Nanomaterial delivery carriers and methods of use thereof

Abstract

The self-assembled nanomaterial comprises Janus-based nanotubes comprising at least one compound of Formulae I-XII or a pharma- ceutically acceptable salt thereof.Compositions comprising the Janus-based nanotubes are also described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This disclosure claims priority to and the benefit of U.S. Provisional Application No. 63 / 326,462, filed April 1, 2022, and entitled "NANOMATERIAL DELIVERY VEHICLE AND METHOD OF USE THEREOF," the entire contents of which are incorporated herein by reference for all purposes.

[0002] Statement on Federally Funded Research This invention was made with government support under Grant No. W81XWH-21-1-0274 awarded by the U.S. Army Medical Research and Development Command, Grant No. AR072027 awarded by the National Institutes of Health, and Grant Nos. 1905785 and 2025362 awarded by the National Science Foundation. The government has certain rights in this invention.

[0003] Incorporation by Reference In accordance with 37 Code of Federal Regulations § 1.52(e)(5), the sequence information contained in the electronic file name "UCT0291PCT_Sequence_Listing_ST26.xml", created on March 28, 2023, and having a size of 88.3 KB, is hereby incorporated by reference in its entirety. [Background technology]

[0004] Many methods for delivering biologically active materials have been developed, which vary by the type and target of the biologically active material. Examples of various delivery vehicles that have been used, particularly for RNA delivery, include viral vectors, lipid nanoparticles, cationic polymers, liposomes, polymeric nanoparticles, carbon nanoparticles, superparamagnetic iron oxide nanoparticles (SPIONs), gold nanoparticles, silver nanoparticles, metal-organic frameworks, cell-penetrating peptides, black phosphorus nanosheets, and DNA nanostructures. Delivery vectors such as lipid nanoparticles can deliver RNA, but they have been reported to have low endosomal escape, which reduces efficacy. In addition, the cationic lipid content of lipid nanoparticles tends to promote a proinflammatory phenotype. These limitations make it difficult to achieve high efficacy RNA delivery for therapeutic applications, hindering clinical application. Summary of the Invention [Problem to be solved by the invention]

[0005] There remains a need to improve the delivery of biologically active materials such as small molecules, nucleic acids, proteins, gene silencing or gene editing tools, and / or other large molecule biomaterials. [Means for solving the problem]

[0006] The present specification discloses a self-assembled nanomaterial comprising a Janus base nanotube (JBNT) comprising at least one compound represented by Formulae I-XII or a pharma- ceutically acceptable salt thereof. [ka] [ka] [ka] During the ceremony, R 1 is H or CH 3 and R 2 is (CH 2 ) j , (CH 2 CH 2 O) k , or (CH 2 CH 2 NH) m wherein j, k, and m are independently an integer of 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 linker group; T is a biologically active or targeting molecule / moiety; R 4 is a coating material.

[0007] The present specification also discloses an injectable composition comprising the above-mentioned self-assembled nanomaterial and a pharma- ceutically acceptable carrier.

[0008] The above-mentioned features and other features are illustrated in the figures and detailed description that follow. [Brief description of the drawings]

[0009] The following figures are exemplary embodiments in which like elements are numbered alike.

[0010] [Figure 1 (1)] Figures 1A, 1B, 1C and 1D show targeting peptide-JBNP for active targeting: Figure 1A is a schematic diagram of LysJBNP-targeting peptide, and Figure 1B shows the structure of targeting peptide-LysJBNP. [Figure 1 (2)] Figures 1A, 1B, 1C and 1D show targeting peptide-JBNP for active targeting. Figure 1C shows TEM images of LysJBNP without targeting peptide and LysJBNP with targeting peptide RLDPTSYLRTFWC. [Figure 1 (3)]Figures 1A, 1B, 1C and 1D show the targeting peptide-JBNP for active targeting. Figure 1D is an absorbance vs. wavelength (nm) graph showing the ultraviolet-visible (UV-Vis) targeting of peptide-Lys-JBNT. [Figure 2 (1)] Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H show the assembly and delivery of LysJBNP. Figure 2A is a schematic diagram of LysJBNP delivery. [Figure 2 (2)] Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H show the assembly and delivery of LysJBNPs. Figure 2B is a graph showing the Zeta potential analysis and Figure 2C is a graph showing the UV-VIS analysis. [Figure 2 (3)] Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H show the assembly and delivery of LysJBNP. Figure 2D shows a TEM image of LysJBNP, and Figure 2E shows the results of a gel shift assay. [Figure 2 (4)] Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H show the organization and delivery of LysJBNPs: Figure 2F is a CLSM z-stack image of siRNA-AlexaFluor®-488 delivered by LysJBNPs, and Figure 2G is an image showing the inhibition of LysJBNP uptake. [Figure 2 (5)] Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H show the organization and delivery of LysJBNP. Figure 2H is a graph showing the quantitative analysis of JBNP uptake by C28 / I2 human chondrocytes. [Figure 3(1)] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K and 3L show the organization and delivery of Arg-JBNP. Figure 3A shows TEM images of Arg-JBNP-miR140, Arg-JBNP-Cas9mRNA and Arg-JBNP-albumin, and Figure 3B shows TEM image analysis of the size and width of Arg-JBNP-miRNA. [Figure 3 (2)]Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K and 3L show the assembly and delivery of Arg-JBNP. Figure 3C shows the zeta potential analysis of Arg-JBNP-miR140, and Figure 3D shows the gel shift assay. [Figure 3 (3)] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, and 3L show the organization and delivery of Arg-JBNP. Figure 3E shows the zeta potential analysis of Arg-JBNP-Cas9mRNA, and Figure 3F shows the CLSM z-stack images of siRNA-AF488 delivered by Arg-JBNP. [Figure 3 (4)] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K and 3L show the assembly and delivery of Arg-JBNP. Figure 3G shows flow cytometry analysis. [Figure 3 (5)] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K and 3L show the organization and delivery of Arg-JBNP. Figure 3H shows the fluorescence image of Cy5.5-eGFP-mRNA delivery via Arg-JBNP or Lipo, and Figure 3I shows the fluorescence image of BSA-AF488 delivery via Arg-JBNP. [Figure 3 (6)] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K and 3L show the assembly and delivery of Arg-JBNP. Figure 3J shows the time-dependent delivery of Cas9eGFP. [Figure 3 (7)] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K and 3L show the assembly and delivery of Arg-JBNP. Figure 3K shows a fluorescent image of Arg-JBNP-eGFP-Cas9mRNA delivery. [Figure 3 (8)] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K and 3L show the assembly and delivery of Arg-JBNP. Figure 3L shows the time dependence of flow cytometry analysis. [Figure 4(1)]Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G and 4H show endosomal escape of JBNP. Figure 4A is an image showing endosomal escape of Arg-JBNP and Lys-JBNP, Figure 4B is an image showing the time-dependent endosomal escape of Lys-JBNP, and Figure 4C is an image showing the inhibition of the proton sponge effect. [Figure 4 (2)] Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G and 4H show endosomal escape of JBNP. Figure 4D is a graph showing the results of an acid-base titration curve, and Figure 4E is an image showing endosomal escape of JBNP and LNP. [Figure 4 (3)] Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G and 4H show endosomal escape of JBNP. Figure 4F is a graph showing quantification of colocalization, and Figure 4G is an image showing antiviral activity associated with JBNP delivery. [Figure 4 (4)] Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G and 4H show endosomal escape of JBNP. Figure 4H is a graph showing inhibition of gene expression of JBNP and LNP. [Figure 5(1)] Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G and 5H show JBNPs with protecting molecules. Figure 5A shows a TEM image of polyethylene glycol (PEG)-JBNT. [Figure 5 (2)] Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G and 5H show JBNP with protective molecules, and Figures 5B and 5C show UV-VIS analysis. [Figure 5 (3)] Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G and 5H show JBNP with protective molecules, and Figures 5B and 5C show UV-VIS analysis. [Figure 5 (4)] Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G and 5H show JBNP with protective molecules. Figure 5D shows a TEM image of PEG-JBNP-siRNA. [Figure 5 (5)] Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G and 5H show JBNP with protective molecules. Figure 5E shows UV-VIS analysis. [Figure 5 (6)]Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G and 5H show JBNPs with protecting molecules. Figure F shows the zeta potential analysis. [Figure 5 (7)] Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G and 5H show JBNP with protective molecules. Figure G shows the cellular uptake of Arg-JBNP and PEG-JBNP by flow cytometry. [Figure 5 (8)] Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G and 5H show JBNPs with protective molecules. Figure 5H shows the flow cytometry percent uptake analysis and Figure I shows the flow cytometry MFI analysis. [Figure 6(1)] Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J and 6K show multifunctional JBNTs. Figure 6A is a graph showing the UV-Vis spectra of ArgJBNT / GlyJBNT / AspJBNT. [Figure 6 (2)] Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J and 6K show multifunctional JBNTs. Figure 6B is a graph showing the calculated UV-Vis spectra of total / co-assembled ArgJBNT, GlyJBNT and AspJBNT. [Figure 6 (3)] Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J and 6K show multifunctional JBNTs. Figure 6C is a graph comparing the sizes of ArgJBNT / AspJBNT / GlyJBNT / co-assembly (Mix), ArgJBNT (Arg), GlyJBNT (Gly) and AspJBNT (Asp). [Figure 6 (4)] Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J and 6K show multifunctional JBNT. Figure 6D is a graph showing the zeta potential of ArgJBNT / AspJBNT / GlyJBNT / co-assembly (Mix), ArgJBNT (Arg), GlyJBNT (Gly) and AspJBNT (Asp). [Figure 6 (5)]Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J and 6K show multifunctional JBNT. Figure 6E shows TEM images of ArgJBNT / AspJBNT / GlyJBNT / HisJBNT co-assembly, ArgJBNT, GlyJBNT and AspJBNT, and Figure 6F shows UV-Vis analysis. [Figure 6 (6)] Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J and 6K show multifunctional JBNTs. Figure 6G shows the zeta potential. [Figure 6 (7)] Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J and 6K show multifunctional JBNTs. Figure 6H is a TEM image of co-assembled ArgJBNT, PEGLysJBNT and SPLysJBNT, and Figure 6I is a TEM image of Arg-JBNP, PEG-JBNP and SP-PEG-JBNP. [Figure 6 (8)] Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J and 6K show multifunctional JBNT. Figure 6J shows flow cytometry analysis of multifunctional JBNP. [Figure 6 (9)] Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J and 6K show multifunctional JBNTs. Figure 6K is a graph of mean fluorescence intensity obtained by flow cytometry. [Figure 7(1)] In FIG. 7A, transferrin-JBNP-mRNA was delivered into cells, resulting in expression of Cas9eGFP mRNA. [Figure 7(2)] Figure 7B shows that peptide WYRGRL (SEQ ID NO: 47) can target type II collagen in the knee joint with WYRGRL-ArgJBNP. Figure 7C shows quantification of the signal by IVIS® Spectral in vivo imaging. [Figure 8(1)] FIG. 8A shows the results of an apoptosis assay using LysJBNP-doxorubicin (LysJBNP-DOX). [Figure 8 (2)] Figure 8B shows quantification of apoptotic cells from the results in Figure 8 A. Figure 8C shows images showing inhibition of spheroid formation. [Figure 8 (3)] Figure 8D shows the measured diameter of the spheroids, and Figure 8E shows the spheroids detected / stained with apoptotic markers. [Figure 8 (4)] FIG. 8F is a flow cytometry analysis examining apoptosis. [Figure 8 (5)] FIG. 8G is a quantification of the apoptosis data of FIG. 8F. [Figure 8 (6)] FIG. 8H shows representative fluorescence images of JBNP-mediated codelivery of small molecule drugs and siRNA. [Figure 8 (7)] FIG. 8I shows data from an apoptosis assay examining codelivery. [Figure 8 (8)] Figure 8J shows quantification data of the apoptosis assay of Figure 9I. Figure 8K shows microscopic images of delivery of LysJBNP-DOX or LysJBNP-DOX-siRNA to ovarian cancer spheroids. [Figure 8 (9)] Figure 8L shows the time-dependent delivery of JBNP-DOX-siRNA to spheroids, and Figure 8M shows representative images of apoptosis staining treated with control, JBNP-DOX, and JBNP-DOX-siRNA. [Figure 8 (10)] FIG. 8N is a plot of an apoptosis assay examining control, JBNP-DOX and JBNP-DOX-siRNA treatments. [Figure 8 (11)] FIG. 8O is a quantification of the apoptosis data of FIG. 8N. [Figure 9(1)] FIG. 9A shows the biodistribution of ArgJBNP-siRNA. [Figure 9 (2)] Figure 9B shows the IgG immune response of ArgJBNP-siRNA compared to saline, and Figure 9C shows the IgM immune response of Arg-JBNP-siRNA compared to saline. [Figure 9 (3)] Figure 9D is a heat map of the delivery efficiency of ArgJBNP-mRNA. Figure 9E is a heat map of the cell viability when treated with ArgJBNP-mRNA. [Figure 9 (4)] FIG. 9F is a heat map of delivery (efficiency x cell viability). [Figure 9 (5)] FIG. 9G is a graph generated to optimize the formulation of ArgJBNP-mRNA using response surface methodology (RSM). [Figure 9 (6)] Figure 9H is a surface plot of the data shown in Figure 9F. Figure 9I is a graph showing the uptake mechanism of ArgJBNP-mRNA. [Figure 9 (7)] Figure 9J shows data from pKa assays performed on ArgJBNP and LysJBNP. Figure 9K shows UV-VIS analysis of ArgJBNT, RNA and ArgJBNP-RNA. [Figure 9 (8)] FIG. 9L is a graph showing cell viability data for various concentrations of ArgJBNP, lipid nanoparticles (LNP), polyethyleneimine nanoparticles (PEI), and single-walled carbon nanotubes (SWNT). [Figure 9 (9)] Figure 9M shows the gel shift assay data of the control treatment, and Figure 9N shows the flow cytometry analysis shown in Figure 9O. [Figure 9 (10)] FIG. 9O is the flow cytometry data for the graph in FIG. 9N. [Figure 9 (11)] Figure 9P shows data from a stability study of ArgJBNP-mRNA. Figure 9Q shows data from a UV-VIS analysis of the time-dependent stability of ArgJBNp-mRNA. [Figure 9 (12)] Figure 9R shows the Z-average size and PDI values ​​of the time-dependent stability of ArgJBNp-mRNA. Figure 9S shows the zeta potential data of the time-dependent stability of ArgJBNp-mRNA. [Figure 9 (13)] Figure 9T shows time-dependent delivery of Cas9 mRNA into ArgJBNP and Lipofectamine® 2000 (Waltham, Massachusetts, USA). Figure 9U shows representative time-dependent delivery of guide RNA (gRNA) via ArgJBNP or Lipofectamine® 2000 (Waltham, Massachusetts, USA). [Figure 9 (14)]Figure 9V shows representative time-dependent data of gene editing in RFP-expressing human umbilical vein endothelial cells (RFP-HUVEC) cells using red fluorescent protein (RFP). [Figure 9 (15)] Figure 9W shows representative fluorescence images of kinetic studies performed by delivering Cas9-eGFP mRNA or gRNA-ATTO550 to C28 / I2 cells, and Figure 9X is a 3D analysis of three articulated treatments. [Figure 9 (16)] FIG. 9Y shows representative fluorescent images of gene editing of Ail4 chondrocytes by multi-joint treatment. [Figure 9 (17)] FIG. 9Z shows flow cytometry data examining the three treatments in Ail4 chondrocytes. [Figure 10(1)] FIG. 10A is a schematic diagram of ArgJBNP-CRISPR. [Figure 10(2)] FIG. 10B shows signals from IVIS® Spectral in vivo imaging of specific organs. [Figure 10(3)] Figure 10C is a quantification of the IVIS® Spectrum in vivo imaging data of Figure 10A. Figure 10D is a fluorescent image of a liver section stained with 4',6-diamidino-2-phenylindole (DAPI). [Figure 10(4)] FIG. 10E shows representative images of liver sections from Ail4 mice stained with hematoxylin and eosin (H&E). [Figure 11(1)] FIG. 11A shows the biodistribution of ArgJBNP-mCherry mRNA in joint organs. [Figure 11 (2)] Figure 11B is a graph showing the distribution of IVIS® Spectrum in vivo imaging data of joint organs. Figure 11C is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein corona analysis. [Figure 11 (3)] FIG. 11D shows liquid chromatography tandem mass spectrometry (LC / MS / MS) data of the protein corona. [Figure 11 (4)]Additionally, FIG. 11E shows representative images of H&E stained tissues to examine the immunogenicity of ArgJBNp-mRNA. [Figure 11 (5)] FIG. 11F shows the relative changes in IgG and IgM upon treatment with ArgJBNp-mRNA. [Figure 11 (6)] FIG. 11G shows the relative changes in white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and platelets (PLT) from complete blood counts (CBC) performed in BALB / cJ mice injected with ArgJBNp-mRNA. [Figure 11 (7)] FIG. 11H shows the percent weight change of four BALB / cJ mice injected with ArgJBNp-mRNA. [Figure 12(1)] FIG. 12A shows UV-VIS analysis of ArgJBNp-RNA. [Figure 12 (2)] Figure 12B shows immunocytochemistry (ICC) images of Rab5 and Rab7, and Figure 12C shows representative images of ArgJBNp-mRNA treated with inhibitors (bafilomycin or chloroquine). [Figure 13(1)] FIG. 13A shows the effect of PEG on cell viability. [Figure 13(2)] Figure 13B shows the biodistribution of PEG-JBNP-siRNA compared to JBNP-siRNA and ArgJBNP-siRNA. Figure 13C shows quantification of IVIS® Spectrum in vivo imaging data of ArgJBNP, LysJBNP and PEG-JBNP in the liver. [Figure 14(1)] Figure 14A shows the combined PEG effect and cartilage targeting ability of WYRGRL-PEG-JBNP-mRNA. Figure 14B shows a representative Safranin O stained section of a knee after administration of WYRGRL-PEG-JBNP-mRNA. [Figure 14(2)] FIG. 14C is a representative H&E stained section of a knee after administration of WYRGRL-PEG-JBNP-mRNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present specification discloses self-assembled nanomaterials including Janus nanotubes composed of units having a single ring system. The self-assembled nanomaterials exhibit low cytotoxicity, low immunogenicity, and minimal side effects in vivo, and may be advantageously used to deliver biologically active materials. The self-assembled nanomaterials also exhibit improved endosomal escape, thereby increasing efficacy.

