Lipid compounds and methods of making and using same

Abstract

Disclosed herein are lipid compounds and compositions comprising lipid compounds, as well as methods for producing and using the same. The disclosure also relates to a method for delivering a drug into a cell by introducing a therapeutically effective amount of the composition, lipid nanoparticles, pharmaceutically acceptable composition, or hydrogel matrix disclosed herein into the cell. The disclosure also discloses a method for promoting wound repair in a subject by administering a therapeutically effective amount of the composition, lipid nanoparticles, pharmaceutically acceptable composition, or hydrogel matrix disclosed herein to the subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 387,546, filed December 15, 2022, the disclosure of which is expressly incorporated herein by reference.

[0002] Statement on Federally Sponsored Research This invention was made with United States government support under Grant No. GM144117 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

[0003] This application relates generally to lipid formulations that can be used for drug delivery and screening. [Background technology]

[0004] Efficient delivery of mRNA for vaccines, gene therapy, and drug delivery is both a key step and a challenge in the application of mRNA therapeutics. Despite promising data from ongoing clinical trials, several issues currently limit the widespread use of oligonucleotides in therapeutic and diagnostic settings. First, free RNA is susceptible to nuclease digestion in plasma, accelerating the degradation of therapeutic agents. Second, these oligonucleotides often cannot reach the intracellular compartments where the relevant translation machinery resides.

[0005] As a result, lipid nanoparticles formed by cationic lipid and other lipid components are used as a possible means to pass through these barriers in delivery and increase the cellular uptake of oligonucleotide.However, the effectiveness of these delivery systems is generally due to the composition structure of the lipid molecules that form the basis, so there is a need for new compositions and methods for delivering mRNA to cells to treat various pathologies.Therefore, there is a continuing need for improved cationic lipids and lipid nanoparticles for the delivery of oligonucleotide. Summary of the Invention

[0006] In accordance with the purpose of the disclosed compositions and methods as embodied and broadly described herein, the disclosed subject matter relates to compositions, and methods of making and using same.

[0007] In some examples, the compound is defined by Formula I or a pharmaceutically acceptable salt thereof: [ka] [In the formula, p is an integer from 0 to 5, n is an integer from 1 to 10, each m, if present, is independently an integer from 1 to 10; R 1 , R 2 , and R 3 are independently OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and R 4 is a substituted or unsubstituted C8-C 18 is alkyl, Each R 5 -L, when present, independently represents hydrogen, OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R a are, if present, independently substituted or unsubstituted C8-C 18 is alkyl, L in each case 1 are independently substituted or unsubstituted alkyl, carbonyl, ester, amide, carbamate, amine, ether, carbonate, thioether, thioester, or urea.

[0008] In some examples, the compound is defined by Formula II or a pharmaceutically acceptable salt thereof: [ka] [In the formula, p is an integer from 0 to 5, n is an integer from 1 to 10, m, if present, is an integer from 1 to 10; R 1 and R 2 are independently OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R 4 are independently substituted or unsubstituted C8-C 18 is alkyl, Each R 5 -L, when present, independently represents hydrogen, OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R a are, if present, independently substituted or unsubstituted C8-C 18 is alkyl, L in each case 1 is independently a substituted or unsubstituted alkyl, carbonyl, ester, amide, carbamate, amine, ether, carbonate, thioether, thioester, or urea; Each R 6 are independently substituted or unsubstituted C1-C7 alkyl.

[0009] In some examples, the compound is defined by Formula III or a pharmaceutically acceptable salt thereof: [ka] [In the formula, p is an integer from 0 to 5, n is an integer from 1 to 10, m, if present, is an integer from 1 to 10; R 1 are independently OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R 4 are independently substituted or unsubstituted C8-C 18 is alkyl, Each R 5 -L, when present, independently represents hydrogen, OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R a are, if present, independently substituted or unsubstituted C8-C 18 is alkyl, L 1 is, if present, a substituted or unsubstituted alkyl, carbonyl, ester, amide, carbamate, amine, ether, carbonate, thioether, thioester, or urea; Each R 6 are independently substituted or unsubstituted C1-C7 alkyl.

[0010] In some examples, the compound is defined by Formula IV or a pharmaceutically acceptable salt thereof: [ka] [In the formula, p is an integer from 0 to 5, n is an integer from 1 to 10, m, if present, is an integer from 1 to 10; Each R 4 are independently substituted or unsubstituted C8-C 18 is alkyl, Each R 5-L, when present, independently represents hydrogen, OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R a are, if present, independently substituted or unsubstituted C8-C 18 is alkyl, Each R 6 are independently substituted or unsubstituted C1-C7 alkyl.

[0011] In some examples, the compound is defined by Formula V or a pharmaceutically acceptable salt thereof: [ka] [In the formula, p is an integer from 0 to 5, n is an integer from 1 to 10, each m, if present, is independently an integer from 1 to 10; R 1 and R 3 are independently OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R 4 are independently substituted or unsubstituted C8-C 18 is alkyl, Each R 5 -L, when present, independently represents hydrogen, OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R a are, if present, independently substituted or unsubstituted C8-C 18 is alkyl, L in each case 1is independently a substituted or unsubstituted alkyl, carbonyl, ester, amide, carbamate, amine, ether, carbonate, thioether, thioester, or urea; Each R 6 are independently substituted or unsubstituted C1-C7 alkyl.

[0012] Also disclosed herein are compositions comprising any of the compounds disclosed herein. Also disclosed herein are compositions comprising any of the compounds disclosed herein further comprising an agent (e.g., mRNA). In some examples, the agent comprises RNA. In some examples, the agent comprises mRNA. In some examples, the agent comprises a polynucleotide encoding a macrophage polarization factor. In some examples, the agent comprises a polynucleotide encoding interleukin-4. In some examples, the agent is encapsulated in a nanoparticle.

[0013] Also provided herein are methods of making any of the compounds and compositions disclosed herein.

[0014] Also provided herein are lipid nanoparticles comprising any of the compounds disclosed herein, a non-cationic lipid, a polyethylene glycol-lipid, and a sterol. In some examples, the non-cationic lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), DPPC (1,2-dipalmitoyl The sterol may comprise a cholesterol-based lipid, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphotidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-5 / 7-glycero-3-phospho-(1'-rac-glycerol) (DOPG), or a combination thereof. In some examples, the sterol may comprise a cholesterol-based lipid, and the molar ratio of the non-cationic lipid may be 20% to 50%. In some examples, the molar ratio of the compound may be 5% to 60%. In some examples, the molar ratio of the sterol may be 20% to 50%. In some examples, the molar ratio of the PEG-lipid may be 0.1% to 2%. In some examples, the molar ratio of the compound is 20% to 30%, the molar ratio of the non-cationic lipid is 35% to 45%, the molar ratio of the sterol is 35% to 45%, and the molar ratio of the polyethylene glycol-lipid is 0.1% to 1%. In some examples, the weight fraction of the drug is 5% to 20%.

[0015] Also disclosed herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of any of the compounds and compositions disclosed herein. Also disclosed herein are lipid nanoparticles and hydrogel matrices encapsulating drugs as disclosed herein.

[0016] Also provided herein is a method for delivering an agent into a cell, comprising: Also disclosed are methods that include introducing into cells any of the compositions, nanoparticles, pharmaceutically acceptable compositions, or hydrogel matrices described herein.

[0017] Also provided herein is a method for promoting wound repair in a subject, comprising: Also disclosed are methods that include administering to a subject any of the compositions, nanoparticles, pharmaceutically acceptable compositions, or hydrogel matrices described herein.

[0018] Additional advantages of the disclosed compounds, compositions, and methods will be set forth in part in the description that follows, and in part will be apparent from the description. The advantages of the disclosed compounds, compositions, and methods will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosed compounds, compositions, and methods as claimed.

[0019] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects of the present disclosure and, together with the description, serve to explain the principles of the disclosure. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows the in vitro delivery efficiency of trisulfide lipid nanoparticles in Hep3B cells using firefly luciferase. [Figure 2] 1 shows the in vivo delivery efficiency of trisulfide lipid nanoparticles in C57BL / 6 mice using firefly luciferase. [Figure 3A]Figure 1 shows the design and synthesis of a library of trisulfide-derived ionizable lipids for the treatment of diabetic wounds. Figure 2 shows a representative synthetic route to TS2, a trisulfide-derived ionizable lipid. Figure 3 shows a list of head amines and acrylate tails used in combinatorial Michael addition reactions. The acrylate tails are named according to the length of the hydrocarbon domain and the type of ester. [Figure 3B] We demonstrate the in vivo delivery efficiency of trisulfide lipid nanoparticles in C57BL / 6 mice using firefly luciferase. We demonstrate how hydrogel-loaded synthetic TS2-IL4 lipid nanoparticles (LNPs) accelerate diabetic wound healing by scavenging ROS and modulating macrophage phenotype at the wound site. [Figure 4A] Screening, optimization, and characterization of trisulfide-derived lipid nanoparticles (TS LNPs) are shown. The mRNA delivery effect of TS LNPs in macrophages is shown. Bioluminescence intensity was normalized to MC3 LNPs (n=3). [Figure 4B]

[0023] Figure 1 shows the screening, optimization, and characterization of trisulfide-derived lipid nanoparticles (TS LNPs). Figure 2 shows a table of the first round optimal formulation of TS2 LNPs. [Figure 4C] Screening, optimization, and characterization of trisulfide-derived lipid nanoparticles (TS LNPs) are shown. Orthogonal assays to determine the influence of each lipid component in four levels of TS2 LNPs are shown (n=3). [Figure 4D] Screening, optimization, and characterization of trisulfide-derived lipid nanoparticles (TS LNPs). Relative bioluminescence intensity of the first round of optimization is shown (n=3). [Figure 4E]

[0023] Figure 1 shows the screening, optimization, and characterization of trisulfide-derived lipid nanoparticles (TS LNPs). Figure 2 shows a table of the second round optimal formulation of TS2 LNPs. [Figure 4F] Screening, optimization, and characterization of trisulfide-derived lipid nanoparticles (TS LNPs). Relative bioluminescence intensity of the second round of optimization is shown (n=3). [Figure 4G] Screening, optimization, and characterization of trisulfide-derived lipid nanoparticles (TS LNPs). Characterization of TS2 LNPs, including size, PDI, encapsulation efficiency, and zeta potential, is shown (n=3). [Figure 4H] Screening, optimization, and characterization of trisulfide-derived lipid nanoparticles (TS LNPs) are shown. A cryo-TEM image of TS2 LNPs is shown. Scale bar: 50 nm. All data are presented as mean ± SD. Statistical significance was calculated by one-way ANOVA with unpaired two-tailed Student's t-test af and Tukey's post-hoc test d. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 5A] Figure 1 shows that TS2-IL4 LNPs induce macrophage polarization and enhance fibroblast viability by scavenging ROS in vitro. Representative flow cytometry analysis of M1 macrophage marker CD86+ after different treatments is shown. [Figure 5B] Figure 1 shows that TS2-IL4 LNPs induce macrophage polarization and enhance fibroblast viability by scavenging ROS in vitro. Quantification of the expression of the M1 macrophage marker CD86+ after different treatments is shown (n=3). [Figure 5C] Figure 1 shows that TS2-IL4 LNPs induce macrophage polarization and enhance fibroblast viability by scavenging ROS in vitro. Representative flow cytometry analysis of M2 macrophage marker CD206+ after different treatments is shown. [Figure 5D] Figure 1 shows that TS2-IL4 LNPs induce macrophage polarization and enhance fibroblast viability by scavenging ROS in vitro. Quantification of the expression of the M2 macrophage marker CD206+ after different treatments is shown (n=3). [Figure 5E]Figure 1 shows that TS2-IL4 LNPs induce macrophage polarization and enhance fibroblast viability by scavenging ROS in vitro. Representative live / dead cell staining CLSM images of fibroblasts after different treatments are shown. Green: live cells, red: dead cells. Scale bar: 100 μm. [Figure 5F] Figure 1 shows that TS2-IL4 LNPs induce macrophage polarization and enhance fibroblast viability by scavenging ROS in vitro. Quantification of cell viability after different treatments is shown (n=3). [Figure 5G] Figure 1 shows that TS2-IL4 LNPs induce macrophage polarization and enhance fibroblast viability by scavenging ROS in vitro. CLSM images of intracellular ROS levels in fibroblasts after different treatments are shown. Scale bar: 100 μm. [Figure 5H] Figure 1 shows that TS2-IL4 LNPs induce macrophage polarization and enhance fibroblast viability by scavenging ROS in vitro. Flow cytometry analysis of intracellular ROS generation after different treatments is shown. All data are presented as mean ± SD. Statistical significance was calculated by one-way ANOVA with Tukey's post-hoc test. ns *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 6A] Figure 1 shows that TS2-IL4 LNPs loaded into hydrogels accelerated wound healing in diabetic mice. Representative bioluminescence imaging of wounds in different groups is shown. [Figure 6B] Figure 1 shows that TS2-IL4 LNPs loaded into hydrogels accelerated wound healing in diabetic mice. Quantification of bioluminescence intensity at wound sites in different groups is shown (n=6). [Figure 6C] Representative photographic images of diabetic wounds with different treatments on days 0, 3, 6, 9, 12, 15, and 18 are shown, showing that hydrogel-loaded TS2-IL4 LNPs accelerated wound healing in diabetic mice. [Figure 6D]Figure 1 shows that TS2-IL4 LNPs loaded in hydrogels accelerated wound healing in diabetic mice. Relative wound area (n=6) in each group is shown. [Figure 6E] Figure 1 shows that TS2-IL4 LNPs loaded into hydrogels accelerated wound healing in diabetic mice. Time to complete wound closure in different treatment groups is shown. [Figure 6F] Hydrogel-loaded TS2-IL4 LNPs accelerated wound healing in diabetic mice. Representative images of H&E staining (top) and Masson staining (bottom) of wounds 18 days after treatment are shown. Scale bar: 500 μm. [Figure 6G] Figure 6 shows that TS2-IL4 LNPs loaded into hydrogels accelerated wound healing in diabetic mice. Quantitative analysis of epidermal thickness in each treatment group after 18 days of treatment (n=6) is shown. All data are presented as mean ± SD. Statistical significance was calculated by one-way ANOVA with Tukey's post-hoc test in Figures 6B, 6D, and 6G and the log-rank test in e. *P<0.05, 6D<0.01, ***P<0.001, ****P<0.0001. [Figure 7A] Flow cytometry analysis and immunofluorescence staining of wound tissue 18 days after treatment. A representative flow cytometry analysis of CD86 expression in wounds 18 days after treatment, gated on F4 / 80+, CD86+ cells, is shown. [Figure 7B] Flow cytometry analysis and immunofluorescence staining of wound tissue 18 days after treatment. Quantification of the percentage of M1 phenotype macrophages (F4 / 80+ CD86+), (n=3). [Figure 7C] Flow cytometry analysis and immunofluorescence staining of wound tissue 18 days after treatment. A representative flow cytometry analysis of CD206 expression in wounds 18 days after treatment, gated on F4 / 80+, CD206+ cells, is shown. [Figure 7D]Flow cytometry analysis and immunofluorescence staining of wound tissue 18 days after treatment. Quantification of the percentage of M2 phenotype macrophages (F4 / 80+CD206+), (n=3). [Figure 7E] Flow cytometry analysis and immunofluorescence staining of wound tissues 18 days after treatment. Representative images of immunofluorescence staining of M1 phenotype macrophages in wounds from different groups are shown. Scale bar: 50 μm. [Figure 7F] Flow cytometry analysis and immunofluorescence staining of wound tissues on day 18 after treatment. Quantification of immunofluorescence staining of M1 phenotype macrophages (CD86+: green, nuclei: blue) in wounds from different groups (n=6). [Figure 7G] Flow cytometry analysis and immunofluorescence staining of wound tissues 18 days after treatment. Representative images of immunofluorescence staining of M2 phenotype macrophages in wounds from different groups are shown. Scale bar: 50 μm. [Figure 7H] Flow cytometry analysis and immunofluorescence staining of wound tissues 18 days after treatment are shown. Quantification of immunofluorescence staining of M1 phenotype macrophages (CD206+: green, nuclei: blue) in wounds from different groups (n=6) is shown. All data are presented as mean ± SD. Statistical significance was calculated by one-way ANOVA with Tukey's post-hoc test. **P<0.01, ***P<0.001, ****P<0.0001. [Figure 8A] Figure 1 shows that TS2-IL4 LNPs loaded into hydrogels accelerated wound healing in diabetic mice. Representative bioluminescence imaging of mouse wounds in different groups is shown. [Figure 8B] Figure 1 shows that TS2-IL4 LNPs loaded into hydrogels accelerated wound healing in diabetic mice. Quantification of bioluminescence intensity at wound sites in different groups is shown (n=6). [Figure 8C] Representative photographic images of diabetic wounds with different treatments on days 0, 3, 6, 9, 12, 15, and 18 are shown, showing that hydrogel-loaded TS2-IL4 LNPs accelerated wound healing in diabetic mice. [Figure 8D] Figure 1 shows that TS2-IL4 LNPs loaded into hydrogels accelerated wound healing in diabetic mice. The relative wound area in each group is shown (n=6). [Figure 8E] Figure 8 shows that TS2-IL4 LNPs loaded into hydrogels accelerated wound healing in diabetic mice. The time to complete wound closure for different treatments is shown. All data are presented as mean ± SD. Statistical significance was calculated by one-way ANOVA with Tukey's post-hoc test in Figures 8B and 8D and by the log-rank test in Figure 8E. *P<0.05, 6D<0.01, ***P<0.001, ****P<0.0001. [Figure 9] The expression levels of IL4 at different time points after incubating TS2-IL4 LNPs with RAW264.7 cells are shown (n=3). [Figure 10] Quantitative analysis of the mean fluorescence intensity of ROS with different treatments is shown (n=3). [Figure 11] Figure 1 shows the mRNA delivery efficiency of TS2 LNPs, ALC 0315 LNPs, and SM102 LNPs in macrophages. Bioluminescence intensity was normalized to the MC3 LNP group (n=3). [Figure 12] (A) Representative images and quantification of immunofluorescent staining of α-SMA+ in wounds from different groups (α-SMA+: green, nuclei: blue). (B) Representative images and quantification of immunofluorescent staining of CD31+ in wounds from different groups (CD31+: red, nuclei: blue). Scale bar: 50 μm. DETAILED DESCRIPTION OF THE INVENTION

[0022] The compounds, compositions and methods described herein may be understood more readily by reference to the following detailed description of certain aspects of the disclosed subject matter and the examples included therein.

[0023] Before the present compounds, compositions, and methods are disclosed and described, it is to be understood that the embodiments described below are not limited to particular synthetic methods or to particular reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0024] Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the subject matter of this disclosure pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0025] General definition In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0026] As used herein, the articles "a," "an," and "the" mean "at least one" unless the context in which the article is used clearly indicates otherwise.

[0027] As used herein, the term "nucleic acid" refers to a polymer of nucleotides, eg, deoxyribonucleotides or ribonucleotides.

[0028] As used herein, the terms "ribonucleic acid" and "RNA" refer to a polymer composed of ribonucleotides.

[0029] As used herein, the terms "deoxyribonucleic acid" and "DNA" refer to a polymer composed of deoxyribonucleotides.

[0030] The term "oligonucleotide" refers to a single- or double-stranded nucleotide multimer having a length of about 2 to about 100 nucleotides. Suitable oligonucleotides can be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22:1859-1862 (1981), or by the triester method by Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both of which are incorporated herein by reference, or by other chemical methods using either a commercially available automated oligonucleotide synthesizer or VLSIPS™ technology. When an oligonucleotide is referred to as "double-stranded," those skilled in the art will understand that a pair of oligonucleotides typically exists in a hydrogen-bonded helical arrangement, such as in association with DNA. In addition to 100% complementary forms of double-stranded oligonucleotides, the term "double-stranded" as used herein is also meant to refer to forms that include structural features such as bulges and loops, which are more fully described in biochemistry textbooks such as Stryer, Biochemistry, Third Ed., (1988), which is incorporated herein by reference for all purposes.

[0031] The term "polynucleotide" refers to a single- or double-stranded polymer composed of nucleotide monomers. In some embodiments, a polynucleotide is generally greater than 100 nucleotides in length and is composed of up to about 8,000 or more nucleotide monomers.

[0032] The term "polypeptide" refers to a compound composed of a single chain of D- or L-amino acids, or a mixture of D- and L-amino acids linked by peptide bonds.

[0033] The term "complementary" refers to the topological compatibility or match of the interacting surfaces of a probe molecule and its target. Thus, a target and its probe can be described as complementary, and further, the properties of the contact surfaces are complementary to each other.

[0034] The term "hybridization" refers to the process of establishing non-covalent, sequence-specific interactions between two or more complementary strands of nucleic acid resulting in a single hybrid, when two strands are referred to as a duplex.

[0035] The term "annealing" refers to the process by which single-stranded nucleic acid sequences pair by hydrogen bonding to complementary sequences to form double-stranded nucleic acid sequences, including the reformation (renaturation) of complementary strands that have been separated by heat (thermal denaturation).

[0036] The term "melting" refers to the denaturation of a double-stranded nucleic acid sequence by high temperature, which separates the double strand into two single strands by breaking the hydrogen bonds between the strands.

[0037] The term "target" refers to a molecule that has affinity for a particular probe. Targets can be natural or artificial molecules. Targets can be used in their native state or as aggregates with other species.

[0038] The term "promoter" or "regulatory element" refers to a region or sequence determinant located upstream or downstream from the transcription start site and involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters need not be of bacterial origin; for example, promoters derived from viruses or other organisms can be used in the compositions, systems, or methods described herein. The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and polyU sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). Regulatory elements can also direct expression in a time-dependent manner, such as a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue- or cell-type-specific. In some embodiments, the vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters.Examples of pol II promoters include, but are not limited to, the retroviral Ruth's sarcoma virus (RSV) LTR promoter (optionally containing the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally containing the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. The term "regulatory element" also encompasses enhancer elements such as WPRE, the CMV enhancer; the R-U5' segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA, Vol. 78(3), pp. 1527-31, 1981). It will be understood by those skilled in the art that the design of the expression vector can depend on factors such as the choice of the host cell to be transformed, the desired expression level, and the like.

[0039] The term "recombinant" refers to a human-engineered nucleic acid (e.g., polynucleotide) or a copy or complement of a human-engineered nucleic acid (e.g., polynucleotide), or, with respect to a protein (i.e., a "recombinant protein"), to the protein (e.g., polynucleotide) encoded by a recombinant nucleic acid. In embodiments, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., polynucleotide) can include a promoter that is heterologous to the second nucleic acid (e.g., polynucleotide) as a result of human manipulation (e.g., by the methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette can include nucleic acids (e.g., polynucleotides) that are combined such that the nucleic acids (e.g., polynucleotides) are highly unlikely to be found in nature. For example, a human-engineered restriction enzyme site or a plasmid vector sequence may flank or separate the promoter from the second nucleic acid (e.g., polynucleotide). Those skilled in the art will recognize that nucleic acids (e.g., polynucleotides) can be engineered in many ways and are not limited to the above examples.

