Nanomaterial delivery carriers and methods of use thereof

Abstract

The self-assembled nano material comprises a Janus-based nano tube, wherein the Janus-based nano tube comprises at least one compound represented by a formula I to a formula XII, or a pharmaceutically acceptable salt of the compound. Also described are compositions comprising the Janus-based nanotubes, comprising a biologically active molecule covalently or non-covalently attached to the self-assembled nanomaterial.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to and the benefit of U.S. Provisional Application No. 63 / 326,462, filed on April 1, 2022, entitled “Nanomaterial Delivery Vehicles and Methods of Use Thereof,” which is incorporated herein by reference in its entirety for all purposes.

[0003] Federal Research Statement

[0004] This invention was made with government support from the Medical Research and Development Command under Grant W81XWH-21-1-0274, the National Institutes of Health under Grant AR072027, and the National Science Foundation under Grants 1905785 and 2025362. The government has certain rights in this invention.

[0005] Incorporation by reference

[0006] Pursuant to 37 C.FR 1.52(e)(5), the sequence information contained in the electronic file named: UCT0291PCT_Sequence_Listing_ST26.xml (size 88.3 KB; created on: March 28, 2023) is incorporated herein by reference in its entirety. Background Art

[0007] At present, multiple methods for delivering bioactive materials have been developed, and according to the type and target of bioactive materials, change. The example of various delivery carriers used (particularly for RNA delivery) includes viral vectors, lipid nanoparticles, cationic polymers, liposomes, polymer nanoparticles, carbon nanoparticles, superparamagnetic iron oxide nanoparticles (SPION), gold nanoparticles, silver nanoparticles, metal organic frameworks, cell penetrating peptides, black phosphorus nanosheets and DNA nanostructures. Delivery carriers, such as lipid nanoparticles, can deliver RNA, but report that its endosome escape rate is low, thereby reducing the therapeutic effect. In addition, the cationic lipid content of lipid nanoparticles tends to produce proinflammatory phenotypes. These limitations make it challenging to obtain the efficient RNA delivery for therapeutic applications, and hinder clinical application.

[0008] There remains a need for improved delivery of biologically active materials, such as small molecules, nucleic acids, proteins, and gene silencing or gene editing tools, and / or other macromolecular biological materials. Summary of the invention

[0009] The present invention discloses a self-assembled nanomaterial, comprising a Janus-based nanotube (JBNT), wherein the Janus-based nanotube comprises at least one compound represented by Formula I to XII, or a pharmaceutically acceptable salt thereof:

[0010]

[0011]

[0012]

[0013] in,

[0014] R 1 is H or CH 3 ;

[0015] R 2 for (CH 2 ) j , (CH 2 CH 2 O) k , or (CH 2 CH 2 NH) m , wherein j, k, and m are independently integers from 1 to 200;

[0016] R 3 is an α-amino acid, a β-amino acid, an α-polypeptide or a β-polypeptide;

[0017] L is a bond or a linking group;

[0018] T is a biologically active molecule or a targeting molecule / fragment; and

[0019] R 4 It is the wrapping material.

[0020] The invention also discloses an injectable composition comprising the self-assembling nano material and a pharmaceutically acceptable carrier.

[0021] The above described and other features are exemplified by the following figures and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The following drawings are of exemplary embodiments, wherein like elements are numbered alike.

[0023] Figure 1A , 1B , 1C and 1D show the targeting peptide-JBNP used for active targeting. Figure 1A is a schematic diagram of LysJBNP-targeting peptide; Figure 1B The structure of the targeting peptide-LysJBNP is shown; Figure 1C TEM images of LysJBNP without targeting peptide and LysJBNP with targeting peptide RLDPTSYLRTFWC are included; Figure 1D The graph shows the relationship between the absorption and wavelength (nm) of ultraviolet visible light (UV-Vis) targeting of peptide-Lys-JBNT.

[0024] Figure 2A , 2B , 2C, 2D, 2E, 2F, 2G, and 2H show the assembly and delivery of LysJBNP. Figure 2A Schematic diagram of LysJBNP delivery; Figure 2B A zeta potential analysis graph is shown; Figure 2C UV-VIS analysis graphs are shown; Figure 2D TEM images of LysJBNPs are shown; Figure 2E Results of the gel retardation assay are shown; Figure 2F For siRNA delivered by LysJBNP- -CLSM z-stack image of 488; Figure 2G is an image showing inhibition of LysJBNP uptake; Figure 2H Figure 2 is a graph showing the quantitative analysis of JBNP uptake by C28 / I2 human chondrocytes.

[0025] Figure 3A , 3B , 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, and 3L show the assembly and delivery of Arg-JBNP. Figure 3A TEM images of Arg-JBNP-miR140, Arg-JBNP-Cas9mRNA, and Arg-JBNP-albumin; Figure 3B TEM image analysis showing the size and width of Arg-JBNP-miRNA; Figure 3C The zeta potential analysis of Arg-JBNP-miR140 is shown; Figure 3D Gel retardation assay is shown; Figure 3E The zeta potential analysis of Arg-JBNP-Cas9mRNA is shown; Figure 3F CLSM z-stack images of siRNA-AF488 delivered by Arg-JBNP are shown; Figure 3G Flow cytometric analysis is shown; Figure 3H Fluorescence images of Cy5.5-eGFP-mRNA delivery by Arg-JBNP or Lipo are shown; Fig. 3I Fluorescence images of BSA-AF488 delivery via Arg-JBNPs are shown; Figure 3J The time-dependent delivery of Cas9eGFP was shown; Figure 3K Fluorescence images of Arg-JBNP-eGFP-Cas9mRNA delivery are shown; Figure 3L The time dependence of flow cytometric analysis is shown.

[0026] Figure 4A , 4B , 4C, 4D, 4E, 4F, 4G, and 4H show the endosomal escape of JBNP. Figure 4A Included are images showing endosomal escape of Arg-JBNP and Lys-JBNP; Figure 4B Included are images showing the time-dependent endosomal escape of Lys-JBNP; Figure 4C Included are images showing suppression of the proton sponge effect; Figure 4D is a graph showing the results of an acid-base titration curve; Figure 4E Included are images showing endosomal escape of JBNP and LNP; Figure 4F Figures showing the quantification of colocalization; Figure 4G Included are images showing the antiviral activity associated with JBNP delivery; Figure 4H is a graph showing the inhibition of JBNP and LNP gene expression.

[0027] Figure 5A , 5B , 5C, 5D, 5E, 5F, 5G, and 5H show JBNPs with protecting molecules. Figure 5A TEM images of polyethylene glycol (PEG)-JBNTs are shown; Figure 5B and 5C UV-VIS analysis is shown; Figure 5D TEM images of PEG-JBNP-siRNA are shown; Figure 5E shows UV-VIS analysis; Panel F shows zeta potential analysis; Figure 5G Cellular uptake of Arg-JBNP and PEG-JBNP determined by flow cytometry is shown; Figure 5H Percent uptake analysis by flow cytometry is shown; Fig.5I MFI analysis by flow cytometry is shown.

[0028] Fig. 6A , 6B , 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J, and 6K show the multifunctional JBNT. Fig. 6A shows the UV-Vis spectra of ArgJBNT / GlyJBNT / AspJBNT; Figure 6B The UV-Vis spectra of the calculated total / co-assembled ArgJBNT, GlyJBNT, and AspJBNT are shown; Figure 6C A size comparison diagram of ArgJBNT / AspJBNT / GlyJBNT / co-assembly (mixed), ArgJBNT (Arg), GlyJBNT (Gly), and AspJBNT (Asp); Fig.6D: The zeta potential diagrams of ArgJBNT / AspJBNT / GlyJBNT / co-assembly (mixed), ArgJBNT (Arg), GlyJBNT (Gly), and AspJBNT (Asp) are shown; Fig. 6E TEM images of ArgJBNT / AspJBNT / GlyJBNT / HisJBNT co-assembly, ArgJBNT, GlyJBNT, and AspJBNT; Fig. 6F UV-Vis analysis is shown; Figure 6G Zeta potential analysis is shown; Figure 6H TEM images of the co-assembly of ArgJBNT, PEGLysJBNT, and SPLysJBNT; Fig.6I TEM images of Arg-JBNP, PEG-JBNP, and SP-PEG-JBNP; Figure 6J Flow cytometric analysis showing multifunctional JBNP; Figure 6K The mean fluorescence intensity obtained by flow cytometry.

[0029] Fig. 7A , transferrin-JBNP-mRNA was delivered into cells and Cas9eGFP mRNA was expressed. Figure 7B It was shown that the peptide WYRGRL (SEQ ID NO: 47) can target type II collagen in the knee joint via WYRGRL-ArgJBNP. Figure 7C Shows Spectrum signal quantification for in vivo imaging.

[0030] Fig. 8A The results of a cell apoptosis assay using LysJBNP-doxorubicin (LysJBNP-DOX) are shown. Figure 8B Shows the Fig. 8A Quantification of apoptotic cells in the results. Figure 8C Figure 2 is an image showing inhibition of spheroid formation. Fig.8D The measured sphere diameters are shown. Fig. 8E Spheroids detected / stained with apoptosis markers are shown. Figure 8F Flow cytometric analysis for detection of cell apoptosis. Figure 8G Yes Figure 8F Quantification of apoptosis data. Figure 8H Representative fluorescence images of co-delivery of small molecule drugs and siRNA via JBNP. Fig.8I Data from apoptosis assays examining co-delivery are shown. Figure 8J For Fig.8I Quantitative data of apoptosis assay. Figure 8KMicroscopic images of the delivery of LysJBNP-DOX or LysJBNP-DOX-siRNA to ovarian cancer spheroids are shown. Figure 8L Time-dependent delivery of JBNP-DOX-siRNA to spheroids is shown. Figure 8M Representative images of apoptosis staining of cells treated with control, JBNP-DOX, and JBNP-DOX-siRNA are shown. Figure 8N This is a graph showing apoptosis tests for cells treated with control, JBNP-DOX, and JBNP-DOX-siRNA. Fig.8O for Figure 8N Quantification of apoptosis data.

[0031] Fig.9A The biodistribution of ArgJBNP-siRNA is shown. Fig. 9B IgG immune responses of ArgJBNP-siRNA compared with saline are shown. Fig. 9C Shown is the IgM immune response of Arg-JBNP-siRNA compared with saline. Fig.9D Heat map of ArgJBNP-mRNA delivery efficiency. Fig.9E Figure 2 is a heat map of cell viability when treated with ArgJBNP-mRNA. Fig.9F Heat map of delivery (efficiency x cell viability). Figure 9G This is a graph drawn using response surface methodology (RSM) to optimize the ArgJBNP-mRNA formulation. Figure 9H yes Fig.9F Surface plot of the data shown. Fig.9I Figure 2 is a diagram showing the mechanism of ArgJBNP-mRNA uptake. Figure 9J Data from pKa analysis of ArgJBNP and LysJBNP are shown. Figure 9K UV-VIS analysis of Arg JBNT, RNA and Arg JBNP-RNA is shown. Figure 9L Figure 2 is a graph showing cell viability data for different concentrations of ArgJBNP, lipid nanoparticles (LNP), polyethyleneimine nanoparticles (PEI), and single-walled carbon nanotubes (SWNT). Figure 9M Gel block test data for the reference treatment are shown. Figure 9N Shows Fig.9O Flow cytometric analysis as indicated. Fig.9O for Figure 9N Flow cytometry data. Figure 9P Stability testing data of ArgJBNP-mRNA are shown. Figure 9Q UV-VIS analysis data of ArgJBNp-mRNA stability over time are shown. Figure 9RThe Z-average particle size and PDI value of ArgJBNp-mRNA stability over time. Figure 9S Shown are zeta potential data for the stability of ArgJBNp-mRNA over time. Figure 9T ArgJBNP and Lipofectamine TM 2000 (Waltham, MA, USA) for time-dependent delivery of Cas9 mRNA. Figure 9U showed that ArgJBNP or Lipofectamine TM Representative time-dependent delivery of guide RNA (gRNA) by 2000 (Waltham, MA, USA). Figure 9V Representative time-dependent data for gene editing using red fluorescent protein (RFP) in RFP-expressing human umbilical vein endothelial cells (RFP-HUVEC) are shown. Figure 9W Representative fluorescence images of kinetic studies of delivery of Cas9-eGFP mRNA or gRNA-ATTO 550 to C28 / I2 cells are shown. Figure 9X 3D analysis for the three indicated treatments. Figure 9Y Representative fluorescent images of gene editing in Ai14 chondrocytes with the indicated treatments are shown. Figure 9Z Shown are flow cytometry data testing the three treatments in Ai14 chondrocytes.

[0032] Fig. 10A Schematic diagram of ArgJBNP-CRISPR. Fig. 10B Shows specific organs Spectrum in vivo imaging signals. Fig. 10C for Fig. 10A middle Quantification of Spectrum in vivo imaging data. Fig. 10D Fluorescence images of liver sections stained with 4',6-diamidino-2-phenylindole (DAPI). Fig.10E Representative images of hematoxylin and eosin (H&E) staining of Ai14 mouse live sections are shown.

[0033] Fig.11A The biodistribution of ArgJBNP-mCherry mRNA in the indicated organs is shown. Fig. 11B To show the specific organ Distribution map of Spectrum in vivo imaging data. Fig. 11C Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for protein corona analysis. Fig.11DThe liquid chromatography-tandem mass spectrometry (LC / MS / MS) analysis data of the protein corona are shown. In addition, Fig.11E Representative images of H&E-stained tissues are shown to detect the immunogenicity of ArgJBNp-mRNA. Fig.11F Relative changes in IgG and IgM after treatment with ArgJBNp-mRNA are shown. Fig.11G Relative changes in white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and platelets (PLT) from complete blood cell count (CBC) of BALB / cJ mice after ArgJBNp-mRNA injection are shown. Fig.11H Shown are the percent changes in body weight of four BALB / cJ mice injected with ArgJBNp-mRNA.

[0034] Fig. 12A UV-VIS analysis of ArgJBN p-RNA is shown. Fig. 12B Immunocytochemistry (ICC) images of Rab5 and Rab7 are shown. Fig. 12C Representative images of cells treated with ArgJBNp-mRNA and inhibitors (bafilomycin or chloroquine) are shown.

[0035] Fig.13A The effect of PEG on cell viability is shown. Fig. 13B The biodistribution of PEG-JBNP-siRNA compared to JBNP-siRNA and ArgJBNP-siRNA is shown. Fig. 13C The results show that Arg-JBNP, Lys-JBNP and PEG-JBNP in the liver Quantification of Spectrum in vivo imaging data.

[0036] Fig.14A The combined PEG effect and cartilage targeting ability of WYRGRL-PEG-JBNP-mRNA were demonstrated. Fig. 14B Representative Safranin-O-stained sections of knee joints after receiving WYRGRL-PEG-JBNP-mRNA. Fig. 14C Representative H&E-stained sections of knee joints after receiving WYRGRL-PEG-JBNP-mRNA. DETAILED DESCRIPTION

[0037] The present invention discloses a self-assembled nanomaterial comprising Janus nanotubes, which is composed of units having a monocyclic system. The self-assembled nanomaterial has low cytotoxicity, low immunogenicity, exhibits minimal side effects in vivo, and can be advantageously used to deliver bioactive materials. The self-assembled nanomaterial also exhibits improved endosomal escape, resulting in high efficacy.

[0038] The self-assembled nanomaterial disclosed in the present invention includes a targeting fragment targeting a specific receptor on the cell surface to promote its specific and selective uptake. The co-assembly of different Janus nanotubes with different functional groups and their use in different amounts can design self-assembled nanomaterials with desired properties. These properties can be adjusted according to cell delivery, in vivo circulation time, passive or active targeting, subcellular targeting, improved cellular uptake and enhanced endosomal escape.