[0012] The self-assembled nanomaterials disclosed herein contain targeting moieties that target specific receptors on the cell surface and facilitate their specific and selective uptake. The co-assembly of different Janus nanotubes with different functional groups and their use in different amounts allows the design of self-assembled nanomaterials with desired properties. These properties may be tailored based on cellular delivery, circulation time in vivo, passive or active targeting, intracellular targeting, improved cellular uptake, and enhanced endosomal escape.

[0013] Throughout this specification and the appended claims, the words "comprise," "include," "have," and variations thereof, such as "comprises," "comprising," "includes," "including," "has," "having," etc., are intended to be inclusive; that is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context permits. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0014] The terms "first", "second", etc., as used herein do not denote any particular order, but are merely for convenience to denote multiple layers, etc.

[0015] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each individual value within that range, and each individual value is incorporated herein as if it were set forth individually herein. Ranges may be expressed herein 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 the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about" or "approximately," it is understood that the particular value forms another embodiment. It will be further understood that both endpoints of each range are disclosed both in conjunction with the other endpoint, as well as independently of the other endpoint.

[0016] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Additionally, all methods described herein having two or more steps may be performed by two or more individuals or entities. Thus, one individual or entity may perform method step (a), another individual or entity may perform method step (b), and yet another individual or entity may perform method step (c), and so on. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to make the invention easier to understand and does not limit the scope of the invention unless expressly stated.

[0017] Units, prefixes and symbols are shown in the form accepted by the International System of Units (SI).

[0018] Groupings of alternative elements or embodiments of the invention disclosed herein should not be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or excluded from a group for reasons of convenience and / or patentability. When any such inclusion or exclusion occurs, the specification is deemed to include the group as modified, so that it satisfies the description of all Markush groups used in the appended claims.

[0019] The headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the specification or claims, which can be obtained by reference to the entire specification, and therefore, the terms defined immediately below are more fully defined by reference to the entire specification.

[0020] The drawings are for the purpose of illustrating preferred embodiments of the invention and are not intended to limit the invention thereto.

[0021] As used herein, "about" or "approximately" means inclusive of the stated value and within an acceptable range of deviation from the particular value as determined by one of ordinary skill in the art, taking into account the measurement in question and the error associated with the measurement of the particular quantity (i.e., the limitations of the measurement system). For example, "about" can mean within one or more standard deviations of the stated value, or within ±10% or 5% of the stated value.

[0022] The compositions, methods and articles can alternatively comprise, consist of, or consist essentially of any suitable materials, steps or ingredients disclosed herein. The compositions, methods and articles can additionally or alternatively be formulated to be free, or substantially free, of any materials (or species), steps or ingredients that are not necessary to achieve the function or purpose of the compositions, methods and articles.

[0023] All ranges disclosed herein are inclusive of the endpoints, which are independently combinable with each other (e.g., the range "up to 25 wt.%, or more specifically, 5 wt.% to 20 wt.%" includes the endpoints and all intermediate values ​​of the range, such as "5 wt.% to 20 wt.%). "Combination" includes blends, mixtures, alloys, reaction products, and the like. Terms such as "first," "second," and the like are not used to denote any order, quantity, or importance, but rather to distinguish one element from another. The terms "a," "an," and "the" do not denote a limitation of quantity and should be construed to include both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. "Or" means "and / or" unless otherwise stated.

[0024] References throughout the specification to "some embodiments," "one embodiment," etc., mean that a particular element described with respect to that embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. It should also be understood that the described elements can be combined in any suitable manner in the various embodiments. "Combinations thereof" are open and include any combination that includes at least one of the recited components or features, optionally together with similar or equivalent components or features that are not recited.

[0025] As used herein, the term "administering" refers to the actual physical introduction of a composition into or onto (as appropriate) a host or cell. Any method of introducing a composition into a host or cell is contemplated by the present invention, and the method is not, and should not be construed as being dependent on a particular means of introduction. Means of introduction are well known to those of skill in the art and are exemplified herein.

[0026] As used herein, "optional" or "optionally" means that the event or circumstance described following the term may or may not occur, and such description encompasses instances where the event or circumstance occurs or does not occur.

