared with I-26 (Figure 5a). As the distance between the amide bond and the tertiary amine increases, the bond-threading induction effect is limited, and thus the pK a As expected, adding one carbon atom to the linker increased the apparent pK aThe pK of I-27-LNPs increased by 1 unit (Figures 5c and 5e). I-27-LNPs showed significantly higher mRNA expression than I-26-LNPs (Figure 5b) while maintaining high encapsulation efficiency and desirable size distribution (Figures 5d and 5e). This result supports the apparent pK a Optimization of the pH range was supported by the apparent pK a is more favorable for intramuscular administration. As a result, the I-27 structure was selected for subsequent optimization of the amine core.

[0242] Adding hydroxyl groups to the amine core matches the performance of SM-102 Given that inclusion of a hydroxyl in the alkyl tail improved mRNA expression, I-27 lipids were synthesized bearing a hydroxyl group positioned two (I-28) or three (I-29) carbons away from the tertiary amine of the core (Figure 6a). All LNPs met the characterization criteria (Figure 6d, Figure 6e, Figure 6f), and the addition of the hydroxyl core significantly improved mRNA expression (Figure 6b). Notably, I-28 achieved a total emission flux comparable to SM-102. As the efficacy of LNPs has been shown to depend on the chemical modification of the mRNA, n 1 The experiment with LNPs delivering mRNA modified with -methyl-pseudouridine was reproduced and gave the same results (Figure 6c) [Melamed, JR et al., Lipid nanoparticle chemistry determines how nucleoside base modifications alter mRNA delivery, J. Controlled Release 341, 206-214, 2022]. Therefore, I-28 was chosen for intramuscular mRNA delivery.

[0243] I-28 produced LNPs with 2.5-fold less lipid than SM-102 (Figure 7d) and formed larger particles (Figures 7a-c). It was predicted, and empirically confirmed, that I-28 would encapsulate more mRNA copies per particle than SM-102 (Figure 7e, Figure 7f, and Table 11). Therefore, we decided to test whether I-28 would be a suitable delivery material for multi-RNA payloads. [Table 11]

[0244] Intramuscular CRISPR-Cas9 gene editing The ability of I-28-LNPs to perform intramuscular gene editing via codelivery of Cas9 mRNA and sgRNA was evaluated. We utilized genetically engineered mice that express tdTomato fluorescent protein upon removal of a LoxP-flanked stop cassette. Once the stop cassette is deleted by the CRISPR-Cas9 system, the gene-edited cells become fluorescent and detectable by the imaging system (Figure 8a). I-28-LNPs were formulated with Cas9 mRNA and sgTOM (4 / 1 mRNA / sgRNA) (Tables 12 and 13). The N / P ratio was increased from 5 to 10 to achieve encapsulation efficiency (>97%) that met the design criteria. Mice were injected intramuscularly with 2 mg / kg total RNA on days 0 and 2 and imaged on day 5 (Figure 8b). This dosing regimen was chosen based on previous reports to overcome high levels of background tissue fluorescence [Cheng, Q. et al., Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing, Nat. Nanotechnol. 15, 313-320, 2020; Wei, T., Cheng, Q., Min, Y.-L., Olson, EN & Siegwart, DJ, Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing, Nat. Commun. 11, 3232, 2020]. Statistically significant tdTomato fluorescence was detected in the LNP-treated leg compared to the untreated leg. This indicates that I-28-LNPs can be used to perform intramuscular gene editing and, more generally, to deliver large multi-RNA payloads (Figure 8c). Successful gene editing was also confirmed by orthogonal techniques, analyzing muscle tissue for the presence of tdTomato by histology and confocal microscopy (Figure 8d). [Table 12] [Table 13]

[0245] Consideration An ionizable lipid structure was devised that contained three variable regions that allowed for fine-tuning of LNP properties. Instead of performing a large-scale, resource-intensive screen of each possible structure, an iterative design strategy was employed that led to the discovery of the potent ionizable lipid I-28. I-28-LNPs fulfill the set design criteria, as they have a size of ≈100 nm, a PDI<0.12, and an encapsulation efficiency >95%, achieving intramuscular mRNA expression comparable to that of SM-102. As a result of its trivalency, I-28 uses 2.5-fold less lipid than SM-102 at the same N / P ratio, while encapsulating more mRNA copies per particle. We then tested whether I-28-LNPs could deliver large combinatorial RNA payloads. As a proof of concept, intramuscular gene editing by co-delivery of Cas9 mRNA and sgRNA in reporter mice was successful, albeit at high doses.