[0039] In this specification and the appended claims, the words "comprise", "include" and "have" and variations thereof, such as "comprises", "comprising", "includes", "including", "has" and "having" are to be interpreted inclusively. That is, where the context permits, these words are intended to express that other elements or integers not specifically recited may be included. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0040] The terms first, second, etc. used in the present invention do not mean to indicate any particular order, but are just for convenience to indicate, for example, a plurality of layers.

[0041] The description of numerical ranges herein is intended merely as a shorthand method of individually referring to each individual value falling within the range, and unless otherwise specified herein, each individual value is included in the specification as if it were individually described herein. In the present invention, ranges may be expressed as from "about" (or "approximately") one particular value, and / or to "about" (or "approximately") another particular value. When such a range is expressed, another embodiment includes from a particular value and / or to another particular value. Similarly, when a numerical value is expressed as an approximation, by using the preposition "about" or "approximately", it will be understood that the particular value constitutes another embodiment. It will be further understood that the endpoints of each range are both disclosed relative to the other endpoint and disclosed independently of the other endpoint.

[0042] All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly contradicted by the context. In addition, all methods described herein and having more than one step can be performed by more than one person or entity. Thus, one person or one entity can perform step (a) of the method, another person or another entity can perform step (b) of the method, and yet another person or another entity can perform step (c) of the method, and so on. The use of any and all embodiments, or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and does not limit the scope of the present invention otherwise requested.

[0043] Units, prefixes, and symbols are expressed in their form accepted by the International System of Units (SI).

[0044] The grouping of optional elements or embodiments of the present invention disclosed by the present invention should not be construed as restriction.Each group member can be mentioned and advocated individually or with any combination of other members of the group or other elements found herein.For convenience and / or patentability reasons, it is expected that one or more members of the group can be included in the group or deleted from the group.When any such inclusion or deletion occurs, the specification is deemed to comprise the modified group at this, thereby satisfying the written description of all Markush groups used in the appended claims.

[0045] The headings used herein are for organizational purposes only and are not meant to limit the scope of the specification or claims, which can be obtained by reference to the specification as a whole. Therefore, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0046] The illustrations are for the purpose of describing preferred embodiments of the present invention and are not intended to limit the present invention thereto.

[0047] As used herein, "about" or "approximately" is inclusive of the stated value and means within an acceptable range of deviation of the specified value determined by one of ordinary skill in the art taking into account the measurement and the errors associated with the measurement of the specified quantity (i.e., the limitations of the measurement system). For example, "about" can mean within one or more standard deviations, or within ±10% or 5% of the stated value.

[0048] Compositions, methods and articles may optionally comprise, consist of or consist essentially of any suitable material, step or component disclosed herein. Compositions, methods and articles may additionally or optionally be formulated to be free of or essentially free of any material (or type), step or component that is not necessary to achieve the function or purpose of the composition, method and article.

[0049] All ranges disclosed in the present invention include endpoints, and the endpoints can be combined independently of each other (for example, the range of "up to 25 wt.%, or more specifically, 5 wt.% to 20 wt.%" includes the endpoints and all intermediate values ​​of the range of "5 wt.% to 25 wt.%, etc.). "Composition" includes blends, mixtures, alloys, reaction products, etc. The terms "first", "second", etc. do not indicate any order, quantity or importance, but are used to distinguish one element from another. The terms "a", "an", and "the" do not indicate a number limitation and should be interpreted as including the singular and the plural, unless otherwise specified herein or clearly contradictory to the context. Unless otherwise explicitly stated, "or" means "and / or". "Some embodiments", "one embodiment", etc. mentioned throughout the specification refer to the specific elements related to the embodiment included in at least one embodiment described in the present invention, and may or may not exist in other embodiments. In addition, it should be understood that the elements can be combined in any suitable manner in various embodiments. "Combinations thereof" are open and include any combination of at least one listed component or feature, optionally with unlisted similar or equivalent components or features.

[0050] As used herein, the term "administering" refers to the actual physical introduction of a composition into a host or cell, or onto a host or cell, as the case may be. Any and all methods of introducing a composition into a host or cell are contemplated according to the present invention; the method is not dependent on any particular means of introduction and should not be construed as such. Methods of introduction are well known to those skilled in the art and are also exemplified herein.

[0051] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not occur.

[0052] As used in the present invention, the term "pharmaceutically acceptable" refers to a composition that is physiologically tolerable and does not generally produce an allergic or similar adverse reaction when administered to a subject (preferably a human subject). Preferably, as used in the present invention, the term "pharmaceutically acceptable" refers to a drug approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, more specifically in humans.

[0053] As used herein, the terms "treat," "treating," and "treatment" include inhibiting a pathological condition, disorder, or disease, e.g., arresting or reducing the development of a pathological condition, disorder, or disease, or clinical symptoms thereof; or alleviating a pathological condition, disorder, or disease, e.g., causing regression of a pathological condition, disorder, or disease, or clinical symptoms thereof. These terms also include cure and cure. Treatment refers to any manner in which the symptoms of a pathological condition, disorder, or disease are ameliorated or otherwise beneficially altered. Preferably, the subject in need of such treatment is a mammal, preferably a human.

[0054] Chemical Definition

[0055] Compounds are described using standard nomenclature. For example, any position not substituted by any specified group is understood to have its valence filled by the specified bond or hydrogen atom. A dash ("-") that is not between two letters or symbols is used to indicate the point of attachment of a substituent. For example, -CHO is attached through the carbon of the carbonyl group.

[0056] Pharmaceutically acceptable salts include salts that retain the biological effectiveness and properties of the compound and are not biologically or otherwise unwelcome. Pharmaceutically acceptable base addition salts can be prepared from inorganic bases and organic bases. By way of example only, salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary amines, secondary amines and tertiary amines, such as alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenylamines, dienylamines, trialnylamines, substituted alkenylamines, di(substituted alkenyl)amines, tri(substituted alkenyl)amines, cycloalkylamines, di(cycloalkyl)amines, tri(cycloalkyl)amines, substituted cycloalkylamines, disubstituted cycloalkylamines, trisubstituted cycloalkylamines, cycloalkenylamines, di(cycloalkenyl)amines )amines, tri(cycloalkenyl)amines, substituted cycloalkenylamines, disubstituted cycloalkenylamines, trisubstituted cycloalkenylamines, arylamines, diarylamines, triarylamines, heteroarylamines, diheteroarylamines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed diamines and triamines, wherein at least two substituents on the amine are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocycle, etc. Also included are amines in which two or three substituents together with the amino nitrogen form a heterocycle or heteroaryl.

[0057] Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, such as alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenylamines, dienylamines, trialnylamines, substituted alkenylamines, di(substituted alkenyl)amines, tri(substituted alkenyl)amines, cycloalkylamines, di(cycloalkyl)amines, tri(cycloalkyl)amines, substituted cycloalkylamines, disubstituted cycloalkylamines, trisubstituted cycloalkylamines, cycloalkenylamines, di(cycloalkenyl)amines, )amines, tri(cycloalkenyl)amines, substituted cycloalkenylamines, disubstituted cycloalkenylamines, trisubstituted cycloalkenylamines, arylamines, diarylamines, triarylamines, heteroarylamines, diheteroarylamines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed diamines and triamines, wherein at least two substituents on the amine are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocycle, etc. Also included are amines in which two or three substituents together with the amino nitrogen form a heterocycle or heteroaryl.

[0058] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues (e.g., amines); alkali or organic salts of acidic residues (e.g., carboxylates); and the like. Pharmaceutically acceptable salts include conventional non-toxic salts and quaternary ammonium salts of the parent compound (e.g., formed from non-toxic inorganic or organic acids). For example, conventional non-toxic acid salts include those derived from inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, etc.); and those derived from organic acids (e.g., acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, HOOC-(CH 2 ) n -COOH (wherein n is 0-4) etc.)

[0059] The term "amino acid" refers to a molecule containing both an amino group and a carboxyl group. Exemplary amino acids include, but are not limited to, the D- and L-isomers of naturally occurring amino acids, and non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. As used herein, the term amino acid includes, but is not limited to, α-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.

[0060] The term "α-amino acid" refers to a molecule that contains both an amino group and a carboxyl group bound to the carbon designated as the α-carbon.

[0061] The term "β-amino acid" refers to a molecule that contains both the amino and carboxyl groups in the β configuration.

[0062] The term "naturally occurring amino acid" refers to any of the 20 amino acids commonly found in peptides synthesized in nature and known by the single-letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.

[0063] The following table lists a summary of the properties of naturally occurring amino acids:

[0064]

[0065]

[0066] "Hydrophobic amino acids" include small hydrophobic amino acids and large hydrophobic amino acids. "Small hydrophobic amino acids" refer to glycine, alanine, proline and their analogs, and "large hydrophobic amino acids" refer to valine, leucine, isoleucine, phenylalanine, methionine, tryptophan and their analogs. "Polar amino acids" refer to serine, threonine, asparagine, glutamine, cysteine, tyrosine and their analogs. "Charged amino acids" refer to lysine, arginine, histidine, aspartic acid, glutamic acid and their analogs.

[0067] The term "amino acid analogue" refers to a molecule that is structurally similar to an amino acid and that can replace an amino acid in forming a peptidomimetic macrocycle. Amino acid analogs include, but are not limited to, 3-amino acids, and amino acids in which an amino or carboxyl group is replaced by a similar reactive group (e.g., a primary amine is replaced by a secondary or tertiary amine, or a carboxyl group is replaced by an ester).

[0068] The term "unnatural amino acid" refers to an amino acid that is not one of the 20 amino acids commonly found in peptides synthesized in nature and is known by the single-letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. Unnatural amino acids or amino acid analogs include, but are not limited to, the following structures:

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078] Amino acid analogs include β-amino acid analogs. Examples of β-amino acid analogs include, but are not limited to, the following: cyclic β-amino acid analogs; β-alanine; (R)-β-phenylalanine; (R)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid; (R)-3-amino-4-(1-naphthyl)-butyric acid; (R)-3-amino-4-(2,4-dichlorophenyl)-butyric acid; (R)-3-amino-4-(2-chlorophenyl)-butyric acid; (R)-3-amino-4-(2-cyanophenyl)-butyric acid; (R)-3-amino-4-(2-fluorophenyl)-butyric acid; (R)-3-amino-4-(2-furanyl)-butyric acid; (R)-3-amino-4-(2-methylphenyl)-butyric acid; (R)-3-amino-4-(2-naphthyl)-butyric acid (R)-3-amino-4-(2-thienyl)-butyric acid; (R)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-(3,4-dichlorophenyl)-butyric acid; (R)-3-amino-4-(3,4-difluorophenyl)-butyric acid; (R)-3-amino-4-(3-benzothienyl)-butyric acid; (R)-3-amino-4-(3-chlorophenyl)-butyric acid; (R)-3-amino-4-(3-cyanophenyl)-butyric acid; (R)-3-amino-4-(3-fluorophenyl)-butyric acid; (R)-3-amino-4-(3-methylphenyl)-butyric acid; (R)-3-amino-4-(3-pyridyl)-butyric acid; (R)-3- Amino-4-(3-thienyl)-butyric acid; (R)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-(4-bromophenyl)-butyric acid; (R)-3-amino-4-(4-chlorophenyl)-butyric acid; (R)-3-amino-4-(4-cyanophenyl)-butyric acid; (R)-3-amino-4-(4-fluorophenyl)-butyric acid; (R)-3-amino-4-(4-iodophenyl)-butyric acid; (R)-3-amino-4-(4-methylphenyl)-butyric acid; (R)-3-amino-4-(4-nitrophenyl)-butyric acid; (R)-3-amino-4-(4-pyridyl)-butyric acid; (R)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid Acid; (R)-3-amino-4-pentafluorophenylbutyric acid; (R)-3-amino-5-hexenoic acid; (R)-3-amino-5-hexynoic acid; (R)-3-amino-5-phenylpentanoic acid; (R)-3-amino-6-phenyl-5-hexenoic acid; (S)-1,2,3,4-tetrahydroisoquinoline-3-acetic acid; (S)-3-amino-4-(1-naphthyl)-butyric acid; (S)-3-amino-4-(2,4-dichlorophenyl)-butyric acid; (S)-3-amino-4-(2-chlorophenyl)-butyric acid; (S)-3-amino-4-(2-cyanophenyl)-butyric acid; (S)-3-amino-4-(2-fluorophenyl)-butyric acid; (S)-3-amino-4-(2-furanyl)-butyric acid;(S)-3-amino-4-(2-methylphenyl)-butyric acid; (S)-3-amino-4-(2-naphthyl)-butyric acid; (S)-3-amino-4-(2-thienyl)-butyric acid; (S)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-(3,4-dichlorophenyl)-butyric acid; (S)-3-amino-4-(3,4-difluorophenyl)-butyric acid; (S)-3-amino-4-(3-benzothienyl)-butyric acid; (S)-3-amino-4-(3-chlorophenyl)-butyric acid; (S)-3-amino-4-(3-cyanophenyl)-butyric acid; (S)-3-amino-4-(3-fluorophenyl)-butyric acid; (S)-3-amino-4-(3-methylphenyl)-butyric acid )-butyric acid; (S)-3-amino-4-(3-pyridyl)-butyric acid; (S)-3-amino-4-(3-thienyl)-butyric acid; (S)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-(4-bromophenyl)-butyric acid; (S)-3-amino-4-(4-chlorophenyl)-butyric acid; (S)-3-amino-4-(4-cyanophenyl)-butyric acid; (S)-3-amino-4-(4-fluorophenyl)-butyric acid; (S)-3-amino-4-(4-iodophenyl)-butyric acid; (S)-3-amino-4-(4-methylphenyl)-butyric acid; (S)-3-amino-4-(4-nitrophenyl)-butyric acid; (S)-3-amino-4-(4-pyridyl) -butyric acid; (S)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-pentafluorophenylbutyric acid; (S)-3-amino-5-hexenoic acid; (S)-3-amino-5-hexynoic acid; (S)-3-amino-5-phenylpentanoic acid; (S)-3-amino-6-phenyl-5-hexenoic acid; 1,2,5,6-tetrahydropyridine-3-carboxylic acid; 1,2,5,6-tetrahydropyridine-4-carboxylic acid; 3-amino-3-(2-chlorophenyl)-propionic acid; 3-amino-3-(2-thienyl)-propionic acid; 3-amino-3-(3-bromophenyl)-propionic acid; 3-amino-3-(4-chlorophenyl)-propionic acid; 3-amino-3-(4-methoxyphenyl)-propionic acid; 3-amino-4, 4,4-Trifluorobutyric acid; 3-aminoadipic acid; D-β-phenylalanine; β-leucine; L-β-homoalanine; L-β-homoaspartic acid γ-benzyl ester; L-β-homoglutamic acid δ-benzyl ester; L-β-homoisoleucine; L-β-homoleucine; L-β-homomethionine; L-β-homophenylalanine; L-β-homoproline; L-β-homotryptophan; L-β-homovaline; L-Nω-benzyloxycarbonyl-β-homolysine; Nω-L-β-homoarginine; O-benzyl-L-β-homohydroxyproline; O-benzyl-L-β-homoserine; O-benzyl-L-β-homothreonine; O-benzyl-L-β-homotyrosine; γ-trityl-L-β-homoasparagine; (R)-β-phenylalanine;L-β-homoaspartic acid γ-tert-butyl ester; L-β-homoglutamic acid δ-tert-butyl ester; L-Nω-β-homolysine; Nδ-trityl-L-β-homoglutamine; Nω-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-β-homoarginine; O-tert-butyl-L-β-homohydroxyproline; O-tert-butyl-L-β-homoserine; O-tert-butyl-L-β-homothreonine; O-tert-butyl-L-β-homotyrosine; 2-aminocyclopentanecarboxylic acid; and 2-aminocyclohexanecarboxylic acid. ;