[0027] As used herein, the term "pharmaceutical acceptable" refers to a composition that is physiologically tolerable and typically does not cause an allergic or similar adverse reaction when administered to a subject, preferably a human subject. Preferably, as used herein, the term "pharmaceutical acceptable" means approved by a regulatory agency of the Federal or State government or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more specifically, in humans.

[0028] As used herein, the terms "treat", "treating" and "treatment" include inhibiting a pathological condition, disease or illness, such as preventing or reducing the onset of the pathological condition, disease or illness or its clinical symptoms, and alleviating a pathological condition, disease or illness, such as causing regression of the pathological condition, disease or illness or its clinical symptoms. These terms also include therapy and cure. Treatment means any manner in which the symptoms of a pathological condition, disease or illness are ameliorated or beneficially altered. Preferably, the subject in need of such treatment is a mammal, preferably a human.

[0029] chemical definition Compounds are described using standard nomenclature. For example, any position not substituted by a indicated group is understood to have its valence saturated with the indicated bond or hydrogen atom. A dash "-" that is not between two letters or symbols indicates the point of attachment of the substituent. For example, -CHO is attached through the carbon of a carbonyl group.

[0030] Pharmaceutically acceptable salts include salts that retain the biological effectiveness and properties of the compounds and are not biologically or otherwise undesirable. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts, and the like. Salts derived from organic bases include salts of primary, secondary, and tertiary amines (e.g., alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenylamines, dialkenylamines, trialkenylamines, 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, tri ... )amines, substituted cycloalkenylamines, disubstituted cycloalkenylamines, trisubstituted cycloalkenylamines, arylamines, diarylamines, triarylamines, heteroarylamines, diheteroarylamines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed diamines and triamines where at least two of the 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, heterocyclic, etc. Also included are amines where two or three of the substituents together with the amino nitrogen form a heterocyclic or heteroaryl group.

[0031] Salts derived from organic bases include primary, secondary and tertiary amines (e.g., alkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines, di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenyl amines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines, di(substituted alkenyl)amines, tri(substituted alkenyl)amines, cycloalkyl amines, di(cycloalkyl)amines, tri(cycloalkyl)amines, substituted cycloalkyl amines, disubstituted cycloalkyl amines, trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl)amines, )amines, substituted cycloalkenylamines, disubstituted cycloalkenylamines, trisubstituted cycloalkenylamines, arylamines, diarylamines, triarylamines, heteroarylamines, diheteroarylamines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed diamines and triamines where at least two of the 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, heterocyclic, etc. Also included are amines where two or three of the substituents together with the amino nitrogen form a heterocyclic or heteroaryl group.

[0032] Examples of pharma- ceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, etc. Pharmaceutically acceptable salts include the conventional non-toxic salts as well as the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, as well as salts derived from 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, mesylic acid, esylic acid, besylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, HOOC-(CH2) n and salts prepared from organic acids such as —COOH (n is 0-4).

[0033] The term "amino acid" refers to a molecule that contains both an amino group and a carboxyl group. Exemplary amino acids include, but are not limited to, both D and L isomers of naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. The term "amino acid" as used herein includes, but is not limited to, α-amino acids, natural amino acids, unnatural amino acids, and amino acid analogs.

[0034] The term "α-amino acid" refers to a molecule that contains both an amino group and a carboxyl group bonded to a carbon called the α carbon.

[0035] The term "β-amino acid" refers to a molecule that contains both an amino group and a carboxyl group in the β configuration.

[0036] The term "naturally occurring amino acid" refers to any one of the twenty amino acids commonly found in naturally synthesized peptides and known by the one-letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.

[0037] The table below summarizes the properties of the natural amino acids. [Table 1]

[0038] "Hydrophobic amino acids" include small hydrophobic amino acids and large hydrophobic amino acids. "Small hydrophobic amino acids" are glycine, alanine, proline, and analogs thereof. "Large hydrophobic amino acids" are valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and analogs thereof. "Polar amino acids" are serine, threonine, asparagine, glutamine, cysteine, tyrosine, and analogs thereof. "Charged amino acids" are lysine, arginine, histidine, aspartic acid, glutamic acid, and analogs thereof.

[0039] The term "amino acid analog" refers to a molecule that is structurally similar to an amino acid and can be substituted for it in the formation of a peptidomimetic macrocycle. Amino acid analogs include, but are not limited to, 3-amino acids and amino acids in which the amino or carboxy group is replaced with a group of similar reactivity (e.g., replacement of a primary amine with a secondary or tertiary amine, or replacement of a carboxy group with an ester).

[0040] The term "non-naturally occurring amino acid" refers to an amino acid that is not one of the 20 amino acids commonly found in naturally synthesized peptides and known by the one-letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. Non-natural amino acids or amino acid analogs include, but are not limited to, structures according to the following:

[0041]

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[0042] Amino acid analogs include β-amino acid analogs. Examples of β-amino acid analogs include 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-amino ... no-4-(2-fluorophenyl)-butyric acid, (R)-3-amino-4-(2-furyl)-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-trifluorophenyl ... (R)-3-amino-4-pentafluoro-phenylbutyric 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-tetrahydro-isoquinoline-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-furyl)-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, (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 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-pentafluoro-phenylbutyric 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-homoapartic 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-t-butyl ester, L-β-homoglutamic acid δ-t-butyl ester, L-Nω-β-homolysine, Nδ-trityl-L-β-homoglutamine, Nω-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L-β-homoarginine, Ot-butyl-L-β-homohydroxy-proline, Ot-butyl-L-β-homoserine, Ot-butyl-L-β-homothreonine, Ot-butyl-L-β-homotyrosine, 2-aminocyclopentanecarboxylic acid, and 2-aminocyclohexanecarboxylic acid.

[0043] Amino acid analogs include analogs of alanine, valine, glycine, or leucine. Examples of amino acid analogs of alanine, valine, glycine, and leucine include α-methoxyglycine, α-allyl-L-alanine, α-aminoisobutyric acid, α-methyl-leucine, 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-thien ... -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-t-butyl-D-alanine, β-t-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·dicyclohexylammonium salt, 4-fluoro-D-phenylglycine, 4-fluoro-L-phenylglycine, 4-hydroxy-D-phenylglycine, 5,5,5-trifluoro-leucine, 6-aminohexanoic acid, cyclopentyl-D-Gly-OH·dicyclohexylammonium salt, cyclopentyl-Gly-OH·dicyclohexylammonium salt, D-α,β-Diaminopropionic acid, D-α-aminobutyric acid, D-α-t-butylglycine, D-(2-thienyl)glycine, D-(3-thienyl)glycine, D-2-aminocaproic acid, D-2-indanylglycine, D-allylglycine dicyclohexylammonium 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 Lysine, 2-amino-β-(dimethylamino)-propionic acid, L-α,β-diaminopropionic acid, L-α-aminobutyric acid, L-α-t-butylglycine, L-β-thienylglycine, L-2-amino-β-(dimethylamino)propionic acid, L-2-aminocaproic acid dicyclohexyl-ammonium 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-allyloxy) (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-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,Examples of amino acids that may be used include, but are not limited to, 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-methoxy-Bzl)-Gly-OH.

[0044] Amino acid analogs include analogs of arginine or lysine. Examples of amino acid analogs of arginine and lysine include citrulline, L-2-amino-3-guanidinopropanoic 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-diaminoheptanedioic 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 (symmetric), Lys(ivDde)-OH, Lys(Me) 2 These include, but are not limited to, -OH·HCl, Lys(Me3)-OH chloride, Nω-nitro-D-arginine and Nω-nitro-L-arginine.

[0045] 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, 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-diaminopimelic acid, L-α-aminosuberic acid, D-2-aminoadipic acid, D-α-aminosuberic acid, α-aminopimelic acid, iminodiacetic acid, L-2-aminoadipic acid, threo-β-methyl-aspartic acid, γ-carboxy-D-glutamic acid γ,γ-di-t-butyl ester, γ-carboxy-L-glutamic acid γ,γ-di-t-butyl ester, Glu(OAll)-OH, L-Asu(OtBu)-OH, and pyroglutamic acid.

[0046] Amino acid analogs include analogs of cysteine ​​and methionine. Examples of amino acid analogs of cysteine ​​and methionine include 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 ... Examples of suitable cysteine ​​analogs include, but are not limited to, 1-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, t-butyl-D-cysteine, trityl-L-homocysteine, trityl-D-penicillamine, cystathionine, homocystine, L-homocystine, (2-aminoethyl)-L-cysteine, seleno-L-cystine, cystathionine, Cys(StBu)-OH, and acetamidomethyl 1-D-penicillamine.

[0047] Amino acid analogs include analogs of phenylalanine and tyrosine. Examples of amino acid analogs of phenylalanine and tyrosine include 3-methyl-phenylalanine, 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-dichloro-phenylalanine, 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-Trihydroxy-phenylalanine, 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, ethyl-tyrosine, and methyltyrosine.

[0048] Amino acid analogs include analogs of proline. Examples of amino acid analogs of proline include, but are not limited to, 3,4-dehydroproline, 4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid, and trans-4-fluoroproline.

[0049] Amino acid analogs include serine and threonine analogs. 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-ethoxybutanoic acid, 2-amino-3-methoxybutanoic 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-hydroxybutanoic acid, and α-methylserine.

[0050] Amino acid analogs include analogs of tryptophan. Examples of amino acid analogs of tryptophan include α-methyl-tryptophan, β-(3-benzothienyl)-D-alanine, β-(3-benzothienyl)-L-alanine, 1-methyl-tryptophan, 4-methyl-tryptophan, 5-benzyloxy-tryptophan, 5-bromo-tryptophan, 5-chloro-tryptophan, 5-fluoro-tryptophan, 5-hydroxy-tryptophan, 5-hydroxy-L-tryptophan, 5-methoxy-tryptophan, 5-methoxy-L-tryptophan, 5-methyl-tryptophan, 6-bromo-tryptophan, 6-chloro-D-alanine, 1-methyl-tryptophan, 4-methyl-tryptophan, 5-benzyloxy-tryptophan, 5-bromo-tryptophan, 5-chloro-tryptophan, 5-fluoro-tryptophan, 5-hydroxy-tryptophan, 5-hydroxy-L-tryptophan, 5-methoxy-tryptophan, 5-methyl-tryptophan, 6-bromo-tryptophan, 6-chloro-D-alanine, 1-methyl-tryptophan, 4-methyl-tryptophan, 5-benzyloxy-tryptophan, 5-bromo-tryptophan, 5-chloro-D-alanine, 5-fluoro-tryptophan, 5-hydroxy-tryptophan, 5-hydroxy-L-tryptophan, 5-methoxy-tryptophan, 5-methyl-tryptophan, 6-bromo-tryptophan, 6-chloro-D-alanine, 1-methyl-tryptophan, 4-methyl-tryptophan, 5-benzyloxy-tryptophan, 5-bromo-trypto -tryptophan, 6-chloro-tryptophan, 6-fluoro-tryptophan, 6-methyl-tryptophan, 7-benzyloxy-tryptophan, 7-bromo-tryptophan, 7-methyl-tryptophan, D-1,2,3,4-tetrahydro-norharman-3-carboxylic acid, 6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid, 7-azatryptophan, L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid, 5-methoxy-2-methyl-tryptophan, and 6-chloro-L-tryptophan.