[0040] The term "expression cassette" refers to a nucleic acid construct that, when introduced into a host cell, results in the transcription and / or translation of an RNA or polypeptide, respectively. In embodiments, an expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., a polynucleotide) can include a promoter that is heterologous to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In some embodiments, an expression cassette comprising a terminator (or termination sequence) operably linked to a second nucleic acid (e.g., a polynucleotide) can include a terminator that is heterologous to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation. In some embodiments, the expression cassette comprises a promoter operably linked to a second nucleic acid (e.g., polynucleotide) and a terminator operably linked to a second nucleic acid (e.g., polynucleotide) as a result of human manipulation. In some embodiments, the expression cassette comprises an endogenous promoter. In some embodiments, the expression cassette comprises an endogenous terminator. In some embodiments, the expression cassette comprises a synthetic (or non-naturally occurring) promoter. In some embodiments, the expression cassette comprises a synthetic (or non-naturally occurring) terminator.

[0041] The term "identity" or percent "identity" with respect to two or more nucleic acid or polypeptide sequences is determined using the BLAST or BLAST 2.0 sequence comparison algorithms using the default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI "Substantially identical" refers to two or more sequences or subsequences that are identical or have a specified percentage of amino acid residues or nucleotides that are identical (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region. Such sequences are said to be "substantially identical." This definition refers to or can be applied to the complement of a subject sequence. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. As described below, preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably, over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent amino acid sequence identity (%) is defined as the percentage of amino acids in a candidate sequence that are identical to those in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment to determine percent sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared, can be determined by known methods.

[0042] For sequence comparison, generally, one sequence serves as a reference sequence, and it is compared with test sequence.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and if necessary, partial sequence coordinates are designated, and sequence algorithm program parameters are designated.Preferably, default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the sequence identity percentage of test sequence and reference sequence based on program parameters.

[0043] A preferred example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms (described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov / ). This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that, when aligned with a word of the same length in a database sequence, match the word or meet a specified positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, cumulative scores are calculated using the parameters M (reward score for a pair of matching residues, always greater than 0) and N (penalty score for mismatching residues, always less than 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value, when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses by default a word length of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix (Henikoff and Henikoff (1989), Proc Natl Acad Sci USA, 89:10915) alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0044] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0045] The phrase "codon optimization," when referring to genes or coding regions of a nucleic acid molecule intended for transformation into various hosts, refers to modifying the codons in the genes or coding regions of that polynucleic acid molecule to reflect the typical codon usage of a selected organism, without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, more than one, or a significant number of codons with one or more codons that are more frequently used in the genes of the selected organism.

[0046] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA of a presequence or secretory leader is operably linked to DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous with each other, and, in the case of a secretory leader, contiguous and in reading phase. However, operably linked nucleic acids (e.g., enhancer and coding sequence) need not be contiguous. Linkage is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice. In embodiments, a promoter is operably linked to a coding sequence if it is capable of affecting expression of a protein from that coding sequence (e.g., regulating relative to the absence of the promoter) (i.e., when the coding sequence is under the transcriptional control of the promoter).

[0047] The term "nucleobase" refers to the portion of a nucleotide that possesses Watson / Crick base pairing functionality. The most common naturally occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T), possess hydrogen-bonding functionality that sequence-specifically links one nucleic acid strand to another.

[0048] As used throughout, "subject" (or "host") means an individual. Thus, a "subject" can include, for example, domestic animals such as cats, dogs, etc., livestock (e.g., cows, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animals. The subject can be a mammal, such as a primate or a human.

[0049] The term "about" as used herein in reference to a measurable value, such as an amount, percentage, etc., is meant to encompass a variation of ±20%, ±10%, ±5%, or ±1% from the measurable value.

[0050] A nucleic acid sequence is "heterologous" to a second nucleic acid sequence if it is derived from a foreign species or, if derived from the same species, is artificially altered from its original form. For example, a heterologous promoter (or heterologous 5' untranslated region (5'UTR)) operably linked to a coding sequence refers to a coding sequence from a species different from that from which the promoter is derived, and, if derived from the same species, refers to a coding sequence that differs from a naturally occurring allelic variant.

[0051] As used herein, the terms "treating" or "treatment" of a subject include administering a drug to a subject with the intent to cure, ameliorate, alleviate, relieve, alter, treat, ameliorate, improve, stabilize, or affect a disease or disorder, or the symptoms of a disease or disorder. The terms "treat" and "treatment" can also refer to reducing the severity and / or frequency of symptoms, eliminating symptoms and / or underlying causes, and ameliorating or repairing damage.

[0052] As used herein, the term "molar ratio" refers to the stoichiometric amount of moles of each component. For example, the molar ratio can be used to define the relative amounts of individual lipid components in a lipid nanoparticle.

[0053] As used herein, the term "prevention" of a disease, disorder, or undesired physiological event in a subject refers to the prevention of the disease, disorder, or undesired physiological event, or the prevention of the symptoms of the disease, disorder, or undesired physiological event.

[0054] An "effective amount" of a drug refers to an amount of the drug sufficient to provide the desired effect. The amount of a drug that is "effective" will vary from subject to subject, depending on many factors, such as the subject's age and general condition, the specific drug(s), and so forth. Therefore, it is not always possible to specify a quantified "effective amount." However, an appropriate "effective amount" for any subject can be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, the "effective amount" of a drug may refer to an amount that encompasses both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of a drug required to achieve a therapeutic effect may vary depending on factors such as the subject's age, sex, and weight. Dosage regimens may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0055] A "pharmaceutically acceptable" ingredient may refer to an ingredient that is not biologically or otherwise undesirable, i.e., the ingredient may be incorporated into a pharmaceutical formulation of the present invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other ingredients of the formulation containing the ingredient. When used in reference to human administration, the term generally means that the ingredient has met the necessary standards of toxicological and manufacturing testing or that the ingredient is included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration.

[0056] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") generally refers to a carrier or excipient useful in preparing a safe and non-toxic pharmaceutical or therapeutic composition, and includes carriers acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as further described herein.

[0057] A "therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, such as, for example, treating a disorder or other undesirable physiological condition, and prophylactic effects, such as, for example, preventing a disorder or other undesirable physiological condition. These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, or when a particular agent is specifically identified, the term includes the agent itself as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, conjugates, active metabolites, isomers, fragments, analogs, and the like.

[0058] As used herein, the terms "controlled release" or "controlled release drug delivery" or "sustained release" refer to the release or administration of a drug from a given dosage form in a controlled manner to achieve desired pharmacokinetic properties in vivo. An aspect of "controlled" drug delivery is the ability to manipulate the formulation and / or dosage form to establish desired kinetics of drug release.

[0059] As used herein, the phrases "concurrent administration," "co-administration," "simultaneous administration," or "administering at the same time" mean that the compounds are administered at the same time or shortly after each other.

[0060] The term "antibody" is used broadly herein and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term "antibody" also includes fragments or polymers of those immunoglobulin molecules, as well as human or humanized forms of immunoglobulin molecules or fragments thereof. Antibodies can be tested for their desired activity using the in vitro assays described herein or by similar methods, and then their in vivo therapeutic and / or prophylactic activity is tested according to known clinical trial methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Those skilled in the art will recognize the equivalent classes in mice. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0061] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of antibody molecules. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired antagonistic activity.

[0062] The disclosed monoclonal antibodies can be produced using any method that produces monoclonal antibodies. For example, the disclosed monoclonal antibodies can be prepared using the hybridoma method, such as the method described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0063] Monoclonal antibodies may also be produced by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be produced and screened using phage display techniques, as described, for example, in U.S. Pat. No. 5,804,440 (Burton et al.) and U.S. Pat. No. 6,096,441 (Barbas et al.).

[0064] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques well known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in WO 94 / 29348, published December 22, 1994, and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment. Pepsin treatment yields fragments with two antigen-binding sites that are still capable of cross-linking antigen.

[0065] As used herein, the terms "antibody or antigen-binding fragment thereof" or "antibody or fragment thereof" encompass fragments such as F(ab')2, Fab', Fab, Fv, sFv, and scFv, including chimeric and hybrid antibodies and hybrid fragments having dual or multiple antigen or epitope specificities. Thus, fragments of antibodies that retain the ability to bind to their specific antigens are provided. For example, fragments of antibodies that retain binding activity are included within the meaning of the term antibody or antigen-binding fragment thereof. Such antibodies and fragments can be produced by techniques well known in the art and screened for specificity and activity according to the methods described in the Examples and general methods for generating and screening antibodies for specificity and activity (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Publications, New York (1988)).

[0066] The meaning of "antibody or antigen-binding fragment thereof" also includes conjugates of antibody fragments and antigen-binding proteins (single-chain antibodies). The meaning of "antibody or antigen-binding fragment thereof" also includes immunoglobulin single variable domains, such as, for example, nanobodies.

[0067] Fragments may also contain insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not linked to other sequences, so long as the activity of the antibody or fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. These modifications may provide additional properties, such as removing / adding amino acids capable of disulfide bonding, extending biological lifespan, or altering secretion characteristics. In either case, the antibody or antibody fragment must retain biologically active properties, such as specific binding to its cognate antigen. Functional or active regions of an antibody or antibody fragment can be identified by mutagenesis of specific regions of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to those skilled in the art and may include site-directed mutagenesis of nucleic acids encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992).

[0068] As used herein, the term "antibody" or "antibodies" can also refer to human antibodies and / or humanized antibodies. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans and, therefore, can provoke an unwanted immune response when administered to humans. Thus, the use of human or humanized antibodies in these methods serves to reduce the likelihood that antibodies administered to humans will provoke an unwanted immune response.

[0069] The term "Toll-like receptor" (TLR) refers to a member of a family of receptors that bind to pathogen-associated molecular patterns (PAMPs) and promote immune responses in mammals. Ten mammalian TLRs are known, e.g., TLRs 1-10. The term "Toll-like receptor agonist" (TLR agonist) refers to a molecule that binds to a TLR. Synthetic TLR agonists are compounds designed to bind to and activate a TLR. Exemplary synthetic TLR agonists provided herein include "TLR-7 agonists," "TLR agonists," "TLR-3 agonists," and "TLR-9 agonists." TLR agonists include imiquimod, resiquimod, broprimine and loxorubine, gardquimod, CL075, SM324405, UC1V150, CU-T12-9, or derivatives thereof.

[0070] chemical definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0071] The organic moieties referred to in defining variable positions within the general formulae described herein (e.g., the term "halogen") are generic to the individual substituents contained within the organic moiety. The prefix C before the group or moiety n -C m indicates in each case the possible number of carbon atoms in the group or moiety that follows.

[0072] As used herein, the term "ion" refers to any molecule, part of a molecule, cluster of molecules, molecular complex, moiety, or atom that contains or can be made to contain a charge (positive, negative, or both simultaneously in one molecule, cluster of molecules, molecular complex, or moiety (e.g., a zwitterion)). Methods for generating charge in a molecule, part of a molecule, cluster of molecules, molecular complex, moiety, or atom are disclosed herein and can be accomplished by methods known in the art, such as, for example, protonation, deprotonation, oxidation, reduction, alkylation, acetylation, esterification, deesterification, hydrolysis, etc.

[0073] The term "anion" is a type of ion and is included within the meaning of the term "ion." An "anion" is any molecule, part of a molecule (e.g., a zwitterion), cluster of molecules, molecular complex, moiety, or atom that contains or can be made to contain a net negative charge. The term "anion precursor" is used herein specifically to refer to a molecule that can be converted to an anion via a chemical reaction (e.g., deprotonation).

[0074] The term "cation" is a type of ion and is included within the meaning of the term "ion." A "cation" is any molecule, portion of a molecule (e.g., a zwitterion), cluster of molecules, molecular complex, moiety, or atom that contains or can be made to contain a net positive charge. The term "cation precursor" is used herein specifically to refer to a molecule that can be converted to a cation via a chemical reaction (e.g., protonation or alkylation).

[0075] As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Additionally, the terms "substituted" or "substituted with" include the implicit proviso that such substitution is subject to the permissible valencies of the substituted atom and substituent, and that the substitution results in a stable compound (e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, etc.).

[0076] "Z 1 "," "Z 2 "," "Z 3 " and "Z 4 " is used herein as a generic term to represent various specific substituents. These symbols can be any substituent not limited to the substituents disclosed herein, and when they are defined as specific substituents in one instance, they may be defined as several other substituents in another instance.

[0077] As used herein, the term "aliphatic" refers to a non-aromatic hydrocarbon group, including branched and unbranched alkyl, alkenyl, or alkynyl groups.

[0078] As used herein, the term "alkyl" refers to a saturated, straight-chain or branched saturated hydrocarbon moiety. Unless otherwise specified, C-C 24 (e.g., C1-C 22 , C1-C 20 , C1-C 18 , C1-C 16, C1-C 14 , C1-C 12 , C1-C 10 , C1-C8, C1-C6, or C1-C4 alkyl groups are contemplated. Examples of alkyl groups include methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethyl butyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. Alkyl substituents may be unsubstituted or substituted with one or more chemical moieties. Alkyl groups may be substituted with one or more groups, including, but not limited to, hydroxyl, halogen, acetal, acyl, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate, carbamate, ketone, nitro, phosphonyl, silyl, sulfoxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below, provided that the substituents are sterically compatible and chemical bonding and strain energy rules are satisfied.

[0079] Although "alkyl" is used throughout this specification generally to refer to both unsubstituted and substituted alkyl groups, substituted alkyl groups are specifically referred to herein by identifying the particular substituent(s) on the alkyl group. For example, the term "halogenated alkyl" or "haloalkyl" specifically refers to an alkyl group substituted with one or more halides (halogens; e.g., fluorine, chlorine, bromine, or iodine). The term "alkoxyalkyl" specifically refers to an alkyl group substituted with one or more alkoxy groups, as described below. The term "alkylamino" specifically refers to an alkyl group substituted with one or more amino groups, as described below. When "alkyl" is used in one example and a specific term such as "alkylalcohol" is used in another example, this does not imply that the term "alkyl" does not also refer to the specific term such as "alkylalcohol."

[0080] This practice is also used for other groups described herein. That is, a term such as "cycloalkyl" refers to both unsubstituted and substituted cycloalkyl moieties, but the substituted moieties can be further specifically identified herein; for example, a particular substituted cycloalkyl can be referred to specifically as, for example, an "alkylcycloalkyl." Similarly, a substituted alkoxy can be specifically referred to as, for example, a "halogenated alkoxy," a particular substituted alkenyl can be, for example, an "alkenylalcohol," and the like. Again, the practice of using a general term such as "cycloalkyl" and a specific term such as "alkylcycloalkyl" is not intended to mean that the general term does not also include the specific term.

[0081] As used herein, the term "alkenyl" refers to an unsaturated, straight-chain or branched hydrocarbon moiety containing a double bond. Unless otherwise specified, C-C 24 (For example, C2-C 22 , C2-C 20 , C2-C 18 , C2-C 16 , C2-C 14 , C2-C 12 , C2-C10C-C, C-C, or C-C) alkenyl groups are contemplated. Alkenyl groups may contain multiple unsaturated bonds.Examples include ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1-ethyl-2-propenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 3-methyl-1-pentenyl, 4-methyl-1-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 1-methyl -3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethylyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-1-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl thenyl, 2,3-dimethyl-1-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-1-butenyl, 3,3-dimethyl-2-butenyl, 1-ethyl-1-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-1-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl, 1-ethyl-2-methyl-1-propenyl, and 1-ethyl-2-methyl-2-propenyl.The term "vinyl" refers to a group having the structure -CH=CH2, 1-propenyl refers to a group having the structure -CH=CH-CH3, and 2-propenyl refers to a group having the structure -CH2-CH=CH2. (Z. 1 Z 2 )C=C(Z 3 Z 4 ), are intended to include both the E and Z isomers. This can be assumed in structural formulas herein where an asymmetric alkene is present, or can be explicitly indicated by the bond symbol C═C. Alkenyl substituents can be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate, carbamate, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfoxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below, provided that the substituents are sterically compatible and chemical bonding and strain energy rules are satisfied.

[0082] As used herein, the term "alkynyl" refers to a straight or branched chain hydrocarbon moiety containing a triple bond. Unless otherwise specified, C-C 24 (For example, C2-C 24 , C2-C 20 , C2-C 18 , C2-C 16 , C2-C 14 , C2-C 12 , C2-C 10, C2-C8, C2-C6, or C2-C4) alkynyl groups are contemplated. The alkynyl group may contain multiple unsaturated bonds. Examples include C2-C6-alkynyl, such as ethynyl, 1-propynyl, 2-propynyl (or propargyl), 1-butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 3-methyl-1-butynyl, 1-methyl-2-butynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 3-methyl-1-pentynyl, 4 Alkynyl substituents include 1-methyl-1-pentynyl, 1-methyl-2-pentynyl, 4-methyl-2-pentynyl, 1-methyl-3-pentynyl, 2-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 3,3-dimethyl-1-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl, and 1-ethyl-1-methyl-2-propynyl. Alkynyl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate, carbamate, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfoxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

[0083] As used herein, the term "aryl," and derivative terms such as aryloxy, refer to a group containing a monovalent aromatic carbocyclic group of 3 to 50 carbon atoms. An aryl group can contain a single ring or multiple fused rings. In some embodiments, an aryl group includes a C6-C 10Aryl groups are included. Examples of aryl groups include, but are not limited to, benzene, phenyl, biphenyl, naphthyl, tetrahydronaphthyl, phenylcyclopropyl, phenoxybenzene, and indanyl. The term "aryl" also includes "heteroaryl," which is defined as a group containing an aromatic group with at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The term "non-heteroaryl," also included within the term "aryl," defines a group containing an aromatic group without a heteroatom. Aryl substituents can be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate, carbamate, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfoxo, sulfonyl, sulfone, sulfoxide, or thiol, as described herein. The term "biaryl" is a special type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups joined through a fused ring structure, as in naphthalene, or through one or more carbon-carbon bonds, as in biphenyl.

[0084] As used herein, the term "cycloalkyl" refers to a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term "heterocycloalkyl" refers to a cycloalkyl group, as defined above, in which at least one of the ring carbon atoms is replaced with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkyl and heterocycloalkyl groups can be substituted or unsubstituted. Cycloalkyl and heterocycloalkyl groups can be substituted with one or more groups, including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate, carbamate, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfoxo, sulfonyl, sulfone, sulfoxide, or thiol, as described herein.

[0085] As used herein, the term "cycloalkenyl" refers to a non-aromatic carbon-based ring consisting of at least three carbon atoms and having at least one double bond (i.e., C=C). Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term "heterocycloalkenyl" refers to a type of cycloalkenyl group, as defined above, and is included within the meaning of the term "cycloalkenyl," in which at least one of the ring carbon atoms is replaced with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkenyl and heterocycloalkenyl groups can be substituted or unsubstituted. The cycloalkenyl and heterocycloalkenyl groups can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate, carbamate, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfoxo, sulfonyl, sulfone, sulfoxide, or thiol, as described herein.

[0086] The term "cyclic group" is used herein to refer to aryl groups, non-aryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems (e.g., monocyclic, bicyclic, tricyclic, polycyclic, etc.) that can be substituted or unsubstituted. A cyclic group can contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.

[0087] As used herein, the term "acyl" refers to a group of the formula -C(O)Z 1 In the formula, Z 1may be hydrogen, hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups as described above. As used herein, the term "acyl" may be used interchangeably with "carbonyl." Throughout this specification, "C(O)" or "CO" is an abbreviation for C=O.

[0088] As used herein, the term "acetal" refers to a compound of the formula (Z 1 Z 2 )C(=OZ 3 )(=OZ 4 ) wherein Z 1 , Z 2 , Z 3 , and Z 4 may independently be hydrogen, halogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups as described above.

[0089] As used herein, the term "alkyl alcohol" refers to a compound of formula Z 1 OH, wherein Z 1 may be any of the alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups described above. Examples of alkyl alcohols include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, tert-butyl alcohol, and the like.

[0090] As used herein, the term "alkoxy" as used herein refers to an alkyl group attached through a single, terminal ether bond, i.e., an "alkoxy" group has the formula Z 1 -O-, where Z 1 is unsubstituted or substituted alkyl as defined above. Unless otherwise specified, Z 1 But C1-C24 (e.g., C1-C 22 , C1-C 20 , C1-C 18 , C1-C 16 , C1-C 14 , C1-C 12 , C1-C 10 Alkoxy groups are contemplated, such as C1-C8, C1-C6, C1-C4) alkyl groups. Examples include methoxy, ethoxy, propoxy, 1-methyl-ethoxy, butoxy, 1-methyl-propoxy, 2-methyl-propoxy, 1,1-dimethyl-ethoxy, pentoxy, 1-methyl-butyloxy, 2-methyl-butoxy, 3-methyl-butoxy, 2,2-dimethyl-propoxy, 1-ethyl-propoxy, hexoxy, 1,1-dimethyl-propoxy, 1,2-dimethyl-propoxy, 1-methyl-pentoxy, 2-methyl-pentoxy, 3- Examples include methyl-pentoxy, 4-methyl-penoxy, 1,1-dimethyl-butoxy, 1,2-dimethyl-butoxy, 1,3-dimethyl-butoxy, 2,2-dimethyl-butoxy, 2,3-dimethyl-butoxy, 3,3-dimethyl-butoxy, 1-ethyl-butoxy, 2-ethylbutoxy, 1,1,2-trimethyl-propoxy, 1,2,2-trimethyl-propoxy, 1-ethyl-1-methyl-propoxy, and 1-ethyl-2-methyl-propoxy.

[0091] The term "aldehyde" as used herein is represented by the formula -C(O)H. Throughout this specification, "C(O)" is a shorthand notation for C=O.

[0092] As used herein, the term "amine" or "amino" refers to a group of the formula -NZ 1 Z 2 Z 3 wherein Z 1 , Z 2 , and Z 3 may each be a substituent as described herein, for example, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0093] As used herein, the term "amide" or "amido" refers to a group of the formula -C(O)NZ 1 Z 2 wherein Z 1 and Z 2 may each be a substituent as described herein, for example, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0094] As used herein, the term "anhydride" refers to a compound of formula Z 1 C(O)OC(O)Z 2 wherein Z 1 and Z 2 may independently be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0095] As used herein, the term "cyclic anhydride" is represented by the formula: [ka] In the formula, Z 1 may be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0096] The term "azide" as used herein is represented by the formula -N=N=N.

[0097] The term "carboxylic acid" as used herein is represented by the formula -C(O)OH.

[0098] As used herein, a "carboxylate" or "carboxyl" group is represented by the formula --C(O)O--.

[0099] As used herein, a "carbonate" group has the formula Z 1 OC(O)OZ 2 It is expressed as:

[0100] The term "cyano" as used herein is represented by the formula --CN.