[0079] Amino acid analogs include analogs of alanine, valine, glycine or leucine. Examples of amino acid analogs of alanine, valine, glycine and leucine include, but are not limited to, the following: α-methoxyglycine; α-allyl-L-alanine; α-aminoisobutyric acid; α-methylleucine; β-(1-naphthyl)-D-alanine; β-(1-naphthyl)-L-alanine; β-(2-naphthyl)-D-alanine; β-(2-naphthyl)-L-alanine; 1-(2-pyridyl)-D-alanine; β-(2-pyridyl)-L-alanine; β-(2-thienyl)-D-alanine; β-(2-thienyl)-L-alanine; β-(3-benzothienyl)-D-alanine; β-(3-benzothienyl)-L-alanine; β-(3-pyridyl)-D-alanine; β-(3-pyridyl)-L-alanine; β-(4-pyridyl)-D-alanine; β-(4-pyridyl)-L-alanine; 1-chloro-L-alanine; 1 -Cyano-L-alanine; 3-cyclohexyl-D-alanine; 3-cyclohexyl-L-alanine; 3-cyclopenten-1-yl-alanine; 3-cyclopentyl-alanine; 3-cyclopropyl-L-Ala-OH dicyclohexylammonium salt; β-tert-butyl-D-alanine; β-tert-butyl-L-alanine; γ-aminobutyric acid; L-α,β-diaminopropionic acid; 2,4-dinitro-phenylglycine; 2,5-dihydro-D-phenylglycine; 2- Amino-4,4,4-trifluorobutyric acid; 2-fluoro-phenylglycine; 3-amino-4,4,4-trifluorobutyric acid; 3-fluoro-valine; 4,4,4-trifluoro-valine; 4,5-dehydro-L-Leu-OH dicyclohexyl ammonium salt; 4-fluoro-D-phenylglycine; 4-fluoro-L-phenylglycine; 4-hydroxy-D-phenylglycine; 5,5,5-trifluoro-leucine; 6-aminohexanoic acid; cyclopentyl-D-glycyl-OH.Dicyclohexyl ammonium salt; cyclopentyl-Gly-OH dicyclohexyl ammonium salt; cyclopentyl-Gly-OH dicyclohexyl ammonium salt; D-α,β-diaminopropionic acid; D-α-aminobutyric acid; D-α-tert-butylglycine; D-(2-thienyl)glycine; D-(3-thienyl)glycine; D-2-aminohexanoic acid; D-2-indanylglycine; D-allylglycine dicyclohexyl ammonium salt; D-cyclohexylglycine; D-norvaline; D-phenylglycine; β-aminobutyric acid; β-aminoisobutyric acid; (2-bromophenyl)glycine; (2-methoxyphenyl)glycine; (2-methylphenyl)glycine; (2-thiazolyl)glycine; (2-thienyl)glycine acid; 2-amino-β-(dimethylamino)-propionic acid; L-α,β-diaminopropionic acid; L-α-aminobutyric acid; L-α-tert-butylglycine; L-β-thienylglycine; L-2-amino-β-(dimethylamino)-propionic acid; L-2-aminocaproic acid dicyclohexylammonium salt; L-2-indanylglycine; L-allylglycine dicyclohexylammonium salt; L-cyclohexylglycine; L-phenylglycine; L-propargylglycine; L-norvaline; N-α-aminomethyl-L-alanine; D-α,γ-diaminobutyric acid; L-α,γ-diaminobutyric acid; β-cyclopropyl-L-alanine; (N-β-(2,4-dinitrophenyl))-L-α, β-Diaminopropionic acid; (N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,β-diaminopropionic acid; (N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,β-diaminopropionic acid; (N-β-4-methyltrityl)-L-α,β-diaminopropionic acid; (N-β-allyloxycarbonyl)-L-α,β-diaminopropionic acid; (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,γ-diaminobutyric acid; (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,γ-diaminobutyric acid (N-γ-4-methyltrityl)-D-α,γ-diaminobutyric acid; (N-γ-4-methyltrityl)-L-α,γ-diaminobutyric acid; (N-γ-allyloxycarbonyl)-L-α,γ-diaminobutyric acid; D-α,γ-diaminobutyric acid; 4,5-dehydro-L-leucine; cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH; D-allylglycine; D-homocyclohexylalanine; L-1-pyrenylalanine; L-2-aminocaproic acid; L-allylglycine; L-homocyclohexylalanine; and N-(2-hydroxy-4-methoxybenzyl)-Gly-OH.

[0080] Amino acid analogs include analogs of arginine or lysine. Examples of amino acid analogs of arginine and lysine include, but are not limited to, the following: citrulline; L-2-amino-3-guanidinopropionic acid; L-2-amino-3-ureidopropionic acid; L-citrulline; Lys(Me) 2 -OH; Lys(N 3 )-OH; Nδ-benzyloxycarbonyl-L-ornithine; Nω-nitro-D-arginine; Nω-nitro-L-arginine; α-methyl-ornithine; 2,6-diaminopimelic acid; L-ornithine; (Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-D-ornithine; (Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-L-ornithine; (Nδ-4-methyltrityl)-D-ornithine; (Nδ-4-methyltrityl)-L-ornithine; D-ornithine; L-ornithine; Arg(Me)(Pbf)-OH; Arg(Me) 2 -OH(asymmetric); Arg(Me) 2 -OH(symmetrical);Lys(ivDde)-OH;Lys(Me) 2 -OH.HCl; Lys(Me3)-OH chloride; Nω-nitro-D-arginine; and Nω-nitro-L-arginine.

[0081] Amino acid analogs include analogs of aspartic acid or glutamic acid. Examples of amino acid analogs of aspartic acid and glutamic acid include, but are not limited to, the following: α-methyl-D-aspartic acid; α-methyl-glutamic acid; α-methyl-L-aspartic acid; γ-methylene-glutamic acid; (N-γ-ethyl)-L-glutamine; [N-α-(4-aminobenzoyl)]-L-glutamic acid; 2,6-diaminopimelate; L-α-aminosuberic acid; D-2-aminoadipic acid; D-α-aminosuberic acid; α-aminopimelate; iminodiacetic acid; L-2-aminoadipic acid; threo-β-methyl-aspartic acid; γ-carboxy-D-glutamic acid γ,γ-di-tert-butyl ester; γ-carboxy-L-glutamic acid γ,γ-di-tert-butyl ester; Glu(OAll)-OH; L-Asu(OtBu)-OH; and pyroglutamic acid.

[0082] Amino acid analogs include analogs of cysteine ​​and methionine. Examples of amino acid analogs of cysteine ​​and methionine include, but are not limited to, Cys(farnesyl)-OH, Cys(farnesyl)-OMe, α-methylmethionine, Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH, 2-amino-4-(ethylthio)butyric acid, buthionine, buthionine-sulfoximine, ethionine, methionine methylsulfonium chloride, selenomethionine, cysteic acid, [2-(4-pyridyl)ethyl]-DL-penicillamine, [2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine, 4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine

[00136] The invention further comprises the following: 4-aminoethyl-L-pyrimidine, 4-methylbenzyl-L-penicillamine, benzyl-D-cysteine, benzyl-L-cysteine, benzyl-DL-homocysteine, carbamoyl-L-cysteine, carboxyethyl-L-cysteine, carboxymethyl-L-cysteine, diphenylmethyl-L-cysteine, ethyl-L-cysteine, methyl-L-cysteine, tert-butyl-D-cysteine, trityl-L-homocysteine, trityl-D-penicillamine, cystathionine, homocystine, L-homocystine, (2-aminoethyl)-L-cysteine, L-selenocysteine, cystathionine, Cys(StBu)-OH, and acetamidomethyl-D-penicillamine.

[0083] Amino acid analogs include analogs of phenylalanine and tyrosine.Examples of amino acid analogs of phenylalanine and tyrosine include 3-methylphenylalanine, 3-hydroxyphenylalanine, α-methyl-3-methoxy-DL-phenylalanine, α-methyl-D-phenylalanine, α-methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 2,4-dichlorophenylalanine, 2-(trifluoromethyl)-D-phenylalanine, 2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine, 2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine, 2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine, 2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine, 2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine, 2-Methyl-L-phenylalanine, 2-nitro-D-phenylalanine, 2-nitro-L-phenylalanine, 2,4,5-trihydroxyphenylalanine, 3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine, 3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine, 3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine, 3,5,3'-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine, 3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine, 3-(trifluoromethyl)-D-phenylalanine, 3 -(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine, 3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine, 3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine, 3-chloro-L-tyrosine, 3-cyano-D-phenylalanine, 3-cyano-L-phenylalanine, 3-fluoro-D-phenylalanine, 3-fluoro-L-phenylalanine, 3-fluoro-tyrosine, 3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine, 3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine, 3-methyl-L-phenylalanine, 3-nitro-D-phenylalanine, 3-nitro-L-phenylalanine, 3-nitro-L-tyrosine, 4-(trifluoromethyl)- D-phenylalanine, 4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine, 4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine, 4-benzoyl-L-phenylalanine, 4-bis(2-chloroethyl)amino-L-phenylalanine, 4-bromo-D-phenylalanine, 4-bromo-L-phenylalanine, 4-chloro-D-phenylalanine, 4-chloro-L-phenylalanine, 4-cyano-D-phenylalanine, 4-cyano-L-phenylalanine, 4-fluoro-D-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-D-phenylalanine, 4-iodo-L-phenylalanine, homophenylalanine, thyroxine, 3,3-diphenylalanine, thyronine, ethyltyrosine, and methyltyrosine.

[0084] Amino acid analogs include analogs of proline. Examples of amino acid analogs of proline include, but are not limited to, 3,4-dehydroproline, 4-fluoroproline, cis-4-hydroxyproline, thiazolidine-2-carboxylic acid, and trans-4-fluoroproline.

[0085] Amino acid analogs include analogs of serine and threonine. Examples of amino acid analogs of serine and threonine include, but are not limited to, 3-amino-2-hydroxy-5-methylhexanoic acid, 2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutyric acid, 2-amino-3-methoxybutyric acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutyric acid, and α-methylserine.

[0086] Amino acid analogs include analogs of tryptophan. Examples of amino acid analogs of tryptophan include, but are not limited to, the following: α-methyltryptophan; β-(3-benzothienyl)-D-alanine; β-(3-benzothienyl)-L-alanine; 1-methyltryptophan; 4-methyltryptophan; 5-benzyloxytryptophan; 5-bromotryptophan; 5-chlorotryptophan; 5-fluorotryptophan; 5-hydroxytryptophan; 5-hydroxy-L-tryptophan; 5-methoxytryptophan; 5-methoxy-L-tryptophan; 5-methyltryptophan; 6-bromotryptophan tryptophan; 6-chloro-D-tryptophan; 6-chlorotryptophan; 6-fluorotryptophan; 6-methyltryptophan; 7-benzyloxytryptophan; 7-bromotryptophan; 7-methyltryptophan; D-1,2,3,4-tetrahydro-n-harmanine-3-carboxylic acid; 6-methoxy-1,2,3,4-tetrahydro-n-harmanine-1-carboxylic acid; 7-azatryptophan; L-1,2,3,4-tetrahydro-n-harmanine-3-carboxylic acid; 5-methoxy-2-methyltryptophan; and 6-chloro-L-tryptophan.

[0087] In some embodiments, the amino acid analog is racemic. In some embodiments, the D isomer of the amino acid analog is used. In some embodiments, the L isomer of the amino acid analog is used. In other embodiments, the amino acid analog includes a chiral center of R or S configuration. In other embodiments, the amino group of the β-amino acid analog is replaced by a protecting group, such as tert-butyloxycarbonyl (BOC group), 9-fluorenylmethoxycarbonyl (FMOC), tosyl, etc. In other embodiments, the carboxylic acid functional group of the β-amino acid analog is protected, for example, as an ester derivative thereof. In some embodiments, a salt of the amino acid analog is used.

[0088] A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide without losing or substantially losing its essential biological or biochemical activity (e.g., receptor binding or activation). An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of a polypeptide, results in the loss or substantial loss of the essential biological or biochemical activity of the polypeptide.

[0089] "Conservative amino acid substitution" refers to the replacement of an amino acid residue by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., K, R, H), amino acids with acidic side chains (e.g., D, E), amino acids with uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), amino acids with non-polar side chains (e.g., A, V, L, I, P, F, M, W), amino acids with β-branched side chains (e.g., T, V, I), and amino acids with aromatic side chains (e.g., Y, F, W, H). Thus, for example, an amino acid residue in a polypeptide that is predicted to be non-essential can be replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g., replacing methionine with norleucine) or substitutions based on other properties (e.g., replacing phenylalanine with 2-thienylalanine).

[0090] The term "peptide" refers to one or more amino acid residues bound together. The term "polypeptide" refers to a linear organic polymer composed of a large number of amino acid residues (20 or more) bound together in a chain, constituting a portion (or all) of a protein molecule.

[0091] The term "cyclic peptide" refers to a polypeptide chain comprising a cyclic sequence of bonds. For example, in any aspect or embodiment described herein, the cyclic peptide includes but is not limited to the following structure:

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108] The term "α-polypeptide" refers to an α-amino acid derived polypeptide.

[0109] The term "β-polypeptide" refers to a β-amino acid derived polypeptide.

[0110] The term "phosphate" refers to an ester of phosphoric acid, with a central phosphoric acid molecule having an alkyl or aromatic substituent. For example, in any aspect or embodiment described herein, phosphate includes but is not limited to the following structure:

[0111]

[0112]

[0113] The term "aliphatic" or "aliphatic group" refers to a hydrocarbon group that can be straight chain (i.e., unbranched), branched, or cyclic (including fused, bridged, and spiro-fused polycyclics), and can be fully saturated or can contain one or more unsaturated units. Suitable aliphatic groups include, but are not limited to, linear or branched alkyl, alkenyl, and alkynyl groups and mixtures thereof. As used herein, the term "aliphatic" or "aliphatic group" also includes partially substituted analogs of these fragments, in which at least one hydrogen atom of the aliphatic group is substituted by an atom other than carbon or hydrogen.

[0114] The term "linker" refers to a chemical group that is linked to one or more other chemical groups through at least one covalent bond.

[0115] Although the present invention has been described with reference to exemplary embodiments, it will be appreciated by those skilled in the art that various changes may be made and elements thereof may be substituted with equivalents without departing from the scope of the present invention. In addition, many modifications may be made to adapt specific circumstances or materials to the teachings of the present invention without departing from the basic scope of the present invention. Therefore, the present invention is not intended to be limited to the specific embodiments disclosed as the best mode contemplated for implementing the present invention, but the present invention will include all embodiments falling within the scope of the appended claims. Unless otherwise specified herein or clearly contradictory to the context, any combination of the elements in all possible variations thereof is included in the present invention.

[0116] Self-assembled nanomaterials

[0117] The self-assembled nanomaterials of the present invention include Janus-based nanotubes (JBNTs). JBNTs are composed of structural units based on a monocyclic system and self-assemble into nanomaterials, which can be used for drug delivery and stents. In particular, JBNTs provide a solution for delivering bioactive molecules to specific cells and / or tissues. In one embodiment, JBNTs include bioactive molecules covalently or non-covalently attached to JBNTs. Further, JBNTs can be connected / coupled to targeting fragments to promote the active targeting of self-assembled nanomaterials including bioactive molecules to specific cells and / or tissues.