[0051] 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 contains a chiral center that is in the R or S configuration. In still other embodiments, the amino group of the β-amino acid analog is substituted with a protecting group, such as tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), tosyl, and the like. In yet other embodiments, the carboxylic acid functionality 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.

[0052] A "non-essential" amino acid residue is one that can be altered from the wild-type sequence of a polypeptide without eliminating or substantially eliminating an essential biological or biochemical activity of the polypeptide (e.g., receptor binding or activation, etc.). An "essential" amino acid residue is one that, when altered from the wild-type sequence of a polypeptide, eliminates or substantially eliminates an essential biological or biochemical activity of the polypeptide.

[0053] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having 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), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), non-polar side chains (e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I), and aromatic side chains (e.g., Y, F, W, H). Thus, a predicted non-essential amino acid residue in a polypeptide is replaced, for example, with another amino acid residue from the same side chain family. Other examples of permissible substitutions are based on isosteric considerations (e.g., norleucine for methionine) or other properties (e.g., 2-thienylalanine for phenylalanine).

[0054] The term "peptide" refers to one or more linked amino acid residues. The term "polypeptide" refers to a linear organic polymer consisting of many amino acid residues (20 or more) linked in a chain, which forms part (or the entirety) of a protein molecule.

[0055] The term "cyclic peptide" refers to a polypeptide chain that includes a cyclic sequence of bonds. For example, in any aspect or embodiment described herein, a cyclic peptide includes, but is not limited to, a structure according to the following:

[0056] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0057] The term "α-polypeptide" refers to a polypeptide derived from α-amino acids.

[0058] The term "β-polypeptide" refers to a polypeptide derived from β-amino acids.

[0059] The term "phosphate ester" refers to an ester of phosphoric acid in which the central phosphate molecule has alkyl or aromatic substituents. For example, in any aspect or embodiment described herein, the phosphate ester includes, but is not limited to, the structure according to the following: [ka]

[0060] The term "aliphatic" or "aliphatic group" refers to a linear (i.e., unbranched), branched, or cyclic (including fused, bridged, and spiro-fused polycyclic) hydrocarbon moiety, which may be fully saturated or contain one or more units of unsaturation. Suitable aliphatic groups include, but are not limited to, linear or branched alkyl groups, alkenyl groups, alkynyl groups, and mixtures thereof. As used herein, the term "aliphatic" or "aliphatic group" also encompasses partially substituted analogs of these moieties in which at least one of the hydrogen atoms of an aliphatic group is replaced with an atom other than carbon or hydrogen.

[0061] The term "linker" refers to a chemical group that links to one or more other chemical groups via at least one covalent bond.

[0062] Although the present invention has been described with reference to exemplary embodiments, those skilled in the art will recognize that various changes and equivalent substitutions can be made to the elements thereof without departing from the scope of the invention. In addition, many modifications can be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope of the invention. Therefore, it is not intended that the invention be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but it is intended that the invention include all embodiments falling within the scope of the appended claims. All combinations of the above-described elements in all possible variations are encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0063] Self-organized nanomaterials The self-assembled nanomaterials of the present disclosure include Janus-based nanotubes (JBNTs). JBNTs consist of structural units based on a single ring system and self-assemble into nanomaterials that can be used for drug delivery and scaffolding. In particular, JBNTs provide a solution for delivering biologically active molecules to specific cells and / or tissues. In one embodiment, JBNTs include biologically active molecules covalently or non-covalently attached to the JBNTs. Furthermore, JBNTs may be coupled / conjugated to targeting moieties to facilitate active targeting of the self-assembled nanomaterials including biologically active molecules to specific cells and / or tissues.

[0064] JBNT is a biocompatible and biodegradable material with relatively low cytotoxicity and immunogenicity. JBNT also combines the advantages of lipid nanoparticles and cationic polymers to improve endosomal escape and enhance efficacy. Advantageously, JBNT can efficiently enter cells via macropinocytosis (using the same mechanism as lipid nanoparticles) and efficiently escape endosomes via the "proton sponge" effect, the same mechanism as cationic polymers. Thus, JBNT provides excellent delivery of biologically active molecules and exhibits extremely low cytotoxicity.

[0065] In one embodiment, the self-assembled nanomaterial comprises Janus-based nanotubes comprising at least one compound represented by Formulae I-XII or a pharma- ceutically acceptable salt thereof.

[0066] [ka] [ka] [ka] [ka] In the formula, R 1 is H or CH 3 and R 2 is (CH 2 ) j , (CH 2 CH 2 O) k , or (CH 2 CH 2 NH) m 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 linker group; T is a biologically active molecule or a targeting molecule / moiety; R 4 is a coating material. In one embodiment, T is a targeting molecule.

[0067] In any aspect or embodiment described herein, the targeting molecule or moiety includes or is a peptide, a cyclic peptide, a small molecule, another molecular structure referred to herein as a targeting molecule or moiety, or a combination thereof.

[0068]

[0043] In any aspect or embodiment describe herein, the coating material comprises a polymer, a peptide, a polypeptide, a lipid-based material, a phosphate ester, or a biomimetic membrane.

[0069] In one embodiment, L is a linker group and is selected from an acid-cleavable group, a reducible disulfide group, an α-amino acid, a β-amino acid, an α-polypeptide, a β-polypeptide, an enzyme-cleavable group, a stimuli-responsive group, or a combination thereof. The acid-cleavable group may comprise an N-acylhydrazone, a carbonate group, or an ester group. The reducible disulfide linker may comprise N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), or 4-(4'-acetylphenoxy)butanoic acid (AcBut), a Val-Cit dipeptide, a Phe-Lys dipeptide, an α-methyl-substituted disulfide, an engineered cysteine ​​residue, or a thiol-containing maytansinoid. The stimuli-responsive linker may comprise a trans-cyclooctene linker or a thioether-containing linker. The enzyme-cleavable linker may include GPLGOAGQ (SEQ ID NO: 89), GDEVEAPKGC (SEQ ID NO: 90), citrulline-valine, a glycosidase-cleavable linker, a β-glucoronidase-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 (para-aminobenzylcarbamate), Val-Cit-PABC-containing linker, Glu-Val-Cit-containing linker or a Vai-Ala-containing linker.

[0070] In one embodiment, T is a targeting molecule, the targeting molecule being selected from a biologically active molecule, an amphiphilic polymer, an aptamer, a peptide, a protein, a polysaccharide, a polyunsaturated fatty acid, or a carbohydrate. The choice of targeting molecule depends on the cells and / or tissues to which the self-assembled nanomaterial is delivered.

[0071] 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; and (v) glycerides such as glycerides, glycerols, and glycerols. v) targeting molecules for infected cells / tissues, such as RGD, c(CMGRC) (SEQ ID NO: 17), PHSRN (SEQ ID NO: 18), LHRD (SEQ ID NO: 19), antigenic peptides, endogenous peptides, cell penetrating peptides, VP22, RPRAPARSASRPRRPVE (SEQ ID NO: 20), sC18, GLRKRLRKFRNKIKEK (SEQ ID NO: 21), Pept1, and PLILLRLLRGQF (SEQ ID NO: 22); and (vi) transferrin, OX26, CAQK ( (vii) targeting molecules for blood-brain barrier (BBB) ​​penetration, 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), Tumor targeting molecules such as EGF, VEGF, LFA-1 and apolipoprotein AI, and (viii) 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 (x) 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) 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 2mitochondrial membrane targeted molecules such as (1,7-bis-4-hydroxy-3-methoxyphenyl-1,6-heptadiene-3,5-dione)-triphenylphosphine, 1,5-dioctadecyl-L-glutamyl 2-histidyl-hexahydrobenzoate-SPC-L, MSVLTPLLLRGLTGSARRLPVPRAKIHWLC (SEQ ID NO: 49), GKRK (SEQ ID NO: 50), and D[KLAKLAK]2 (SEQ ID NO: 51); and (xvii) KKKRKV (SEQ ID NO: 52), KRPAATKKAGQAKKKKL (SEQ ID NO: 53), HIV-1 TAT, GRKKRRQRRRPQ (SEQ ID NO: 54), R8, RRRRRRRR (SEQ ID NO: 55), penetratin, RQIKIWFQNRRMKWKK (SEQ ID NO: 56), HA2 peptide, GDIMGEWGNEIFGAIAAGFLG (SEQ ID NO: 57), GALA (SEQ ID NO: 58), WEAALAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 59), Pas, FFLIPKG (SEQ ID NO: 60), THRPPMWSPWVWP (SEQ ID NO: 61), Angiopep2, TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 62), glutathione, (γE)CG, CDX, FKESWREARGTRIERG (SEQ ID NO: 63), chlorotoxin, MCMPCFTTDHQMARKCDDC CGGKGRGKCYGPQCLCR (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 ID NO: 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),(xviii) nuclear targeting molecules such as 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); and (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 macrophage (TAM) targeting peptides such as YEQDPWGVKWWY (SEQ ID NO: 78), CSPGAKVRC (SEQ ID NO: 79); (xxii) regulatory T lymphocyte (Treg) targeting peptides such as CGNKRTRGC (SEQ ID NO: 25); and (xxiii) myeloid-derived immunosuppressive cell (MDSC) targeting peptides such as MEWSLEKGYTIK (SEQ ID NO: 80).

[0072] In any aspect or embodiment described herein, T is a biologically active molecule.

[0073] In any aspect or embodiment described herein, T is a targeting molecule, and the self-assembled nanomaterial further comprises a biologically active molecule covalently or non-covalently attached to the self-assembled nanomaterial. In any aspect or embodiment described herein, the self-assembled nanomaterial comprises a biologically active molecule non-covalently attached (associated) to the JBNT. In any aspect or embodiment described herein, the biologically active molecule is at least partially encapsulated by the JBNT. For example, in any aspect or embodiment described herein, 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 biologically active molecule is encapsulated by the JBNT.

[0074] Non-limiting examples of biologically active agents include nucleic acids, proteins, peptides, and small molecule drugs. In one embodiment, the biologically active agent is a nucleic acid, such as mRNA, guide RNA (gRNA or sgRNA), crRNA, tracrRNA, tRNA, ssDNA, dsDNA, cDNA, or a combination thereof.

[0075] In any aspect or embodiment described herein, R 2is a coating material comprising a polymer, peptide, polypeptide, lipid-based material, or a biomimetic membrane. The coating material can serve to protect the self-assembled nanomaterial from specific or non-specific removal from the body by cells and / or organs. In any aspect or embodiment described herein, the polymeric coating material includes, but is not limited to, polyethylene glycol (PEG), chitosan, hyaluronic acid, poloxamer, polyvinyl alcohol, polysaccharides, neutrally charged polyamino acids, negatively charged polyamino acids, or combinations thereof. In any aspect or embodiment described herein, the peptide coating material includes, but is not limited to, a "self" peptide (a peptide generated by proteolysis of a self-protein in a cell expressing an MHC class I or II molecule, e.g., a TCR-peptide-MHC class II peptide), an anti-thrombotic peptide (e.g., a CD31 agonist peptide), 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 herein, the polypeptide (protein) coating material includes, but is not limited to, phytochelatin, GCK15, PD-L1, CD47, CD24, β2-microglobulin, bovine serum albumin (BSA), hydrophobin, clusterin / ApoJ, fibrinogen, or a combination thereof. In any aspect or embodiment described herein, the lipid-based coating material includes, but is not limited to, natural waxes (e.g., carnauba wax, candelilla wax, rice wax, beeswax), petroleum-based waxes (e.g., paraffin wax, polyethylene wax), petroleum-based oils, mineral oils, vegetable oils, acetoglycerides, fatty acids, resins (e.g., shellac, wood rosin), or a combination thereof.In any aspect or embodiment described herein, the biomimetic membrane coating material includes, but is not limited to, membranes of red blood cells (RBCs), white blood cells (WBCs), cancer cells, mesenchymal stem cells, platelets, beta cells, or combinations thereof. Combinations including at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or more) of the foregoing coating materials may also be used.