[0101] As used herein, the term "ester" refers to an ester of the formula -Z 1 OC(O)Z 2 or -Z 1 C(O)OZ 2 wherein Z 1 and Z 2 may independently be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0102] As used herein, the term "ether" refers to a group of the formula Z 1 OZ 2 wherein Z 1 and Z 2 may independently be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0103] As used herein, the term "epoxy" or "epoxide" refers to a cyclic ether having a three atom ring and can be represented by the following formula: [ka] In the formula, Z 1 , Z 2 , Z 3 , and Z 4 may independently be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0104] As used herein, the term "ketone" refers to a ketone having the formula ZC(O)Z 2 wherein Z 1 and Z 2 may independently be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0105] The terms "halide" or "halogen" or "halo" as used herein refer to fluorine, chlorine, bromine, and iodine.

[0106] The term "hydroxyl" as used herein is represented by the formula --OH.

[0107] The term "nitro" as used herein is represented by the formula -NO2.

[0108] The term "phosphonyl" as used herein refers to a group of the formula -P(O)(OZ 1 )2, wherein Z 1 may be hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0109] As used herein, the term "silyl" refers to a group of the formula -SiZ 1 Z 2 Z 3 In the formula, Z 1 , Z 2 , and Z 3 may independently be hydrogen, an alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0110] The term "sulfonyl" or "sulfone" refers to a group of the formula -S(O)Z 1is used herein to refer to a sulfooxo group represented by the formula: 1 may be hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as defined above.

[0111] As used herein, the term "sulfide" includes groups of the formula -S-.

[0112] The term "thiol" as used herein is represented by the formula --SH.

[0113] As used herein, "R 1 "," "R 2 "," "R 3 "," "R n " etc. (where n is any integer) may independently have one or more of the above groups. For example, R 1 When is a straight-chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be replaced with a hydroxyl group, an alkoxy group, an amine group, an alkyl group, a halide, or the like. Depending on the group selected, the first group can be incorporated into the second group, or alternatively, the first group can be pendant (i.e., attached) to the second group. For example, in the phrase "an alkyl group comprising an amino group," the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) selected will determine whether the first group is embedded in or attached to the second group.

[0114] Unless stated to the contrary, formulas containing chemical bonds shown only with solid lines, rather than wedges or dashed lines, contemplate each possible stereoisomer or mixture of stereoisomers (e.g., each enantiomer, each diastereomer, each meso compound, racemic mixture, or scalemic mixture).

[0115] compound Disclosed herein are compounds and methods for making and using them. For example, disclosed herein are compositions comprising a compound defined by Formula I, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, p is an integer from 0 to 5, n is an integer from 1 to 10, each m, if present, is independently an integer from 1 to 10; R 1 , R 2 , and R 3 are independently OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and R 4 is a substituted or unsubstituted C8-C 18 is alkyl, Each R 5 -L, when present, independently represents hydrogen, OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R a are, if present, independently substituted or unsubstituted C8-C 18 is alkyl, L in each case 1 are independently substituted or unsubstituted alkyl, carbonyl, ester, amide, carbamate, amine, ether, carbonate, thioether, thioester, or urea.

[0116] In some examples of Formula I, n is 1 to 7. In some examples of Formula I, n is 1 to 6. In some examples of Formula I, n is 1 to 5. In some examples of Formula I, n is 1 to 4. In some examples of Formula I, n is 1 to 3. In some examples of Formula I, n is 1.

[0117] In some examples of Formula I, m is 1 to 7. In some examples of Formula I, m is 1 to 6. In some examples of Formula I, m is 1 to 5. In some examples of Formula I, m is 1 to 4. In some examples of Formula I, m is 1 to 3. In some examples of Formula I, m is 1. In some examples of Formula I, R 5 is a substituted or unsubstituted C1-C5 alkyl. In some examples of Formula I, R 5 is methyl. In some examples of Formula I, R 5 is a substituted or unsubstituted C1-C5 alkyl alcohol. In some examples of Formula I, R 5 is an unsubstituted C1-C5 alkyl alcohol. In some examples of Formula I, R 5 is —CHOH. In some examples of Formula I, R 5 is OH. In some examples of Formula I, R 5 is hydrogen. In some examples of Formula I, R 5 -L 1 -SSSR a is.

[0118] In some examples of Formula I, R 4 is a substituted or unsubstituted C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula I, R 4 is substituted C8~C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula I, R 4 is a C8-C substituted with one or more substituents selected from the group consisting of amines, amides, esters, ethers, and carbonates. 18 In some examples of Formula I, R 4 is unsubstituted C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12In some examples of Formula I, R 4 is an unsubstituted branched chain C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula I, R 4 is an unsubstituted straight chain C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 alkyl).

[0119] In some examples of Formula I, p is 0 to 4. In some examples of Formula I, p is 0 to 3. In some examples of Formula I, p is 0 to 2. In some examples of Formula I, p is 0 to 1. In some examples of Formula I, p is 0. In some examples of Formula I, p is 1. In some examples of Formula I, p is 2 or more (e.g., 2, 3, 4, 5). In some examples of Formula I, when p is 2 or more, each R 5 In some examples of Formula I, when p is 2 or greater, at least one R 5 are different. In some examples of Formula I, when p is 2 or greater, each m is the same. In some examples of Formula I, when p is 2 or greater, at least one m is different.

[0120] In some examples of Formula I, R1 is OH. In some examples of Formula I, R1 is a substituted or unsubstituted C1-C5 alkyl. In some examples of Formula I, R1 is a substituted C1-C5 alkyl. In some examples of Formula I, R1 is an unsubstituted C1-C5 alkyl. In some examples of Formula I, R1 is a substituted or unsubstituted C1-C5 alkyl alcohol. In some examples of Formula I, R1 is a substituted C1-C5 alkyl alcohol. In some examples of Formula I, R1 is an unsubstituted C1-C5 alkyl alcohol. In some examples of Formula I, R1 is -L 1 -SSSR a is.

[0121] In some examples of Formula I, R2 is OH. In some examples of Formula I, R2 is a substituted or unsubstituted C1-C5 alkyl. In some examples of Formula I, R2 is a substituted C1-C5 alkyl. In some examples of Formula I, R2 is an unsubstituted C1-C5 alkyl. In some examples of Formula I, R2 is a substituted or unsubstituted C1-C5 alkyl alcohol. In some examples of Formula I, R2 is a substituted C1-C5 alkyl alcohol. In some examples of Formula I, R2 is an unsubstituted C1-C5 alkyl alcohol. In some examples of Formula I, R2 is -L 1 -SSSR a is.

[0122] In some examples of Formula I, R3 is OH. In some examples of Formula I, R3 is a substituted or unsubstituted C1-C5 alkyl. In some examples of Formula I, R3 is a substituted C1-C5 alkyl. In some examples of Formula I, R3 is an unsubstituted C1-C5 alkyl. In some examples of Formula I, R3 is a substituted or unsubstituted C1-C5 alkyl alcohol. In some examples of Formula I, R3 is a substituted C1-C5 alkyl alcohol. In some examples of Formula I, R3 is an unsubstituted C1-C5 alkyl alcohol. In some examples of Formula I, R3 is -L 1 -SSSR a is.

[0123] In some examples of Formula I, each R a If present, are independently substituted or unsubstituted C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula I, each R a If present, independently substitute C8 to C 18 In some examples of Formula I, each R a are, if present, independently unsubstituted C8-C 18 It is alkyl.

[0124] In some examples of Formula I, each occurrence of L 1is independently substituted or unsubstituted alkyl. In some examples of Formula I, L at each occurrence 1 is independently carbonyl. In some examples of Formula I, L at each occurrence is 1 is independently an ester. In some examples of Formula I, L at each occurrence is 1 is independently an amide. In some examples of Formula I, L at each occurrence is 1 is independently a carbamate ester. In some examples of Formula I, L at each occurrence 1 is independently an amine. In some examples of Formula I, L at each occurrence 1 is independently an ether. In some examples of Formula I, L at each occurrence is 1 is independently a carbonate ester. In some examples of Formula I, L at each occurrence 1 is independently a thioether. In some examples of Formula I, L at each occurrence is 1 is independently a thioester. In some examples of Formula I, L at each occurrence is 1 are independently urea. 1 In some examples of Formula I having groups, the compounds contain combinations of the above (eg, both a carbonate ester and a substituted or unsubstituted alkyl).

[0125] In some examples of Formula I, each L 1 is the formula -Z 1 C(O)OZ 2 -, wherein Z 1 and Z 2 are each independently substituted or unsubstituted C1 to C 10 In some examples of Formula I, Z is alkyl. 1 and Z 2 are each independently substituted or unsubstituted C1-C5 alkyl. In some examples of Formula I, Z 1 is —(C2H4)—. In some examples of Formula I, Z 2 is an unsubstituted C1-C7 alkyl. In some examples of Formula I, Z 2 teeth, [ka] is selected from.

[0126] In some examples, the compound is defined by Formula II or a pharmaceutically acceptable salt thereof: [ka] [In the formula, p is an integer from 0 to 5, n is an integer from 1 to 10, m, if present, is an integer from 1 to 10; R 1 and R 2 are independently OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R 4 are independently substituted or unsubstituted C8-C 18 is alkyl, Each R 5 -L, when present, independently represents hydrogen, OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R a are, if present, independently substituted or unsubstituted C8-C 18 is alkyl, L in each case 1 is independently a substituted or unsubstituted alkyl, carbonyl, ester, amide, carbamate, amine, ether, carbonate, thioether, thioester, or urea; Each R 6 are independently substituted or unsubstituted C1-C7 alkyl.

[0127] In some examples of Formula II, n is 1 to 7. In some examples of Formula I, n is 1 to 6. In some examples of Formula I, n is 1 to 5. In some examples of Formula II, n is 1 to 4. In some examples of Formula I, n is 1 to 3. In some examples of Formula II, n is 1.

[0128] In some examples of Formula II, m is 1 to 7. In some examples of Formula II, m is 1 to 6. In some examples of Formula II, m is 1 to 5. In some examples of Formula II, m is 1 to 4. In some examples of Formula II, m is 1 to 3. In some examples of Formula II, m is 1. In some examples of Formula II, R 5 is a substituted or unsubstituted C1-C5 alkyl. In some examples of Formula II, R 5 is methyl. In some examples of Formula II, R 5 is a substituted or unsubstituted C1-C5 alkyl alcohol. In some examples of Formula II, R 5 is an unsubstituted C1-C5 alkyl alcohol. In some examples of Formula II, R 5 is —CHOH. In some examples of Formula II, R 5 is OH. In some examples of Formula II, R 5 is hydrogen. In some examples of Formula II, R 5 -L 1 -SSSR a is.

[0129] In some examples of Formula II, each R 4 are independently substituted or unsubstituted C8 to C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula II, each R 4 are independently substituted C8 to C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula II, each R 4is a C8-C substituted with one or more substituents independently selected from the group consisting of amines, amides, esters, ethers, and carbonates; 18 In some examples of Formula II, each R 4 are independently unsubstituted C8 to C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula II, each R 4 are independently unsubstituted branched C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula II, each R 4 are independently unsubstituted linear C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula II, each R 4 In some examples of Formula II, at least one R 4 are different.

[0130] In some examples of Formula II, p is 0 to 4. In some examples of Formula II, p is 0 to 3. In some examples of Formula II, p is 0 to 2. In some examples of Formula II, p is 0 to 1. In some examples of Formula II, p is 0. In some examples of Formula II, p is 1. In some examples of Formula II, p is 2 or more (e.g., 2, 3, 4, 5). In some examples of Formula II, when p is 2 or more, each R 5 In some examples of Formula II, when p is 2 or greater, at least one R 5 are different. In some examples of Formula II, when p is 2 or greater, each m is the same. In some examples of Formula II, when p is 2 or greater, at least one m is different.

[0131] In some examples of Formula II, R1 is OH. In some examples of Formula II, R1 is a substituted or unsubstituted C1-C5 alkyl. In some examples of Formula II, R1 is a substituted C1-C5 alkyl. In some examples of Formula II, R1 is an unsubstituted C1-C5 alkyl. In some examples of Formula II, R1 is a substituted or unsubstituted C1-C5 alkyl alcohol. In some examples of Formula II, R1 is a substituted C1-C5 alkyl alcohol. In some examples of Formula II, R1 is an unsubstituted C1-C5 alkyl alcohol. In some examples of Formula II, R1 is -L 1 -SSSR a is.

[0132] In some examples of Formula II, R2 is OH. In some examples of Formula II, R2 is a substituted or unsubstituted C1-C5 alkyl. In some examples of Formula II, R2 is a substituted C1-C5 alkyl. In some examples of Formula II, R2 is an unsubstituted C1-C5 alkyl. In some examples of Formula II, R2 is a substituted or unsubstituted C1-C5 alkyl alcohol. In some examples of Formula II, R2 is a substituted C1-C5 alkyl alcohol. In some examples of Formula II, R2 is an unsubstituted C1-C5 alkyl alcohol. In some examples of Formula II, R2 is -L 1 -SSSR a is.

[0133] In some examples of Formula II, each R a If present, are independently substituted or unsubstituted C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula II, each R a If present, independently substitute C8 to C 18 In some examples of Formula II, each R a are, if present, independently unsubstituted C8-C 18 It is alkyl.

[0134] In some examples of Formula II, L in each instance 1 is independently substituted or unsubstituted alkyl. In some examples of Formula II, L at each occurrence 1 is independently carbonyl. In some examples of Formula II, L at each occurrence 1 is independently an ester. In some examples of Formula II, L at each occurrence is 1 is independently an amide. In some examples of Formula II, L at each occurrence is 1 is independently a carbamate ester. In some examples of Formula II, L at each occurrence 1 is independently an amine. In some examples of Formula II, L at each occurrence 1 is independently an ether. In some examples of Formula II, L at each occurrence 1 is independently a carbonate ester. In some examples of Formula II, L in each instance 1 is independently a thioether. In some examples of Formula II, L at each occurrence 1 is independently a thioester. In some examples of Formula II, L at each occurrence is 1 are independently urea. 1 In some examples of Formula II having groups, the compounds contain combinations of the above (eg, both a carbonate ester and a substituted or unsubstituted alkyl).

[0135] In some examples of Formula II, each L 1 is the formula -Z 1 C(O)OZ 2 -, wherein Z 1 and Z 2 are each independently substituted or unsubstituted C1 to C 10 In some examples of Formula II, Z is alkyl. 1 and Z 2 are each independently substituted or unsubstituted C1-C5 alkyl. In some examples of Formula II, Z 1 is —(C2H4)—. In some examples of Formula II, Z 2 is an unsubstituted C1-C7 alkyl. In some examples of Formula II, Z 2 teeth, [ka] is selected from.

[0136] In some examples of Formula II, each R 6 is independently an unsubstituted C1-C7 alkyl. In some examples of Formula II, each R 6 is independently a linear C1-C7 alkyl. In some examples of Formula II, each R 6 is independently a branched C1-C7 alkyl. In some examples of Formula II, each R 6 are each independently substituted or unsubstituted C1-C5 alkyl. In some examples of Formula II, each R 6 is —(C2H4)—. In some examples of Formula II, each R 6 is independently an unsubstituted C1-C7 alkyl. In some examples of Formula II, each R 6 is, independently, [ka] In some examples of Formula II, each R 6 In some examples of Formula II, at least one R 6 are different.

[0137] In some examples, the compound is defined by Formula III or a pharmaceutically acceptable salt thereof: [ka] [In the formula, p is an integer from 0 to 5, n is an integer from 1 to 10, m, if present, is an integer from 1 to 10; R 1 are independently OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R4 are independently substituted or unsubstituted C8-C 18 is alkyl, Each R 5 -L, when present, independently represents hydrogen, OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R a are, if present, independently substituted or unsubstituted C8-C 18 is alkyl, L 1 is, if present, a substituted or unsubstituted alkyl, carbonyl, ester, amide, carbamate, amine, ether, carbonate, thioether, thioester, or urea; Each R 6 are independently substituted or unsubstituted C1-C7 alkyl.

[0138] In some examples of Formula III, n is 1 to 7. In some examples of Formula III, n is 1 to 6. In some examples of Formula III, n is 1 to 5. In some examples of Formula III, n is 1 to 4. In some examples of Formula III, n is 1 to 3. In some examples of Formula III, n is 1.

[0139] In some examples of Formula III, m is 1 to 7. In some examples of Formula III, m is 1 to 6. In some examples of Formula III, m is 1 to 5. In some examples of Formula III, m is 1 to 4. In some examples of Formula III, m is 1 to 3. In some examples of Formula III, m is 1. In some examples of Formula III, R 5 is a substituted or unsubstituted C1-C5 alkyl. In some examples of Formula III, R 5 is methyl. In some examples of Formula III, R 5 is a substituted or unsubstituted C1-C5 alkyl alcohol. In some examples of Formula III, R 5is an unsubstituted C1-C5 alkyl alcohol. In some examples of Formula III, R 5 is —CHOH. In some examples of Formula III, R 5 is OH. In some examples of Formula III, R 5 is hydrogen. In some examples of Formula III, R 5 -L 1 -SSSR a is.

[0140] In some examples of Formula III, each R 4 are independently substituted or unsubstituted C8 to C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula III, each R 4 are independently substituted C8 to C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula III, each R 4 is a C8-C substituted with one or more substituents independently selected from the group consisting of amines, amides, esters, ethers, and carbonates; 18 In some examples of Formula III, each R 4 are independently unsubstituted C8 to C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula III, each R 4 are independently unsubstituted branched C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula III, each R 4 are independently unsubstituted linear C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula III, each R4 In some examples of Formula III, at least one R 4 are different.

[0141] In some examples of Formula III, p is 0 to 4. In some examples of Formula III, p is 0 to 3. In some examples of Formula III, p is 0 to 2. In some examples of Formula III, p is 0 to 1. In some examples of Formula III, p is 0. In some examples of Formula III, p is 1. In some examples of Formula III, p is 2 or more (e.g., 2, 3, 4, 5). In some examples of Formula III, when p is 2 or more, each R 5 In some examples of Formula III, when p is 2 or greater, at least one R 5 are different. In some examples of Formula III, when p is 2 or greater, each m is the same. In some examples of Formula III, when p is 2 or greater, at least one m is different.

[0142] In some examples of Formula III, R1 is OH. In some examples of Formula III, R1 is a substituted or unsubstituted C1-C5 alkyl. In some examples of Formula III, R1 is a substituted C1-C5 alkyl. In some examples of Formula III, R1 is an unsubstituted C1-C5 alkyl. In some examples of Formula III, R1 is a substituted or unsubstituted C1-C5 alkyl alcohol. In some examples of Formula III, R1 is a substituted C1-C5 alkyl alcohol. In some examples of Formula III, R1 is an unsubstituted C1-C5 alkyl alcohol. In some examples of Formula III, R1 is -L 1 -SSSR a is.

[0143] In some examples of Formula III, each R a If present, are independently substituted or unsubstituted C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula III, each Ra If present, independently substitute C8 to C 18 In some examples of Formula III, each R a If present, unsubstituted C8-C 18 It is alkyl.

[0144] In some examples of Formula III, L 1 is a substituted or unsubstituted alkyl. In some examples of Formula III, L 1 is carbonyl. In some examples of Formula III, L 1 is an ester. In some examples of Formula III, L 1 is an amide. In some examples of Formula III, L 1 is a carbamate ester. In some examples of Formula III, L 1 is an amine. In some examples of Formula III, L 1 is an ether. In some examples of Formula III, L 1 is a carbonate ester. In some examples of Formula III, L 1 is a thioether. In some examples of Formula III, L 1 is a thioester. In some examples of Formula III, L 1 is urea.

[0145] In some examples of Formula III, L 1 is the formula -Z 1 C(O)OZ 2 -, wherein Z 1 and Z 2 are each independently substituted or unsubstituted C1 to C 10 In some examples of Formula III, Z is alkyl. 1 and Z 2 are each independently substituted or unsubstituted C1-C5 alkyl. In some examples of Formula III, Z 1 is —(C2H4)—. In some examples of Formula III, Z 2 is an unsubstituted C1-C7 alkyl. In some examples of Formula III, Z 2 teeth, [ka] is selected from.

[0146] In some examples of Formula III, each R 6 is independently an unsubstituted C1-C7 alkyl. In some examples of Formula III, each R 6 is independently a linear C1-C7 alkyl. In some examples of Formula III, each R 6 is independently a branched C1-C7 alkyl. In some examples of Formula III, each R 6 are each independently substituted or unsubstituted C1-C5 alkyl. In some examples of Formula III, each R 6 is —(C2H4)—. In some examples of Formula III, each R 6 is independently an unsubstituted C1-C7 alkyl. In some examples of Formula III, each R 6 is, independently, [ka] In some examples of Formula III, each R 6 In some examples of Formula III, at least one R 6 are different.

[0147] In some examples, the compound is defined by Formula IV or a pharmaceutically acceptable salt thereof: [ka] [In the formula, p is an integer from 0 to 5, n is an integer from 1 to 10, m, if present, is an integer from 1 to 10; Each R 4 are independently substituted or unsubstituted C8-C 18 is alkyl, Each R 5-L, when present, independently represents hydrogen, OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R a are, if present, independently substituted or unsubstituted C8-C 18 is alkyl, Each R 6 are independently substituted or unsubstituted C1-C7 alkyl.

[0148] In some examples of Formula IV, n is 1 to 7. In some examples of Formula IV, n is 1 to 6. In some examples of Formula IV, n is 1 to 5. In some examples of Formula IV, n is 1 to 4. In some examples of Formula IV, n is 1 to 3. In some examples of Formula IV, n is 1.

[0149] In some examples of Formula IV, m is 1 to 7. In some examples of Formula IV, m is 1 to 6. In some examples of Formula IV, m is 1 to 5. In some examples of Formula IV, m is 1 to 4. In some examples of Formula IV, m is 1 to 3. In some examples of Formula IV, m is 1. In some examples of Formula IV, R 5 is a substituted or unsubstituted C1-C5 alkyl. In some examples of Formula IV, R 5 is methyl. In some examples of Formula IV, R 5 is a substituted or unsubstituted C1-C5 alkyl alcohol. In some examples of Formula IV, R 5 is an unsubstituted C1-C5 alkyl alcohol. In some examples of Formula IV, R 5 is —CHOH. In some examples of Formula IV, R 5 is OH. In some examples of Formula IV, R 5 is hydrogen. In some examples of Formula IV, R 5 -L 1 -SSSR a is.

[0150] In some examples of Formula IV, each R 4 are independently substituted or unsubstituted C8 to C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula IV, each R 4 are independently substituted C8 to C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula IV, each R 4 is a C8-C substituted with one or more substituents independently selected from the group consisting of amines, amides, esters, ethers, and carbonates; 18 In some examples of Formula IV, each R 4 are independently unsubstituted C8 to C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula IV, each R 4 are independently unsubstituted branched C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula IV, each R 4 are independently unsubstituted linear C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula IV, each R 4 In some examples of Formula IV, at least one R 4 are different.

[0151] In some examples of Formula IV, p is 0 to 4. In some examples of Formula IV, p is 0 to 3. In some examples of Formula IV, p is 0 to 2. In some examples of Formula IV, p is 0 to 1. In some examples of Formula IV, p is 0. In some examples of Formula IV, p is 1. In some examples of Formula IV, p is 2 or greater (e.g., 2, 3, 4, 5). In some examples of Formula IV, when p is 2 or greater, each R 5 In some examples of formula IV, when p is 2 or greater, at least one R 5 are different. In some examples of Formula IV, when p is 2 or greater, each m is the same. In some examples of Formula IV, when p is 2 or greater, at least one m is different.