[0118] JBNT is a biocompatible, biodegradable material with relatively low cytotoxicity and low immunogenicity. JBNT also combines the advantages of lipid nanoparticles and cationic polymers for improved endosomal escape and high efficacy. Advantageously, JBNT effectively enters cells through macropinocytosis (using the same mechanism as lipid nanoparticles) and can effectively escape from endosomes through the "proton sponge" effect (the same mechanism as cationic polymers). Therefore, JBNT can achieve excellent delivery of bioactive molecules and exhibit extremely low cytotoxicity.

[0119] In one embodiment, the self-assembled nanomaterial comprises a Janus-based nanotube, wherein the Janus-based nanotube comprises at least one compound represented by Formula I to XII, or a pharmaceutically acceptable salt thereof.

[0120]

[0121]

[0122]

[0123] Among them, R 1 is H or CH 3 ; R 2 for (CH 2 )j , (CH 2 CH 2 O) k , or (CH 2 CH 2 NH) m , wherein j, k, and m are independently 1-200; R 3 is an α-amino acid, a β-amino acid, an α-polypeptide or a β-polypeptide; L is a bond or a linking group; T is a biologically active molecule or a targeting molecule; and R 4 In one embodiment, T is a targeting molecule.

[0124] In any aspect or embodiment described in the present invention, the targeting molecule or fragment includes or is a peptide, a cyclic peptide, a small molecule, another molecular structure mentioned in the present invention as a targeting molecule or fragment, or a combination thereof.

[0125] In any aspect or embodiment described herein, the encapsulating material comprises a polymer, a peptide, a polypeptide, a lipid-based material, a phosphate ester or a biomimetic membrane.

[0126] In one embodiment, L is a linking group, selected from acid cleavable groups, reducible disulfide groups, α-amino acids, β-amino acids, α-polypeptides, β-polypeptides, enzyme cleavable groups, stimulus response groups or combinations thereof. Acid cleavable groups may include N-acylhydrazones, carbonate groups or ester groups. Reducible disulfide linkers may include N-succinimidyl-4-(2-pyridyldithio) pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), or 4-(4'-acetylphenoxy) butyric acid (AcBut), Val-Cit dipeptide, Phe-Lys dipeptide, α-methyl substituted disulfide, engineered cysteine ​​residues or sulfhydryl-containing maytansine compounds. Stimuli-responsive linkers may include trans-cyclooctene linkers or thioether-containing linkers. The enzyme-cleavable linker can include GPLGOAGQ (SEQ ID NO:89), GDEVEAPKGC (SEQ ID NO:90), citrulline-valine, a glycosidase-cleavable linker, a β-glucuronidase-cleavable linker, a β-galactosidase-cleavable linker, a phosphatase-cleavable linker, a pyrophosphate-containing linker, a dipeptide-containing linker, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Phe-Lys-PABC (p-aminobenzylcarbamate), a Val-Cit-PABC-containing linker, a Glu-Val-Cit-containing linker, or a Val-Ala-containing linker.

[0127] In one embodiment, T is a targeting molecule selected from a bioactive molecule, an amphiphilic polymer, an aptamer, a peptide, a protein, a polysaccharide, a polyunsaturated fatty acid or a carbohydrate. The selection of the targeting molecule depends on the cells and / or tissues to which the self-assembled nanomaterial is to be delivered.

[0128] In any aspect or embodiment described herein, non-limiting examples of targeting molecules include: (i) small molecules, such as folic acid, thiamine and dimercaptosuccinic acid; (ii) proteins, such as bovine serum albumin (BSA), transferrin, antibodies, nanobodies, lectins, cytokines, fibrinogen and thrombin; (iii) polysaccharides, such as hyaluronic acid, chitosan, dextran, oligosaccharides and heparin; (iv) polyunsaturated fatty acids, such as palmitic acid and phospholipids; (v) targeting molecules for infection of cell targeting molecules / tissues, such as RGD, c(CMGRC) (SEQ ID NO: 17), PHSRN (SEQ ID NO: 18), LHRD (SEQ ID NO: 19), antigenic peptides, internalization peptides, cell penetrating peptides, VP22, RPRAPARSASRPRRPVE (SEQ ID NO: 20), sC18, GLRKRLRKFRNKIKEK (SEQ ID NO: 21), Pept1, and PLILLRLLRGQF (SEQ ID NO: 22). NO:22); (vi) targeting molecules for blood-brain barrier (BBB) ​​penetration, such as transferrin, OX26, CAQK (SEQ ID NO:23) and lactoferrin; (vii) tumor targeting molecules, such as F3, KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO:24), Lyp-1, CGNKRTRGC (SEQ ID NO:25), CREKA (SEQ ID NO:26), Bld-3, CSNRDARRC (SEQ ID NO:27), AHNP (SEQ ID NO:28), YCDGFYACYMDV (SEQ ID NO:29), SP204 (KQFSALPFNFYT peptide; SEQ ID NO:30), EGF, VEGF, LFA-1 and apolipoprotein AI; (viii) targeting molecules for infarcted cardiac tissue and / or atherosclerosis-related diseases, such as SP204 (KQFSALPFNFYT;(SEQ ID NO:30), PLGLAGGWGERDGS (SEQ ID NO:31), GGGGYDRVTIHPF (SEQ ID NO:32), VHSPNKK (SEQ ID NO:33), VHPKQHR (SEQ ID NO:34), VLTTGLPALISWIKRKRQQ (SEQ ID NO:35), NNSKSHT (SEQ ID NO:36), VHPKQHRAEEAK (SEQ ID NO:37), C*NNSKSHTC*C (SEQ ID NO:38), VHPK (SEQ ID NO:39), VHPKQHRGGSKGC (SEQ ID NO:40), Ab (429), antibodies specific for VCAM-1 (e.g., Ab M / K-2.7), PECAM-1, ICAM-1 (Ab R6.5), or LFA-1 integrin; (ix) white fat targeting molecules, such as SP204 and CKGGRAKDC (SEQ ID NO:41); (x) alveolar targeting molecules, such as WGA; (xi) intestinal targeting proteins, such as UEA-1; (xii) membrane dipeptidase targeting molecules, such as GFE and CGFECVRQCPERC (SEQ ID NO:42); (xiii) endoplasmic reticulum (ER) targeting molecules, such as KDEL (SEQ ID NO:43) peptide, SEKDEL (SEQ ID NO:44), Eriss and MRYMILGLLALAAVCSA (SEQ ID NO:45) peptide; (xiv) chondrocyte targeting peptides, such as RLDPTSYLRTFW (SEQ ID NO:46); (xv) cartilage targeting peptides, such as WYRGRL (SEQ ID NO:47); (xvi) mitochondrial membrane targeting molecules, such as RGD-4C-GG-D (KLAKLAK); 2 (SEQ ID NO:48), D-Arg-Dmt-Lys-Phe-NH 2 、Phe-D-Arg-Phe-Lys-NH 2 、D-Arg-Dmt-Orn-Phe-NH 2 、D-Arg-(2'6'-dimethylTyr)-Lys-Phe-NH 2, (1,7-bis-4-hydroxy-3-methoxyphenyl-1,6-heptadiene-3,5-dione)-triphenyl-phosphine, 1,5-dioctadecyl-L-glutamyl 2-histidyl-hexahydrobenzoic acid-SPC-L, MSVLTPLLLRGLTGSARRLPVPRAKIHWLC (SEQ ID NO: 49), GKRK (SEQ ID NO: 50), and D[KLAKLAK]2 (SEQ ID NO: 51);(xvii) nuclear targeting molecules, such as KKKRKV (SEQ ID NO: 52), KRPAATKKAGQAKKKKL (SEQ ID NO: 53), HIV-1TAT, GRKKRRQRRRPQ (SEQ ID NO: 54), R8, RRRRRRRR (SEQ ID NO: 55), penetrant peptide, RQIKIWFQNRRMKWKK (SEQ ID NO: 56), HA2 peptide, GDIMGEWGNEIFGAIAAGFLG (SEQ ID NO: 57), GALA (SEQ ID NO: 58), WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 59), Pas, FFLIPKG (SEQ ID NO: 60), THRPPMWSPWVWP (SEQ ID NO: 61), Angiopep2, TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 62), NO: 62), glutathione, (γE)CG, CDX, FKESWREARGTRIERG (SEQ ID NO: 63), chlorotoxin, MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR (SEQ ID NO: 64), MiniAP-4, c(DLATEPAL[Dap]) (SEQ ID NO: 65), g7, GFTGFLS (glucose) (SEQ ID NO:66), RV29, YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO:67), iRGD, CRGDKRGPDEC (SEQ ID NO:68), IL-13p, TAMRAVDKLLLHLKKLFREGQFNRNFESIIICRDRT (SEQ ID NO:69), CGEMGWVRC (SEQ IDNO:70), Lyp-1, c(CGNKRTRGC)(SEQ ID NO:25), DOPAC-MYIEALDKYAC-COOH(SEQ ID NO:71), Pro-Lys-Lys-Lys-Arg-Lys-Val (SEQ ID NO:72), Ala-Ala-Phe-Glu-Asp-Leu-Arg-Val-Leu-Ser (SEQ ID NO:73), and Lys-Arg-Pro-Ala-Ala-Thr-Lys-Lys-Arg-Gly-Qln-Arg-Lys-Lys-Lys-Lys (SEQ ID NO:74); (xviii) MMP targeting peptides, such as GPLGIAGQ (SEQ ID NO:75);(xix) transferrin receptor targeting peptides, such as THRPPMWSPVWP (SEQ ID NO: 76); (xx) synovial membrane targeting peptides, such as SFHQFARATLAS (SEQ ID NO: 77); (xxi) tumor-associated macrophages (TAMs) targeting peptides, such as YEQDPWGVKWWY (SEQ ID NO: 78), CSPGAKVRC (SEQ ID NO: 79); (xxii) regulatory T lymphocytes (Tregs) targeting peptides, such as CGNKRTRGC (SEQ ID NO: 25); (xxiii) myeloid-derived suppressor cells (MDSCs) targeting peptides, such as MEWSLEKGYTIK (SEQ ID NO: 80). ;

[0129] In any aspect or embodiment described herein, T is a biologically active molecule.

[0130] In any aspect or embodiment described in the present invention, T is a targeting molecule, and the self-assembled nanomaterial further comprises a bioactive molecule covalently or non-covalently attached to the self-assembled nanomaterial. In any aspect or embodiment described in the present invention, the self-assembled nanomaterial comprises a bioactive molecule non-covalently attached to (connected to) JBNT. In any aspect or embodiment described in the present invention, the bioactive molecule is at least partially encapsulated by JBNT. For example, in any aspect or embodiment described in the present invention, at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 80%, or 100% of the bioactive molecules are encapsulated by JBNT.

[0131] Non-limiting examples of bioactive agents include nucleic acids, proteins, polypeptides, and small molecule drugs. In one embodiment, the bioactive agent is a nucleic acid such as mRNA, guide RNA (gRNA or sgRNA), crRNA, tracrRNA, tRNA, ssDNA, dsDNA, cDNA, or a combination thereof.

[0132] In any aspect or embodiment described herein, R 2It is a wrapping material comprising a polymer, peptide, polypeptide, lipid-based material or biomimetic membrane. Wrapping materials can help protect the self-assembled nano material from specific or nonspecific removal of cells and / or organs from the body. In any aspect or embodiment described in the present invention, the polymer wrapping material includes but is not limited to polyethylene glycol (PEG), chitosan, hyaluronic acid, poloxamer, polyvinyl alcohol, polysaccharide, neutrally charged poly (amino acid), negatively charged poly (amino acid) or its combination. In any aspect or embodiment described in the present invention, peptide encapsulation materials include but are not limited to "self" peptides (peptides produced by proteolytic degradation of self proteins in cells expressing MHC class I or class II molecules, such as TCR peptides-MHC class II peptides), antithrombotic peptides (e.g., CD-31 agonist peptides), CALNN (SEQ ID NO: 81), CCVVT (SEQ ID NO: 82), CLPFFD (SEQ ID NO: 83), (γE) C (γE) C (γE) CG (SEQ ID NO: 84), GCGGCGGKGGCGGCG (SEQ ID NO: 85), GNYTCEVTELTREGETIIELK (SEQ ID NO: 86), hexahistidine, or a combination thereof. In any aspect or embodiment described in the present invention, polypeptide (protein) encapsulation materials include but are not limited to phytochelatins, GCK15, PD-L1, CD47, CD24, β-2-microglobulin, bovine serum albumin (BSA), hydrophobic protein, clusterin / ApoJ, fibrinogen, or a combination thereof. In any aspect or embodiment described in the present invention, lipid-based encapsulating materials include, but are not limited to, natural waxes (e.g., carnauba wax, candelilla wax, rice bran wax, beeswax), petroleum-based waxes (e.g., paraffin and polyethylene wax), petroleum-based oils, mineral oils, vegetable oils, acetylated glycerides, fatty acids, resins (e.g., shellac and wood rosin), or combinations thereof. In any aspect or embodiment described in the present invention, biomimetic membrane encapsulating materials include, but are not limited to, red blood cell (RBC) membranes, white blood cell (WBC) membranes, cancer cell membranes, mesenchymal stem cell membranes, platelet membranes, beta cell membranes, or combinations thereof. Combinations comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or more) of the aforementioned encapsulating materials may also be used.

[0133] In any aspect or embodiment described in the present invention, the self-assembled nanomaterial may include a single type of JBNT, or may include more than one JBNT. In any aspect or embodiment described in the present invention, the self-assembled nanomaterial is co-assembled by more than one (e.g., multiple) JBNTs, and each (JBNT) has different properties from each other. For example, in any aspect or embodiment described in the present invention, different types of JBNTs may have properties that increase the hydrophobicity, stability and / or self-assembly of the self-assembled nanomaterial. These properties can be designed to affect cell delivery, including proton sponge effect, in vivo circulation time, passive or active targeting, subcellular targeting, improved cellular uptake and / or enhanced endosomal escape of self-assembled nanomaterials.

[0134] In any aspect or embodiment described herein, the Janus-based nanotubes include at least one compound represented by Formula I to Formula IV.

[0135] In any aspect or embodiment described herein, the Janus-based nanotube comprises at least one compound represented by Formula V to Formula VIII.

[0136] In any aspect or embodiment described herein, the Janus-based nanotubes include at least one compound represented by Formula IX to Formula XIII.

[0137] In any aspect or embodiment described herein, the Janus-based nanotubes include the following combination:

[0138] Compounds represented by Formula I to Formula IV and compounds represented by Formula V to Formula VIII;

[0139] Compounds represented by Formula I to Formula IV and compounds represented by Formula IX to Formula XIII;

[0140] Compounds represented by Formula V to Formula VIII and compounds represented by Formula IX to Formula XIII;

[0141] Compounds represented by Formula I to Formula IV, compounds represented by Formula V to Formula VIII, and compounds represented by Formula IX to Formula XIII; or

[0142] A combination of them.

[0143] In any aspect or embodiment described herein, the total amount of JBNT present in the self-assembled nanomaterial ranges from 0.1 weight % (wt%) to 99.9 wt%, or from 1 wt% to 90 wt%, based on the total weight of the self-assembled nanomaterial. In any aspect or embodiment described herein, the total concentration of JBNT in the self-assembled nanomaterial ranges from 1 microgram / milliliter (μg / mL) to 1 gram / milliliter (g / mL).