[0076] In any aspect or embodiment described herein, the self-assembled nanomaterial may include a single type of JBNT or may include multiple types of JBNP. In any aspect or embodiment described herein, the self-assembled nanomaterial is co-assembled from two or more (e.g., multiple) JBNTs, each having different properties from each other. For example, in any aspect or embodiment described herein, the different types of JBNTs can have properties that enhance the hydrophobicity, stability, and / or self-assembly of the self-assembled nanomaterial. These properties may be designed to affect cellular delivery, including the proton sponge effect, circulation time in vivo, passive or active targeting, intracellular targeting, improved cellular uptake, and / or enhanced endosomal escape of the self-assembled nanomaterial.

[0077] In any aspect or embodiment described herein, the Janus-based nanotube comprises at least one compound represented by Formulae I-IV.

[0078] In any aspect or embodiment described herein, the Janus-based nanotube comprises at least one compound represented by Formulae V-VIII.

[0079] In any aspect or embodiment described herein, the Janus-based nanotube comprises at least one compound represented by Formulas IX-XIII.

[0080]

[0131] In any aspect or embodiment describe herein, the Janus-based nanotube comprises: A combination of a compound represented by formula I to IV and a compound represented by formula V to VIII. A combination of a compound represented by formula I to IV and a compound represented by formula IX to XIII. A combination of a compound represented by formulas V to VIII and a compound represented by formulas IX to XIII. A combination of a compound represented by formula I to IV, a compound represented by formula V to VIII, and a compound represented by formula IX to XIII, or Combinations of these Includes.

[0081] In any aspect or embodiment described herein, the total amount of JBNT present in the self-assembled nanomaterial ranges from 0.1 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).

[0082] In any aspect or embodiment described herein, the self-assembled nanomaterial is in the form of fibers 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 μm.

[0083] In any aspect or embodiment describe herein, the pH of the self-assembled nanomaterial is from 1 to 10.

[0084] In any aspect or embodiment described herein, the self-assembled nanomaterial may have a single compartment structure or a multi-compartment structure. As used herein, in any aspect or embodiment described herein, a single compartment nanomaterial includes a single population of self-assembled nanomaterial, i.e., a single type of JBNT and one or more ECM molecules attached to the JBNT. As used herein, in any aspect or embodiment described herein, a multi-compartment nanomaterial includes two or more populations of self-assembled nanomaterial that form a multi-compartment structure by electrostatic multi-layer assembly. For example, in any aspect or embodiment described herein, the opposing electrostatic charges of the first and second populations of JBNTs can facilitate the assembly of the multi-compartment nanomaterial. The first and second populations are assembled in sequence, such that one population forms an inner compartment and the second population forms an outer compartment.

[0085] In any aspect or embodiment described herein, the self-assembled nanomaterials can be optionally combined with extracellular matrix (ECM) molecules. In any aspect or embodiment described herein, the self-assembled nanomaterials described herein are tunable materials that include Janus-based nanotubes and ECM. In any aspect or embodiment described herein, the JBNTs can be assembled with different ECMs to form different nanomatrix materials, e.g., scaffolds, for different cells / tissues. In any aspect or embodiment described herein, these nanomatrix materials can have a single compartment structure or a multi-compartment structure. In any aspect or embodiment described herein, the nanomatrix materials can be fabricated with multifunctional layers or compartments to achieve various functions (e.g., cell growth support, drug release). In any aspect or embodiment described herein, the ECM molecule includes, but is 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 molecule (ICAM1-5), integrin, proteoglycan, (aggrecan), glycosaminoglycan (e.g., hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate), glycoprotein, or a combination thereof.

[0086] In any aspect or embodiment described herein, the weight ratio of JBNT to ECM molecules is between 1000:1 and 1:1.

[0087]

[0023] In any aspect or embodiment described herein, the coating material comprises polyethylene glycol (PEG).

[0023] In any aspect or embodiment described herein, the coating material comprises PEG and the self-assembled nanomaterial comprises a targeting molecule.

[0088] In any aspect or embodiment described herein, JBNP or JBNT comprises a targeting molecule comprising the amino acid sequences WYRGRL and miR140 (WYRGRL-JBNP-miR140). In any aspect or embodiment described herein, WYRGRL-JBNP-miR140 is used and effective in treating, preventing, or ameliorating one or more symptoms of osteoarthritis.

[0089] In any aspect or embodiment described herein, JBNP or JBNT comprises a 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 ameliorate one or more symptoms of osteoarthritis, such as inflammation (e.g., inflammation of the knee joint).

[0090] In any aspect or embodiment described herein, the JBNP or JBNT comprises an IL-1R siRNA (JBNP-IL-1R siRNA). In any aspect or embodiment described herein, the JBNP-IL-1R siRNA is used and effective in treating, preventing, or ameliorating one or more symptoms of osteoarthritis, such as inflammation (e.g., inflammation of the knee joint).

[0091] In any aspect or embodiment described herein, JBNP or JBNT comprises IL-1RA mRNA (JBNP-IL-1RA mRNA). In any aspect or embodiment described herein, JBNP-IL-1RA mRNA is used and effective in treating, preventing or ameliorating one or more symptoms of osteoarthritis, such as inflammation (e.g., inflammation of the knee joint). In any aspect or embodiment described herein, the IL-1RA mRNA sequence is [ka] It is.

[0092] In any aspect or embodiment described herein, the JBNP or JBNT comprises an IL-1RA peptide mRNA (JBNP-IL-1RA peptide mRNA). In any aspect or embodiment described herein, the JBNP-IL-1RA peptide mRNA is used and effective in treating, preventing or ameliorating one or more symptoms of osteoarthritis, such as inflammation (e.g., inflammation of the knee joint). In any aspect or embodiment described herein, the IL-1RA peptide mRNA sequence is [ka] It is.

[0093] In any aspect or embodiment described herein, JBNT or JBNP includes a lysine or arginine side chain. In any aspect or embodiment described herein, JBNT or JBNP includes a lysine or arginine side chain to target the self-assembled nanomaterial to the liver.

[0094] In any aspect or embodiment described herein, JBNT or JBNP comprises a histidine side chain. In any aspect or embodiment described herein, JBNT or JBNP comprises a histidine side chain to target the self-assembled nanomaterial to an organ other than the liver.

[0095] 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, and is used in a method of delivering the JBNT or JBNP to cartilage.

[0096] In any aspect or embodiment described herein, the JBNP comprises an agent that is an mRNA, optionally with a targeting molecule and optionally with a reporter or label (e.g., tdTomato mRNA, Cy5, AF488, etc.). In any aspect or embodiment described herein, the JBNP comprises an agent that is an mRNA, optionally with a targeting molecule and optionally with a reporter / label (e.g., tdTomato mRNA, Cy5, AF488, etc.) that is delivered to the liver, kidney, brain, lung, spleen, lymph node, bone, muscle, heart, pancreas, intestine, solid tumor, or combinations thereof (JBNP-mRNA).

[0097] 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.

[0098] In any aspect or embodiment described herein, the JBNP comprises a CRISPR (e.g., Cas9 mRNA and gRNA) and a tumor targeting moiety. In any aspect or embodiment described herein, the JBNP comprising a CRISPR (e.g., Cas9 mRNA and gRNA) and a tumor targeting moiety is used in a method of delivering and editing DNA in tumor cells.

[0099] In any aspect or embodiment described herein, JBNT and JBNP, when administered, do not elicit acute toxicity, an innate immune response, an adaptive immune response, or a combination thereof.

[0100] In any aspect or embodiment described herein, the side chain of JBNT is modified to promote certain types of cell renewal. For example, in any aspect or embodiment described herein, lysine-based JBNT is used to promote cellular uptake of JBNP via micropinocytosis. As a further example, in any aspect or embodiment described herein, arginine-based JBNT is used to promote cellular renewal of JBNP via clathrin-dependent endocytosis.

[0101] In any aspect or embodiment described herein, the JBNP comprises a lysine-based JBNT, which provides significantly improved endosomal escape, apparently due to a proton sponge effect.

[0102] In any aspect or embodiment described herein, the JBNP comprises an arginine-based JBNT, which provides significantly enhanced endosomal escape, apparently due to a pore-forming effect in escape from early endosomes.

[0103] In any aspect or embodiment described herein, the JBNPs of the present disclosure are less cytotoxic than lipid nanoparticles, polymeric nanoparticles (e.g., poly-l-lysine (PLL) nanoparticles or polyethyleneimine (PEI) nanoparticles), single-walled nanotubes, or combinations thereof.

[0104] In any aspect or embodiment described herein, the JBNP comprises Cas9mRNA-eGFP and gRNA-ATTO550.

[0105] Pharmaceutical Compositions The self-assembled nanomaterials disclosed herein can be formulated as an injectable composition. In any aspect or embodiment described herein, the injectable composition comprises the self-assembled nanomaterial and a pharma- ceutically acceptable carrier. In any aspect or embodiment described herein, the self-assembled nanomaterials can be administered parenterally, subcutaneously, intravenously, intramuscularly, intrasternally in a sterile medium, or in the form of a sterile injectable aqueous or oily suspension by injection techniques. Advantageously, in any aspect or embodiment described herein, auxiliary agents such as local anesthetics, preservatives, buffers, etc. can be dissolved in the vehicle.

[0106] The pH of the pharmaceutical composition comprising the self-assembled nanomaterial may be physiological pH.

[0107] The present disclosure is further illustrated by the following non-limiting examples.

[0108] The present disclosure is further illustrated by the following non-limiting examples. EXAMPLES

[0109] Materials: All reagents and solvents disclosed herein were obtained from commercial suppliers, including Sigma-Aldrich, Alfa Aesar, Fisher Scientific, and Thermo Fisher, and were used without further purification.

[0110] Example 1 A compound designated ArgJBNT was prepared as follows. [ka]

[0111] Synthesis of compound A3: Pre-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, followed by addition of solid NaBH(OAc)3 (49 mg) was added to the solution, and the resulting slurry was stirred at room temperature for 24 hours. After completion, the reaction mixture was quenched with water and extracted with dichloromethane. The organic layers were combined and washed with brine. MgSO 4 After drying at rt, filtration and evaporation of the solvent under reduced pressure, the crude product A3 (156 mg) was obtained and used in the next step without further purification.

[0112] Synthesis of compound ArgJBNT: Compound A3 (156 mg) was added to a 94% TFA / thioanisole (2.8 mL) solution. After stirring at room temperature for 72 hours, the solution was diluted with diethyl ether (Et 2 0) was added. A white precipitate was formed and was precipitated by centrifugation. After discarding the supernatant, the white precipitate was diluted with Et 2 The crude product was obtained by washing with O. The crude product was purified using HPLC to yield compound ArgJBNT (37 mg, 70%). 1H NMR (500 MHz, DC1 / 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: 409.2055, found: 409.2078.