[0246] Independent optimization of the tail, linker, and core regions of ionizable lipids has yielded several findings regarding the influence of structural features on LNP performance. First, incorporation of hydroxyl groups next to tertiary amines in both the core and tail significantly improved mRNA expression (Figures 3 and 6). Without being limited to theory, this may be due to hydrogen bonding increasing lipid-mRNA interactions, although its role in cellular uptake and endosomal release remains unclear. Second, studies of lipids with two or three carbon linkers have revealed that pK a It was found that mRNA expression in muscle improved when the pH approached 7 (Figure 5). Interestingly, both I-28 and SM-102 LNPs had apparent pK values ​​in the range of 6.6–6.9. a, reinforcing its relevance as a design parameter for intramuscular mRNA delivery (Table 12) [Hassett, KJ et al., Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines, Mol. Ther. Nucleic Acids 15, 1-11, 2019]. Third, the lipophilicity of the ionizable lipid (based on the predicted LogD at pH 7.4) is related to the pK a Although the ionizable lipids that achieved the highest encapsulation efficiency had a relatively low LogD of 3.5-6.5, another study reported that the optimal LogD range for systemic delivery is 10-14 (Table 13) [Rajappan, K. et al., Property-Driven Design and Development of Lipids for Efficient Delivery of siRNA, J.Med.Chem.63,12992-13012,2020]. Thus, LogD is a function of the hydrogen bonds and pK a This is a parameter that does not affect intramuscular mRNA expression as much as [Table 14]

[0247] After discovering the high efficacy of I-28, a preliminary study on its IL-6 response was conducted. IL-6 is an acute inflammatory cytokine that may enhance vaccine performance [Alameh, M.-G. et al., Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses, Immunity 54, 2877-2892.e7, 2021]. Compared to the SM-102 formulation used in the vaccine, the I-28 formulation induced a similar IL-6 response. (Figure 9)

[0248] In summary, a new ionizable lipid family was designed and an optimized ionizable lipid I-28 was identified for efficient intramuscular delivery of mRNA with low lipid doses. This study reported structure-function discoveries in terms of hydrogen bonding, ionization behavior, and lipophilicity that lay a solid foundation for the design of LNPs for intramuscular mRNA delivery. Using a similar iterative design strategy, the impact of ionizable lipid structure on LNP stability, in vivo clearance, tolerability, and immunogenicity can be explored, enabling the next generation of mRNA vaccines and therapeutics.

Claims

1. A dendron or a salt and / or solvate thereof, The first generation of the dendron has the formula (1): 【Chemistry 1】 wherein each X １ are the same, 【Chemistry 2】 selected from the group consisting of: The second generation of the dendron has formula (2): 【Transformation 3】 wherein each X ２ are the same and are selected from the group consisting of C 1-40 alkyl and C 2-40 alkenyl, wherein each alkyl and each alkenyl is optionally interrupted by one or more groups selected from the group consisting of —S—S—, —C(O)—, —OC(O)—, —C(O)O—, —OC(O)O—, —NR 3 C(O)O—, —OC(O)NR 3 —, —C(O)S—, —SC(O)—, —NR 3 C(O)—, —C(O)NR 3 —, —NR 3 C(O)NR 4 —, and —C(NC 1-20 alkyl)-, wherein said alkyl and said alkenyl are optionally substituted by one or more substituents selected from the group consisting of halo, NR 5 R 5′ , and OH, and R 3 , R 4 , R 5 , and R 5′ are each independently hydrogen or C 1-10 alkyl; The third generation of the dendron has formula (3): 【Chemistry 4】 wherein each X ３ are the same and are selected from the group consisting of C 1-40 alkyl and C 2-40 alkenyl, wherein each alkyl and each alkenyl is optionally interrupted by one or more groups selected from the group consisting of —S—S—, —C(O)—, —OC(O)—, —C(O)O—, —OC(O)O—, —NR 3 C(O)O—, —OC(O)NR 3 —, —C(O)S—, —SC(O)—, —NR 3 C(O)—, —C(O)NR 3 —, —NR 3 C(O)NR 4 —, and —C(NC 1-20 alkyl)-, wherein said alkyl and said alkenyl are optionally substituted by one or more substituents selected from the group consisting of halo, NR 5 R 5′ , and OH, and R 3 , R 4 , R 5 , and R 5′ are each independently hydrogen or C 1-10 alkyl; In formula (1), formula (2), and formula (3), R １ represents C1-10 alkyl, C2-10 alkenyl, C ２ and C1-4 alkylene-S-S-C1-4 alkyl, each of which is unsubstituted or selected from the group consisting of OH, OC1-15 alkyl, C(O)OC1-15 alkyl, and NR ２ R ２’ and / or one or more of the fluoro, phenyl, heteroaryl, heterocycloalkyl, and cycloalkyl groups may be further substituted with one to four of C 1-4 alkyl and C 1-4 fluoroalkyl; ２ and R ２’ are independently H and C １－１０ selected from the group consisting of alkyl; Each L 1 , L 2 and L 3 is the same or different and is a linking group, wherein each linking group is 【Transformation 5】 wherein d, e, f, g, u, v, w, x, and y are each independently 1, 2, 3, 4, 5, or 6; each available hydrogen atom bonded to a carbon may be independently replaced with a fluorine atom; A dendron or a salt and / or solvate thereof.