[0152] In some examples of Formula IV, each R a If present, are independently substituted or unsubstituted C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula IV, each R a If present, independently substitute C8 to C 18 In some examples of Formula IV, each R a are, if present, independently unsubstituted C8-C 18 It is alkyl.

[0153] In some examples of Formula IV, each R 6 is independently an unsubstituted C1-C7 alkyl. In some examples of Formula IV, each R 6 is independently a linear C1-C7 alkyl. In some examples of Formula IV, each R 6 is independently a branched C1-C7 alkyl. In some examples of Formula IV, each R 6 are each independently substituted or unsubstituted C1-C5 alkyl. In some examples of Formula IV, each R 6 is —(C2H4)—. In some examples of Formula IV, each R 6is independently an unsubstituted C1-C7 alkyl. In some examples of Formula IV, each R 6 is, independently, [ka] In some examples of Formula IV, each R 6 In some examples of Formula IV, at least one R 6 are different.

[0154] In some examples, the compound is defined by Formula V or a pharmaceutically acceptable salt thereof: [ka] [In the formula, p is an integer from 0 to 5, n is an integer from 1 to 10, each m, if present, is independently an integer from 1 to 10; R 1 and R 3 are independently OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R 4 are independently substituted or unsubstituted C8-C 18 is alkyl, Each R 5 -L, when present, independently represents hydrogen, OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R a are, if present, independently substituted or unsubstituted C8-C 18 is alkyl, L in each case 1is independently a substituted or unsubstituted alkyl, carbonyl, ester, amide, carbamate, amine, ether, carbonate, thioether, thioester, or urea; Each R 6 are independently substituted or unsubstituted C1-C7 alkyl.

[0155] In some examples of Formula V, n is 1 to 7. In some examples of Formula V, n is 1 to 6. In some examples of Formula V, n is 1 to 5. In some examples of Formula V, n is 1 to 4. In some examples of Formula V, n is 1 to 3. In some examples of Formula V, n is 1.

[0156] In some examples of Formula V, m is 1 to 7. In some examples of Formula V, m is 1 to 6. In some examples of Formula V, m is 1 to 5. In some examples of Formula V, m is 1 to 4. In some examples of Formula V, m is 1 to 3. In some examples of Formula V, m is 1. In some examples of Formula V, R 5 is a substituted or unsubstituted C1-C5 alkyl. In some examples of Formula V, R 5 is methyl. In some examples of Formula V, R 5 is a substituted or unsubstituted C1-C5 alkyl alcohol. In some examples of Formula V, R 5 is an unsubstituted C1-C5 alkyl alcohol. In some examples of Formula V, R 5 is —CHOH. In some examples of Formula V, R 5 is OH. In some examples of Formula V, R 5 is hydrogen. In some examples of Formula V, R 5 -L 1 -SSSR a is.

[0157] In some examples of Formula V, R1 is OH. In some examples of Formula V, R1 is a substituted or unsubstituted C1-C5 alkyl. In some examples of Formula V, R1 is a substituted C1-C5 alkyl. In some examples of Formula V, R1 is an unsubstituted C1-C5 alkyl. In some examples of Formula V, R1 is a substituted or unsubstituted C1-C5 alkyl alcohol. In some examples of Formula V, R1 is a substituted C1-C5 alkyl alcohol. In some examples of Formula V, R1 is an unsubstituted C1-C5 alkyl alcohol. In some examples of Formula V, R1 is -L 1 -SSSR a is.

[0158] In some examples of Formula V, R3 is OH. In some examples of Formula V, R3 is a substituted or unsubstituted C1-C5 alkyl. In some examples of Formula V, R3 is a substituted C1-C5 alkyl. In some examples of Formula V, R3 is an unsubstituted C1-C5 alkyl. In some examples of Formula V, R3 is a substituted or unsubstituted C1-C5 alkyl alcohol. In some examples of Formula V, R3 is a substituted C1-C5 alkyl alcohol. In some examples of Formula V, R3 is an unsubstituted C1-C5 alkyl alcohol. In some examples of Formula V, R3 is -L 1 -SSSR a is.

[0159] In some examples of Formula V, each R 4 are independently substituted or unsubstituted C8 to C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula V, each R 4 are independently substituted C8 to C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula V, each R 4 is a C8-C substituted with one or more substituents independently selected from the group consisting of amines, amides, esters, ethers, and carbonates;18 In some examples of Formula V, each R 4 are independently unsubstituted C8 to C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula V, each R 4 are independently unsubstituted branched C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula V, each R 4 are independently unsubstituted linear C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12 In some examples of Formula V, each R 4 In some examples of Formula V, at least one R 4 are different.

[0160] In some examples of Formula V, p is 0 to 4. In some examples of Formula V, p is 0 to 3. In some examples of Formula V, p is 0 to 2. In some examples of Formula V, p is 0 to 1. In some examples of Formula V, p is 0. In some examples of Formula V, p is 1. In some examples of Formula V, p is 2 or more (e.g., 2, 3, 4, 5). In some examples of Formula V, when p is 2 or more, each R 5 In some examples of Formula V, when p is 2 or greater, at least one R 5 are different. In some examples of Formula V, when p is 2 or greater, each m is the same. In some examples of Formula V, when p is 2 or greater, at least one m is different.

[0161] In some examples of Formula V, each R a If present, are independently substituted or unsubstituted C8-C 18 Alkyl (e.g., C 10 ~C 14 Alkyl or C 12In some examples of Formula V, each R a If present, independently substitute C8 to C 18 In some examples of Formula V, each R a are, if present, independently unsubstituted C8-C 18 It is alkyl.

[0162] In some examples of formula V, L in each case 1 is independently substituted or unsubstituted alkyl. In some examples of Formula V, L at each occurrence 1 is independently carbonyl. In some examples of Formula V, L at each occurrence 1 is independently an ester. In some examples of Formula V, L at each occurrence 1 In some examples of Formula V, L at each occurrence is an amide. 1 is independently a carbamate ester. In some examples of Formula V, L in each instance 1 is independently an amine. In some examples of Formula V, L at each occurrence 1 is independently an ether. In some examples of Formula V, L at each occurrence 1 is independently a carbonate ester. In some examples of Formula V, L in each instance 1 is independently a thioether. In some examples of Formula V, L at each occurrence 1 is independently a thioester. In some examples of Formula V, L at each occurrence 1 are independently urea. 1 In some examples of Formula V having groups, the compounds contain combinations of the above (eg, both a carbonate ester and a substituted or unsubstituted alkyl).

[0163] In some examples of Formula V, each L 1 is the formula -Z 1 C(O)OZ 2 -, wherein Z 1 and Z 2 are each independently substituted or unsubstituted C1 to C 10 In some examples of Formula V, Z is alkyl. 1 and Z 2are each independently substituted or unsubstituted C1-C5 alkyl. In some examples of Formula V, Z 1 is —(C2H4)—. In some examples of Formula V, R 2 is a substituted C1-C7 alkyl. In some examples of Formula V, Z 2 teeth, [ka] is selected from.

[0164] In some examples of Formula V, each R 6 is independently an unsubstituted C1-C7 alkyl. In some examples of Formula V, each R 6 is independently a linear C1-C7 alkyl. In some examples of Formula V, each R 6 is independently a branched C1-C7 alkyl. In some examples of Formula V, each R 6 are each independently substituted or unsubstituted C1-C5 alkyl. In some examples of Formula V, each R 6 is —(C2H4)—. In some examples of Formula V, each R 6 is independently an unsubstituted C1-C7 alkyl. In some examples of Formula V, each R 6 is, independently, [ka] In some examples of Formula V, each R 6 In some examples of Formula V, at least one R 6 are different.

[0165] In some embodiments, the compound is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0166] In some embodiments, the compound is [ka] is selected from the group consisting of:

[0167] In some embodiments, the compound is [ka] is selected from the group consisting of:

[0168] In some examples, the compound includes: [ka]

[0169] In some examples, the compound includes: [ka]

[0170] In some examples, the compound includes: [ka]

[0171] nanoparticles Also disclosed herein are lipid nanoparticles (eg, one or more lipid particles) comprising any of the compounds disclosed herein.

[0172] In one aspect, the present disclosure provides a compound of any one of formulas I-V, Nanoparticles are provided that include a non-cationic lipid, a polyethylene glycol lipid, and a sterol.

[0173] In one aspect, the present disclosure provides nanoparticles comprising a compound of formula I, a non-cationic lipid, a polyethylene glycol-lipid, and a sterol.

[0174] In one aspect, the present disclosure provides nanoparticles comprising a compound of formula II, a non-cationic lipid, a polyethylene glycol-lipid, and a sterol.

[0175] In one aspect, the present disclosure provides nanoparticles comprising a compound of formula III, a non-cationic lipid, a polyethylene glycol-lipid, and a sterol.

[0176] In one aspect, the present disclosure provides nanoparticles comprising a compound of formula IV, a non-cationic lipid, a polyethylene glycol-lipid, and a sterol.

[0177] In one aspect, the present disclosure provides nanoparticles comprising a compound of formula V, a non-cationic lipid, a polyethylene glycol-lipid, and a sterol.

[0178] In one aspect, the present disclosure provides nanoparticles comprising a compound of the formula:

[0179] Various compounds of Formulas I-V are described in the Compounds section above. In some embodiments, the nanoparticles comprise a compound of Formulas I-V in a molar ratio of about 10% to about 40%. In some embodiments, the nanoparticles comprise a compound of Formulas I-V in a molar ratio of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%. In some embodiments, the nanoparticles comprise a compound of Formulas I-V in a molar ratio of about 20%. In some examples, the molar ratio of the compound is 5% to 60%.

[0180] In some embodiments, the nanoparticles comprise a non-cationic lipid. In some embodiments, the non-cationic lipid interacts with the lipid as a helper lipid. In some embodiments, the non-cationic lipid includes 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), DPPC (1,2-dipalmitoyl-sn- Non-cationic lipids may include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-5 / 7-glycero-3-phospho-(1'-rac-glycerol) (DOPG), or combinations thereof. In one embodiment, the non-cationic lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the non-cationic lipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). In one embodiment, the non-cationic lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the non-cationic lipid is 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE). Although several non-cationic lipids are described herein, additional non-cationic lipids can be used in combination with the compounds disclosed herein.

[0181] In some examples, the molar ratio of non-cationic lipid is 20% to 50%. In some embodiments, the nanoparticles comprise non-cationic lipid at a molar ratio of about 10% to about 40%. In some embodiments, the nanoparticles comprise non-cationic lipid at a molar ratio of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%. In one embodiment, the nanoparticles comprise non-cationic lipid at a molar ratio of about 30%.

[0182] In some embodiments, the nanoparticles contain polyethylene glycol lipids (PEG lipids). The PEG lipids are incorporated to form a hydrophilic outer layer and stabilize the particles. Non-limiting examples of polyethylene glycol lipids include PEG-modified lipids such as PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Representative polyethylene glycol lipids include DMG-PEG, DLPE-PEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG. In one embodiment, the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG). In one embodiment, the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol-2000 (DMG-PEG2000). DMG-PEGXXXX means 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol-XXXX, where XXXX represents the molecular weight of the polyethylene glycol moiety, for example, DMG-PEG2000 or DMG-PEG5000.

[0183] In some embodiments, the nanoparticles comprise polyethylene glycol-lipid at a molar ratio of about 0% to about 5%. In some examples, the PEG-lipid molar ratio is 0.1% to 2%. In some embodiments, the nanoparticles comprise polyethylene glycol-lipid at a molar ratio of about 0%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 1.5%, about 2%, about 3%, about 4%, or about 5%. In one embodiment, the nanoparticles comprise polyethylene glycol-lipid at a molar ratio of about 0.75%.

[0184] In some embodiments, nanoparticles comprise sterol.Sterol is well known to those skilled in the art and generally refers to a compound that has a perhydrocyclopentanophenanthrene ring system and has one or more OH substituents.Examples of sterol include, but are not limited to, cholesterol, campesterol, ergosterol, sitosterol, etc.

[0185] In some embodiments, the sterol is selected from cholesterol-based lipids. In some embodiments, the one or more cholesterol-based lipids are selected from cholesterol, PEGylated cholesterol, DC-Choi (N,N-dimethyl-N-ethylcarboxamido cholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, or a combination thereof.

[0186] Sterols can be used to modulate the permeability and fluidity of particles based on their function in cell membranes, hi one embodiment, the sterol is cholesterol.

[0187] In some embodiments, the nanoparticles comprise a sterol in a molar ratio of about 25% to about 50%. In some examples, the molar ratio of the sterol is 20% to 50%. In some embodiments, the nanoparticles comprise a sterol in a molar ratio of about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In one embodiment, the nanoparticles comprise a sterol in a molar ratio of about 40%.

[0188] In one embodiment, the present disclosure provides a method for the preparation of a compound of Formulas I-V in a composition comprising 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG 2000 and cholesterol.

[0189] In one embodiment, the present disclosure provides a method for preparing a medicament for the preparation of a compound of Formulas I-V, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG 2000 and cholesterol.

[0190] In one embodiment, the present disclosure provides a method for the preparation of a compound of Formulas I-V in a composition comprising 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG 2000 and cholesterol.

[0191] In some examples, the molar ratio of the compound is 20% to 30%, the molar ratio of the non-cationic lipid is 35% to 45%, the molar ratio of the sterol is 35% to 45%, and the molar ratio of the polyethylene glycol-lipid is 0.1% to 1%.

[0192] In one embodiment, the nanoparticles further comprise a drug. In one embodiment, the nanoparticles further comprise a therapeutic agent. In one embodiment, the nanoparticles further comprise a diagnostic agent.

[0193] The agent delivered into cells may be a polynucleotide. Polynucleotides or oligonucleotides that can be introduced according to the methods herein include all types of DNA, cDNA, and RNA sequences. For example, the polynucleotide may be double-stranded DNA, single-stranded DNA, complex DNA, encapsulated DNA, naked RNA, encapsulated RNA, messenger RNA (mRNA), tRNA, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), antisense RNA (asRNA), and combinations thereof. The polynucleotide may also be a DNA construct, such as an expression vector, encoding a desired gene product (e.g., a gene product homologous or heterologous to the target into which it is introduced). In one embodiment, the agent is mRNA.

[0194] Nanoparticles can be of any shape (e.g., spherical, rod-like, square, elliptical, triangular, polygonal, etc.). In some examples, nanoparticles can have a regular shape, an irregular shape, an isotropic shape, an anisotropic shape, or a combination thereof. In some examples, nanoparticles are substantially spherical in shape.

[0195] Lipid particles can have an average particle size. "Average particle size" and "mean particle size" are used interchangeably herein and generally refer to the statistical average particle size of particles in a particle population. For example, the average particle size of a plurality of particles having a substantially spherical shape can include the average diameter of a plurality of particles. For particles having a substantially spherical shape, the diameter of the particle can refer to, for example, the hydrodynamic diameter. As used herein, the hydrodynamic diameter of a particle refers to the maximum linear distance between two points on the surface of the particle. The average particle size can be measured using methods well known in the art, such as by scanning electron microscopy, transmission electron microscopy, and / or dynamic light scattering.

[0196] The lipid particles can have an average particle size of, for example, 30 nanometers (nm) or more (e.g., 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 110 nm or more, 120 nm or more, 130 nm or more, 140 nm or more, 150 nm or more, 160 nm or more, 170 nm or more, 180 nm or more, 190 nm or more, 200 nm or more, 225 nm or more, 250 nm or more, 275 nm or more, 300 nm or more, 325 nm or more, 350 nm or more, 375 nm or more, 400 nm or more, 425 nm or more, 450 nm or more, 475 nm or more, 500 nm or more, 550 nm or more, 600 nm or more, 650 nm or more, 700 nm or more, or 750 nm or more). In some examples, the lipid particles can have an average particle size of 800 nm or less (e.g., 750 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 475 nm or less, 450 nm or less, 425 nm or less, 400 nm or less, 375 nm or less, 350 nm or less, 325 nm or less, 300 nm or less, 275 nm or less, 250 nm or less, 225 nm or less, 200 nm or less, 190 nm or less, 180 nm or less, 170 nm or less, 160 nm or less, 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, 110 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, or 40 nm or less). The average particle size of the lipid particles can range from any of the minimum values ​​above to any of the maximum values ​​above. For example, the lipid particles can have an average particle size of 30 nm to 800 nm (e.g., 30 nm to 425 nm, 425 nm to 800 nm, 30 nm to 200 nm, 200 nm to 400 nm, 400 nm to 600 nm, 600 nm to 800 nm, 50 nm to 800 nm, 30 nm to 750 nm, or 50 nm to 750 nm).

[0197] Regarding particle size distribution characteristics, the parameter used to define the size range of lipid particles is called the "polydispersity index" (PDI). The term "polydispersity" (or "dispersity" as recommended by IUPAC) is used to describe the degree of heterogeneity in the particle size distribution. PDI essentially represents the distribution of particle size populations within a particular sample. The PDI value ranges from 0.0 (for a completely homogeneous sample) to 1.0 (for a highly polydisperse sample with multiple particle size populations).

[0198] In some examples, the lipid particles have a molecular weight of 0.5 or less (e.g., 0.49 or less, 0.48 or less, 0.47 or less, 0.46 or less, 0.45 or less, 0.44 or less, 0.43 or less, 0.42 or less, 0.41 or less, 0.40 or less, 0.39 or less, 0.38 or less, 0.37 or less, 0.36 or less, 0.35 or less, 0.34 or less, 0.33 or less, 0.32 or less, 0.31 or less, 0.30 or less, 0.29 or less, 0.28 or less, 0.27 or less, 0.26 ... 0.25 or less, 0.24 or less, 0.23 or less, 0.22 or less, 0.21 or less, 0.20 or less, 0.19 or less, 0.18 or less, 0.17 or less, 0.16 or less, 0.15 or less, 0.14 or less, 0.13 or less, 0.12 or less, 0.11 or less, 0.10 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less).

[0199] In some cases, lipid particles may be substantially monodisperse. As used herein, "monodisperse" and "uniform particle size distribution" generally refer to a population of particles in which all particles have the same or approximately the same particle size. As used herein, monodisperse distribution refers to a particle distribution in which 80% of the distribution (e.g., 85% of the distribution, 90% of the distribution, or 95% of the distribution) is within 25% of the median particle size (e.g., within 20% of the median particle size, within 15% of the median particle size, within 10% of the median particle size, or within 5% of the median particle size).

[0200] composition As described herein, compositions containing an active compound and certain excipients can be useful in various medical and non-medical applications. For example, pharmaceutical compositions containing an active compound and excipients can be useful for delivering an effective amount of a drug to a subject in need thereof. Functional food compositions containing an active compound and excipients can be useful for delivering an effective amount of a functional food, such as a dietary supplement, to a subject in need thereof. Cosmetic compositions containing an active compound and excipients can be formulated as creams, ointments, balms, pastes, films, or liquids, and can be useful for makeup, hair products, and materials useful for personal hygiene. Compositions containing an active compound and excipients can be useful in non-medical applications, such as emulsions or emulsifiers, and can be useful, for example, as food ingredients, for fire fighting, surface disinfection, oil cleaning, etc.

[0201] In certain embodiments, the composition further comprises an agent described herein. For example, in certain embodiments, the agent is a small molecule, an organometallic compound, a nucleic acid, a protein, a peptide, a polynucleotide, a metal, a targeted agent, an isotopically labeled compound, a drug, a vaccine, an immunological agent, or an agent useful in bioprocessing. In certain embodiments, the agent is a polynucleotide. In certain embodiments, the polynucleotide is DNA or RNA. In certain embodiments, the RNA is RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA. In certain embodiments, the polynucleotide and the one or more active compounds are not covalently linked. In some examples, the weight fraction of the agent is 5% to 20%.

[0202] In one aspect, the disclosure provides a composition comprising a compound of Formulas IV and a drug.

[0203] In one aspect, the disclosure provides a composition comprising nanoparticles comprising a compound of Formulas IV and a drug.

[0204] In another embodiment, the present specification provides a composition comprising nanoparticles comprising a compound of Formulas I-V and a drug, wherein the drug comprises mRNA encoding at least one antigenic polypeptide or an immunogenic fragment thereof capable of inducing an immune response against the antigenic polypeptide.

[0205] In some embodiments, mRNA encoding at least one antigenic polypeptide or immunogenic fragment thereof capable of inducing an immune response against the antigenic polypeptide is encapsulated in the nanoparticles.

[0206] In some aspects, the present specification discloses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and nanoparticles comprising mRNA of at least one antigenic polypeptide or an immunogenic fragment thereof capable of inducing an immune response against the antigenic polypeptide.

[0207] In some embodiments, the present specification discloses a hydrogel matrix encapsulating the lipid nanoparticles described herein and a drug. The terms "hydrogel" and "hydrogel matrix" are used interchangeably and generally refer to cross-linked, water-insoluble, and water-containing materials. In the context of drug delivery systems, hydrogels can include biocompatible and non-toxic materials.

[0208] Drugs The agents delivered by the compounds, compositions, and systems described herein can be therapeutic, diagnostic, or prophylactic agents. Any compound to be administered to a subject can be delivered using the particles or nanoparticles described herein. The agent can be an organic molecule (e.g., a therapeutic agent, a drug), an inorganic molecule, a nucleic acid, a protein, an amino acid, a peptide, a polypeptide, a polynucleotide, a targeting agent, an isotopically labeled organic or inorganic molecule, a vaccine, an immunological agent, or the like.

[0209] In certain embodiments, the pharmaceutical agent is a pharmaceutically active organic molecule, e.g., a drug, such as an antibiotic, antiviral, anesthetic, steroid, anti-inflammatory, antitumor, anticancer agent, antigen, vaccine, antibody, decongestant, antihypertensive, sedative, contraceptive, progestin, anticholinergic, analgesic, antidepressant, antipsychotic, beta-adrenergic blocker, diuretic, cardiovascular activator, vasoactive agent, nonsteroidal anti-inflammatory agent, nutritional agent, etc.

[0210] In certain embodiments of the present disclosure, the agent to be delivered may be a mixture of agents.

[0211] Diagnostic agents include gases, metals, commercially available imaging agents used in positron emission tomography (PET), computer-assisted tomography (CAT), single-photon emission computed tomography, X-ray, fluoroscopy, and magnetic resonance imaging (MRI), and contrast agents. Examples of materials suitable for use as contrast agents in MRI include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium. Examples of materials useful for CAT and X-ray imaging include iodine-based materials.