[0144] In any aspect or embodiment described herein, the self-assembled nanomaterial is in the form of a fibril having an average diameter of 5 nm to 500 nm, or 5 nm to 250 nm, or 10 nm to 100 nm, or 20 nm to 60 nm, and an average length of 75 nm to 100 mm, or 100 nm to 10 mm, or 100 nm to 5 mm, or 100 nm to 1 mm, or 125 nm to 500 nm.

[0145] In any aspect or embodiment of the present invention, the pH value of the self-assembled nanomaterial is 1 to 10.

[0146] In any aspect or embodiment described in the present invention, the self-assembled nanomaterial can have a single or multiple compartment structure. As used in the present invention, in any aspect or embodiment described in the present invention, the single-chamber nanomaterial includes a single group of self-assembled nanomaterials, i.e., a single type of JBNT and one or more ECM molecules attached to the JBNT. As used in the present invention, in any aspect or embodiment described in the present invention, the multi-chamber nanomaterial includes two or more groups of self-assembled nanomaterials that are assembled layer by layer by electrostatics to form a multi-chamber structure. For example, in any aspect or embodiment described in the present invention, the opposite electrostatic charges on the first group and the second group of JBNTs can drive the assembly of the multi-chamber nanomaterial. By assembling the first group and the second group in sequence, one group will form an internal compartment and the second group will form an external compartment.

[0147] In any aspect or embodiment described in the present invention, the self-assembled nanomaterial can be selectively combined with extracellular matrix (ECM) molecules. In any aspect or embodiment described in the present invention, the self-assembled nanomaterial described in the present invention is an adjustable material including Janus-based nanotubes and ECM. In any aspect or embodiment described in the present invention, JBNT can be assembled with different ECMs to form different nanomatrix materials, such as scaffolds, for different cells / tissues. In any aspect or embodiment described in the present invention, these nanomatrix materials can have a single or multiple compartment structures. In any aspect or embodiment described in the present invention, the nanomatrix material can be made to have multifunctional layers or compartments to achieve various functions (e.g., supporting cell growth, drug release). In any aspect or embodiment described herein, ECM molecules include, but are not limited to, hydroxyapatite, fibronectin, Matn1, MAtn3, laminin, collagen (e.g., type I collagen, type II collagen), elastin, vitronectin, fibrillin, perlecan, fibrinogen, osteonectin, tenascin, thrombospondin, intercellular adhesion molecules (ICAM1-5), integrins, proteoglycans (aggrecan), glycosaminoglycans (e.g., hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate), glycoproteins, or combinations thereof.

[0148] In any aspect or embodiment described herein, the weight ratio of JBNT to ECM molecules is from 1000:1 to 1:1.

[0149] In any aspect or embodiment described herein, the encapsulating material comprises polyethylene glycol (PEG). In any aspect or embodiment described herein, the encapsulating material comprises PEG and the self-assembling nanomaterial comprises a targeting molecule.

[0150] In any aspect or embodiment described herein, JBNP or JBNT comprises a targeting molecule comprising the amino acid sequence WYRGRL and miR140 (WYRGRL-JBNP-miR140). In any aspect or embodiment described herein, WYRGRL-JBNP-miR140 is used and effectively treats, prevents or ameliorates one or more symptoms of osteoarthritis.

[0151] In any aspect or embodiment described herein, JBNP or JBNT comprises CRISPR targeting sgIL-1R (JBNP-CRISPR-sgIL-1R). In any aspect or embodiment described herein, JBNP-CRISPR-sgIL-1R is used to treat, prevent or improve one or more symptoms of osteoarthritis, such as inflammation (e.g., knee inflammation).

[0152] In any aspect or embodiment described herein, JBNP or JBNT comprises IL-1RA mRNA (JBNP-IL-1RAmRNA). In any aspect or embodiment described herein, JBNP-IL-1RAmRNA is used and effectively treats, prevents or ameliorates one or more symptoms of osteoarthritis, such as inflammation (e.g., knee inflammation). In any aspect or embodiment described herein, the mRNA sequence of IL-1RA is: (SEQ ID NO: 87).

[0153] In any aspect or embodiment described herein, JBNP or JBNT comprises IL-1RA peptide mRNA (JBNP-IL-1RA peptide mRNA). In any aspect or embodiment described herein, JBNP-IL-1RA peptide mRNA is used and effectively treats, prevents or improves one or more symptoms of osteoarthritis, such as inflammation (e.g., knee inflammation). In any aspect or embodiment described herein, the mRNA sequence of IL-1RA peptide is: ATGCGGCCTAGCGGCAGAAAGTCTAGCAAGATGCAGGCCTTTAGAATCTGGGACGTTAATCAGAAAACCTTCTACCTCAGAAACAACCAGCTGGTCGCCGGCTACCTGCAGGGCCCCAACGTGAATCTGGAAGAGAAGATCGACGTGGTGCCAATCGAGCCTCACGCCCTGTTTCTGGGCATCCACGGCGGAAAAATGTGCCTGTCTTGTGTGAAGT CCGGAGATGAGACAAGACTGCAACTGGAAGCCGTGAACATTACAGACCTGAGCGAGAACCGGAAGCAGGACAAGCGGTTCGCCTTCATCAGAAGCGACAGCGGCCCTACAACCAGCTTCGAGAGCG CCGCTTGCCCCGGCTGGTTTCCTGTGCACCGCCATGGAAGCTGATCAGCCTGTGTCCCTGACCAACATGCCTGATGAAGGCGTGATGGTGACCAAGTTCTACTTCCAGGAGGACGAGATGTGA(SEQ ID NO:88).

[0154] In any aspect or embodiment described herein, the JBNT or JBNP comprises lysine or arginine side chains. In any aspect or embodiment described herein, the JBNT or JBNP comprises lysine or arginine side chains to target the self-assembling nanomaterial to the liver.

[0155] In any aspect or embodiment described herein, the JBNT or JBNP comprises histone side chains. In any aspect or embodiment described herein, the JBNT or JBNP comprises histidine side chains to target the self-assembling nanomaterial to organs other than the liver.

[0156] In any aspect or embodiment described herein, the JBNT or JBNP comprises a cartilage targeting molecule, such as WYRGRL or RLDPTSYLRTFW. In any aspect or embodiment described herein, the JBNT or JBNP comprises a cartilage targeting molecule, such as WYRGRL or RLDPTSYLRTFW, for use in a method of delivering the JBNT or JBNP to cartilage.

[0157] In any aspect or embodiment described herein, the JBNP comprises an mRNA agent, optionally with a targeting molecule, and optionally with a reporter or tag (e.g., tdTomato mRNA, Cy5, AF488, etc.). In any aspect or embodiment described herein, the JBNP comprises an mRNA agent (JBNP-mRNA), optionally with a targeting molecule, and optionally with a reporter / tag (e.g., tdTomato mRNA, Cy5, AF488, etc.), which can be delivered to the liver, kidney, brain, lung, spleen, lymph node, bone, muscle, heart, pancreas, intestine, solid tumor, or a combination thereof.

[0158] In any aspect or embodiment described herein, the JBNP comprises Cas9 mRNA and sgRB1. In any aspect or embodiment described herein, the JBNP comprising Cas9 mRNA and sgRB1 is used in a method of delivering sgRB1 to (and editing) hepatocytes.

[0159] In any aspect or embodiment described herein, the JBNP comprises CRISPR (e.g., Cas9 mRNA and gRNA) and a tumor targeting fragment. In any aspect or embodiment described herein, the JBNP comprising CRISPR (e.g., Cas9 mRNA and gRNA) and a tumor targeting fragment is used in a method of delivering and editing tumor cell DNA.

[0160] In any aspect or embodiment described herein, when administered, JBNT and JBNP do not induce acute toxicity, innate immune response, adaptive immune response, or a combination thereof.

[0161] In any aspect or embodiment of the invention, the side chain of JBNT is modified to promote cell renewal of a specific type. For example, in any aspect or embodiment of the invention, lysine-based JBNT is used to promote cellular uptake of JBNP via micropinocytosis. By further example, in any aspect or embodiment of the invention, arginine-based JBNT is used to promote cellular uptake of JBNP via clathrin-mediated endocytosis.

[0162] In any aspect or embodiment described herein, the JBNP comprises a lysine-based JBNT, which appears to have significantly better endosomal escape due to the proton sponge effect.

[0163] In any aspect or embodiment described herein, the JBNP comprises an arginine-based JBNT, which appears to have significantly enhanced endosomal escape due to a pore-forming effect to escape early endosomes.

[0164] In any aspect or embodiment described herein, the JBNP of the present invention has lower cytotoxicity than lipid nanoparticles, polymer nanoparticles (e.g., poly-L-lysine (PLL) nanoparticles or polyethyleneimine (PEI) nanoparticles), single-walled nanotubes, or a combination thereof.

[0165] In any aspect or embodiment described herein, the JBNP comprises Cas9mRNA-eGFP and gRNA-ATTO550.

[0166] Pharmaceutical composition

[0167] The self-assembling nanomaterial disclosed in the present invention can be formulated as an injectable composition. In any aspect or embodiment described in the present invention, the injectable composition includes the self-assembling nanomaterial and a pharmaceutically acceptable carrier. In any aspect or embodiment described in the present invention, the self-assembling nanomaterial can be administered parenterally in a sterile medium in the form of a sterile injectable aqueous or oily suspension, subcutaneously, intravenously, intramuscularly, intrasternally or by infusion technology. Advantageously, in any aspect or embodiment described in the present invention, an adjuvant (e.g., a local anesthetic, a preservative and a buffer) can be dissolved in a carrier.

[0168] The pH of the pharmaceutical composition including the self-assembling nanomaterial may be physiological pH.

[0169] The present invention is further illustrated by the following non-limiting examples.

[0170] The present invention is further illustrated by the following non-limiting examples.

[0171] Example

[0172] Materials: All reagents and solvents disclosed in the present invention were obtained from commercial suppliers and used without further purification. Commercial suppliers include Sigma-Aldrich, Alfa Aesar, Fisher Scientific, and Thermo Fisher.

[0173] Example 1

[0174] The preparation method of the designated compound ArgJBNT is as follows.

[0175]

[0176] Synthesis of compound A3. The previously synthesized compounds A1 (96 mg) and A2 (154 mg) were dissolved in 1,2-dichloroethane (DCE, 4 mL). N,N-diisopropylethylamine (DIPEA, 78 μL) was added to the reaction mixture and stirred at room temperature for 15 min. Solid NaBH(OAc) was then added to the solution. 3 (49 mg). The resulting slurry was then stirred at room temperature for 24 hours. After completion of the reaction, the reaction mixture was quenched with water and extracted with dichloromethane. The organic layers were combined and washed with brine. MgSO 4 After drying, filtration and evaporation of the solvent under reduced pressure, the crude product A3 (156 mg) was obtained, which was used in the next step without further purification.

[0177] Synthesis of Compound ArgJBNT. Compound A3 (156 mg) was added to a 94% TFA / methylphenyl sulfide (2.8 mL) solution. After stirring at room temperature for 72 hours, diethyl ether (Et 2 O). A white precipitate was formed and then centrifuged. After the supernatant was poured off, Et 2 O to wash the white precipitate to obtain a crude product. The crude product was purified by HPLC to obtain compound ArgJBNT (37 mg, 70%). 1H NMR (500 MHz, DCl / D2O) δ 4.62–4.43 (m, 2H), 4.11 (dd, J=7.4, 4.8 Hz, 1H), 3.58–3.45 (m, 2H), 3.26–3.19 (m, 2H), 3.03 (s, 3H), 2.07–1.94 (m, 2H), 1.80–1.60 (m, 2H). HRMS (ESI) [M+H]+ calculated value is 409.2055, found at 409.2078.

[0178] Reaction Scheme 1 illustrates the formation of compound ArgJBNT.

[0179] Solution 1

[0180]

[0181] Example 2

[0182] The preparation method of the designated compound GlyJBNT is as follows.

[0183]

[0184] Synthesis of compound A5. The previously synthesized compound A1 (96 mg) and the commercially available compound A4 (40 mg) were dissolved in 1,2-dichloroethane (DCE, 4 mL). N,N-diisopropylethylamine (DIPEA, 78 μL) was added to the reaction mixture and stirred at room temperature for 15 min. Then, solid NaBH(OAc) was added to the solution. 3 (49 mg). The resulting slurry was then stirred at room temperature for 24 hours. After completion of the reaction, the reaction mixture was quenched with water and extracted with dichloromethane. The organic layers were combined and washed with brine. MgSO 4 After drying, filtration and evaporation of the solvent under reduced pressure, the crude product A5 (102 mg) was used in the next step without further purification.

[0185] Synthesis of compound GlyJBNT. Compound A5 (102 mg) was added to a 94% TFA / methylphenyl sulfide (2.8 mL) solution. After stirring at room temperature for 72 hours, diethyl ether (Et 2 O). A white precipitate was formed and then centrifuged. After the supernatant was poured off, Et 2 The white precipitate was washed with 0 to obtain a crude product. The crude product was purified by HPLC to obtain compound GlyJBNT (34 mg, 85%). 1 H NMR(500MHz,DMSO-d6)δ11.83(br,m,1H),8.88(br,m,2H),8.54(br,m,1H),8.13(br,m,1H),7.7 0(br,m,1H),4.34(br,m,2H),3.88(br,m,2H),3.26(br,m,2H),2.93(br,m,3H).HRMS(ESI)[M+H] + Calculated at 310.2158, found at 310.1208.

[0186] Reaction Scheme 2 illustrates the formation of compound GlyJBNT.

[0187] Solution 2

[0188]

[0189] Example 3

[0190] The preparation method of the designated compound AspJBNT is as follows.

[0191]

[0192] Synthesis of compound A7. The previously synthesized compound A1 (96 mg) and the commercially available compound A6 (68 mg) were dissolved in 1,2-dichloroethane (DCE, 4 mL). N,N-diisopropylethylamine (DIPEA, 78 μL) was added to the reaction mixture and stirred at room temperature for 15 min. Then, solid NaBH(OAc) was added to the solution. 3 (49 mg). The resulting slurry was then stirred at room temperature for 24 hours. After completion of the reaction, the reaction mixture was quenched with water and extracted with dichloromethane. The organic layers were combined and washed with brine. MgSO 4 After drying, filtration and evaporation of the solvent under reduced pressure, the crude product A7 (110 mg) was used in the next step without further purification.

[0193] Synthesis of Compound AspJBNT Compound A7 (110 mg) was added to a 94% TFA / methylphenyl sulfide (2.8 mL) solution. After stirring at room temperature for 72 hours, diethyl ether (Et 2 O). A white precipitate was formed and then centrifuged. After the supernatant was poured off, Et 2 The white precipitate was washed with 0 to obtain a crude product. The crude product was purified by HPLC to obtain compound AspJBNT (47 mg, 98%). 1 H NMR(500MHz,DMSO-d6)δ11.77(br,m,1H),8.51(br,m,1H),8.06(br,m,1H),7.55(br,m, 1H),4.34(br,m,2H),4.16(br,m,1H),2.92(br,m,3H),2.85(br,m,2H).HRMS(ESI)[M+H] + Calculated at 368.1313, found at 368.1278.

[0194] Reaction Scheme 3 illustrates the formation of compound AspJBNT.

[0195] Solution 3

[0196]

[0197] Example 4

[0198] The preparation method of the designated compound HisJBNT is as follows.