[0113] Reaction Scheme 1 shows the formation of the compound ArgJBNT. [ka]

[0114] Example 2 The compound designated GlyJBNT was prepared as follows. [ka]

[0115] Synthesis of compound A5: Pre-synthesized compound A1 (96 mg) and 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, followed by addition of solid NaBH(OAc) 3 (49 mg) was added to the solution, and the resulting slurry was stirred at room temperature for 24 hours. After completion, the reaction mixture was quenched with water and extracted with dichloromethane. The organic layers were combined and washed with brine. MgSO 4 After drying at rt, filtration and evaporation of the solvent under reduced pressure, the crude product A5 (102 mg) was used in the next step without further purification.

[0116] Synthesis of compound GlyJBNT: Compound A5 (102 mg) was added to a 94% TFA / thioanisole (2.8 mL) solution. After stirring at room temperature for 72 hours, the solution was diluted with diethyl ether (Et 2 0) was added. A white precipitate was formed and was precipitated by centrifugation. After discarding the supernatant, the white precipitate was diluted with Et 2 The crude product was obtained by washing with O. The crude product was purified using HPLC to yield 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), 7.70(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 value: 310.2158, measured value: 310.1208.

[0117] Reaction Scheme 2 shows the formation of the compound GlyJBNT. [ka]

[0118] Example 3 The compound designated AspJBNT was prepared as follows. [ka]

[0119] Synthesis of compound A7: Pre-synthesized compound A1 (96 mg) and 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, followed by addition of solid NaBH(OAc) 3 (49 mg) was added to the solution, and the resulting slurry was stirred at room temperature for 24 hours. After completion, the reaction mixture was quenched with water and extracted with dichloromethane. The organic layers were combined and washed with brine. MgSO 4 After drying at rt, filtration and evaporation of the solvent under reduced pressure, the crude product A7 (110 mg) was used in the next step without further purification.

[0120] Synthesis of Compound AspJBNT: Compound A7 (110 mg) was added to a 94% TFA / thioanisole (2.8 mL) solution. After stirring at room temperature for 72 hours, the solution was diluted with diethyl ether (Et 2 0) was added. A white precipitate was formed and was precipitated by centrifugation. After discarding the supernatant, the white precipitate was diluted with Et 2 The crude product was obtained by washing with O. The crude product was purified using HPLC to yield 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), 7.5 5(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 value: 368.1313, measured value: 368.1278.

[0121] Reaction Scheme 3 shows the formation of the compound AspJBNT. [ka]

[0122] Example 4 The compound designated HisJBNT was prepared as follows. [ka]

[0123] Synthesis of compound A9: Pre-synthesized compound A1 (96 mg) and 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, followed by addition of solid NaBH(OAc) 3 (49 mg) was added to the solution, and the resulting slurry was stirred at room temperature for 24 hours. After completion, the reaction mixture was quenched with water and extracted with dichloromethane. The organic layers were combined and washed with brine. MgSO 4 After drying at rt, filtration and evaporation of the solvent under reduced pressure, the crude product A9 (17 mg) was used in the next step without further purification.

[0124] Synthesis of compound HisJBNT: Compound A7 (17 mg) was added to a 94% TFA / thioanisole (0.44 mL) solution. After stirring at room temperature for 72 hours, the solution was diluted with diethyl ether (Et 2 0) was added. A white precipitate was formed and was precipitated by centrifugation. After discarding the supernatant, the white precipitate was diluted with Et 2 The crude product was obtained by washing with O. The crude product was purified using HPLC to yield compound HisJBNT (8 mg, 14%). 1 H NMR(500MHz,DMSO-d6)δ11.77(br,m,1H), 8.51(br,m,1H), 8.06(br,m,1H), 7.88(br,m,1H), 7.5 5(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 value: 390.1633, measured value: 390.1601.

[0125] Reaction Scheme 4 shows the formation of the compound HisJBNT. [ka]

[0126] Example 5 A compound designated PEGLysJBNT was prepared as follows. [ka]

[0127] Synthesis of compound PEGLysJBNT: NaHCO 3 The aqueous solution (0.8 mL, 0.01 M) was added to a solution of MS(PEG)24 methyl-PEG-NHS-ester (5 mg) in DMF (0.2 mL). After 24 h, the reaction mixture was purified using HPLC to yield the compound PEGLysJBNT (1 mg, 33%). HRMS (ESI) [M+H] + Calculated value: 1479.8390, measured value: 1479.8654.

[0128] Reaction Scheme 5 shows the formation of PEGlysJBNT. [ka]

[0129] Example 6 The compound designated LPLysJBNT was prepared as follows. [ka]

[0130] Synthesis of compound LPLysJBNT: NaHCO of previously synthesized LysJBNT (1 mg) 3The aqueous solution (0.8 mL, 0.01 M) was added to a solution of N-α-maleimidomethyl succinimide ester (AMAS, 1 mg) in DMF (0.2 mL). After 24 h, human collagen-targeting peptide RLDPTSYLRTFWC (7 mg) was added to the reaction mixture and reacted for another 24 h. The reaction mixture was purified using HPLC to produce compound LPLysJBNT (5 mg, 100%). HRMS (ESI) [M+2H] 2+ Calculated value: 1095.5226, measured value: 1095.5038.

[0131] Reaction Scheme 5 shows the formation of LPLysJBNT. [ka]

[0132] Example 7 A compound designated SPLysJBNT was prepared as follows. [ka]

[0133] Synthesis of compound SPLysJBNT: NaHCO of previously synthesized LysJBNT (1 mg) 3 The aqueous solution (0.8 mL, 0.01 M) was added to a solution of N-α-maleimidomethyl succinimide ester (AMAS, 1 mg) in DMF (0.2 mL). After 24 h, the targeting peptide WYRGRLC (4 mg) targeting human collagen was added to the reaction mixture and waited another 24 h. The reaction mixture was purified using HPLC to produce the compound SPLysJBNT (3 mg, 100%). HRMS (ESI) [M+3H] 3+ Calculated value: 490.8984, measured value: 490.8820.

[0134] Reaction Scheme 5 shows the formation of SPLysJBNT. [ka]

[0135] Example 8: Multifunctional Janus-based Nanoparticles (JBNPs) Multifunctional JBNT: Multifunctional JBNT was combined in an appropriate molar ratio at room temperature for 24 hours to promote the self-assembly of JBNT. To prepare JBNT consisting of PEGLysJBNT and ArgJBNT, the molar ratio of PEGLysJBNT / ArgJBNT is 0.05:1. To prepare JBNT consisting of peptides LysJBNT, PEGLysJBNT and ArgJBNT, the molar ratio of peptides LysJBNT / PEGLysJBNT / ArgJBNT is 0.05:0.05:1.

[0136] Multifunctional JBNPs. Multifunctional JBNPs were prepared by mixing the multifunctional JBNT with cargo molecules (e.g., RNA, biologically active small molecules, proteins) in appropriate molar ratios in nuclease-free water, followed by sonication for 2 min 30 s at room temperature at 100% amplitude in a sonicator. The concentrations of the different components are c(JBNT) = 1 mg / mL, c(siRNA) = 5 μM, c(mRNA) = 0.05 mg / mL, c(protein) = 0.1 mg / mL. The ratios (volume to volume) of each cargo are 4:1 (Arg-JBNT:siRNA), 20:1 (Arg-JBNT:mRNA), and 3:1 (Arg-JBNT:protein).

[0137] Arg-JBNP-siRNA was assembled by mixing Arg-JBNT (1 mg / ml) and siRNA-AF488 (5 μM) in a 4:1 volume-to-volume ratio in nuclease-free water. Arg-JBNP-mRNA was assembled by mixing Arg-JBNT (1 mg / ml) and Cas9mRNA (0.05 mg / ml) in a 20:1 volume-to-volume ratio. Arg-JBNP-albumin was assembled by mixing Arg-JBNT (1 mg / ml) and albumin (0.1 mg / ml) in a 3:1 volume-to-volume ratio. The mixture of ArgJBNT and cargo in nuclease-free water was then sonicated for 2 min 30 s at 100% amplitude in a sonicator.

[0138] Characterization of JBNP. The particle size and zeta potential of JBNP were measured by dynamic light scattering (Zetasizer), and the morphology was observed by transmission electron microscopy (TEM). Gel shift assays were performed on 0.8% low melting point agarose gels followed by electrophoresis. UV-Vis absorption spectra were recorded on a NanoDrop One. The buffering capacities of JBNP and polymers, and JBNP and cationic polymers, at the same 0.08 μmol, were titrated by adding 2 μL of 10 mM HCl or 10 mM NaOH to investigate.

[0139] A Nikon A1 scanning confocal laser microscope was used for fluorescence imaging.

[0140] Example 9: Characterization and testing Lys-JBNP delivery: Assembled Lys-JBNP (30 μg / ml) was transfected into C28 / I2 cells and then incubated at 37°C, 5% CO 2 The cells were incubated with 0.5% ethanol for 24 or 48 hours. The cells were then fixed with 4% formaldehyde, treated with Triton® X-100, and stained with rhodamine phalloidin (30 min) and DAPI (10 min). The uptake of siRNA-AlexaFluor® 488 (AllStars Neg. siRNA488, Qiagen) was quantified by flow cytometry 24 or 48 hours after transfection into the cells. For siRNA knockdown studies, assembled Lys-JBNP (30 μg / ml) was used to deliver GAPDH siRNA (On-TARGETplus Human GAPDH siRNA, Horizon Discovery) for 24 hours. Lipofectamine® 2000 (Invitrogen) was used as a control according to the manufacturer's protocol. GAPDH gene expression was analyzed by RT-PCR.

[0141] Endosomal escape. In the endosomal escape study, Lysotracker® Red was added to cells followed by fixation with 4% formaldehyde. The degree of colocalization of Lysotracker® Red and JBNP was quantified based on the Pearson correlation coefficient (R) using Image J software according to the colocalization threshold and the coloc2 plugin.

[0142] Cellular uptake mechanism studies. C28 / I2 human chondrocytes were exposed to several different inhibitors for 1 h, specifically, chlorpromazine (Cpz) hydrochloride (100 μM, 30 min), methyl-β-cyclodextrin (Mβcd, 1 mM, 30 min), cytochrome D (CytD, 4 μM, 1 h), latrunculin A (2 μM, 30 min), bafilomycin A1 (200 nM, 30 min), and chloroquine (10 μM, 30 min).

[0143] Antiviral studies. RGD fiber-modified adenovirus expressing GFP was added to human embryonic fibroblasts. JBNPs or LNPs containing eGFP siRNA were contacted with the infected cells for 24 h. A fluorescent microscope was used to capture cell images.

[0144] Results of Examples 1 to 9 JBNT successfully delivers RNA with enhanced endosomal escape and high biocompatibility. As shown in FIG. 1A, a targeting moiety (peptide) is conjugated to JBNT and co-assembles with JBNT to provide a targeting peptide-JBNT. In particular, JBNT is combined with a targeting peptide under conditions that promote binding of the peptide to JBNT. In general, the binding of the targeting peptide is a covalent bond. 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 FIG. 1B.

[0145] By optimizing the molar ratios of JBNT, targeting peptide and biologically active molecule, it was possible to generate targeting peptide-JBNP (i) LysJBNT, ii) RLDPTSYLRTFWC peptide-PEGLysJBNT, iii) LysJBNP, iv) RLDPTSYLRTFWC peptide-PEGLysJBNP) as shown in FIG. 1C and with the absorbance profile shown in FIG. 1D.