[0212] Therapeutic and prophylactic agents include, but are not limited to, antibiotics, dietary supplements, and vaccines. Vaccines can include isolated proteins or peptides, inactivated organisms and viruses, killed organisms and viruses, genetically modified organisms or viruses, cell extracts, and at least one antigenic polypeptide or immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response against the antigenic polypeptide). Therapeutic and prophylactic agents may be combined with interleukins, interferons, cytokines, and adjuvants, such as cholera toxin, alum, and Freund's adjuvant. Prophylactic drugs include Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans, Borrelia burgdorferi, CamphylobacterAntigens of bacterial organisms such as Bacillus jejuni, and antigens of viruses such as smallpox virus, influenza A and B viruses, respiratory syncytial virus, parainfluenza virus, measles virus, HIV virus, varicella-zoster virus, herpes simplex virus types 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps virus, rabies virus, rubella virus, coxsackievirus, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, hepatitis A, B, C, D, and E viruses, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial These include antigens of fungal, protozoan, and parasitic organisms such as T. trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, etc. These antigens may be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof.

[0213] In some examples, the agent comprises RNA. In some examples, the agent comprises mRNA. In some examples, the agent comprises a polynucleotide encoding a macrophage polarization factor, such as interleukin-4 (IL-4) or interleukin-13 (IL-13). In some examples, the agent comprises a polynucleotide encoding TNF-α, IL-1β, IL-6, IL-12, IL-23, CXCL1, CXCL3, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, CXCL13, CXCL16, CXCR3, CCL2, CCL3, CCL4, CCL5, CCL8, CCL11, CCL15, CCL19, CCL20, IL-10, TGF-β, IL-1RA, IL-4, IL-13, CCL1, CCL2, CCL5, CXCL10, CCL13, CCL14, CCL17, CCL18, CCL22, CCL23, CCL24, CCL26, CXCL16, CCR2, CCR3, CCR4, or nitric oxide. In some examples, the agent comprises a polynucleotide encoding interleukin-4. In some examples, the agent is encapsulated in nanoparticles. In some embodiments, the agent is a ribonucleic acid (RNA) (e.g., mRNA) polynucleotide having an open reading frame encoding at least one (e.g., at least 2, 3, 4, or 5) antigenic polypeptide or immunogenic fragment thereof (e.g., immunogenic fragment capable of inducing an immune response against the antigenic polypeptide). The above sequences are well known in the art and can be found, for example, at ncbi.nlm.nih.gov.

[0214] In some embodiments, the nucleic acids disclosed herein comprise at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide comprises a chemically modified nucleobase, a chemically modified ribose, a chemically modified phosphodiester bond, or a combination thereof.

[0215] In one embodiment, the at least one chemically modified nucleotide is a chemically modified nucleobase.

[0216] In one embodiment, the chemically modified nucleobase is 5-formylcytidine (5fC), 5-methylcytidine (5meC), 5-methoxycytidine (5moC), 5-hydroxycytidine (5hoC), 5-hydroxymethylcytidine (5hmC), 5-formyluridine (5fU), 5-methyluridine (5-meU), 5-methoxyuridine (5moU), 5-carboxymethylesteruridine (5camU), pseudouridine (Ψ), N 1 -Methylpseudouridine (me 1 Ψ), N 6 -Methyladenosine (me 6 A), or thienoguanosine ( th G).

[0217] In some embodiments, the chemically modified nucleobase is 5-methoxyuridine (5moU). In some embodiments, the chemically modified nucleobase is pseudouridine (Ψ). In some embodiments, the chemically modified nucleobase is N 1 -Pseudouridine (me 1 Ψ).

[0218] The structures of these modified nucleobases are shown below: [ka] In one embodiment, at least one chemically modified nucleotide is a chemically modified ribose.

[0219] In one embodiment, the chemically modified ribose is selected from 2'-O-methyl (2'-O-Me), 2'-fluoro (2'-F), 2'-deoxy-2'-fluoro-beta-D-arabino-nucleic acid (2'F-ANA), 4'-S, 4'-SFANA, 2'-azido, UNA, 2'-O-methoxy-ethyl (2'-O-ME), 2'-O-allyl, 2'-O-ethylamine, 2'-O-cyanoethyl, locked nucleic acid (LAN), methylene-cLAN, N-MeO-amino BNA, or N-MeO-aminooxy BNA. In one embodiment, the chemically modified ribose is 2'-O-methyl (2'-O-Me). In one embodiment, the chemically modified ribose is 2'-fluoro (2'-F).

[0220] The structures of these modified riboses are shown below: [ka] In one embodiment, at least one chemically modified nucleotide is a chemically modified phosphodiester bond.

[0221] In one embodiment, the chemically modified phosphodiester bond is selected from phosphorothioate (PS), boranophosphate, phosphodithioate (PS2), 3',5'-amide, N3'-phosphoramidate (NP), phosphodiester (PO), or 2',5'-phosphodiester (2',5'-PO). In one embodiment, the chemically modified phosphodiester bond is phosphorothioate.

[0222] The structures of these modified phosphodiester bonds are shown below: [ka]

[0223] Manufacturing method Also provided herein are methods for producing any of the compounds or compositions disclosed herein. Also disclosed herein are methods for producing any of the lipid particles disclosed herein. Also provided herein are methods for producing any of the pharmaceutical compositions disclosed herein.

[0224] The compounds described herein can be prepared by a variety of methods well known to those skilled in the art of organic synthesis, or by variations thereof that will be recognized by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art.

[0225] Variations of the compounds described herein include the addition, deletion, or movement of various components described for each compound. Similarly, if one or more chiral centers are present in a molecule, the chirality of the molecule can be altered. Furthermore, the synthesis of a compound can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups, can be determined by those skilled in the art. Protecting group chemistry is described, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0226] Starting materials and reagents used in preparing the disclosed compounds and compositions may be purchased from Katchem (Prague, Czech Republic), Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott These reagents are available from manufacturers such as Fieser and R. Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc.These compounds are prepared by methods known to those skilled in the art, following procedures described in references such as (Berkeley, 1989). Other materials, such as pharmaceutical excipients, disclosed herein may be obtained from commercial sources.

[0227] The reactions to produce the compounds described herein can be carried out in a solvent that can be selected by one skilled in the art of organic synthesis. The solvent can be substantially non-reactive to the starting materials (reactants), intermediates, or products under the conditions (i.e., temperature and pressure) at which the reaction is carried out. The reaction can be carried out in one solvent or a mixture of multiple solvents. The formation of the product or intermediate can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13C) infrared spectroscopy, spectrophotometry (e.g., ultraviolet-visible), or mass spectrometry, or chromatography, e.g., high performance liquid chromatography (HPLC) or thin layer chromatography.

[0228] How to use Also provided herein are methods of using any of the compounds or compositions disclosed herein.

[0229] In one aspect, the present description provides a method for delivering an agent (e.g., a polynucleotide) into a cell, the method comprising: The method includes introducing into a cell a composition comprising nanoparticles, the nanoparticles comprising a compound of any one of Formulas I-V, a non-cationic lipid, a polyethylene glycol-lipid, a sterol, and a drug.

[0230] In one aspect, the present description provides a method for delivering an agent into a cell, the method comprising: Intracellularly, nanoparticles comprising a compound of formula I, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, p is an integer from 0 to 5, n is an integer from 1 to 10, each m, if present, is independently an integer from 1 to 10; R 1 , R 2 , and R 3 are independently OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and R 4 is a substituted or unsubstituted C8-C 18 is alkyl, Each R 5 -L, when present, independently represents hydrogen, OH, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkyl alcohol, or -L 1 -SSSR a and Each R a are, if present, independently substituted or unsubstituted C8-C 18 is alkyl, L in each case 1 are independently substituted or unsubstituted alkyl, carbonyl, ester, amide, carbamate, amine, ether, carbonate, thioether, thioester, or urea; Polyethylene glycol-lipids, Sterols and and a drug, and introducing into a cell a composition comprising nanoparticles comprising the drug.

[0231] In some embodiments, nanoparticles comprising any of the compounds described in the Compounds section above are used in the methods herein for delivery of agents into cells.

[0232] In some embodiments, the agent is a polynucleotide. In some embodiments, the agent is RNA. In some embodiments, the agent is mRNA. In some embodiments, the agent is a therapeutic, diagnostic, or prophylactic agent.

[0233] In some embodiments, provided herein are methods for delivering a polynucleotide. In some embodiments, provided herein are methods for delivering a polynucleotide (e.g., mRNA) to correct a mutation in a genome. For example, mRNA can be delivered to correct a mutation that causes hemophilia (due to a mutation in a gene encoding factor VIII (F8, hemophilia A) or factor IX (F9, hemoglobin B)). In some embodiments, provided herein are methods for delivering a polynucleotide (e.g., mRNA) to provide expression of the mRNA in a cell (and translation to produce a protein). In some embodiments, provided herein are methods for delivering a polynucleotide (e.g., mRNA) to induce an immune response in a subject. In some embodiments, the RNA (e.g., mRNA) polynucleotide has an open reading frame encoding at least one (e.g., at least 2, 3, 4, or 5) hMPV, PIV, RSV, MeV, and / or Beta-CoV (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigenic polypeptide, or any combination of two or more antigenic polypeptides.

[0234] In one aspect, the methods described herein are directed to the treatment of cancer, such as, among others, melanoma, lung cancer (such as lung adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, non-small cell carcinoma, small cell carcinoma, mesothelioma, etc.); breast cancer (such as ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma, serous cavity breast carcinoma, etc.); colorectal cancer (such as colon carcinoma, rectal carcinoma, colorectal adenocarcinoma); anal cancer; pancreatic cancer (such as pancreatic adenocarcinoma, pancreatic islet cell carcinoma, neuroendocrine tumors, etc.); prostate cancer; prostate adenocarcinoma; ovarian cancer (such as ovarian epithelial or surface epithelial-stromal tumors, including serous tumors, endometrioid tumors and mucinous cystadenocarcinomas, sex cord-stromal tumors, Tumors); Liver and bile duct cancer (hepatocellular carcinoma, cholangiocarcinoma, hemangioma, etc.); Esophageal cancer (esophageal adenocarcinoma and squamous cell carcinoma, etc.); Oral cavity and oropharyngeal squamous cell carcinoma; Salivary gland adenoid cystic carcinoma; Bladder cancer; Bladder carcinoma; Uterine cancer (including endometrial adenocarcinoma, eye, uterine serous carcinoma, uterine clear cell carcinoma, uterine sarcoma, leiomyosarcoma, mixed Mullerian tumor); Glioma, glioblastoma, medulloblastoma, and other tumors of the brain; Kidney cancer (renal cell carcinoma, clear cell carcinoma, Wilms' tumor, etc.); Head and neck cancer (squamous cell carcinoma, etc.); Gastric cancer (gastric carcinoma, gastric adenocarcinoma, gastrointestinal stromal tumor); Testicular tumor; Germ cell tumor; Neuroendocrine tumor Tumors; cervical cancer; carcinoids of the gastrointestinal tract, breast, and other organs; signet ring cell carcinoma; mesenchymal tumors such as sarcomas, fibrosarcoma, hemangiomas, hemangiomatosis, hemangiopericytomas, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, leiomyosarcoma, skin, melanoma, neck, retinoblastoma, head and neck cancer, pancreatic, brain, thyroid, testicular, kidney, bladder, soft tissue, adrenal, urethral, ​​penile cancer, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma Used to treat malignant fibrous histiocytoma, lymphangiosarcoma, mesothelioma, squamous cell carcinoma; epidermoid carcinoma, malignant skin adnexal tumor, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, adrenal nephroma, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, anaplastic glioma; glioblastoma multiforme, neuroblastoma, medulloblastoma, malignant meningioma, malignant neurilemmoma, neurofibrosarcoma, parathyroid carcinoma, medullary thyroid carcinoma, bronchial carcinoid, pheochromocytoma, pancreatic islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phyllodes, salivary carcinoma, thymic carcinoma, and vaginal carcinoma.

[0235] In some embodiments, the compositions and methods described herein are useful for treating or preventing cancer. In some cases, the cancer is a circulating cancer cell (circulating tumor cell). In some cases, the cancer is a metastatic cancer cell.

[0236] In some aspects, the methods described herein are useful for promoting wound repair in a subject. For example, administration of any of the compositions, nanoparticles, pharmaceutically acceptable compositions, or hydrogel matrices described herein can be used to promote wound repair in a diabetic wound in a subject. Diabetic wounds can include acute diabetic wounds and chronic diabetic wounds. Acute diabetic wounds generally refer to wounds that undergo normal healing over time. Chronic diabetic wounds generally do not follow the same healing progression as acute diabetic wounds. For example, chronic diabetic wounds can include, but are not limited to, pressure ulcers, diabetic ulcers, venous ulcers, and arterial ulcers.

[0237] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0238] In some embodiments, the antibody or antigen-binding fragment thereof and the nanoparticles are administered by intramuscular injection or are administered systemically.

[0239] In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises an additional immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is selected from an anti-CD40 antibody, an anti-PDL1 antibody, an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination thereof.

[0240] In one embodiment, the immunotherapeutic agent is an anti-PD-L1 antibody. In one embodiment, the anti-PD-L1 antibody is selected from atezolizumab, durvalumab, or avelumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab (MPDL3280A) (Roche). In one embodiment, the anti-PD-L1 antibody is durvalumab (MEDI4736). In one embodiment, the anti-PD-L1 antibody is avelumab (MS0010718C).

[0241] In one embodiment, the immunotherapeutic agent is a programmed death protein 1 (PD-1) inhibitor or an inhibitor of programmed death protein ligand 1 or 2. PD-1 inhibitors are known in the art and include, for example, nivolumab (BMS), pembrolizumab (Merck), pidilizumab (CureTech / Teva), AMP-244 (Amplimmune / GSK), BMS-936559 (BMS), and MEDI4736 (Roche / Genentech).

[0242] In one embodiment, the immunotherapeutic agent is an anti-PD1 antibody. In one embodiment, the anti-PD1 antibody is nivolumab. In one embodiment, the anti-PD1 antibody is pembrolizumab.

[0243] In one embodiment, the immunotherapeutic agent is an anti-CTLA4 antibody. In some embodiments, the anti-CTLA4 antibody is ipilimumab.

[0244] In some embodiments, the additional therapeutic agent is an anti-neoplastic agent, such as avilason acetate, avitrexate (metrecelate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), Abraxane, ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, trastuzumab emtansine (Ado-Trastuzumab Emtansine), Adriamycin (doxorubicin hydrochloride), Adrucil (fluorouracil), afatinib maleate, Afinitor (everolimus), Aquinzeo (netupitant / palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, alemtuzumab, Alimta (pemetrexed disodium), Aloxi (palonosetron hydrochloride), ambochlorin (chlorambucil), aminolevulinic acid, anastrozole, aprepitan , Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanon (nelarabine), arsenic trioxide, Arzera (ofatumumab), asparaginase erwinia chrysanthemum, Avastin (bevacizumab), axitinib, azacitidine, BEACOPP, Besenam (carmustine), Beleodac (belinostat), belinstat, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, Bexxar (tositumomab and iodine I 131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Bilincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, busulfan, Busulfex (busulfan), cabazitaxel, cabozantinib s-malate, CAF, Camptosar (alemtuzumab), Camptosar Taxol (irinotecan hydrochloride), capecitabine, CAPOX, carboplatin, carboplatin-taxol, carfilzomib, Carmbris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CeeNU (lomustine), ceritinib, Cerbidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab,Chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), chloral (clofarabine), CMF, Cometriq (cabozantinib s-malate), COPP, COPP-ABV, Cosmegen (dactinomycin), Crizolib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine, liposomal, Cytosar U (cytarabine), cyclo Xan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin diftitox, denosumab, DepoCyt (liposomal cytarabine), DepoFoam (liposomal cytarabine), dexrazoxane hydrochloride, dinutuximab, docetaxel, Doxil (doxorubicin hydrochloride liposomal), doxorubicin hydrochloride, doxorubicin hydrochloride liposomal, Dox-SL (doxorubicin Hydrochloride liposomal), DTIC-Dome (dacarbazine), Efudex (fluorouracil), ERYTECH (rasburicase), Elence (epirubicin hydrochloride), Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinase (asparaginase), etopofos (epileptic acid), toposide phosphate), etoposide, etoposide phosphate, evacet (doxorubicin hydrochloride liposomal), everolimus, Evista (raloxifene hydrochloride), exemestane, Fairston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate Fluoroplex (fluorouracil), fluorouracil, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab,FOLFIRINOX, FOLFOX, Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivert vaccine), Gardasil 9 (recombinant HPV nonvalent vaccine), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Giotrif (afatinib dimaleate), Glivec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant, HPV nonvalent vaccine, recombinant, HPV quadrivalent vaccine, recombinant, Hycamtin (topotecan hydrochloride), HyperCVAD, Ibrance (palbociclib), ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, IFEX (ifosfamide), ifosfamide, ifosfamid, imatinib mesylate, Imbruvica (ibrutinib), imiquimod, interferon alfa-2b, recombinant, Intron A (recombinant interferon alfa-2b), iodine I 131 Thiositomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, ISTODAX (romidepsin), ixabepilone, Ixempra (ixabepilone), Jakavi (ruxolitinib), Jevtana (cabazitaxel), Kadcyla (trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembroline) Ibuprofen (izumab), Kyprolis (carfilzomib), lanreotide acetate, lapatinib tosilate hydrate, lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, Levulan (aminolevulinic acid), Linfolizin (chlorambucil), Lipodox (doxorubicin hydrochloride liposomal),Liposomal cytarabine, lomustine, Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lupron Depot-3 Month (leuprolide acetate), Lupron Depot-4 Month Month (leuprolide acetate), Lynparza (olaparib), Marqibo (vincristine sulfate liposomal), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, Megase (megestrol acetate), megestrol acetate, Mekinist (trametinib), mercaptopurine, mesna, Mesnex (mesna), metazolastone (temozolomide), methotrexate Methotrexate, Methotrexate LPF (methotrexate), Mexate (methotrexate), Mexate-AQ (methotrexate), Mitomycin C, Mitoxantrone Hydrochloride, Mitozylex (mitomycin C), MOPP, Mosvir (plelixafor), Mustargen (mechlorethamine hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mylosar (azacytidine), Mylothrix (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), navelbine (vinorelbine tartrate), nelarabine, Neosar (cyclophosphamide), netupitant and palonosetron hydrochloride, Neupogen (filgrastim), Nexavar (sorafenib tosylate), nilotinib, nivolumab, Nolvadex (tamoxifen citrate), N-plate (romiplostim) ), obinutuzumab, odomuzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, omacetaxine mepesuxinate, Oncaspar (pegaspargase), ondansetron hydrochloride, Ontak (denileukin diftitox), OPPA oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, palifermin, palonosetron hydrochloride,Palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, pegaspargase, PEG-interferon alfa-2b, PEG-Intron (PEG-interferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag olamine), proben Di(sipuleucel-T), Purintoru (mercaptopurine), Purixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonvalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, R-EPOCH, Revlimid (lenamidomide), Rheumatrex (methotrexate), Rituxan (rituximab), rituximab, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), ruxolitinib phosphate, Sclerosol intrapleural aerosol Intrapleural Aerosol (talc), siltuximab, sipuleucel-T, Somatuline Depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), STANFORD V, sterile talc powder (talc), Steritalc (talc), Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (pegylated interferon alfa-2b), Sylvant (siltuximab), Synovir (thalidomide), Synribo (omacetaxine mepesuxinate), TAC, Tafinlar (dabrafenib), talc, tamoxifen citrateTarabin PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thiotepa, Toposar, (etoposide), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and iodine I 131 tositumomab, Totect (dexrazoxane hydrochloride), TPF, tramelenib, trastuzumab, Treanda (bendamustine hydrochloride), Trisenox (arsenic trioxide), Tycarb (lapatinib ditosilate), Unituxin (dinutuximab), vandetanib, VAMP, Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Versal (vin Vinblastine sulfate), vemurafenib, Bepcid (etoposide), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomal, vinorelbine tartrate, VIP vismodegib, Voraxaze (glucarpidase), vorinostat, Votrie (pazopanib hydrochloride), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), Xeliri, Xelox, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Zaltrap (Ziv-aflibercept), Zelboraf (vemurafenib), Zevalin (ibritumumab) The agent may be selected from the group consisting of Mabtiuxetan, Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib), and Zytiga (abiraterone acetate).

[0245] In one embodiment, provided herein is a method of treating an inflammatory disorder, such as an autoimmune disease, in a subject. The method comprises administering to the subject a therapeutically effective amount of a compound, a combination of compounds, or a composition provided herein, or a pharmaceutically acceptable form thereof, or a pharmaceutical composition provided herein. Examples of autoimmune diseases include, but are not limited to, acute disseminated encephalomyelitis (ADEM), Addison's disease, antiphospholipid syndrome (APS), aplastic anemia, autoimmune hepatitis, autoimmune skin diseases, celiac disease, Crohn's disease, diabetes mellitus (type 1), Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, lupus erythematosus, multiple sclerosis, myasthenia gravis, myoclonus syndrome (OMS), optic neuritis, and orthothyroiditis. These disorders include adenitis, emphysema, polyarthritis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, Reiter's syndrome, Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis"), warm autoimmune hemolytic anemia, Wegener's granulomatosis, alopecia areata (e.g., inflammatory alopecia), Chagas' disease, chronic fatigue syndrome, autonomic neuropathy, endometriosis, hidradenitis suppurativa, interstitial cystitis, neuromyotonia, sarcoidosis, scleroderma, ulcerative colitis, vitiligo, and vulvodynia. Other disorders include bone resorption disorders and thrombosis.

[0246] Inflammation takes many forms, including but not limited to acute, adhesive, atrophic, catarrhal, chronic, cirrhotic, diffuse, disseminated, exudative, fibrinous, fibrosing, focal, granulomatous, hyperplastic, hypertrophic, interstitial, metastatic, necrotizing, obstructive, parenchymal, plastic, productive, proliferative, pseudomembranous, suppurative, sclerosing, seroplastic, serous, simple, specific, subacute, suppurative, toxic, traumatic, and / or ulcerative inflammation.