[0199]

[0200] Synthesis of compound A9. The previously synthesized compound A1 (96 mg) and the commercially available compound A8 (56 mg) were dissolved in 1,2-dichloroethane (DCE, 4 mL). N,N-diisopropylethylamine (DIPEA, 100 μL) was added to the reaction mixture and stirred at room temperature for 15 min. Then, solid NaBH(OAc) was added to the solution. 3 (49 mg). The resulting slurry was then stirred at room temperature for 24 hours. After completion of the reaction, the reaction mixture was quenched with water and extracted with dichloromethane. The organic layers were combined and washed with brine. MgSO 4 After drying, filtration and evaporation of the solvent under reduced pressure, the crude product A9 (17 mg) was used in the next step without further purification.

[0201] Synthesis of compound HisJBNT. Compound A7 (17 mg) was added to a 94% TFA / methylphenyl sulfide (0.44 mL) solution. After stirring at room temperature for 72 hours, diethyl ether (Et 2 O). A white precipitate was formed and then centrifuged. After the supernatant was poured off, Et 2 The white precipitate was washed with 0 to obtain a crude product. The crude product was purified by HPLC to obtain compound HisJBNT (8 mg, 14%). 1 HNMR(500MHz,DMSO-d6)δ11.77(br,m,1H),8.51(br,m,1H),8.06(br,m,1H),7.88(br,m,1H),7.55(br,m, 1H),7.14(br,m,1H),4.34(br,m,2H),4.16(br,m,1H),2.92(br,m,3H),2.85(br,m,2H).HRMS(ESI)[M+H] + Calculated at 390.1633, found at 390.1601.

[0202] Reaction Scheme 4 illustrates the formation of compound HisJBNT.

[0203] Solution 4

[0204]

[0205] Example 5

[0206] The preparation method of the designated compound PEGLysJBNT is as follows.

[0207]

[0208] Synthesis of compound PEGLysJBNT. 3A solution of the synthesized LysJBNT (1 mg) in aqueous solution (0.8 mL, 0.01 M) was added to a DMF solution (0.2 mL) of MS(PEG)24 methyl-PEG-NHS-ester (5 mg). After 24 hours, the reaction mixture was purified by HPLC to obtain the compound PEGLysJBNT (1 mg, 33%). HRMS (ESI) [M+H] + Calculated at 1479.8390, found at 1479.8654.

[0209] Reaction Scheme 5 illustrates the formation of compound PEGLysJBNT.

[0210] Solution 5

[0211]

[0212] Example 6

[0213] The preparation method of the designated compound LPLysJBNT is as follows.

[0214]

[0215] Synthesis of compound LPLysJBNT. 3 A solution of previously synthesized LysJBNT (1 mg) in aqueous solution (0.8 mL, 0.01 M) was added to a DMF solution (0.2 mL) of N-α-maleimidomethyl succinimide ester (AMAS, 1 mg). After 24 hours, the peptide RLDPTSYLRTFWC (7 mg) targeting human collagen was added to the reaction mixture and reacted for another 24 hours. The reaction mixture was purified using HPLC to give the compound LPLysJBNT (5 mg, 100%). HRMS (ESI) [M+2H] 2+ Calculated as 1095.5226, found at 1095.5038.

[0216] Reaction Scheme 6 illustrates the formation of compound LPLysJBNT.

[0217] Solution 6

[0218]

[0219] Example 7

[0220] The preparation method of the designated compound SPLysJBNT is as follows.

[0221]

[0222] Synthesis of compound SPLysJBNT. 3A solution of previously synthesized LysJBNT (1 mg) in aqueous solution (0.8 mL, 0.01 M) was added to a DMF solution (0.2 mL) of N-α-maleimidomethyl succinimide ester (AMAS, 1 mg). After 24 hours, the targeting peptide WYRGRLC (4 mg) targeting human collagen was added to the reaction mixture and waited for another 24 hours. The reaction mixture was purified using HPLC to obtain the compound SPLysJBNT (3 mg, 100%). HRMS (ESI) [M+3H] 3+ Calculated at 490.8984, found at 490.8820.

[0223] Reaction Scheme 7 illustrates the formation of compound SPLysJBNT.

[0224] Solution 7

[0225]

[0226] Example 8: Multifunctional Janus-based Nanoparticles (JBNP)

[0227] Multifunctional JBNT. Multifunctional JBNT was combined at appropriate molar ratios at room temperature for 24 h to promote the self-assembly of JBNT. To prepare JBNT composed of PEGLysJBNT and ArgJBNT, the molar ratio of PEGLys / ArgJBNT was 0.05:1. To prepare JBNT composed of peptide-LysJBNT, PEGLysJBNT and ArgJBNT, the molar ratio of peptide-LysJBNT / PEGLys / ArgJBNT was 0.05:0.05:1.

[0228] Multifunctional JBNP. Multifunctional JBNP was prepared by mixing cargo molecules (e.g., RNA, bioactive small molecules, proteins) with multifunctional JBNT in an appropriate molar ratio in nuclease-free water, and then sonicating at 100% amplitude for 2 minutes and 30 seconds at room temperature using a sonicator. The concentrations of the different components are as follows: c(JBNT) = 1 mg / mL; c(siRNA) = 5 μM; c(mRNA) = 0.05 mg / mL. c(protein) = 0.1 mg / mL. The ratio (volume ratio) of each cargo: (4:1 ratio (Arg-JBNT: siRNA), (20:1 ratio Arg-JBNT: mRNA), (3:1 Arg-JBNT: protein).

[0229] Arg-JBNP-siRNA was assembled by mixing Arg-JBNT (1 mg / ml) and siRNA-AF488 (5 uM) in nuclease-free water at a volume ratio of 4:1. Arg-JBNP-mRNA was assembled by mixing Arg-JBNT (1 mg / ml) with Cas9mRNA (0.05 mg / ml) at a volume ratio of 20:1. Arg-JBNT-albumin was assembled by mixing Arg-JBNT (1 mg / ml) with albumin (0.1 mg / ml) at a volume ratio of 3:1. The mixture of Arg-JBNT and cargo in nuclease-free water was then sonicated with a sonicator at an amplitude of 100% for 2 minutes and 30 seconds.

[0230] JBNP characterization. Dynamic light scattering (Zetasizer) was used to measure the particle size and zeta potential of JBNP, and the morphology was observed by transmission electron microscopy (TEM). Gel retardation test was performed using 0.8% low melting point agarose gel followed by electrophoresis. UV-Vis absorption spectra were recorded using NanoDropOne. The buffering capacity of JBNP with polymer and JBNP with cationic polymer at the same 0.08 μmol was titrated by adding 2 μL of 10 mM HCl or 10 mM NaOH.

[0231] Fluorescence imaging was performed using a Nikon A1 confocal laser scanning microscope.

[0232] Example 9: Characterization and testing

[0233] Lys-JBNP delivery. The assembled Lys-JBNP (30 μg / ml) was transfected into C28 / I2 cells and then incubated at 37°C and 5% CO 2 The cells were then fixed with 4% formaldehyde and incubated with Triton TM X-100 treatment, and then labeled with rhodamine phalloidin (30 min) and stained with DAPI (10 min). 24 h or 48 h after transfection into cells, siRNA- 488 (AllStars Neg. siRNA488, Qiagen) was uptaken. For siRNA knockdown studies, GAPDH siRNA (On-TARGETplus Human GAPDH siRNA, Horizon Discovery) was delivered using assembled Lys-JBNP (30 μg / ml) for 24 h. Lipofectamine TM 2000 (Invitrogen) was used as a control. The gene expression of GAPDH was analyzed by RT-PCR.

[0234] Endosomal escape studies. For endosomal escape studies, Lysotracker was added to cells prior to fixation with 4% formaldehyde. TM Lysotracker was quantified based on the Pearson correlation coefficient (R) using Image J software, following the colocalization threshold and the coloc2 plugin. TM The extent of colocalization of Red and JBNP.

[0235] Cellular uptake mechanism studies. C28 / I2 human chondrocytes were exposed to several different inhibitors for 1 h, specifically, chlorpromazine hydrochloride (Cpz) (100 μM, 30 min), methyl-β-cyclodextrin (Mβcd, 1 mM, 30 min), cytochrome D (CytD, 4 μM, 1 h), erythromycin A (2 μM, 30 min), bafilomycin A1 (200 nM, 30 min), and chloroquine (10 μM, 30 min).

[0236] Antiviral study: Adenovirus expressing GFP and modified with RGD fibers was added to human lung fibroblasts. JBNP or LNP containing eGFP siRNA was exposed to infected cells for 24 h, and cell images were obtained using a fluorescence microscope.

[0237] Results of Examples 1-9

[0238] JBNT successfully delivered RNA with enhanced endosomal escape and high biocompatibility. Figure 1A As shown, the targeting fragment (peptide) is coupled to JBNT and co-assembled with JBNT to provide a targeting peptide-JBNT. In particular, JBNT and the targeting peptide are combined under conditions that promote binding of the peptide to JBNT. Typically, the binding of the targeting peptide is covalent. The JBNT containing the targeting peptide is contacted with a biologically active molecule (e.g., a nucleic acid (e.g., siRNA), a protein and / or a small molecule drug). The structure of an exemplary targeting peptide-JBNT is shown in Figure 1B shown.

[0239] By optimizing the molar ratio of JBNT, targeting peptide and bioactive molecule, the following can be obtained: Figure 1C The targeting peptide-JBNT shown is (i) LysJBNT ii) RLDPTSYLRTFWC peptide-PEGLysJBNT iii) LysJBNP iv) RLDPTSYLRTFWC peptide-PEG LysJBNP), and has Figure 1D Absorbance characteristics shown.

[0240] like Figures 2A-2HAs shown, JBNPs include bioactive molecules, such as siRNA, proteins and / or small molecule drugs, as cargo and can be used to deliver the bioactive molecules to target cells in vitro or in vivo. Figure 2G-2H The effects of various inhibitors on JBNP uptake by target cells are shown. 3 Cells treated with either , red sea sponge A (Lat), or cytochalasin D (CytD) demonstrated reduced uptake of JBNP as evidenced by decreased intracellular fluorescence.

[0241] Figures 3A-3E The successful delivery of small RNA, mRNA and albumin (protein) using ArgJBNT was shown. Figure 3A TEM images of Arg-JBNP containing miR140 (Arg-JBNP-miR140), Cas9 mRNA (Arg-JBNP-Cas9 mRNA), and albumin (Arg-JBNP-albumin). Figure 3B The results in are based on the analysis of TEM image data and show the average size and width of Arg-JBNP-miRNA. Figure 3C and 3E The zeta potential analysis of Arg-JBNP-miR140 and Arg-JBNP-Cas9 mRNA respectively. Figure 3D shows the results of the gel retardation assay, Figure 3F Shown are CLSM z-stack images of siRNA-AF488 delivered by Arg-JBNPs.

[0242] Figure 3G Figure 2 is a flow cytometric analysis showing cellular uptake of siRNA-AF488. Figure 3H For Arg-JBNP or Fluorescence images of 200 delivered Cy5.5-eGFP-mRNA. Fig. 3I Fluorescence images of BSA-AF488 delivered by Arg-JBNP. Figure 3J A series of flow cytometry images showing the time-dependent uptake of Cas9eGFP.

[0243] The mRNA containing the GFP sequence encoding Cas9 upstream (see Figure 3K ) was added to Arg-JBNP to generate Arg-JBNP-eGFP-Cas9mRNA. Figure 3K Fluorescence images of cells taking up Arg-JBNP-eGFP-Cas9 mRNA are shown in FIG. Figure 3L Time-dependent delivery of Arg-JBNP-eGFP-Cas9 mRNA detected by flow cytometry is shown in FIG.

[0244] The study also showed that JBNPs have enhanced endosomal escape properties compared to lipid nanoparticles and excel in delivering cargo bioactive molecules. Figures 4A-4G middle.

[0245] In addition, if Figures 5A-5H As shown, JBNPs modified with PEG can be protected from nonspecific cellular uptake by undesired cell types. Figure 5A The nanotube structure of PEGLysJBNT in TEM is shown. Figure 5B and C show the comparison of different ratios (0, 0.01:1, 0.02:1, 0.05:1) of PEGLysJBNT / ArgJBNT in UV-Vis. Figure 5D Comparison of different ratios of PEGLysJBNT / ArgJBNT / siRNA in TEM is shown. Figure 5E Comparison of different ratios of PEGLysJBNT / ArgJBNT / siRNA in UV-Vis is shown. Fig. 5F A comparison of different ratios of PEGLysJBNT / ArgJBNT / siRNA (0, 0.01:1:0.33, 0.02:1:0.33, 0.05:1:0.33) by zeta potential using a zetasizer is shown. Figure 5G In this study, we transfected Arg-JBNP or PEG-JBNP for 24 h and then performed flow cytometry. Figure 5H and 5I Flow cytometry analysis is shown. PEGylation is to prevent nonspecific cellular uptake (eg, immune cells and other unwanted cells), and then we want to add targeting peptides to the PEG so that we can achieve active targeting.

[0246] The study also showed that combining multiple JBNTs with different functions (i.e., multifunctional JBNTs) provides JBNTs with multifunctional properties. For example, the PEG effect and the targeting ability of JBNPs can be combined (see Figures 6A-6K ). It is also possible to combine more positively charged JBNTs, such as arginine-JBNT, to improve cellular uptake efficiency (see 3F-3L). Another example could be combining an enhanced endosomal escape effect with JBNT. This can be achieved by combining histidine-JBNT, which has excellent proton sponge effect capabilities (see Figure 4D ).

[0247] Example 10: Detection of the ability of cartilage-targeted JBNP-IL-1RA mRNA to inhibit knee osteoarthritis-related inflammation

[0248] The severity of osteoarthritis (OA) was assessed by MANKIN score, and then treatment (saline, JBNP-IL-1RA mRNA, JBNP-scrambled mRNA, JBNP without mRNA) was administered by intra-articular injection to mice with surgical destabilization of the medial meniscus (DMM) model (3-month-old males) 1 month after surgery. Safranin-O staining was performed on tissue sections and detected by fluorescence microscopy. The following behavioral studies were performed to detect the effect of treatment: Y-Maze spontaneous changes, open field test, object recognition test and conditioned fear. An exemplary non-limiting example of IL-1RA mRNA sequence is: (SEQ ID NO: 87).

[0249] Example 11: Detection of the ability of cartilage-targeted JBNP-IL-1RA peptide mRNA to inhibit inflammation associated with knee osteoarthritis

[0250] The severity of OA was assessed by MANKIN score, and then treatment (saline, JBNP-IL-1RA peptide mRNA, JBNP-scrambled mRNA, JBNP without mRNA) was administered to mice with surgical destabilization of the medial meniscus (DMM) model (3 months old) by intra-articular injection 1 month after surgery. IL-1RA peptide is an interleukin-1 receptor antagonist peptide. Safranin-O staining was performed on tissue sections and detected by fluorescence microscopy. The following behavioral studies were performed to examine the treatment effects: Y-Maze spontaneous changes, open field test, object recognition test and conditioned fear. An exemplary, non-limiting example of an mRNA sequence for an IL-1RA peptide is: ATGCGGCCTAGCGGCAGAAAGTCTAGCAAGATGCAGGCCTTTAGAATCTGGGACGTTAATCAGAAAACCTTCTACCTCAGAAACAACCAGCTGGTCGCCGGCTACCTGCAGGGCCCCAACGTGAATCTGGAAGAGAAGATCGACGTGGTGCCAATCGAGCCTCACGCCCTGTTTCTGGGCATCCACGGCGGAAAAATGTGCCTGTCTTGTGTGAAGTCCGG AGATGAGACAAGACTGCAACTGGAAGCCGTGAACATTACAGACCTGAGCGAGAACCGGAAGCAGGACAAGCGGTTCGCCTTCATCAGAAGCGACAGCGGCCCTACAACCAGCTTCGAGAGCGCCGCTTGCCCCGGCTGGTTCCTGTGCACCGCCATGGAAGCTGATCAGCCTGTGTCCCTGACCAACATGCCTGATGAAGGCGTGATGGTGACCAAGTTCTACTTCCAGGAGGACGAGATGTGA (SEQ ID NO:88).