[0146] As shown in Figures 2A-2H, JBNPs can carry biologically active molecules, such as siRNA, proteins, and / or small molecule drugs, as cargo and can be used to deliver the biologically active molecules to target cells in vitro or in vivo. Figures 2G-2H show the effect of various inhibitors on the uptake of JBNPs by target cells. Cells exposed to low temperature (4 °C) or NaN 3 Cells treated with latrunculin A (Lat) or cytochalasin D (CytD) show reduced uptake of JBNP as evidenced by a decrease in intracellular fluorescence.

[0147] Figures 3A-3E show the successful delivery of small RNA, mRNA, and albumin (protein) using ArgJBNP. Figure 3A shows TEM images of Arg-JBNP containing miR140 (Arg-JBNP-miR140), Arg-JBNP containing Cas9mRNA (Arg-JBNP-Cas9mRNA), and Arg-JBNP containing albumin (Arg-JBNP-Albumin). The results in Figure 3B are based on the analysis of TEM image data and show the average size and width of Arg-JBNP-miRNA. Figures 3C and 3E are zeta potential analyses of Arg-JBNP-miR140 and Arg-JBNP-Cas9mRNA, respectively. Figure 3D shows the results of a gel shift assay, and Figure 3F shows z-stack images by CLSM of siRNA-AF488 delivered by Arg-JBNP.

[0148] Figure 3G is a flow cytometry analysis graph showing the uptake of siRNA-AF488 by cells. Figure 3H is a fluorescence image of Cy5.5-eGFP-mRNA delivery via Arg-JBNP or Lipofectamine® 200. Figure 3I is a fluorescence image of BSA-AF488 delivery via Arg-JBNP. Figure 3J is a series of flow cytometry graphs showing the time-dependent uptake of Cas9eGFP.

[0149] To create Arg-JBNP-eGFP-Cas9mRNA, an mRNA containing a sequence encoding GFP upstream of Cas9 (see Figure 3K) was added to Arg-JBNP. Figure 3K shows a fluorescent image demonstrating the uptake of Arg-JBNP-eGFP-Cas9mRNA by cells. Figure 3L shows the time-dependent delivery of Arg-JBNP-eGFP-Cas9mRNA measured by flow cytometry.

[0150] It was also shown that JBNPs have enhanced endosomal escape properties compared to lipid nanoparticles, and are superior in delivering cargo biologically active molecules. The results are shown in Figures 4A-4G.

[0151] Also, as shown in Figures 5A-5H, JBNPs modified with PEG are protected from non-specific cellular uptake by undesirable cell types. Figure 5A shows the nanotube structure of PEGLysJBNT in TEM. Figures 5B and 5C show a comparison of PEGLysJBNT / ArgJBNT at different ratios (0, 0.01:1, 0.02:1, 0.05:1) using UV-Vis. Figure 5D shows a comparison of PEGLysJBNT / ArgJBNT / siRNA at different ratios (0, 0.01:1:0.33, 0.02:1:0.33, 0.05:1:0.33) using TEM. Figure 5E shows a comparison of PEGLysJBNT / ArgJBNT / siRNA at different ratios (0, 0.01:1:0.33, 0.02:1:0.33, 0.05:1:0.33) using UV-Vis. Figure 5F shows the zeta potential comparison of different ratios (0, 0.01:1:0.33, 0.02:1:0.33, 0.05:1:0.33) of PEGLysJBNT / ArgJBNT / siRNA using a Zetasizer. In Figure 5G, ArgJBNP or PEG-JBNP were transfected for 24 hours, followed by flow cytometry. Figures 5H and 5I show the analysis by flow cytometry. PEGylation is to protect against non-specific cellular uptake (e.g., immune cells and other unwanted cells), and a targeting peptide with PEG is added to achieve active targeting.

[0152] It was also demonstrated that combining multiple JBNTs with different functions (i.e., multifunctional JBNTs) provides JBNTs with multifunctional properties. For example, it is now possible to combine both the PEG effect and the targeting ability of JBNPs (see Figures 6A-6K). It is also possible to combine more positively charged JBNTs, such as arginine-JBNT, to improve the cellular uptake efficiency (see Figures 3F-3L). Another example is combining an enhanced endosomal escape effect into JBNTs. This can be achieved by combining histidine-JBNT, which has excellent proton sponge effect ability (see Figure 4D).

[0153] Example 10: Examination of the ability of cartilage-targeted JBNP-IL-1RA mRNA to inhibit osteoarthritis-associated inflammation in the knee joint Treatments (saline, JBNP-IL-1RA mRNA, JBNP-scrambled mRNA, JBNP without mRNA) are administered by intra-articular injection to surgically destabilized medial meniscus (DMM) model (3-month-old male) mice one month after surgery after the severity of osteoarthritis (OA) is assessed by MANKIN score. Histological sections are stained with Safranin O and examined under a fluorescent microscope. Behavioral studies of Y-maze spontaneous alternation, open field, object recognition test, and fear conditioning are performed to examine the efficacy of the treatments. Illustrative non-limiting examples of mRNA sequences for IL-1RA are: [ka] It is.

[0154] Example 11: Examination of the ability of cartilage-targeted JBNP-IL-1RA peptide mRNA to inhibit osteoarthritis-associated inflammation in the knee joint After the severity of OA is assessed by the MANKIN score, the surgically destabilized medial meniscus (DMM) model (3 months old) mice are administered treatments (saline, JBNP-IL-1RA peptide mRNA, JBNP-scrambled mRNA, JBNP without mRNA) by intra-articular injection one month after surgery. IL-1RA peptide is an interleukin-1 receptor antagonist peptide. Tissue sections are stained with Safranin O and examined under a fluorescent microscope. To examine the efficacy of the treatment, behavioral studies of Y-maze spontaneous alternation, open field, object recognition test and fear conditioning are performed. Illustrative non-limiting examples of the mRNA sequence of IL-1RA peptide are: [ka] It is.

[0155] Example 12: Amino acid side chain modifications can be used to target the self-assembled nanomaterials of the present disclosure to organs of interest The protein corona of the self-assembled nanomaterials and compositions comprising the same described herein can be further modified by modification of the side chains of amino acid residues. The assembled JBNPs are incubated with mouse serum for 30 minutes. Then, the mixture is centrifuged at 13800 relative centrifugal force (RCF) at 4°C for 15 minutes. The supernatant is discarded and the precipitate is washed three times with phosphate-buffered saline. The proteins are separated by SDS-PAGE and the protein bands are confirmed by liquid chromatography tandem mass spectrometry (LC / MC / MS). Lysine and arginine side chains are believed to bind to the apolipoprotein E (APOE) family and target the self-assembled nanomaterials of the present disclosure to the liver. As a further example, histidine side chains in the protein corona composition are believed to target the self-assembled nanomaterials of the present disclosure to organs other than the liver.

[0156] Example 13: JBNP of the present disclosure actively targets cartilage and has an extended half-life when the cartilage targeting peptide WYRGRL is conjugated to JBNP Cartilage targeting peptide WYRGRL (SEQ ID NO: 47) was conjugated to JBNP with siRNA reporter, and WYRGRL-JBNP with assembled siRNA dye was introduced into human chondrocyte C28 / I2 cells. Fluorescence-activated cell sorting (FACS) was performed to quantify the targeting ability of WYRGRL-JBNP to target cartilage. JBNP of the present disclosure actively targets cartilage and has an extended half-life when a cartilage targeting molecule or moiety is conjugated to it (Figure 7B).

[0157] Example 14: Examination of the ability to deliver miR140 with cartilage-targeted ArgJBNP to delay or prevent the progression of osteoarthritis Cartilage targeting can be achieved, for example, via the peptide WYRGRL. OA severity is assessed by MANKIN score before administering treatment (saline, JBNP-miR140, JBNP-scrambled miRNA, or JBNP without miR140) by intra-articular injection to surgically destabilized medial meniscus (DMM) model mice one month after surgery. To test the efficacy of the treatment, behavioral studies of Y-maze spontaneous alternation, open field, object recognition test, and fear conditioning are performed. DAPI, H&E, and Safranin O staining of tissue sections are used to reveal effective targeting and delivery by fluorescence microscopy.

[0158] Example 15: Examination of the ability of cartilage-targeted JBNP-CRISPR targeting sgIL-1R to treat osteoarthritis, slow osteoarthritis progression, and inhibit inflammation Cartilage targeting can be achieved, for example, via the peptide WYRGRL. After the severity of OA is assessed by the MANKIN score, treatments (saline, JBNP-CRISPR-sgIL-1R, JBNP-CRISPR-sgNeg, or JBNP-CRISPR without sgRNA) are administered by intra-articular injection to surgically destabilized medial meniscus (DMM) model mice or 129SVE-M wild-type mice 2 months after surgery. OA progression is examined under a fluorescent microscope by DAPI, H&E, and Safranin O staining of tissue sections. To examine the efficacy of the treatment, behavioral studies of Y-maze spontaneous alternation, open field, object recognition test, and fear conditioning are performed.

[0159] Example 16: Examination of the ability of cartilage-targeted JBNP-IL-1RA siRNA to inhibit osteoarthritis-associated inflammation in the knee joint Cartilage targeting can be achieved, for example, via the peptide WYRGRL. After the severity of OA is assessed by the MANKIN score, treatments (saline, JBNP-IL-1RA siRNA, JBNP-negative siRNA, JBNP without siRNA) are administered by intra-articular injection to surgically destabilized medial meniscus (DMM) model (3-month-old male) mice one month after surgery. Histological sections are stained with Safranin O and examined under a fluorescent microscope. To examine the efficacy of treatment, behavioral studies of Y-maze spontaneous alternation, open field, object recognition test and fear conditioning are performed.

[0160] Example 17: Examination of the ability of cartilage-targeted JBNP-IL-1RA mRNA to inhibit osteoarthritis-associated inflammation in the knee joint Cartilage targeting can be achieved, for example, via the peptide WYRGRL. After the severity of OA is assessed by the MANKIN score, treatments (saline, JBNP-IL-1RA mRNA, JBNP-scrambled mRNA, JBNP without mRNA) are administered by intra-articular injection to surgically destabilized medial meniscus (DMM) model (3-month-old male) mice one month after surgery. Histological sections are stained with Safranin O and examined under a fluorescent microscope. To examine the efficacy of the treatment, behavioral studies of Y-maze spontaneous alternation, open field, object recognition test, and fear conditioning are performed. Illustrative non-limiting examples of mRNA sequences for IL-1RA are: [ka] It is.

[0161] Example 18: Examination of the ability of cartilage-targeted JBNP-IL-1RA peptide mRNA to inhibit osteoarthritis-associated inflammation in the knee joint Cartilage targeting can be achieved, for example, via the peptide WYRGRL. After the severity of OA is assessed by the MANKIN score, treatments (saline, JBNP-IL-1RA peptide mRNA, JBNP-scrambled mRNA, JBNP without mRNA) are administered by intra-articular injection to surgically destabilized medial meniscus (DMM) model (3 months old) mice one month after surgery. IL-1RA peptide is an interleukin-1 receptor antagonist peptide. Tissue sections are stained with Safranin O and examined under a fluorescent microscope. To examine the efficacy of the treatment, behavioral studies of Y-maze spontaneous alternation, open field, object recognition test and fear conditioning are performed. Illustrative non-limiting examples of mRNA sequences for IL-1RA peptide are: [ka] It is.