[0247] Exemplary inflammatory conditions include, but are not limited to, acne, anemia (e.g., aplastic anemia, hemolytic autoimmune anemia), asthma, arteritis (e.g., polyarteritis, temporal arteritis, periarteritis nodosa, Takayasu's arteritis), arthritis (e.g., crystalline arthritis, osteoarthritis, psoriatic arthritis, gout flare, gouty arthritis, reactive arthritis, rheumatoid arthritis, and Reiter's arthritis), ankylosing spondylitis, amylosis, amyotrophic lateral sclerosis, autoimmune diseases, allergies or allergic reactions, atherosclerosis nodosa, tracheal ulcers, and ulcers. bronchiolitis, bursitis, chronic prostatitis, conjunctivitis, Chagas' disease, chronic obstructive pulmonary disease, cermatomyositis, diverticulitis, diabetes (e.g., type 1 diabetes, type 2 diabetes), skin conditions (e.g., psoriasis, eczema, burns, dermatitis, pruritus (itching)), endometriosis, Guillain-Barre syndrome, infections, ischemic heart disease, Kawasaki disease, glomerulonephritis, gingivitis, hypersensitivity, headaches (e.g., migraine, tension headache), intestinal obstruction (e.g., idiopathic thrombocytopenic purpura, interstitial cystitis (painful bladder syndrome), gastrointestinal disorders (e.g., peptic ulcer, regional enteritis, diverticulitis) , gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., selected from eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, and eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, fecal diversion colitis, Behcet's syndrome, and atypical colitis) and inflammatory bowel syndrome (IBS)), lupus, multiple sclerosis, morphea, myasthenia gravis, myocardial ischemia, nephropathic syndrome, pemphigus vulgaris, pernicious anemia, peptic ulcer, multiple sclerosis, urinary tract infection ... These include myositis, primary biliary cirrhosis, encephalopathy-associated neuroinflammation (e.g., Parkinson's disease, Huntington's disease, and Alzheimer's disease), prostatitis, chronic inflammation associated with cranial radiation injury, pelvic inflammatory disease, polymyalgia rheumatica, reperfusion injury, regional enterocolitis, rheumatic fever, systemic lupus erythematosus, scleroderma, scleroderma (scierodoma), sarcoidosis, spondyloarthritis Sjogren's syndrome, thyroiditis, transplant rejection, tendonitis, trauma or injury (e.g., frostbite, chemical irritants, toxins, scars, burns, physical injuries), vasculitis, vitiligo, and Wegener's granulomatosis.In certain embodiments, the inflammatory disorder is selected from arthritis (e.g., rheumatoid arthritis), inflammatory bowel disease, inflammatory bowel syndrome, asthma, psoriasis, endometriosis, interstitial cystitis, and prostatitis. In certain embodiments, the inflammatory condition is an acute inflammatory condition (e.g., inflammation resulting from an infection). In certain embodiments, the inflammatory condition is a chronic inflammatory condition (e.g., conditions resulting from asthma, arthritis, and inflammatory bowel disease). These compounds may also be useful in treating inflammation associated with trauma and non-inflammatory muscle pain. In certain embodiments, the inflammatory disorder is selected from arthritis (e.g., rheumatoid arthritis), inflammatory bowel disease, inflammatory bowel syndrome, asthma, psoriasis, endometriosis, interstitial cystitis, and prostatitis. In certain embodiments, the inflammatory condition is an acute inflammatory condition (e.g., inflammation resulting from an infection). In certain embodiments, the inflammatory condition is a chronic inflammatory condition (e.g., conditions resulting from asthma, arthritis, and inflammatory bowel disease). These compounds may also be useful in treating inflammation associated with trauma and non-inflammatory muscle pain.

[0248] Immune disorders, such as autoimmune disorders, include, but are not limited to, arthritis (degenerative joint diseases such as rheumatoid arthritis, spondyloarthropathy, gouty arthritis, osteoarthritis, systemic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, undifferentiated spondyloarthritis), Behcet's disease, hemolytic autoimmune anemia, multiple sclerosis, amyotrophic lateral sclerosis, amylosis, acute painful shoulder, psoriasis, and juvenile degenerative diseases. age-related arthritis), asthma, atherosclerosis, osteoporosis, bronchitis, tendonitis, bursitis, skin conditions (e.g., psoriasis, eczema, burns, dermatitis pruritus (itching)), bedwetting, eosinophilic diseases, gastrointestinal disorders (e.g., peptic ulcer, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic gastroenteropathy), gastritis, diarrhea, stomach These include esophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, fecal diversion colitis, Behcet's syndrome, inflammatory bowel disease) and irritable bowel syndrome (IBS)), relapsing polychondritis (e.g., atrophic polychondritis and systemic polychondritis), and disorders that are improved by gastrokinetic agents (e.g., ileus, postoperative ileus, and ileus during sepsis; gastroesophageal reflux disease (GORD, or its synonym GERD); eosinophilic esophagitis, gastroparesis such as diabetic gastroparesis; food intolerances and food allergies, and other functional bowel disorders such as non-ulcer dyspepsia (NUD) and non-cardiac chest pain (NCCP, costochondritis, etc.)).

[0249] In some examples, the compound or composition is administered to a subject at a dose of 1 μg per kilogram (kg) of the subject's body weight per day (μg / kg / day) or more (e.g., 2 μg / kg / day or more, 3 μg / kg / day or more, 4 μg / kg / day or more, 5 μg / kg / day or more, 10 μg / kg / day or more, 15 μg / kg / day or more, 20 μg / kg / day or more, 25 μg / kg / day or more, 30 μg / kg / day or more, 35 μg / kg / day or more, 40 μg / kg / day or more, 45 μg / kg / day or more, 50 μg / kg / day or more, 60 μg / kg / day or more, 70 μg / kg / day or more, 80 μg / kg / day or more, 90 μg / kg / day or more, 100 μg / kg / day or more, 125 μg / kg / day or more, 150 μg / kg / day or more, g / kg / day or more, 175 μg / kg / day or more, 200 μg / kg / day or more, 225 μg / kg / day or more, 250 μg / kg / day or more, 300 μg / kg / day or more, 350 μg / kg / day or more, 400 μg / kg / day or more, 450 μg / kg / day or more, 500 μg / kg / day or more, 600 μg / kg / day or more, 700 μg / kg / day or more, 800 μg / kg / day or more, 900 μg / kg / day or more, 1 milligram (mg) / kg / day or more, 2 mg / kg / day or more, 3 mg / kg / day or more, 4 mg / kg / day or more, 5 mg / kg / day or more, 6 mg / kg / day or more, 7 mg / kg / day or more, 8 mg / kg / day or more, or 9 mg / kg / day or more.In some examples, the compound or composition is administered to a subject at a dose of 10 mg per kilogram (kg) of the subject's body weight per day (mg / kg / day) or less (e.g., 9 mg / kg / day or less, 8 mg / kg / day or less, 7 mg / kg / day or less, 6 mg / kg / day or less, 5 mg / kg / day or less, 4 mg / kg / day or less, 3 mg / kg / day or less, 2 mg / kg / day or less, 1 mg / kg / day or less, 900 μg / kg / day or less, 800 μg / kg / day or less, 700 μg / kg / day or less, 600 μg / kg / day or less, 500 μg / kg / day or less, 450 μg / kg / day or less, 400 μg / kg / day or less, 350 μg / kg / day or less, 300 μg / kg / day or less, 250 μg / kg / day or less, or less, 225 μg / kg / day or less, 200 μg / kg / day or less, 175 μg / kg / day or less, 150 μg / kg / day or less, 125 μg / kg / day or less, 100 μg / kg / day or less, 90 μg / kg / day or less, 80 μg / kg / day or less, 70 μg / kg / day or less, 60 μg / kg / day or less, 50 μg / kg / day or less, 45 μg / kg / day or less, 40 μg / kg / day or less, 35 μg / kg / day or less, 30 μg / kg / day or less, 25 μg / kg / day or less, 20 μg / kg / day or less, 15 μg / kg / day or less, 10 μg / kg / day or less, 5 μg / kg / day or less, 4 μg / kg / day or less, 3 μg / kg / day or less, or 2 μg / kg / day or less.

[0250] The amount of the compound or composition administered to a subject can range from any of the minimum values ​​described above to any of the maximum values ​​described above. For example, the compound or composition can be administered to a subject in an amount of 1 microgram (μg) to 10 milligrams (mg) per kilogram (kg) of the subject's body weight per day (e.g., 1 μg / kg / day to 100 μg / kg / day, 100 μg / kg / day to 10 mg / kg / day, 1 μg / kg / day to 100 μg / kg / day, 100 μg / kg / day to 1 mg / kg / day, 1 mg / kg / day to 10 mg / kg / day, 5 μg / kg / day to 10 mg / kg / day, 1 μg / kg / day to 5 mg / kg / day, or 5 to 5 mg / kg / day).

[0251] However, it is understood that the specific dosage level for a particular subject will depend on a variety of factors.

[0252] Such factors include the age, weight, general health, sex, and diet of the subject. Other factors include the time and route of administration, rate of excretion, drug combinations, and the type and severity of the particular disease or disorder.

[0253] Compositions, Formulations, Administration Methods, and Kits In vivo application of the disclosed compounds and compositions containing them can be achieved by any suitable method and technique now or in the future known to those skilled in the art. For example, the disclosed compounds can be formulated in a physiologically or pharmaceutically acceptable form and administered by any suitable route known in the art, including, for example, oral, nasal, rectal, topical, and parenteral administration routes. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection. The administration of the disclosed compounds or compositions can be a single administration or continuous or at varying intervals, as can be easily determined by those skilled in the art.

[0254] The compounds disclosed herein and compositions containing them can also be administered using liposome technology, sustained-release capsules, implantable pumps, and biodegradable containers.These delivery methods can advantageously provide a uniform dose over a long period of time.The compounds can also be administered in their salt derivative form or crystalline form.

[0255] The compounds disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are well known to those skilled in the art and are described in detail in numerous readily available sources. For example, E.W. Martin, Remington's Pharmaceutical Science (1995), describes formulations that can be used in connection with the disclosed methods. Generally, the compounds disclosed herein can be formulated so that an effective amount of the compound is combined with suitable excipients to facilitate effective administration of the compound. The compositions used can also be in a variety of forms. These include solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and application. The compositions can also contain conventional pharmaceutically acceptable carriers and diluents well known to those skilled in the art.

[0256] Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for administration of such dosages for the desired use, the compositions disclosed herein can contain from about 0.1% to 100% by weight of one or more of the subject compounds, based on the total weight of the composition including the carrier or diluent.

[0257] The pharmaceutical carrier used can be, for example, solid, liquid or gas.The examples of solid carrier include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.The examples of liquid carrier include sugar syrup, peanut oil, olive oil and water.The examples of gas carrier include carbon dioxide and nitrogen.

[0258] Suitable formulations for administration include aqueous sterile injection solutions, which may contain, for example, antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the preparation of the sterile liquid carrier, e.g., water, prior to use for injection. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, and the like. It should be understood that, in addition to the excipients specifically mentioned above, the compositions disclosed herein may contain other agents conventional in the art, taking into account the type of formulation in question.

[0259] The compounds disclosed herein, and compositions containing them, can be delivered to cells by direct contact with the cells or via a carrier means. Carrier means for delivering compounds and compositions to cells are known in the art.

[0260] For the treatment of neoplastic disorders, the compounds or compositions disclosed herein can be administered to a patient in need of treatment in combination with other antitumor or anticancer agents, and / or radiation therapy and / or photodynamic therapy, and / or surgical treatment to remove the tumor. These other agents or treatments can be administered simultaneously or at different times as the compounds or compositions disclosed herein. For example, the compounds or compositions disclosed herein can be combined with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin or bleomycin, etoposide or camptothecin, antiangiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and / or other anticancer agents or antibodies, such as GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.), respectively, or immunotherapies such as ipilimumab and bortezomib.

[0261] In certain examples, the compounds and compositions disclosed herein, optionally combined with a pharmaceutically acceptable carrier such as an inert diluent, can be administered locally to one or more anatomical sites, e.g., the site of unwanted cell proliferation (e.g., injected or topically applied to a tumor site or benign skin tumor, e.g., a tumor or skin tumor). The compounds and compositions disclosed herein can be administered systemically, e.g., intravenously or orally, optionally combined with a pharmaceutically acceptable carrier such as an inert diluent, or an assimilable edible carrier for oral delivery. They can be enclosed in hard or soft-shell gelatin capsules, compressed into tablets, or incorporated directly into the food of the patient's diet. For oral therapeutic administration, the active compounds can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

[0262] Tablets, troches, pills, capsules, etc. may also contain binders such as tragacanth gum, acacia, cornstarch, or gelatin; diluents such as dicalcium phosphate; disintegrating agents such as cornstarch, potato starch, or alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, fructose, lactose, or aspartame, or flavorings such as peppermint, wintergreen oil, or cherry flavor. When the unit dosage form is a capsule, in addition to the above-mentioned materials, a liquid carrier such as vegetable oil or polyethylene glycol may be contained. Various other materials may be present as coatings or to modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, or the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetener, methyl and propylparabens as preservatives, a dye, and a flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. Moreover, the active compounds may be incorporated into sustained-release preparations and devices.

[0263] The compounds and compositions disclosed herein, including pharmaceutically acceptable salts, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salts can be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin, and mixtures thereof, as well as in oils. These preparations may contain preservatives to prevent the growth of microorganisms under normal storage and use conditions.

[0264] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. Optionally, the action of microorganisms can be inhibited by various other antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, such as sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0265] Pharmaceutical compositions disclosed herein suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions may be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some instances, the final injectable form may be sterile and effectively fluid for easy injectability. In some instances, the pharmaceutical composition may be stable under the conditions of manufacture and storage, and thus may be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0266] Sterile injectable solutions are prepared by incorporating the compounds and / or agents disclosed herein in the required amount in an appropriate solvent with various other ingredients as listed above, followed by filtered sterilization, if required. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying techniques, which yield a powder that combines the active ingredient present in the previously sterile-filtered solution with any additional desired ingredients.

[0267] The pharmaceutical compositions disclosed herein can be in a form suitable for topical use, such as, for example, an aerosol, a cream, an ointment, a lotion, a dusting powder, a mouthwash, a gargle, a solution, a tincture, etc. In some instances, the compositions can be in a form suitable for use in a transdermal device. In some instances, it may be desirable to combine them with a dermatologically acceptable carrier, which may be solid or liquid, and administer them topically to the skin as a composition. The compounds and agents and compositions disclosed herein can be applied topically to the skin of a subject. These formulations can be prepared by conventional processing methods using either the compounds disclosed herein or pharmaceutically acceptable salts thereof.

[0268] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, etc. Useful liquid carriers include water, alcohol, or glycol, or a water-alcohol / glycol mixture, optionally supplemented with a non-toxic surfactant, to dissolve or disperse the compound at an effective level. Adjuvants such as fragrances and additional antibacterial agents can be added to optimize the properties for a given application. The resulting liquid composition can be applied from absorbent pads, impregnated into bandages and other dressings, or sprayed onto the affected area using a pump-type or aerosol sprayer.

[0269] Thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified cellulose, or modified mineral materials may also be used with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like for application directly to the user's skin.

[0270] The pharmaceutical composition disclosed herein can be in a form suitable for rectal administration, where the carrier is solid.In some cases, the mixture forms a unit-dose suppository.Suitable carriers include cocoa butter and other materials commonly used in the art.Suppositories can be conveniently formed by first mixing the composition with softened or melted carrier, followed by cooling and shaping.

[0271] In addition to the above-mentioned carrier components, the above-mentioned pharmaceutical formulations can optionally contain one or more additional carrier components such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. Furthermore, other adjuvants can be included to make the formulation isotonic with the blood of the intended recipient. Compositions comprising any of the compounds disclosed herein and / or pharmaceutically acceptable salts thereof can also be prepared in powder or liquid concentrate form.

[0272] Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

[0273] The dosage range for administering the composition is sufficient to produce the desired effect that the symptom or disorder is affected.The dosage should not be so large as to cause harmful side effects such as undesired cross-reactions, anaphylactic reactions, etc. Generally, the dosage varies depending on the age, condition, sex, and degree of disease of the patient, and can be determined by those skilled in the art.The dosage can be adjusted by an individual physician if there are any adverse symptoms.The dosage can vary, for example, by administering one or more doses daily for one or several days.

[0274] Also disclosed are kits comprising a compound disclosed herein in one or more containers. The disclosed kits may optionally include a pharmaceutically acceptable carrier and / or diluent. In one embodiment, the kit includes one or more other ingredients, adjuvants, or supplements described herein. In one embodiment, the kit includes instructions or packaging materials describing how to administer the compound or composition of the kit. The containers of the kit can be made of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, the compound and / or agent disclosed herein is provided in the kit as a solid, such as a tablet, pill, or powder form. In another embodiment, the compound and / or agent disclosed herein is provided in the kit as a liquid or solution. In one embodiment, the kit includes an ampoule or syringe containing the compound and / or agent disclosed herein in liquid or solution form.

[0275] In some instances, the kit further comprises at least one agent, and the compound and agent are formulated together.

[0276] In some instances, the compound and agent are packaged together.

[0277] Kits can also include compounds and / or products that are packaged together, formulated together, and / or delivered together with other components. For example, a drug manufacturer, drug reseller, physician, compounding house, or pharmacist can provide a kit that includes a disclosed compound and / or product and other components for delivery to a patient.

[0278] It is contemplated that the disclosed kits can be used in connection with the disclosed methods of making the disclosed compositions, the disclosed methods of using the disclosed compositions, and / or the disclosed compositions.

[0279] Numerous embodiments of the invention have been described. It will, of course, be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0280] The following examples are intended to further illustrate certain aspects of the systems and methods described herein, and are not intended to limit the scope of the claims. [Example]

[0281] The following examples are set forth below to illustrate methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention that would be apparent to one skilled in the art.

[0282] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise specified, parts are parts by weight, temperature is °C or is ambient temperature, and pressure is at or near atmospheric. There are many variations and combinations of measuring conditions, such as component concentrations, temperature, pressure, and other measuring ranges and conditions, that can be used to optimize the described processes.

[0283] Example 1. Lipid derivatives Efficient delivery of mRNA is both a critical step and a challenge for mRNA therapeutic applicants. Although ongoing clinical trials have yielded promising data, the clinical use of mRNA requires the discovery and development of more efficient delivery systems.

[0284] Described herein are lipid derivatives for vaccine, gene therapy, mRNA therapeutics, and drug delivery applications. Synthetic routes and characterization are provided below.

[0285] [ka] [ka]

[0286] [Table 1] [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9]

[0287] General Procedure for the Synthesis of Thiotosylates 5-7, 15-17, and 29 To a dry round-bottom flask was added an alkyl halide (1.0 equiv., 10 mmol) in dry acetonitrile (50 mL). Sodium 4-methylbenzenesulfonothioate (1.0 equiv., 10.5 mmol) and TBAI (1.0 mmol, 0.1 equiv.) were then added, and the reaction was refluxed for 3 h. After complete consumption of the starting material, the solvent was evaporated, and the residue was dissolved in 200 mL of EA and washed with 50 mL of water. The organic layer was then dried over anhydrous Na2SO4, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography.

[0288] [ka] Yield: 90%. 1 H NMR (300 MHz, CDCl3) δ 7.81 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 2.97 (t, J = 7.5 Hz, 2H), 2.45 (s, 3H), 1.66 - 1.51 (m, 2H), 1.36 - 1.13 (m, 14H), 0.88 (t, J = 6.9 Hz, 3H). [ka] Yield: 92%. 1H NMR (300 MHz, CDCl3) δ 7.81 (dd, J = 8.4, 1.8 Hz, 2H), 7.34 (d, J = 7.8 Hz, 2H), 2.97 (t, J = 7.5 Hz, 2H), 2.45 (s, 3H), 1.57 (h, J = 7.2 Hz, 2H), 1.31 - 1.17 (m, 18H), 0.88 (t, J = 6.9 Hz, 3H). [ka] Yield: 89%. 1 H NMR (300 MHz, CDCl3) δ 7.81 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 2.98 (t, J = 7.5 Hz, 2H), 2.45 (s, 3H), 1.59 (q, J = 7.2 Hz, 2H), 1.23 (d, J = 13.1 Hz, 22H), 0.88 (t, J = 6.6 Hz, 3H).

[0289] General procedure for the synthesis of disulfanylacetic acids 9-11 and 30. The sulfonothioate (3 mmol, 1 equiv.) and KSAc (3.9 mmol, 1.3 equiv.) were dissolved in DCM (20 mL) at room temperature and stirred for 6 h. The mixture was then diluted with DCM (100 mL) and washed with saturated aqueous NaCl. The organic phase was dried over anhydrous NaSO, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography.

[0290] [ka] Yield: 89%. 1 H NMR (300 MHz, CDCl3) δ 2.71 (t, J = 7.5 Hz, 2H), 2.44 (s, 3H), 1.62 (p, J = 7.2 Hz, 2H), 1.42 - 1.25 (m, 14H), 0.88 (t, J = 6.6 Hz, 3H).

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[0291] General procedure for the synthesis of alcohols containing trisulfide linkers (18-26, 31). A mixture of thiosulfonate (6.0 mmol, 1.2 equiv.) and disulfanylacetic acid (5.0 mmol, 1.0 equiv.) was dissolved in THF (50 mL), and the resulting solution was cooled to −78 °C under N for 10 min. Sodium methoxide (5 mL, 1 M solution in MeOH, 1.0 equiv.) was added dropwise, and the resulting solution was rapidly stirred for 5 min. The reaction was quenched by the addition of saturated aqueous ammonium chloride (50 mL), the THF was removed under reduced pressure, and the product was extracted with EtOAc (3 × 50 mL). After drying over anhydrous NaSO and evaporation of the solvent, the residue was purified by silica column chromatography using hexane / ethyl acetate as eluent.

[0292] [ka] Yield: 68%. 1 H NMR (300 MHz, CDCl3) δ 3.96 (t, J = 5.7 Hz, 2H), 3.05 (t, J = 5.7 Hz, 2H), 2.95 - 2.85 (m, 2H), 1.86 (s, 1H), 1.81 - 1.68 (m, 2H), 1.46 - 1.21 (m, 14H), 0.88 (t, J = 6.6 Hz, 3H). [ka] Yield: 65%. 1 H NMR (300 MHz, CDCl3) δ 3.96 (q, J = 5.9 Hz, 2H), 3.05 (t, J = 5.7 Hz, 2H), 2.90 (t, J = 7.3 Hz, 2H), 2.06 (td, J = 6.6, 1.9 Hz, 1H), 1.75 (p, J = 7.2 Hz, 2H), 1.45 - 1.35 (m 2H), 1.35- 1.15 (m, 16H), 0.88 (t, J = 6.6 Hz, 3H). [ka] Yield: 58%. 1 H NMR (300 MHz, CDCl3) δ 3.96 (t, J = 5.7 Hz, 2H), 3.05 (t, J = 5.7 Hz, 2H), 2.90 (t, J = 7.2 Hz, 2H), 1.90 (br. s, 1H), 1.80 - 1.68 (m, 2H), 1.46 - 1.18 (m, 22H), 0.88 (t, J = 6.6 Hz, 3H).

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[0293] General procedure for the synthesis of acrylates containing trisulfide linkers (10H, 12H, 14H, 10B6, 10M, 12M, 14M, 10E, 12E, and 14E). The alcohol containing the trisulfide linker (3.0 mmol, 1.0 equiv.) and EtN (6.0 mmol, 2.0 equiv.) were dissolved in 30 mL of CHCl. ​​The solution was cooled to 0 °C under an argon atmosphere, and then a solution of acryloyl chloride (3.6 mmol, 1.2 equiv.) was added dropwise. The resulting mixture was stirred for 2 h, then quenched with water (10 mL) and extracted with CHCl (3 × 20 mL). The combined organic phase was dried over NaSO. The solvent was removed under reduced pressure, and the residue was purified on a silica gel column eluted with hexane and ethyl acetate to give the desired acrylate as an oil.

[0294] [ka] Yield: 92%. 1 H NMR (300 MHz, CDCl3) δ 6.44 (dd, J = 17.4, 1.5 Hz, 1H), 6.14 (dd, J = 17.4, 10.5 Hz, 1H), 5.85 (dd, J = 10.5, 1.5 Hz, 1H), 4.49 (t, J = 6.6 Hz, 2H), 3.14 (t, J = 6.6 Hz, 2H), 2.96 - 2.84 (m, 2H), 1.82 - 1.66 (m, 2H), 1.47 - 1.18 (m, 14H), 0.88 (t, J = 6.6 Hz, 3H).