[0251] Example 12: Amino acid side chain modification can be used to target the self-assembled nanomaterials of the present invention to target organs

[0252] The protein corona of the self-assembled nanomaterials described in the present invention and the compositions comprising it can be further modified by side chain modification of amino acid residues. The assembled JBNP was incubated with mouse serum for 30 minutes. Then, centrifuged at a relative centrifugal force (RCF) of 13800 for 15 minutes at 4°C. The supernatant was discarded and the precipitate was washed three times with phosphate-buffered saline. The proteins were separated by SDS-PAGE and the protein bands were identified by liquid chromatography-tandem mass spectrometry (LC / MS / MS). Lysine and arginine side chains bind to the apolipoprotein E (APOE) family and are believed to target the self-assembled nanomaterials of the present invention to the liver. By further example, it is believed that the histidine side chains in the protein corona composition target the self-assembled nanomaterials of the present invention to organs other than the liver.

[0253] Example 13: The JBNP of the present invention actively targets cartilage and has a prolonged half-life when the cartilage targeting peptide WYRGRL is linked to JBNT

[0254] The cartilage targeting peptide WYRGRL (SEQ ID NO: 47) was linked to JBNP with a siRNA reporter, and the assembled WYRGRL-JBNP and siRNA dye were transfected into human chondrocyte C28 / I2 cells. Fluorescence activated cell sorting (FACS) was performed to quantify the targeting ability of WYRGRL-JBNP to cartilage. The JBNP of the present invention actively targets cartilage and has an extended half-life ( Figure 7B ).

[0255] Example 14: Testing the ability of cartilage-targeted ArgJBNP to deliver miR140 to delay or prevent osteoarthritis progression

[0256] Cartilage targeting can be achieved by, e.g., peptide WYRGRL To achieve. The severity of OA was assessed by MANKIN score, and then treatment (normal saline, JBNP-miR140, JBNP-scrambled miRNA, or JBNP without miR140) was administered to mice with surgical destabilization of the medial meniscus (DMM) model 1 month after surgery by intra-articular injection. The following behavioral studies were performed to detect the therapeutic effect: Y-Maze spontaneous changes, open field test, object recognition test and conditioned fear. DAPI staining, H&E staining and Safranin-O staining of tissue sections were used to demonstrate effective targeting and delivery by fluorescence microscopy.

[0257] Example 15: Testing the ability of cartilage-targeted JBNP-CRISPR to target sgIL-1R to treat osteoarthritis, delay the progression of osteoarthritis, and inhibit inflammation

[0258] Cartilage targeting can be achieved by, e.g., peptide WYRGRL To achieve. The severity of OA was assessed by MANKIN score, and then treatment (saline, JBNP-CRISPR-sgIL-1R, JBNP CRISPR sgNeg, or JBNP-CRISPR without sgRNA) was administered by intra-articular injection to medial meniscus surgical destabilization (DMM) model mice or 129SVE-M wild-type mice 2 months after surgery. DAPI staining, H&E staining, and Safranin-O staining of tissue sections were used to detect OA progression by fluorescence microscopy. The following behavioral studies were performed to examine the therapeutic effects: Y-Maze spontaneous changes, open field test, object recognition test, and conditioned fear.

[0259] Example 16: Detection of the ability of cartilage-targeted JBNP-IL-1R siRNA to inhibit inflammation associated with knee osteoarthritis

[0260] Cartilage targeting can be achieved by, e.g., peptide WYRGRL To achieve. The severity of OA was assessed by MANKIN score, and then treatment (normal saline, JBNP-IL-1R siRNA, JBNP negative siRNA, JBNP without siRNA) was administered by intra-articular injection to mice with surgical destabilization of the medial meniscus (DMM) model (3-month-old male) 1 month after surgery. Safranin-O staining was performed on tissue sections and detected by fluorescence microscopy. The following behavioral studies were performed to examine the treatment effects: Y-Maze spontaneous changes, open field test, object recognition test and conditioned fear.

[0261] Example 17: Detection of the ability of cartilage-targeted JBNP-IL-1RA mRNA to inhibit inflammation associated with knee osteoarthritis

[0262] Cartilage targeting can be achieved by, e.g., peptide WYRGRLThe severity of OA was assessed by MANKIN score, and then treatment (saline, JBNP-IL-1RA mRNA, JBNP-scrambled mRNA, JBNP without mRNA) was administered by intra-articular injection to mice with surgical destabilization of the medial meniscus (DMM) model (3-month-old males) 1 month after surgery. Safranin-O staining was performed on tissue sections and detected by fluorescence microscopy. The following behavioral studies were performed to examine the treatment effects: Y-Maze spontaneous changes, open field test, object recognition test and conditioned fear. An exemplary non-limiting example of IL-1RA mRNA sequence is: (SEQ ID NO: 89).

[0263] Example 18: Detection of the ability of cartilage-targeted JBNP-IL-1RA peptide mRNA to inhibit knee osteoarthritis inflammation

[0264] Cartilage targeting can be achieved by, e.g., peptide WYRGRLAchieved. The severity of OA was assessed by the MANKIN score, and then treatment (normal saline, JBNP-IL-1RA peptide mRNA, JBNP-scrambled mRNA, JBNP without mRNA) was administered to mice with surgical destabilization of the medial meniscus (DMM) model (3 months old) by intra-articular injection 1 month after surgery. IL-1RA peptide is an interleukin-1 receptor antagonist peptide. Safranin-O staining was performed on tissue sections and detected by fluorescence microscopy. The following behavioral studies were performed to examine the treatment effects: Y-Maze spontaneous changes, open field test, object recognition test and conditioned fear. An exemplary, non-limiting example of an mRNA sequence for an IL-1RA peptide is: ATGCGGCCTAGCGGCAGAAAGTCTAGCAAGATGCAGGCCTTTAGAATCTGGGACGTTAATCAGAAAACCTTCTACCTCAGAAACAACCAGCTGGTCGCCGGCTACCTGCAGGGCCCCAACGTGAATCTGGAAGAGAAGATCGACGTGGTGCCAATCGAGCCTCACGCCCTGTTTCTGGGCATCCACGGCGGAAAAATGTGCCTGTCTTGTGTGAAGTCCGG AGATGAGACAAGACTGCAACTGGAAGCCGTGAACATTACAGACCTGAGCGAGAACCGGAAGCAGGACAAGCGGTTCGCCTTCATCAGAAGCGACAGCGGCCCTACAACCAGCTTCGAGAGCGCCGCTTGCCCCGGCTGGTTCCTGTGCACCGCCATGGAAGCTGATCAGCCTGTGTCCCTGACCAACATGCCTGATGAAGGCGTGATGGTGACCAAGTTCTACTTCCAGGAGGACGAGATGTGA (SEQ ID NO:90).

[0265] Example 19: Amino acid side chain modification can be used to target the self-assembled nanomaterials of the present invention to target organs

[0266] The protein corona of the self-assembled nanomaterials described in the present invention and the compositions comprising it can be further modified by side chain modification of amino acid residues. The assembled JBNP was incubated with mouse serum for 30 minutes. Then, centrifuged at 13800RCF for 15 minutes at 4°C. The supernatant was discarded and the precipitate was washed three times with phosphate-buffered saline. The proteins were separated by SDS-PAGE and the protein bands were identified by liquid chromatography-tandem mass spectrometry (LC / MS / MS). Lysine and arginine side chains bind to the apolipoprotein E (APOE) family and are believed to target the self-assembled nanomaterials of the present invention to the liver. By further example, it is believed that the histidine side chains in the protein corona composition target the self-assembled nanomaterials of the present invention to organs other than the liver.

[0267] Example 20: The JBNP of the present invention actively targets cartilage and has a prolonged half-life when the cartilage targeting peptide WYRGRL is linked to JBNT

[0268] The cartilage targeting peptide WYRGRL (SEQ ID NO: 47) was linked to JBNP with an mRNA reporter, and the assembled WYRGRL-JBNP was transfected with the mRNA dye into human chondrocyte C28 / I2 cells. FACS was performed to quantify the targeting ability of WYRGRL-JBNP to cartilage. JBNP actively targets cartilage and has an extended half-life ( Figure 7B and Figure 7C ).

[0269] Example 21: Liver-targeted JBNP of the present invention can deliver drugs to the liver

[0270] BALB / cJ mice were injected intravenously with ArgJBNP-mcherry mRNA. Imaging monitoring of the biodistribution of ArgJBNP-mcherry mRNA ( Fig.11A and Fig. 11B ). ArgJBNP has significant biodistribution in the liver. The transfected liver can then be homogenized. Western blot and real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) are used to detect transfection efficiency.

[0271] Example 22: Testing the ability of the kidney-targeted JBNP of the present invention to deliver drugs to the kidney

[0272] BALB / cJ mice were intravenously injected with kidney-targeted-JBNP-mRNA Cy5. The biodistribution of kidney-targeted-JBNP-mRNA Cy5 was monitored by imaging. Then, the transfected kidneys were homogenized. Western blot and RT-qPCR were used to detect the transfection efficiency.

[0273] Example 23: Testing the ability of the brain-targeted JBNP of the present invention to deliver drugs to the brain

[0274] BALB / cJ mice were intravenously injected with brain-targeted-JBNP-mRNA Cy5. Imaging was used to monitor the biodistribution of brain-targeted JBNP-mRNA Cy5. Then, the transfected brain was homogenized. Western blot and RT-qPCR were used to detect the transfection efficiency.

[0275] Example 24: Testing the ability of the lung-targeted JBNP of the present invention to deliver drugs to the lungs

[0276] BALB / cJ mice were intravenously injected with lung-targeted-JBNP-mRNA Cy5. The biodistribution of lung-targeted-JBNP-mRNA Cy5 was monitored by imaging. Then, the transfected lungs were homogenized. Western blot and RT-qPCR were used to detect the transfection efficiency.

[0277] Example 25: Testing the ability of the spleen-targeted JBNP of the present invention to deliver drugs to the spleen and lymph nodes

[0278] BALB / cJ mice were intravenously injected with spleen-targeted-JBNP-mRNA Cy5. Imaging was used to monitor the biodistribution of spleen-targeted-JBNP-mRNA Cy5. Then, the transfected spleen and lymph nodes were homogenized. Western blot and RT-qPCR were used to detect the transfection efficiency.

[0279] Example 26: Testing the ability of the bone-targeted JBNP of the present invention to deliver drugs to bones

[0280] BALB / cJ mice were intravenously injected with bone-targeted-JBNP-mRNA Cy5. Imaging was used to monitor the biodistribution of bone-targeted-JBNP-mRNA Cy5. Then, the transfected bones were homogenized. Western blot and RT-qPCR were used to detect the transfection efficiency.

[0281] Example 27: Testing the ability of the muscle-targeted JBNP of the present invention to deliver drugs to muscles

[0282] BALB / cJ mice were injected intravenously with muscle-targeted-JBNP-mRNA Cy5. The biodistribution of muscle-targeted-JBNP-mRNA Cy5 was monitored by imaging. Then, the transfected muscles were homogenized. Western blot and RT-qPCR were used to detect the transfection efficiency.

[0283] Example 28: Testing the ability of the cardiac-targeted JBNP of the present invention to deliver drugs to the heart

[0284] BALB / cJ mice were intravenously injected with heart-targeted-JBNP-mRNA Cy5. Imaging was used to monitor the biodistribution of cardiac-targeted-JBNP-mRNA Cy5. Then, the transfected hearts were homogenized. Western blot and RT-qPCR were used to detect the transfection efficiency.

[0285] Example 29: Testing the ability of the pancreas-targeted JBNP of the present invention to deliver drugs to the pancreas

[0286] BALB / cJ mice were intravenously injected with pancreas-targeted-JBNP-mRNA Cy5. Imaging was used to monitor the biodistribution of pancreas-targeted-JBNP-mRNA Cy5. Then, the transfected pancreas was homogenized. Western blot and RT-qPCR were used to detect the transfection efficiency.

[0287] Example 30: Testing the ability of the intestinal-targeted JBNP of the present invention to deliver drugs to the intestine

[0288] BALB / cJ mice were intravenously injected with intestine-targeted-JBNP-mRNA Cy5. Imaging was used to monitor the biodistribution of intestinal-targeted-JBNP-mRNA Cy5. Then, the transfected intestine was homogenized. Western blot and RT-qPCR were used to detect the transfection efficiency.

[0289] Example 31: Testing the ability of the tumor-targeting JBNP of the present invention to deliver drugs to solid tumors

[0290] Tumor-targeted-JBNP-mRNA Cy5 was injected intravenously into xenograft models. Imaging was used to monitor the biodistribution of tumor-targeted-JBNP-mRNA Cy5. Then, the transfected solid tumors were homogenized. Western blot and RT-qPCR were used to detect the transfection efficiency. The mice were monitored for tumor size, body weight, complete blood count (CBC), tumor necrosis factor-α (TNF-α) level, interferon-γ (IFN-γ) level, immunoglobulin G (IgG) level, immunoglobulin M (IgM) level, and toxicity.

[0291] Example 32: Optimizing Dosage and Delivery of JBNP-CRISPR

[0292] The optimal dose and optimal delivery time of ArgJBNP-CRISPR were determined by gene editing in the Ai14 mouse model. ArgJBNP-CRISPR, consisting of Cas9 mRNA (0.25 mg / kg) and sgLOXp, was administered intravenously via tail vein injection or retro-orbital injection for 2-7 days. Imaging monitoring / detection of tandem dimer Tomato (tdTomato) positive signals ( Figures 10A-10D ). Next-generation sequencing (NGS) can be used to quantify gene editing efficiency in target organs.

[0293] Example 33: Testing the ability of JBNP-CRISPR to target and edit DNA in major organs

[0294] The assembled JBNP-CRISPR consisting of Cas9 mRNA and sgRB1 was intravenously injected into BALB / cJ mice for 7-28 days. Sanger sequencing or next-generation sequencing (NGS) was used to explore the RB1 gene editing efficiency.

[0295] Example 34: Testing the ability of JBNP-CRISPR to target and edit DNA in different organs by linking targeting molecules or fragments

[0296] JBNP-CRISPR with a kidney-targeting molecule or fragment can target the kidney and edit the DNA of kidney cells. JBNP-CRISPR with a heart-targeting molecule or fragment can target the heart and edit the DNA of heart cells. JBNP-CRISPR with a spleen-targeting molecule or fragment can target the spleen and edit the DNA of spleen cells. JBNP-CRISPR with a lymph node-targeting molecule or fragment can target the lymph node and edit the DNA of lymph node cells. JBNP-CRISPR with a lung-targeting molecule or fragment can target the lung and edit the DNA of lung cells. JBNP-CRISPR with a muscle-targeting molecule or fragment can target the muscle and edit the DNA of muscle cells. JBNP-CRISPR with a pancreas-targeting molecule or fragment can target the pancreas and edit the DNA of pancreatic cells. JBNP-CRISPR with an intestinal-targeting molecule or fragment can target the intestine and edit the DNA of intestinal cells.

[0297] Example 35: Testing the ability of JBNP-CRISPR to target and edit solid tumor DNA by attaching targeting molecules or fragments

[0298] JBNP-CRISPR with tumor-targeting molecules or fragments can target solid tumors and edit the DNA of tumor cells.