2. R １ but, 【Transformation 6】 2. The dendron of claim 1, selected from the group consisting of:

3. Each L in formula (1) １ are the same, or each L in formula (2) ２ The dendron of claim 1 , wherein:

4. X ２ but, 【Transformation 7】 selected from the group consisting of Each of these is X ２ one or two OH, one or two NH, provided that the total number of carbon atoms in ２ and / or optionally substituted with one or more fluorines, The dendron of claim 1 .

5. The dendron is 【Transformation 8】 【change】 【change】 【change】 or a salt and / or solvate thereof.

6. A nanoparticle comprising one or more dendrons according to claim 1.

7. A colloid comprising one or more dendrons according to claim 1.

8. A supramolecular structure comprising one or more dendrons according to claim 1.

9. A composition comprising one or more dendrons of claim 1 and a therapeutic agent.

10. 10. A method of delivering one or more compounds to a cell or a subject, comprising contacting the cell or subject with the composition of claim 9, The therapeutic agent is selected from the group consisting of unmodified or modified nucleic acids, mitochondria, plasmids, PolyIC and related adjuvants, ribonucleoproteins, proteins, peptides, cells, stains, dyes, and small molecule drugs; method.

11. 11. The method of claim 10, wherein the composition and the therapeutic agent delivered to a cell or subject are present in a weight ratio of dendron(s):therapeutic agent(s) of about 100:1 to about 1:

5.

12. The method of claim 11 , wherein the composition further comprises one or more lipids.

13. 13. The method of claim 12, wherein the one or more lipids are selected from steroids, steroid derivatives, PEG-lipids, and phospholipids, and mixtures thereof.

14. The PEG-lipid may comprise one or more C linked to a linker group having a PEG chain. ６－２４ Alkyl group or C ６－２４ alkenyl group, or C ６－２４ The method of claim 13, wherein the compound comprises a fatty acid group.

15. 10. A method for treating a disease, disorder, or condition, comprising administering to a subject an effective amount of the composition of claim 9, the disease, disorder or condition is selected from the group consisting of an infectious disease, an autoimmune disease, a cancer, a genetic disease, a chronic disease, trauma, wound healing, a traumatic brain injury, a neuromuscular disease, and a gastrointestinal disease; method.

16. 10. The composition of claim 9, wherein the composition has a pKa of about 4 to about 8.

17. The composition of claim 9 further comprising a chemically modified polynucleotide.

18. 18. The composition of claim 17, wherein the composition has a pKa of about 5 to about 7.

19. 18. The composition of claim 17, wherein the nanoparticles have an average diameter of about 50 nm to about 200 nm.

20. A lipid nanoparticle (LNP) comprising the nanoparticle of claim 6.

21. 21. The LNP of claim 20, comprising one or more lipids.

22. 22. The LNP of claim 21, wherein the one or more lipids are selected from steroids, steroid derivatives, PEG-lipids, and phospholipids, and mixtures thereof.

23. The dendron is 【Chemistry 9】 【change】 or a salt and / or solvate thereof.

24. The dendron of formula 1 is 【Chemistry 10】 The dendron of claim 1 ,

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