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[0295] General Procedure for the Michael Addition of Core Amine to Acrylates Containing Trisulfide Linkers A mixture of the core amine and acylate (1.2 equiv.) was warmed to 90 °C and stirred for 12 h. The reaction mixture was purified by CombiFlash system eluting with dichloromethane and DCM / MeOH / NH3·H2O (75:22:3) to give the desired acrylate as an oil.

[0296] [ka] 1 H NMR (300 MHz, CDCl3) δ 4.41 (q, J = 6.6 Hz, 6H), 3.60 (s, 2H), 3.10 (td, J = 6.6, 2.1 Hz, 6H), 2.88 (t, J = 7.2 Hz, 8H), 2.76 (t, J = 7.2 Hz, 4H), 2.72 - 2.30 (m, 12H), 1.81 - 1.60 (m, 8H), 1.42 - 1.20 (m, 42H), 0.88 (t, J = 6.9 Hz, 9H). [ka] 11H NMR (300 MHz, CDCl3) δ 4.40 (q, J = 6.9 Hz, 6H), 3.75 - 3.50 (m, 2H), 3.10 (td, J = 6.6, 2.1 Hz, 6H), 2.88 (t, J = 7.2 Hz, 8H), 2.76 (t, J = 6.9 Hz, 6H), 2.66 - 2.34 (m, 10H), 1.85 - 1.57 (m, 8H), 1.45 - 1.20 (m, 54H), 0.88 (t, J = 6.6 Hz, 9H).

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[0297] In vitro delivery efficiency of trisulfide lipid nanoparticles in Hep3B cells One day before treatment, 2 x 10 cells were plated per well in a white-bottom 96-well plate. 4Hep3B cells were seeded onto the wells. The cells were then incubated with firefly luciferase mRNA-trisulfide lipid nanoparticles (50 ng of mRNA per well, triplicate) for 18 hours. Luminescence intensity was measured and normalized to that of the Lipofectamine™ 3000 reagent-treated wells (Figure 1).

[0298] In vivo delivery efficiency of trisulfide lipid nanoparticles in C57BL / 6 mice. C57BL / 6 mice were intravenously injected with firefly luciferase mRNA-trisulfide lipid nanoparticles (mRNA dose 0.3 mg / kg, 3 mice per group). Six hours after intravenous injection, 150 μl of XenoLight d-luciferin was injected intraperitoneally into each mouse. Eight minutes after injection of the substrate, the mice were euthanized, and major organs were collected and imaged (Figure 2).

[0299] Example 2. Lipid nanoparticle (LNP) delivery of mRNA to modulate the microenvironment to treat acute diabetic wounds Diabetic wounds, a common complication in patients with hyperglycemia, are associated with high morbidity, mortality, and recurrence rates, resulting in significant economic losses worldwide. 1~3 Although wound unloading or growth factor therapy has shown significant reductions in healing time for chronic wounds in clinical trials, their widespread use is hindered by cost and potential side effects. 4~6 There is an urgent need to develop more effective, safe, and convenient methods for managing diabetic wounds.

[0300] A major challenge in diabetic wound healing is the complex microenvironment 7,8 The pathogenesis of chronic, non-healing diabetic wounds is multifaceted, including uncontrolled accumulation of reactive oxygen species (ROS) and persistent inflammation. 9~11 Abnormal accumulation of ROS in the pathological microenvironment of diabetic wounds causes excessive oxidative stress and impaired angiogenesis, leading to delayed wound healing. 12、13 Persistent inflammation in wounds is due to the presence of M1 phenotype macrophages in the microenvironment. 14~16While M1 phenotype macrophages in diabetic wounds continue to release proinflammatory cytokines such as tumor necrosis factor α (TNF-α), M2 phenotype macrophages can promote wound repair through an anti-inflammatory response. 17,18 Therefore, modulation of the wound inflammatory microenvironment, including mitigation of ROS and phenotypic modulation, may be a useful approach for treating diabetic wound healing.

[0301] Lipid nanoparticle (LNP)-mRNA formulations have been developed and are undergoing clinical evaluation for the prevention and treatment of viral infections, cancer, and genetic diseases based on their unique biocompatibility, biodegradability, and delivery efficiency. 19~22 Therefore, it is hypothesized that the development of bioresponsive LNP-mRNA formulations capable of delivering specific therapeutic proteins will reshape the wound microenvironment for the treatment of diabetic wounds. Previous reports on the oxidation of sulfur atoms induced by ROS have shown that ROS-induced oxidation of sulfur atoms can be effectively reversed. 23-25 Inspired by this, we used a combinatorial approach to design and synthesize a library of ionizable lipids containing ROS-sensitive trisulfide linkers (Figure 3A). The ROS-scavenging ability of LNPs formulated with these trisulfide-derived lipids (TS LNPs) may restore redox balance in the pathological microenvironment of diabetic wounds. Furthermore, previous studies have shown that interleukin-4 (IL4) can promote macrophage polarization toward the M2 phenotype, which has been evaluated in preclinical studies. 26~28 Therefore, the development of TS LNPs capable of delivering IL4 mRNA has the potential to promote diabetic wound healing. Specifically, in this example, TS LNPs were used to encapsulate IL4 mRNA. The TS-IL4 mRNA formulation can then be loaded into a hydrogel, which is then used to treat diabetic wounds. After ROS scavenging and immune modulation of macrophage polarization by TS-IL4 LNPs, the wound microenvironment is remodeled, ultimately leading to diabetic wound healing (Figure 3B).

[0302] Materials and Methods Chemicals and Reagents 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2k ), D-Lin-MC3-DMA (MC3), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) were purchased from Avanti Polar Lipids. All other chemicals and solvents were purchased from Sigma-Aldrich.

[0303] cell culture Mouse cell line RAW264.7 cells were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) (Corning) supplemented with 10% FBS at 37°C under a 5% CO2 atmosphere. Fibroblast cells were purchased from ATCC and cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% FBS under a 5% CO2 atmosphere.

[0304] RNA synthesis Firefly luciferase (FLuc) and IL4 linear dsDNA were obtained from Integrated DNA Technologies. The corresponding plasmids were generated using the pUC19 vector in Golden Gate assembly. mRNA was synthesized by a previously reported method. 46 .

[0305] Preparation and characterization of LNPs Ionizable lipids, DOPE, cholesterol, DMG-PEG 2kLNP-mRNA formulations were prepared by mixing an ethanol solution containing α-glucan with a citric acid solution (pH = 3) containing mRNA using a NanoAssemblr (Precision NanoSystems). Size, polydispersity index (PDI), and zeta potential were measured using a NanoZS Zetasizer (Malvern). Encapsulation efficiency was quantified using a RiboGreen assay. Morphology was observed using a Glacios Cryo-TEM (Thermo Scientific). For initial screening, ionizable lipids, DOPE, cholesterol, and DMG-PEG were added. 2K LNP-mRNA was prepared by mixing the lipid compounds at a molar ratio of 20:30:40:0.75, followed by adding FLuc mRNA to the lipid compound at a ratio of 10:1. In the first round of optimization, L16(4) 4 The molar ratio of each lipid component was adjusted based on an orthogonal array. In the second round of optimization, the ratio of mRNA to compound was adjusted to L12(3) 4 Optimization was performed based on an orthogonal array. MC3, ALC-0315, and SM102 LNP-mRNA formulations were prepared. 20 The mRNA delivery efficiency was measured 18 hours after treatment by adding Bright-Glo luciferase substrate (Promega) to the cells and quantified using Cytation 5 (Biotek).

[0306] Assessment of macrophage polarization in vitro Evaluating the effect of TS-LNPs on macrophage polarization. RAW274.7 cells were treated with LPS (10 μg -1 ) for 24 hours. Then, the cells were plated in a 6-well plate with 5 × 10 cells per well. 4Cells were seeded onto the cells. After overnight incubation, the cells were treated with TS2-IL4 LNP, MC3-IL4 LNP, and free IL4 mRNA for 48 hours. Cells treated with LPS alone were used as a control. After all treatments, cells were harvested and then stained with anti-mouse CD86 (Biolegend) and anti-mouse CD206 antibodies (Biolegend) for 30 minutes for flow cytometry analysis. Data were analyzed using FlowJo.

[0307] Assessment of intracellular ROS scavenging The intracellular ROS scavenging ability of TS2 LNP was evaluated in fibroblasts. 47 Briefly, cells were seeded in 12-well plates and cultured for 24 h. Next, the cells were co-incubated with H2O2 (200 μM) and either TS2 LNPs or MC3 LNPs for an additional 24 h. After rinsing three times with PBS, the cells were incubated with DCFDA (10 μM) for 20 min. Intracellular ROS levels were assessed by flow cytometry. Meanwhile, intracellular fluorescence was photographed using a confocal fluorescence microscope. Similarly, cell viability was assessed using a fluorescence-based Live / Dead kit and an MTT kit according to the manufacturer's instructions.

[0308] Assessment of wound healing in diabetic mice All male dl / dl mice (BKS.Cg-Dock7m+ / +Leprdb / J, 00642, 12 weeks) were purchased from the Jackson Laboratory. All mouse studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the Icahn School of Medicine at Mount Sinai. A full-thickness wound model was constructed as previously described. Briefly, dl / dl mice were anesthetized using isoflurane, and then a wound was created in the shaved back skin using a 7 mm biopsy punch (Integra Miltex).

[0309] To investigate the delivery and expression of TS2-mRNA encoding Fluc, mice were randomly divided into three groups and treated with FLuc mRNA, MC3-FLuc LNPs, and TS2-FLuc LNPs. Hydrogels (Advanced BioMatrix) were prepared and loaded with FLuc, MC3-FLuc, and TS2-FLuc according to the manufacturer's instructions. Prior to treatment, the hydrogel and LNPs were mixed at a 1:1 volume ratio for 15 minutes. Then, 20 μL of the hydrogel mixture containing 2 μg of FLuc was administered to the wound. After 6 hours, in vivo bioluminescence imaging was performed.

[0310] To evaluate the wound healing process, mice were treated with free IL4 mRNA, MC3-IL4 LNPs, TS2-IL4 LNPs, SM102-IL4 LNPs, and TS2 LNPs. The hydrogel mixture was prepared as described above, and 20 μL of the hydrogel solution containing 2 μg of IL4 mRNA was administered to the wound. After treatment, a 7 mm donut-shaped silicone splint was attached to the wound area and suspended using 6-0 nylon sutures (Ethicon). The wound area was measured and photographed with a digital camera on the day of surgery and every 3 days until the wound was completely healed.

[0311] Histology and immunofluorescence After treatment, all regenerated dorsal skin was harvested, and one half was snap-frozen and stored at -80°C. The other half was fixed in 4% paraformaldehyde and processed for paraffin embedding. Masson's trichrome staining and H&E were performed on 5 μm sections. Images were collected using a Nikon microscope to observe the stained sections and measure epidermal thickness.

[0312] For in vivo immunofluorescence staining, paraffin-embedded mouse wound sections were deparaffinized and antigen-retrieved (citrate buffer, 10 mM, 0.05% Tween 20, pH 6.0) as described above. Sections were incubated overnight at 4°C with rabbit mAB CD31 (1:200, Abcam ab222783), goat pAB α-SMA (1:200, Abcam ab7817), CD206 (1:100, Biolegend), and CD86 (1:100, Biolegend). After washing with PBS, secondary antibodies, Alexa Fluor 594 donkey anti-rabbit IgG (1:200, Invitrogen, A21207) and Alexa Fluor 488 donkey anti-rabbit IgG (1:800, Invitrogen, A21206), were added and incubated at 37°C for 1 hour. Nuclear counterstaining was performed with 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen). Fluorescent images of each section were acquired using a fluorescence microscope and quantified using ImageJ.

[0313] Assessment of IL4 expression In vitro assays, 1 x 10 6 RAW264.7 cells were seeded in each well of a 6-well plate and treated with 0.2 μg of TS2-IL4 LNP. At different time points, cell supernatants were collected by centrifugation, and IL4 concentrations were determined by ELISA kit (RayBiotech) according to the manufacturer's protocol.

[0314] Skin dissection and flow cytometry To analyze macrophages at the wound site, wound tissue was harvested and analyzed by flow cytometry according to a previously reported method. Briefly, mouse skin consisting of the wound and approximately 0.5 mm of periwound tissue was excised and maintained in ice-cold sterile PBS. The skin tissue was then minced and soaked at 2 mg ml in DMEM (Gibco) containing 10% FBS and 1% P / S on a shaker. -1 of collagenase P (Roche), 2 mg ml -1 of Dispase (Sigma), and 1 mg·ml-1 The cells were placed in a digestion enzyme cocktail containing 100 ml of DNase I (Stemcell Technologies) at 37°C for 30 minutes. During digestion, the extracellular matrix was disrupted every 10 minutes using a glass pipette. The single-cell suspension was passed through a 40 μm cell strainer. After washing with PBS and counting the cells, the single-cell suspension was incubated with fluorescently labeled CD45, CD11b, F4 / 80, CD206, and CD86 antibodies. All samples were detected using a flow cytometer and analyzed by FLOWJO.

[0315] Statistics information Statistical analysis was performed using GraphPad Prism software. For comparisons between multiple data groups, one-way analysis of variance (ANOVA) followed by Tukey's comparison test was performed with thresholds of *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. For comparisons between two data groups, unpaired Student's t-test was performed with thresholds of *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

[0316] result Synthesis and formulation of trisulfide-derived LNPs (TS LNPs) for mRNA delivery. As shown in Figure 3A, the synthetic process for trisulfide-derived ionizable lipids began with the preparation of 10 hydrophobic tails bearing both ester and trisulfide moieties. First, alkylthiosulfonates 4 and 8 were prepared by the substitution reaction of sodium 4-methylbenzenesulfonothioate 2 with alkyl bromides 3 and 7, respectively. Next, alkylthiosulfonothioate 4 was displaced with potassium thioacetate 5 in dichloromethane to give disulfanylacetic acid 6. Introduction of the trisulfide linker to alcohol 9 was achieved by the reaction between disulfanylacetic acid 6 and alkylthiotosylate 8 in the presence of sodium methoxide, following a previously reported procedure. 24Finally, acrylate 12H was obtained by acrylation of alcohol 9, which then underwent a Michael addition reaction with amine A to yield the final product TS2. Both linear and branched tails were incorporated to evaluate their effect on mRNA delivery efficiency. The acrylate tails were named according to the length of the hydrocarbon domain and the type of ester. Tails 10H, 12H, and 14H possessed primary ester linkers, whereas tails 10M, 10B6, 12M, 14M, 10E, 12E, and 14E replaced the primary ester with a branched ester (M refers to a methyl branch, and E refers to an ethyl branch). In total, a diverse library of 70 ROS-sensitive trisulfide lipids was obtained by combinatorial aza-Michael addition reactions using seven aliphatic amines and ten acrylate hydrophobic tails, following a modified procedure previously described in the literature (Table 3). 29 The crude product was purified by silica gel flash chromatography, and the chemical structure of each lipid was determined. 1 It was confirmed by 1 H NMR spectrum and mass spectrometry.

[0317] Trisulfide lipids were formulated with firefly luciferase (Fluc) mRNA, and mRNA delivery efficiency in macrophages was evaluated based on bioluminescence intensity. Among all these LNPs, TS2 LNPs demonstrated superior mRNA delivery efficiency compared to other TS LNPs. Furthermore, the delivery efficiency of TS2 LNPs was 30-fold increased compared to the clinically used MC3 LNP at the same mRNA concentration. Furthermore, most LNPs incorporating TS lipids with amines E (TS41-50), F (TS51-60), or G (TS61-70) as head groups demonstrated higher or equivalent in vitro mRNA delivery efficiency compared to MC3 LNPs, whereas most TS lipids with amines B, C, or D demonstrated lower mRNA delivery efficiency (Figure 4A).

[0318] To further optimize the formulation of TS2 LNPs, we investigated L16(4) to determine the optimal molar ratio of each lipid component. 4An orthogonal array was designed (Figure 4B). Based on the bioluminescence intensity obtained from 16 orthogonal formulations of TS2 LNP, the effects of various molar ratios of individual lipid components on mRNA delivery were systematically investigated (Figures 4C and 4D). The optimal molar ratio, specifically ionizable lipid:DOPE:cholesterol:DMG-PEG, was determined. 2k The optimal molar ratio of DMG-PEG was determined to be 30:50:50:0.5. Compared to the original formulation, the mRNA delivery efficiency showed a 1.7-fold improvement when using the optimal formulation. Subsequently, the mass ratio of ionizable lipid to mRNA was further investigated, and DMG-PEG was used to enhance the mRNA delivery efficiency of TS2 LNPs. 2K The molar ratio of DMG-PEG was adjusted within the orthogonal predicted formulation (Figure 4E). After the second round of optimization, the mass ratio of lipid to mRNA was determined to be 5 (wt / wt), and DMG-PEG 2K The molar ratio of MC3 LNP to TS2 LNP was determined to be 1.5. The bioluminescence intensity of TS2 LNP after the second round of optimization was 1.5-fold higher than that of the top orthogonal formulation (A12) obtained after the first round of optimization. Furthermore, the delivery efficiency of TS2 LNP exceeded that of MC3 LNP by more than 80-fold (Figure 4F). This lead TS2 LNP formulation exhibited a hydrodynamic diameter of approximately 100 nm and a polydispersity index (PDI) of less than 0.15 (Figure 4G). The mRNA encapsulation efficiency was approximately 80%, and the particles exhibited a slight positive charge (Figure 4G). Furthermore, the resulting TS2 LNPs exhibited a spherical morphology when visualized by cryo-transmission electron microscopy (cryo-TEM) (Figure 4H). Therefore, the TS2 LNP (B3) formulation was selected for ex vivo delivery of mRNA to macrophages in the following studies.

[0319] TS2-IL4 LNPs induce macrophage polarization and scavenge intracellular ROS in vitro. Pathological wounds in diabetes are often accompanied by dysregulated inflammation, macrophage invasion, aggregation, motility, and phenotypic changes. 30These abnormalities lead to an increased population of M1 phenotype macrophages and the continuous release of inflammatory factors, thereby inducing persistent inflammation. This leads to difficulty in converting pro-inflammatory M1 phenotype macrophages to anti-inflammatory M2 phenotype macrophages, impeding the healing process and delaying wound healing. 31,32 Previous studies have shown that IL4 induces the polarization of macrophages toward an anti-inflammatory M2 phenotype. 15 In the following experiments, TS2 LNPs containing IL4 mRNA were formulated to reprogram the macrophage phenotype. These results show that IL4 was expressed and secreted by macrophages as early as 6 hours after co-incubation with TS2-IL4 LNPs (Figure 9). Subsequently, lipopolysaccharide (LPS)-stimulated RAW 264.7 cells were incubated with different treatments, including PBS, free IL4 mRNA, MC3-IL4 LNPs, and TS2-IL4 LNPs. The cells were then harvested and analyzed by flow cytometry. These results demonstrate that the expression of M1 markers (CD86) in macrophages treated with TS2-IL4 LNPs was significantly increased. + ) (approximately 31.4%), which was significantly lower than that of macrophages treated with free IL4 mRNA (approximately 44.4%) and MC3-IL4 LNP (approximately 46.7%), respectively (Figures 5A and 5B). In contrast, the expression of a marker of M2 macrophages (CD206 + ) was upregulated after treatment with TS2-IL4 LNPs (approximately 53.0%), which was 1.6-fold and 1.3-fold higher than that of the groups treated with free IL4 mRNA (approximately 32.1%) and MC3-IL4 LNPs (approximately 38.9%) (Figures 5C and 5D). These results indicate that TS2-IL4 LNPs were able to induce a phenotypic shift from M1 to M2 in macrophages through the expression of IL4.

[0320] In the complex wound environment, uncontrolled accumulation of ROS, including hydrogen peroxide (H2O2), induces oxidative stress. 33This stress reduces the viability of endogenous fibroblasts, vascular endothelial cells, and immune cells, and inactivates growth factors within the wound, significantly reducing their healing capacity. 34 To verify whether TS2 LNPs can respond to and scavenge intracellular ROS at the cellular level, we investigated the protective ability of TS2 LNPs against excessive ROS in fibroblasts. First, we recreated the ROS microenvironment in vitro by inducing oxidative stress using H2O2. Live / dead confocal laser scanning microscopy (CLSM) imaging revealed that fibroblasts co-incubated with H2O2 underwent significant cell death. However, higher cell viability was observed after co-incubation of TS2 LNPs with H2O2, comparable to that of the PBS control group (Figure 5E). Notably, the MC3 LNP-treated group also showed a significant number of dead cells. Subsequently, the MTT assay results were consistent with the fluorescence imaging. After 24 h of exposure to H2O2, cell viability was only 43.4%. In contrast, cell viability increased to 88.1% after treatment with TS2 LNPs compared to the MC3-treated group (Figure 5F). These results suggest that TS2 LNPs have the ability to protect fibroblasts from oxidative stress and significantly enhance cell viability.

[0321] Furthermore, intracellular ROS levels were assessed using 2',7'-dichlorodihydrofluorescein diacetate (DCFDA), an indicator of ROS response. As shown in Figure 5G, fibroblasts in the TS2 LNP-treated group exhibited significantly lower green fluorescence than those in the HO and MC3 LNP-treated groups. Quantitatively, the fluorescence intensity in TS2 LNP-treated cells was reduced by 7-fold and 5-fold compared to HO and MC3 treatments, respectively (Figure 10). Further flow cytometry analysis demonstrated similar results regarding the ability of TS2 LNP to scavenge ROS in fibroblasts (Figure 5H). Taken together, TS2 LNP demonstrates its ability for efficient ROS response and antioxidant capacity, thereby promoting the wound healing process in diabetes.

[0322] Hydrogel-loaded TS2-IL4 LNPs accelerated wound healing in db / db mice. Next, this study examined the delivery of mRNA by TS2 LNPs at the site of diabetic wounds in dl / dl mice. A wound model was established by creating a 7-mm circular wound on the back of a dl / dl mouse. 35 To facilitate clinical application, TS2 LNP-Fluc mRNA was loaded into a hydrogel and implanted into wounds for LNP delivery. Bioluminescence imaging results showed that TS2-Fluc LNPs exhibited bioluminescence intensities 5.1-fold and 1868.1-fold higher than those of MC3-Fluc LNPs and free Fluc mRNA, respectively (Figures 6A and 6B). The therapeutic effects of TS2-IL4 LNPs were then evaluated. All mice with wounds were randomly divided into four groups (untreated, IL4 mRNA, MC3-IL4 LNP, and TS2-IL4 LNP). The hydrogel was then implanted into the wound site as described above. The wound site was monitored every 3 days for a total of 18 days. As shown in Figure 6C, TS2-IL4 LNP treatment showed an accelerated wound healing rate from day 9 compared to the other groups. Quantitative results showed that approximately 80% wound closure was achieved by day 12 in the TS2-IL4 LNP-treated group, whereas no significant difference was observed in the MC3-IL4 LNP- and free IL4 mRNA-treated groups compared with the untreated group (Figure 6D). Furthermore, the TS2-IL4 LNP-treated group showed complete closure from day 15 onward, with all wounds closed 18 days after treatment. Notably, more than half of the wounds achieved complete closure by day 15 after treatment with TS2-IL4 LNP, and the closure time function was significantly more efficient than in the other groups (Figure 6E).