[0299] Example 36: The JBNP of the present invention does not cause any acute toxicity

[0300] Acute toxicity was tested by tail vein injection of JBNP doses (optimized doses for each treatment) every 5 days for 20 days. H&E staining was performed on tissue sections of liver, spleen, kidney, heart, pancreas, lung, brain, bone, and muscle. No acute toxicity caused by administration of JBNP was detected in the organs tested ( Figures 11E-11H ).

[0301] Example 37: Testing whether the JBNP of the present invention induces an innate immune response when administered

[0302] BALB / cJ mice were injected intravenously with JBNP every 3 days. For each injection time point, serum (30 μl) was collected from the submandibular tear. IFN-γ, IL-1β, IL-6, IL-10, IL-12 (p70), TNF-α, MCP-1, MIP-2, MIG, IL-2, IL-5, and IL-17 were detected by immunomultiplex assay and enzyme-linked immunosorbent assay (ELISA).

[0303] Example 38: JBNP of the present invention does not induce an adaptive immune response when administered

[0304] Adaptive immunity via IgG and IgM was not observed. JBNP was intravenously injected into BALB / cJ mice every 3 days for 9 days. Serum was collected before and 10 days after receiving JBNP. Then, IgM and IgG levels were measured by ELISA ( Fig. 9B , Fig. 9C and Fig.11F ). The serum IgM and IgG antibody levels on day 10 were compared with the pre-injection baseline. No adaptive immune response was observed after repeated administration of the JBNP of the present invention ( Fig. 9B , Fig. 9C and Fig.11F ).

[0305] Example 39: Detection of the cellular uptake mechanism of JBNP based on the modification or composition of JBNT

[0306] Cells were pretreated with several endocytosis inhibitors, including macropinocytosis inhibitors (red sea sponge A, cytochalasin D), clathrin-mediated inhibitors (chlorpromazine), and caveolae-mediated inhibitors (methyl-b-cyclodextrin). The assembled ArgJBNP-mRNA AF488 was then transfected into the cells. After 24 h of incubation, FACS was used to quantify the AF488 signal and determine the uptake mechanism ( Fig.9I ). Different side chains may have different cellular uptake mechanisms. For example, the cellular uptake of JBNP containing lysine-based JBNT may be through macropinocytosis. In addition, the cellular uptake of JBNP containing arginine-based JBNT may be carried out through clathrin-mediated endocytosis.

[0307] Example 40: The endosomal escape mechanism of JBNPs depends on the modification or composition of JBNT

[0308] The endosomal escape of JBNPs containing different types of JBNTs of the present invention was detected by using LysoTracker TM Endosomes were detected by staining cells in contact with the tested JBNPs with RED. The degree of colocalization was quantified based on the Pearson correlation coefficient (r) using Image J software according to the colocalization threshold and the coloc2 plug-in. Bafilomycin A1 and chloroquine inhibitors were used in pretreatment to demonstrate the proton sponge effect. Calcein was used to measure and model the pore formation of escaped endosomes. JBNPs containing lysine-based JBNTs have significantly better endosomal escape, which seems to be due to the proton sponge effect (US Patent Application Publication No. 20220133893A1). In addition, JBNPs containing arginine-based JBNTs have significantly enhanced endosomal escape, which seems to be due to the pore-forming effect of escaping early endosomes ( Fig. 12B and Fig. 12C ).

[0309] Example 41: The JBNP of the present invention has high biocompatibility and low cytotoxicity

[0310] Human chondrocytes (5000 cells / well) were seeded and incubated overnight. Then, the cell viability of various carriers including JBNP was determined by cell counting kit-8 analysis (Sigma). After 24 hours of co-incubation, the absorbance was measured by microplate reader. The JBNP of the present invention has high biocompatibility and has lower cytotoxicity ( ) than lipid nanoparticles, polymer nanoparticles (e.g., polyethyleneimine nanoparticles (PEI), lipid nanoparticles (LNP) and single-walled carbon nanotubes (SWNT). Figure 9L ).

[0311] Example 42: JBNP of the present invention can be used for CRISPR delivery

[0312] C28 / I2 cells were seeded in 24-well plates (5000 cells / well) and incubated overnight. The co-assembled JBNP-Cas9mRNA-eGFP and gRNA-ATTO550 dye were directly transferred into the wells and transfected for 72 hours. Then, FACS was performed to quantify the co-delivery in the cells ( Figure 9T and Figure 9U ). Co-delivery of Cas9mRNA-eGFP and gRNA-ATTO550 was imaged by confocal laser scanning microscopy ( Figure 9W ).

[0313] Results of Examples 10-42

[0314] An exemplary transferrin-targeted JBNP delivering mRNA to C28 / I2 cells is shown in FIG7E . An exemplary in vivo targeting study is also shown in FIG7E . Figure 7B and Figure 7C Cartilage-targeted WYRGRL-ArgJ BNPs increased the retention time of delivered siRNA in the knee joint.

[0315] Figures 8A-8G It was shown that a small molecule drug (doxorubicin) can be delivered via JBNPs. Fig. 8A and 8B Detection of apoptosis markers in monolayers of transfected cells is shown. Figure 8C and 8D The inhibitory effect on spheroid formation when treated with JBNP-DOX is shown. Figures 8E-8G The delivery of JBNP-DOX to ovarian cancer spheroids is shown. Figures 8H-8J It was shown that a small molecule drug (doxorubicin) could be co-delivered with siRNA to a monolayer of SKOV-3 cells via JBNPs. Figure 8K-8O Co-delivery of DOX and siRNA to ovarian cancer spheroids is shown. Figure 8K and 8L are fluorescent images of DOX and siRNA-AF488 delivered by JBNP. Figure 8M Representative images of spheroids stained with the apoptotic marker Caspase 3 / 7. Figure 8N and 8O FACS plots and quantification of apoptosis analysis are shown.

[0316] Fig.9A The biodistribution of siRNA delivered by ArgJ BNP in BALB / Cj mice is shown. Fig. 9B and 9C Immunogenicity studies of ArgJBNP-siRNA are shown. Figures 9D-9F The ArgJBNP-mRNA formulation was optimized based on delivery efficiency and cell viability. Figure 9G and 9H Response surface methodology (RSM) was used to optimize the ArgJBNP-mRNA formulation. Fig.9I Analysis of the uptake mechanism of ArgJBNP-mRNA. Figure 9J 6-(p-Tolyl)-2-naphthalene-6-sulfonic acid (TNS) assay for pKA analysis of ArgJBNP-mRNA. Figure 9K For UV-VIS analysis. Figure 9L The results of cell viability assay of ArgJBNP are shown. Figure 9M ArgJBNP-mRNA-gRNA gel retardation assay is shown. Figure 9N and9O Flow cytometry analysis is shown, with LysJBNP-mRNA and Lipofectamine TM The delivery efficiency of ArgJBNP-mRNA was tested compared with that of ArgJBNP-mRNA. Figure 9P-9S The excellent stability of ArgJBNP-mRNA at room temperature for 2 weeks was shown. Figure 9T and 9U Shown is the time-dependent delivery of Cas9 mRNA and gRNA in C28 / I2 cells. Figure 9V Time-dependent gene editing of RFP-HUVEC cells analyzed by flow cytometry is shown. Figure 9W Kinetic study of Cas9eGFP mRNA and gRNA delivery by ArgJBNP. Figure 9X 3D rendering of gRNA and Cas9 mRNA delivery into C28 / I2 cells. Figure 9Y and 9Z In the study, ArgJBNP was able to deliver Cas9 mRNA and gRNA to and edit chondrocytes of Ai14 mice. Figures 10A-10D The editing ability of ArgJBNP-CRISPR in vivo was demonstrated. Fig.10E Representative images of H&E-stained sections of the liver after injection of ArgJBNP-CRISPR are shown.

[0317] also, Fig.11A and Fig. 11B The biodistribution of mCherry mRNA delivered by ArgJBNP is shown. Fig. 11C and 11D Protein corona analysis of ArgJBNP-mRNA is shown. Fig. 11C The protein corona was visualized by SDS-PAGE. Fig.11D LC / MS / MS analysis of the ArgJBNP-mRNA protein corona.

[0318] also, Figures 11E-11H The immunogenicity of ArgJBNP-mRNA was tested. Fig.11E Shown are H&E-stained sections of organs after repeated injections of ArgJBNP-scrambled mRNA. Fig.11F IgG and IgM immune responses are shown. Fig.11G A CBC including WBC, RBC, HGB, and PLT counts was performed. Fig.11H Changes in body weight are shown.

[0319] Fig. 12A UV-VIS analysis of ArgJBNP at pH 5.2 and pH 7.4 is shown. Fig. 12B The endosomal escape of ArgJBNP in early endosomes (Rab5) and late endosomes (Rab7) was shown. Fig. 12C Endosomal escape of ArgJBNP by using inhibitors was shown.

[0320] also, Fig.13A Cell viability assays performed on PEG-JBNT are shown. PEGylation prevents nonspecific cellular uptake (eg, immune cells and other undesired cells), and targeting peptides can be used with PEG to achieve active targeting. Fig. 13B and Fig. 13C The biodistribution of siRNA delivered by PEG-JBNP to BALB / cJ mice is shown.

[0321] Exemplary Figures 14A-14C The combined PEG effect and targeting ability of WYRGRL-PEG-JBNP-mRNA-cy5 were shown.

[0322] Compositions, methods and articles may alternatively include, consist of or consist essentially of any suitable material, step or component disclosed herein. Compositions, methods and articles may additionally or alternatively be formulated to be free of or essentially free of any material (or species), step or component that is not necessary to achieve the function or purpose of the composition, method and article.

[0323] Although specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are not currently foreseeable or may not currently be foreseeable may occur to applicants or other persons skilled in the art. Therefore, the appended claims and their possible amendments are intended to include all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. A self-assembled nanomaterial comprising a Janus-based nanotube, wherein the Janus-based nanotube comprises at least one compound represented by Formula I to XII, or a pharmaceutically acceptable salt thereof: in, R 1 is H or CH3; R 2 For (CH2) j 、(CH2CH2O) k , or (CH2CH2NH) m , wherein j, k, and m are independently integers from 1 to 200; R 3 is an α-amino acid, a β-amino acid, an α-polypeptide or a β-polypeptide; L is a bond or a linking group; T is a biologically active molecule or a targeting molecule; and R 4 It is the wrapping material.

2. The self-assembled nanomaterial according to claim 1, wherein the Janus-based nanotube comprises at least one compound represented by Formula I to IV.

3. The self-assembled nanomaterial according to claim 1, wherein the Janus-based nanotube comprises at least one compound represented by Formulas V to VIII.

4. The self-assembled nanomaterial according to claim 1, wherein the Janus-based nanotube comprises at least one compound represented by Formulae IX to XIII.

5. The self-assembled nanomaterial according to claim 1, wherein the Janus-based nanotubes comprise a combination of: Compounds represented by Formula I to Formula IV and compounds represented by Formula V to Formula VIII; Compounds represented by Formula I to Formula IV and compounds represented by Formula IX to Formula XIII; Compounds represented by Formula V to Formula VIII and compounds represented by Formula IX to Formula XIII; Compounds represented by Formula I to Formula IV, compounds represented by Formula V to Formula VIII, and compounds represented by Formula IX to Formula XIII; or Its combination.

6. The self-assembled nanomaterial according to any one of claims 1 to 5, wherein L is a linking group and is selected from an acid cleavable group, a reducible disulfide group, an α-amino acid, a β-amino acid, an α-peptide, a β-peptide, an enzyme cleavable group, and a stimulus responsive group.

7. The self-assembled nanomaterial according to claim 6, wherein: The acid cleavable group is an N-acylhydrazone, a carbonate group or an ester group; The reducible disulfide linker is N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), or 4-(4'-acetylphenoxy)butyric acid (AcBut), Val-Cit dipeptide, Phe-Lys dipeptide, α-methyl substituted disulfide, modified cysteine ​​residues or sulfhydryl-containing maytansine compounds; The stimulus-responsive linker is a trans-cyclooctene linker or a thioether-containing linker; or The enzyme-cleavable linker is GPLGOAGQ (SEQ ID NO: 89), GDEVEAPKGC (SEQ ID NO: 90), citrulline-valine, a glycosidase-cleavable linker, a β-glucuronidase-cleavable linker, a β-galactosidase-cleavable linker, a phosphatase-cleavable linker, a pyrophosphate-containing linker, a dipeptide-containing linker, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Phe-Lys-PABC (p-aminobenzylcarbamate), a Val-Cit-PABC-containing linker, a Glu-Val-Cit-containing linker, or a Val-Ala-containing linker.

8. The self-assembled nano material according to any one of claims 1 to 7, wherein R 4 It is an encapsulating material and is selected from polymers, peptides, polypeptides, lipid-based materials, phosphate esters or biomimetic membranes.

9. The self-assembled nano material according to any one of claims 1 to 8, wherein R 4 Selected from polyethylene glycol, chitosan, hyaluronic acid, poloxamer, polyvinyl alcohol, polysaccharide, neutral polyamino acid, negatively charged polyamino acid, phytochelatin, autopeptide and antithrombotic peptide.

10. The self-assembled nanomaterial according to any one of claims 1 to 8, wherein the T is a targeting molecule and is selected from the group consisting of bioactive molecules, amphiphilic polymers, aptamers, peptides, cyclic peptides, proteins, polysaccharides, polyunsaturated fatty acids and carbohydrates.

11. The self-assembling nanomaterial according to any one of claims 1 to 8, wherein the T is a targeting molecule, and the self-assembling nanomaterial further comprises a bioactive molecule covalently or non-covalently attached to the self-assembling nanomaterial.

12. The self-assembling nanomaterial of claim 10, wherein the bioactive molecule is non-covalently attached to the self-assembling nanomaterial.

13. The self-assembled nanomaterial according to any one of claims 10-11, wherein the bioactive molecule is at least partially encapsulated by the self-assembled nanomaterial.

14. The self-assembled nanomaterial according to any one of claims 1 to 12, wherein the bioactive molecule comprises a nucleic acid, a protein, a peptide, a cyclic peptide, a small molecule drug, or a combination thereof.

15. The self-assembled nanomaterial according to any one of claims 1-13, wherein the bioactive molecule comprises miRNA, siRNA, mRNA, gRNA, crRNA, tracrRNA, tRNA, ssDNA, dsDNA, cDNA, or a combination thereof.

16. The self-assembled nanomaterial according to any one of claims 1 to 14, wherein the Janus-based nanotubes are present in an amount of 0.1 wt% to 99.9 wt% based on the total weight of the self-assembled nanomaterial. 17 . The self-assembled nanomaterial according to claim 1 , wherein the concentration of the Janus-based nanotubes in the self-assembled nanomaterial is 1 μg / mL to 1 g / mL.

18. The self-assembled nanomaterial according to any one of claims 1 to 16, wherein the pH of the self-assembled nanomaterial is 1 to 10.

19. The self-assembled nanomaterial according to any one of claims 1-17, further comprising extracellular matrix (ECM) molecules.

20. The composition of claim 18, wherein the ECM molecules comprise hydroxyapatite, fibronectin, Matn1, Matn3, laminin, cartilage oligomeric matrix protein, collagen, elastin, vitronectin, fibrillin, perlecan, fibrinogen, osteonectin, tenascin, thrombospondin, intercellular adhesion molecules (ICAM1-5), integrins, proteoglycans, glycoproteins, or combinations thereof.

21. An injectable pharmaceutical composition comprising the self-assembling nanomaterial according to any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

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