[0323] To further evaluate the chronic wound healing ability of TS2-IL4 LNPs, hematoxylin and eosin (H&E) staining and Masson's trichrome staining were performed. After 18 days of treatment, the TS2-IL4 LNP-treated group showed complete epidermal layer formation, in contrast to the partial epidermis observed in the MC3-IL4 LNP, free IL4 mRNA, and untreated groups (Figure 6F). Furthermore, the TS2-IL4 LNP-treated group showed the highest epidermal thickness value of 101.4 μm compared to the MC3-IL4 LNP (approximately 64.3 μm), free IL4 mRNA (approximately 43.5 μm), and untreated groups (approximately 34.5 μm), which showed a 1.5-fold, 2.3-fold, and 2.9-fold increase, respectively (Figure 6G). These results indicate that TS2-IL4 LNPs can promote diabetic wound healing.

[0324] Macrophage polarization and angiogenesis at the wound site in dl / dl mice During wound healing, inflammation initiates an entire cascade of events that are significantly influenced by macrophage polarization. M2 phenotype macrophages play a key role in suppressing inflammation and promoting tissue regeneration. 17 Macrophage polarization was examined in the wound sites of the different treatment groups by flow cytometry and immunofluorescence staining. As shown in Figures 7A and 7B, a decrease in the M1 phenotype macrophage population was observed in the free IL4 mRNA, MC3-IL4 LNP, and TS2-IL4 LNP-treated groups. M1 phenotype macrophages (F4 / 80) in the TS2-IL4 LNP-treated wounds were significantly decreased. + CD86 + The proportion of M2 phenotype macrophages (F4 / 80) in the TS2-IL4 LNP-treated group (approximately 19.4%) was also lower than that in the MC3-IL4 LNP-treated group, and was 2.3-fold lower than that in the untreated group. + CD206 + ) (approximately 43.8%) was 1.8-fold, 1.5-fold, and 1.3-fold higher than in the untreated, free IL4 mRNA, and MC3-IL4 LNP groups, respectively, accompanied by increases (Figures 7C and 7D).

[0325] Next, in this experiment, we characterized M2 macrophage polarization by CD206 / CD86 immunofluorescence staining 18 days after treatment. Within the subcutaneous layer of the wound, the TS2-IL4 LNP-treated group exhibited the lowest number of CD86-positive cells (green) compared with the untreated group and the IL4 mRNA and MC3-IL4 LNP-treated groups (Figures 7E and 7F). In contrast, a widespread distribution of CD206-positive cells (green) was observed in the TS2-IL4 LNP group. Furthermore, both the MC3-IL4 LNP and free IL4 mRNA groups showed significantly reduced CD206 expression compared with the TS2-IL4 LNP-treated group (Figures 7G and 7H).

[0326] Finally, α-SMA and CD31 immunofluorescence staining was used to assess angiogenesis in TS2-IL4 LNP-treated wounds at day 18. The well-established impairment of wound contractility and dysregulation of fibroblast-to-myofibroblast transformation were observed throughout the healing process in diabetic mice. 35,36 Considering this, we specifically examined α-SMA expression to determine the potential influence of TS2-IL4 LNP levels on myofibroblasts. Notably, the number of α-SMA-positive cells was significantly increased in the TS2-IL4 LNP-treated group, and was higher than that of the free IL4 mRNA and MC3-IL4 LNP groups (Figures 12A-12B). Furthermore, since angiogenesis has been widely reported to have a positive effect on wound closure, 37,38 We further investigated the formation of new blood vessels (CD31 positive). Compared to the free IL4 mRNA or MC3-IL4 treated groups, the TS2-IL4 LNP-treated wounds contained significantly more CD31 positive cells (Figures 12A-12B). Together, these results demonstrate that TS2-IL4 LNP significantly induces M2 macrophage polarization, promotes angiogenesis, and significantly accelerates the rate of wound closure.

[0327] In a separate experiment, the delivery efficiency of TS2 LNP was tested alongside two FDA-approved LNP formulations, ALC-0315 and SM102 LNP. The results showed that the order of delivery efficiency in RAW264.7 was TS2 LNP > SM102 LNP > ALC-0315 LNP (Figure 11). The results also showed that the mRNA delivery efficiency of SM102 LNP in wound tissue was lower than that of the TS2 LNP vehicle (Figures 8A and 8B). Next, in this study, we compared the wound healing efficacy of TS2-IL4 LNP and SM102-IL4 LNP in diabetic mice. After 18 days of treatment, both the SM102-IL4 LNP group and the TS2 LNP group without IL4 mRNA accelerated wound healing compared to the untreated group, but the TS2-IL4 LNP-treated group showed the fastest healing outcome (Figures 8C-8E). These results also indicate that the acceleration of wound healing may be due to the combined effects of ROS scavenging and macrophage polarization induced by IL4.

[0328] Consideration Due to a complex microenvironment characterized by the presence of excessive ROS and persistent inflammation, many diabetic wounds can develop into chronic wounds. Despite the FDA approval of recombinant growth factor-based products, these do not fundamentally address the challenges posed by the diabetic wound microenvironment. 4 Furthermore, there is a potential risk of cancer associated with their repeated administration. 39 Furthermore, ongoing clinical trials of cell-based therapies for wound healing face challenges such as short half-lives, storage problems, and high costs, resulting in a lack of definitive advantages in promoting healing. 6 To promote diabetic wound healing, it is essential to regulate the wound microenvironment by scavenging excess ROS, attenuating inflammation, and promoting angiogenesis to facilitate wound healing.

[0329] The use of LNP-mRNA formulations to treat diabetic wounds represents a promising therapeutic strategy. Repeated injections of LNPs delivering VEGF mRNA have been reported for the treatment of chronic wounds.40 VEGF expression is beneficial for wound healing by promoting angiogenesis, but uncontrolled angiogenesis due to overexpression of VEGF leads to the formation of abnormal blood vessels, which may contribute to complications such as keloid formation. 41~44 Currently reported LNP formulations do not address the challenges of the wound microenvironment, which may reduce wound healing performance. Therefore, there is an urgent need to develop new strategies to modulate the wound microenvironment and promote wound healing.

[0330] In this example, we hypothesized that developing an LNP-mRNA formulation that scavenges ROS could remodel the wound microenvironment and treat diabetic wounds. To test this approach, we synthesized and formulated ROS-responsive TS LNPs. Using a combinatorial approach, we developed a diverse library of 70 ROS-sensitive trisulfide-ionizable lipids to systematically explore hydrophobic tails for mRNA delivery to macrophages, thereby modulating the wound microenvironment. Researchers have previously reported that the reaction of trisulfides with ROS generates H2S, a potent antioxidant that neutralizes ROS and alleviates oxidative stress. 45 This trisulfide group effectively scavenges excess intracellular ROS, resulting in cytoprotective and potentially anti-inflammatory effects. Furthermore, in highly oxidative microenvironments, the trisulfide linker plays an important role in lipid degradation and subsequent LNP degradation. Cellular assays demonstrated that TS2 LNPs exhibited superior ROS-scavenging ability compared with clinically approved MC3 LNPs. This protective mechanism protected fibroblasts from oxidative stress, resulting in a significant 50% increase in cell viability in high-ROS microenvironments (Figure 5F). Notably, in a diabetic wound model, TS2 LNPs without mRNA loading also promoted wound healing to some extent, which was attributed to the ROS-scavenging ability of the trisulfide group (Figure 8C).

[0331] Because IL4 normally activates signal transducer and activator of transcription 6, promoting the transcription of M2-associated genes while decreasing M1-associated genes, we formulated TS LNPs with IL4 mRNA to induce macrophage polarization in wounds. 28 In cell experiments, we observed that TS2-IL4 LNPs could express and secrete IL4 in macrophages and promote the polarization of M1 macrophages to the M2 phenotype. The percentage of M2 macrophages in the TS2-IL4 LNP group was 1.3-fold and 1.6-fold higher than those in the MC3-IL4 LNP group and the free IL4 mRNA group, respectively (Figure 5D). Similarly, at the wound site, an increased percentage of M2 macrophages was observed in the TS2-IL4 LNP group compared to those in the MC3-IL4 LNP group and the free IL4 mRNA group (Figure 7D). However, in the absence of IL4 mRNA, a reduced wound healing effect was observed (Figure 8C). Consistent with the effect of macrophage polarization to the M2 phenotype, immunofluorescence staining for CD31 and α-SMA demonstrated an increased density of newly formed and more mature blood vessels after TS2-IL4 LNP treatment (Figures 12A-12B).

[0332] In a dl / dl diabetic wound model, the TS2-IL4 LNP-treated group showed an accelerated healing rate compared with the MC3-IL4 LNP-, SM102-IL4 LNP-, free IL4 mRNA-, and TS2 LNP-treated groups (Figures 6C and 8C). This result is primarily due to the combined effects of ROS scavenging and increased presence of M2 macrophages at the wound site, which promotes the formation of intact epithelium, blood vessels, and myofibroblasts.

[0333] conclusion In summary, we developed and tested a formulation based on ROS-sensitive TS LNPs for delivering IL4 mRNA. The designed formulation demonstrated potent ROS-scavenging ability, protected fibroblasts from oxidative stress, and significantly enhanced cell viability. Furthermore, TS2-IL4 LNPs demonstrated the ability to reprogram M1 macrophages to an anti-inflammatory M2 phenotype. In a diabetic mouse wound model, a single administration of TS2-IL4 LNPs to the wound site accelerated wound healing by scavenging ROS and inducing M2 macrophage polarization. In summary, this formulation can be used to treat not only diabetic wounds but also other types of chronic or acute wounds.

[0334] The methods of the appended claims are not limited in scope by the specific methods described herein, which are intended to be illustrative of some aspects of the claims; any methods that are functionally equivalent are intended to be within the scope of the claims. Various modifications of the methods in addition to those shown and described herein are intended to be within the scope of the appended claims. Furthermore, although only certain representative method steps disclosed herein have been specifically described, other combinations of method steps are also intended to be within the scope of the appended claims, even if not specifically recited. Thus, although combinations of steps, elements, components, or components may be explicitly referred to herein, other combinations of steps, elements, components, and components are included, even if not explicitly stated.

[0335] Other advantages, which are obvious and inherent to the present invention, will be apparent to those skilled in the art. It will be understood that certain characteristics and subcombinations are advantageous and may be used without reference to other characteristics and subcombinations. It will be understood that, since many possible embodiments of the invention contemplated by and within the scope of the claims can be practiced without departing from the scope thereof, all matters herein set forth or shown in the accompanying drawings are to be interpreted in an illustrative sense and not in a limiting sense.

Claims

1. A compound defined by Formula I, or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 [In the formula, p is an integer from 0 to 5; n is an integer from 1 to 10, m, if present, is an integer from 1 to 10; R １ , R ２ , and R ３ are independently OH, substituted or unsubstituted C １ ~C ５ Alkyl, substituted or unsubstituted C １ ~C ５ Alkyl alcohol, or -L １ -S-S-S-R ａ and R ４ is a substituted or unsubstituted C ８ ~C １８ is alkyl, Each R ５ are, if present, independently hydrogen, OH, substituted or unsubstituted C １ ~C ５ Alkyl, substituted or unsubstituted C １ ~C ５ Alkyl alcohol, or -L １ -S-S-S-R ａ and Each R ａ When present, independently represents a substituted or unsubstituted C ８ ~C １８ is alkyl, L in each case １ are independently substituted or unsubstituted alkyl, carbonyl, ester, amide, carbamate, amine, ether, carbonate, thioether, thioester, or urea.

2. Each L １ But, formula -Z １ C(O)OZ ２ an ester represented by the formula: １ and Z ２ are each independently substituted or unsubstituted C １ ~C １０ The compound of claim 1, wherein:

3. Z １ and Z ２ are each independently substituted or unsubstituted C １ ~C ５ The compound of claim 2, wherein the compound is alkyl.

4. Each Z １ But -(C ２ H ４ 4. The compound according to claim 2, wherein R is 1 or 2;

5. Each Z ２ are independently unsubstituted C １ ~C ７ The compound according to any one of claims 2 to 4, which is alkyl.

6. Each Z ２ However, independently, branched chain C １ ~C ７ The compound according to any one of claims 2 to 5, which is alkyl.

7. Each Z ２ but, 【Chemistry 2】 The compound according to any one of claims 2 to 6, selected from:

8. 8. The compound of any one of claims 1 to 7, further defined by Formula II, or a pharmaceutically acceptable salt thereof: 【Transformation 3】 [In the formula, p is an integer from 0 to 5; n is an integer from 1 to 10, m, if present, is an integer from 1 to 10; R １ and R ２ are independently OH, substituted or unsubstituted C １ ~C ５ Alkyl, substituted or unsubstituted C １ ~C ５ Alkyl alcohol, or -L １ -S-S-S-R ａ and Each R ４ are independently substituted or unsubstituted C ８ ~C １８ is alkyl, Each R ５ are, if present, independently hydrogen, OH, substituted or unsubstituted C １ ~C ５ Alkyl, substituted or unsubstituted C １ ~C ５ Alkyl alcohol, or -L １ -S-S-S-R ａ and Each R ａ When present, independently represents a substituted or unsubstituted C ８ ~C １８ is alkyl, L in each case １ is independently a substituted or unsubstituted alkyl, carbonyl, ester, amide, carbamate, amine, ether, carbonate, thioether, thioester, or urea; Each R ６ are independently substituted or unsubstituted C １ ~C ７ alkyl].

9. 9. The compound of any one of claims 1 to 8, further defined by Formula III, or a pharmaceutically acceptable salt thereof: 【Chemistry 4】 [In the formula, p is an integer from 0 to 5; n is an integer from 1 to 10, m, if present, is an integer from 1 to 10; R １ are independently OH, substituted or unsubstituted C １ ~C ５ Alkyl, substituted or unsubstituted C １ ~C ５ Alkyl alcohol, or -L １ -S-S-S-R ａ and Each R ４ are independently substituted or unsubstituted C ８ ~C １８ is alkyl, Each R ５ are, if present, independently hydrogen, OH, substituted or unsubstituted C １ ~C ５ Alkyl, substituted or unsubstituted C １ ~C ５ Alkyl alcohol, or -L １ -S-S-S-R ａ and Each R ａ When present, independently represents a substituted or unsubstituted C ８ ~C １８ is alkyl, L １ is, if present, a substituted or unsubstituted alkyl, carbonyl, ester, amide, carbamate, amine, ether, carbonate, thioether, thioester, or urea; Each R ６ are independently substituted or unsubstituted C １ ~C ７ alkyl].

10. 10. The compound of any one of claims 1 to 9, further defined by Formula IV, or a pharmaceutically acceptable salt thereof: 【Transformation 5】 [In the formula, p is an integer from 0 to 5; n is an integer from 1 to 10, m, if present, is an integer from 1 to 10; Each R ４ are independently substituted or unsubstituted C ８ ~C １８ is alkyl, Each R ５ are, if present, independently hydrogen, OH, substituted or unsubstituted C １ ~C ５ Alkyl, substituted or unsubstituted C １ ~C ５ Alkyl alcohol, or -L １ -S-S-S-R ａ and Each R ａ When present, independently represents a substituted or unsubstituted C ８ ~C １８ is alkyl, Each R ６ are independently substituted or unsubstituted C １ ~C ７ alkyl].

11. 11. The compound of any one of claims 1 to 10, further defined by Formula V, or a pharmaceutically acceptable salt thereof: 【Transformation 6】 [In the formula, p is an integer from 0 to 5; n is an integer from 1 to 10, each m, if present, is independently an integer from 1 to 10; R １ and R ３ are independently OH, substituted or unsubstituted C １ ~C ５ Alkyl, substituted or unsubstituted C １ ~C ５ Alkyl alcohol, or -L １ -S-S-S-R ａ and Each R ４ are independently substituted or unsubstituted C ８ ~C １８ is alkyl, Each R ５ are, if present, independently hydrogen, OH, substituted or unsubstituted C １ ~C ５ Alkyl, substituted or unsubstituted C １ ~C ５ Alkyl alcohol, or -L １ -S-S-S-R ａ and Each R ａ When present, independently represents a substituted or unsubstituted C ８ ~C １８ is alkyl, L in each case １ is independently a substituted or unsubstituted alkyl, carbonyl, ester, amide, carbamate, amine, ether, carbonate, thioether, thioester, or urea; Each R ６ are independently substituted or unsubstituted C １ ~C ７ alkyl].

12. The compound according to any one of claims 1 to 11, wherein p is 1 to 5.

13. Each R ５ are independently substituted or unsubstituted C １ ~C ５ The compound of claim 12, wherein the compound is alkyl.

14. Each R ５ The compound according to any one of claims 12 to 13, wherein is methyl.

15. Each R ５ are independently substituted or unsubstituted C １ ~C ５ The compound according to any one of claims 12 to 13, which is an alkyl alcohol.

16. The compound according to any one of claims 1 to 15, wherein p is 1.

17. The compound according to any one of claims 1 to 15, wherein p is 0.

18. Each R ６ are independently unsubstituted C １ ~C ７ The compound according to any one of claims 8 to 17, which is alkyl.

19. Each R ６ However, independently, branched chain C １ ~C ７ The compound according to any one of claims 8 to 18, which is alkyl.

20. Each R ６ However, independently, linear C １ ~C ７ The compound according to any one of claims 8 to 18, which is alkyl.

21. Each R ６ but, 【Transformation 7】 The compound according to any one of claims 8 to 20, independently selected from:

22. In the formula, each R ６ The compound according to any one of claims 8 to 21, wherein

23. At least one R ６ The compound according to any one of claims 8 to 21, wherein are different.

24. The compound according to any one of claims 1 to 23, wherein n is 1 to 7.

25. The compound according to any one of claims 1 to 24, wherein n is 1 to 3.

26. 26. The compound of any one of claims 1 to 25, wherein each m is independently 1 to 3.

27. Each R ４ is unsubstituted C ８ ~C １８ The compound of any one of claims 1 to 26, which is alkyl.

28. Each R ４ is unsubstituted C １０ ~C １４ The compound of any one of claims 1 to 27, which is alkyl.

29. Each R ４ are independently unsubstituted C １２ The compound of any one of claims 1 to 28, which is alkyl.

30. Each R ４ is substituted with one or more substituents selected from the group consisting of amines, amides, esters, ethers, and carbonates; ８ ~C １８ The compound of any one of claims 1 to 26, which is alkyl.

31. Each R ４ The compound according to any one of claims 1 to 30, wherein

32. At least one R ４ The compound according to any one of claims 1 to 30, wherein: 【Request Item 33】 【Chemistry 8-1】 【Chemistry 8-2】 【Chemistry 8-3】 【Chemistry 8-4】 【Chemistry 8-5】 【Chemistry 8-6】 【Chemistry 8-7】 【Chemistry 8-8】 [Chemistry 8-9] 【Chemistry 8-10】 The compound according to any one of claims 1 to 32, selected from the group consisting of: 【Request Item 34】 【Chemistry 9】 The compound of any one of claims 1 to 33, selected from the group consisting of: 【Request Item 35】 【Chemistry 10】 The compound of any one of claims 1 to 33, selected from the group consisting of: 【Request Item 36】 【Chemistry 11】 The compound of any one of claims 1 to 33, comprising: 【Request Item 37】 【Chemistry 12】 The compound of any one of claims 1 to 33, comprising:

38. A composition comprising a compound according to any one of claims 1 to 37 and a drug.

39. 39. The composition of claim 38, wherein the agent comprises a polynucleotide.

40. The composition of any one of claims 38 to 39, wherein the agent comprises RNA.

41. The composition of any one of claims 38 to 40, wherein the agent comprises mRNA.

42. The composition of any one of claims 38 to 41, wherein the agent comprises a polynucleotide encoding a macrophage polarization factor.

43. The composition of any one of claims 38 to 41, wherein the agent comprises a polynucleotide encoding interleukin-4.

44. A method for producing the compound according to any one of claims 1 to 37.

45. A compound according to any one of claims 1 to 37, a non-cationic lipid, Lipid nanoparticles comprising polyethylene glycol (PEG)-lipids and sterols.

46. The lipid nanoparticle of claim 45, further comprising a drug.

47. The lipid nanoparticle of claim 46, wherein the agent comprises a polynucleotide.

48. The lipid nanoparticle of any one of claims 46 to 39, wherein the agent comprises RNA.

49. The lipid nanoparticle of any one of claims 46 to 48, wherein the agent comprises mRNA.

50. The lipid nanoparticle of any one of claims 46 to 49, wherein the agent comprises a polynucleotide encoding a macrophage polarization factor.

51. The lipid nanoparticle product of any one of claims 46 to 50, wherein the agent comprises a polynucleotide encoding interleukin-4.

52. The lipid nanoparticle of any one of claims 46 to 51, wherein the drug is encapsulated in the nanoparticle.

53. The non-cationic lipid may be 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), or DPPC (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine).

53. The lipid nanoparticle of any one of claims 45 to 52, comprising 1,2-dioleoyl-sn-glycero-3-phosphotidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-5 / 7-glycero-3-phospho-(1'-rac-glycerol) (DOPG), or a combination thereof.

54. The lipid nanoparticle of any one of claims 45 to 53, wherein the sterol comprises a lipid whose main component is cholesterol.

55. The lipid nanoparticles according to any one of claims 45 to 54, wherein the molar ratio of the non-cationic lipid is 20% to 50%.

56. The lipid nanoparticle of any one of claims 45 to 55, wherein the molar ratio of the compound is 5% to 60%.

57. The lipid nanoparticles according to any one of claims 45 to 56, wherein the molar ratio of the sterol is between 20% and 50%.

58. The lipid nanoparticle of any one of claims 45 to 57, wherein the PEG-lipid molar ratio is 0.1% to 2%.

59. The lipid nanoparticle of any one of claims 45 to 58, wherein the molar ratio of the compound is 20% to 30%, the molar ratio of the non-cationic lipid is 35% to 45%, the molar ratio of the sterol is 35% to 45%, and the molar ratio of the polyethylene glycol-lipid is 0.1% to 1%.

60. The lipid nanoparticle of any one of claims 45 to 59, wherein the weight fraction of the drug is 5% to 20%.

61. 61. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a compound according to any one of claims 1 to 37, a composition according to any one of claims 38 to 43, or a nanoparticle according to any one of claims 45 to 60.

62. A hydrogel matrix encapsulating the lipid nanoparticles of any one of claims 46 to 60 and the drug.

63. 62. A method for delivering a drug into a cell, the method comprising introducing into the cell a therapeutically effective amount of a composition according to any one of claims 38 to 43, or a lipid nanoparticle according to any one of claims 45 to 60, a pharmaceutically acceptable composition according to claim 61, or a hydrogel matrix according to claim 62.

64. 62. A method for promoting wound repair in a subject, comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 38-43, the lipid nanoparticle of any one of claims 45-60, the pharmaceutically acceptable composition of claim 61, or the hydrogel matrix of claim 62.

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