[0162] Example 19: Amino acid side chain modifications can be used to target the self-assembled nanomaterials of the present disclosure to organs of interest The protein corona of the self-assembled nanomaterials and compositions comprising the same described herein can be further modified by modification of the side chains of amino acid residues. The assembled JBNPs are incubated with mouse serum for 30 minutes. Then, the mixture is centrifuged at 13800 RCF for 15 minutes at 4°C. The supernatant is discarded and the precipitate is washed three times with phosphate buffered saline. The proteins are separated by SDS-PAGE and the protein bands are confirmed by liquid chromatography tandem mass spectrometry (LC / MC / MS). Lysine and arginine side chains are believed to bind to the apolipoprotein E (APOE) family and target the self-assembled nanomaterials of the present disclosure to the liver. As a further example, histidine side chains in the protein corona composition are believed to target the self-assembled nanomaterials of the present disclosure to organs other than the liver.

[0163] Example 20: JBNP of the present disclosure actively targets cartilage and has an extended half-life when the cartilage targeting peptide WYRGRL is conjugated to JBNP Cartilage targeting peptide WYRGRL (SEQ ID NO: 47) was conjugated to JBNP with mRNA reporter, and the assembled WYRGRL-JBNP was introduced into human chondrocyte C28 / I2 cells with mRNA dye. FACS was performed to quantify the targeting ability of WYRGRL-JBNP to target cartilage. JBNP actively targets cartilage and has an extended half-life when a cartilage targeting molecule or moiety is conjugated to it (Figure 7B and Figure 7C).

[0164] Example 21: Liver-targeted JBNPs of the present disclosure can deliver active agents to the liver ArgJBNP-mcherry mRNA was administered intravenously to BALB / cJ mice. The biodistribution of ArgJBNP-mcherry mRNA was monitored by IVIS® imaging (Figures 11A and 11B). ArgJBNP shows significant biodistribution in the liver, and transfected livers can be homogenized. Transfection efficiency is determined by Western blot and real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR).

[0165] Example 22: Examination of the ability of kidney-targeted JBNPs of the present disclosure to deliver agents to the kidney Kidney-targeted JBNP-mRNA Cy5 is administered intravenously to BALB / cJ mice. The biodistribution of kidney-targeted JBNP-mRNA Cy5 is monitored by IVIS® imaging. The transfected kidneys are then homogenized. Transfection efficiency was determined using Western blot and RT-qPCR.

[0166] Example 23: Examination of the ability of brain-targeted JBNPs of the present disclosure to deliver agents to the brain Brain-targeted JBNP-mRNA Cy5 is administered intravenously to BALB / cJ mice. The biodistribution of brain-targeted JBNP-mRNA Cy5 is monitored by IVIS® imaging. The transfected brains are then homogenized. Transfection efficiency is determined using Western blot and RT-qPCR.

[0167] Example 24: Examination of the ability of the lung-targeted JBNPs of the present disclosure to deliver agents to the lung Lung-targeted JBNP-mRNA Cy5 is administered intravenously to BALB / cJ mice. The biodistribution of lung-targeted JBNP-mRNA Cy5 is monitored by IVIS® imaging. Transfected lungs are then homogenized. Transfection efficiency is determined using Western blot and RT-qPCR.

[0168] Example 25: Examination of the ability of spleen-targeted JBNPs of the present disclosure to deliver agents to the spleen and lymph nodes Spleen-targeted JBNP-mRNA Cy5 is administered intravenously to BALB / cJ mice. The biodistribution of spleen-targeted JBNP-mRNA Cy5 is monitored by IVIS® imaging. The transfected spleen and lymph nodes are then homogenized. Transfection efficiency is determined using Western blot and RT-qPCR.

[0169] Example 26: Examination of the ability of bone-targeted JBNPs of the present disclosure to deliver agents to bone Bone-targeted JBNP-mRNA Cy5 is administered intravenously to BALB / cJ mice. The biodistribution of bone-targeted JBNP-mRNA Cy5 is monitored by IVIS® imaging. The transfected bones are then homogenized. Transfection efficiency is determined using Western blot and RT-qPCR.

[0170] Example 27: Examination of the ability of muscle-targeted JBNPs of the present disclosure to deliver agents to muscle Muscle-targeted JBNP-mRNA Cy5 is administered intravenously to BALB / cJ mice. The biodistribution of muscle-targeted JBNP-mRNA Cy5 is monitored by IVIS® imaging. The transfected muscles are then homogenized. Transfection efficiency is determined using Western blot and RT-qPCR.

[0171] Example 28: Examination of the ability of cardiac-targeted JBNPs of the present disclosure to deliver agents to the heart Cardiac-targeted JBNP-mRNA Cy5 is administered intravenously to BALB / cJ mice. The biodistribution of cardiac-targeted JBNP-mRNA Cy5 is monitored by IVIS® imaging. The transfected hearts are then homogenized. Transfection efficiency is determined using Western blot and RT-qPCR.

[0172] Example 29: Examination of the ability of pancreas-targeted JBNPs of the present disclosure to deliver agents to the pancreas Pancreas-targeted JBNP-mRNA Cy5 is administered intravenously to BALB / cJ mice. The biodistribution of pancreas-targeted JBNP-mRNA Cy5 is monitored by IVIS® imaging. The transfected pancreas is then homogenized. Transfection efficiency is determined using Western blot and RT-qPCR.

[0173] Example 30: Examination of the ability of gut-targeted JBNPs of the present disclosure to deliver agents to the gut Intestine-targeted JBNP-mRNA Cy5 is administered intravenously to BALB / cJ mice. Biodistribution of intestine-targeted JBNP-mRNA Cy5 is monitored by IVIS® imaging. Transfected hearts are then homogenized. Transfection efficiency is determined using Western blot and RT-qPCR.

[0174] Example 31: Examination of the ability of tumor-targeted JBNPs of the present disclosure to deliver agents to tumors Tumor-targeted JBNP-mRNA Cy5 is administered intravenously to xenograft models. The biodistribution of tumor-targeted JBNP-mRNA Cy5 is monitored by IVIS® imaging. The transfected solid tumors are then homogenized. Transfection efficiency is determined using Western blot and RT-qPCR. Mice are monitored for tumor size, weight, complete blood count (CBC), tumor necrosis factor alpha (TNF-alpha) levels, interferon gamma (INF-gamma) levels, immunoglobulin G (IgG) levels, immunoglobulin M (IgM) levels, and toxicity.

[0175] Example 32: Optimization of JBNP-CRISPR dosage and delivery The optimized dose and optimized delivery time of ArgJBNP-CRISPR were determined by performing gene editing in the Ai14 murine model. ArgJBNP-CRISPR consisting of Cas9 mRNA (0.25 mg / kg) and sgLOXp were injected intravenously via tail vein injection or retro-orbital injection for 2-7 days. Positive tandem dimer Tomato (tdTomato) signals were monitored / detected by IVIS® imaging (Figures 10A-10D). Next generation sequencing (NGS) can be used to quantify gene editing efficiency in target organs.

[0176] Example 33: Examination of the ability of JBNP-CRISPR to target and edit DNA in major organs The assembled JBNP-CRISPR, consisting of Cas9 mRNA and sgRB1, is intravenously injected into BALB / cJ mice for 7 to 28 days. Sanger sequencing or next-generation sequencing (NGS) is used to study the RB1 gene editing efficiency.

[0177] Example 34: Examination of the ability of JBNP-CRISPR to target and edit DNA in different organs by binding targeting molecules or moieties JBNP-CRISPR with kidney targeting molecule or portion can target kidney and edit DNA of kidney cells. JBNP-CRISPR with heart targeting molecule or portion can target heart and edit DNA of heart cells. JBNP-CRISPR with spleen targeting molecule or portion can target spleen and edit DNA of spleen cells. JBNP-CRISPR with lymph node targeting molecule or portion can target lymph node and edit DNA of lymph node cells. JBNP-CRISPR with lung targeting molecule or portion can target lung and edit DNA of lung cells. JBNP-CRISPR with muscle targeting molecule or portion can target muscle and edit DNA of muscle cells. JBNP-CRISPR with pancreas targeting molecule or portion can target pancreas and edit DNA of pancreatic cells. JBNP-CRISPR with intestine targeting molecule or portion can target intestine and edit DNA of intestine cells.

[0178] Example 35: Examination of the ability of JBNP-CRISPR to target and edit DNA in solid tumors by binding targeting molecules or moieties JBNP-CRISPR carrying a tumor-targeting molecule or moiety can target solid tumors and edit the DNA of tumor cells.

[0179] Example 36: JBNPs of the present disclosure do not cause any acute toxicity Acute toxicity was examined by tail vein injection of JBNP at doses (optimized doses for each treatment) once every 5 days for 20 days. H&E staining of tissue sections of liver, spleen, kidney, heart, pancreas, lung, brain, bone, and muscle was performed. No acute toxicity due to administration of JBNP was detected in any of the examined organs (Figures 11E-11H).

[0180] Example 37: Examination of whether the JBNP of the present disclosure induces a natural immune response when administered JBNP is administered intravenously to BALB / cJ mice once every 3 days. Serum (30 μl) is collected from the submandibular fissure at each injection time point. IFN-gamma, IL-1 beta, IL-6, IL-10, IL-12(p70), TNF-alpha, MCP-1, MIP-2, MIG, IL-2, IL-5, and IL-17 are assayed by immunomultiplex assay and enzyme-linked immunosorbent assay (ELISA).

[0181] Example 38: JBNPs of the present disclosure do not elicit an adaptive immune response when administered No adaptive immunity was observed with IgG and IgM. JBNP was administered intravenously to BALB / cJ mice once every 3 days for 9 days. Serum was collected before and 10 days after administration of JBNP, and IgM and IgG levels were examined by ELISA (FIGS. 9B, 9C, and 11E). Serum IgM and IgG antibody levels on day 10 were compared to baseline levels before injection. No adaptive immune responses were observed after repeated administration of JBNP of the present disclosure (FIGS. 9B, 9C, and 11F).

[0182] Example 39: Examination of the cellular uptake mechanism of JBNP based on the modification or construction of JBNT The cells were pretreated with several endocytosis inhibitors, including macropinocytosis inhibitors (latrunculin A, cytochalasin D), clathrin-dependent inhibitors (chlorpromazine), and caveolae-dependent inhibitors (methyl-b-cyclodextrin). Then, the assembled ArgJBNP-mRNA AF488 was transfected into the cells. After 24 h of incubation, the AF488 signal was quantified using FACS to determine the uptake mechanism (Figure 9I). Different side chains may have different cellular uptake mechanisms. For example, the cellular uptake of JBNPs containing lysine-based JBNTs may occur via macropinocytosis. Furthermore, the cellular uptake of JBNPs containing arginine-based JBNTs may occur via clathrin-dependent endocytosis.

[0183] Example 40: Endosomal escape mechanism of JBNP depends on modification or configuration of JBNT Endosome escape was examined for JBNPs containing different types of JBNT of the present disclosure. Endosomes were examined by staining cells in contact with the JBNPs to be tested using LysoTracker® RED, which stains and visualizes endosomes in cells. 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 plugin. Bafilomycin A1 and chloroquine inhibitors were used for pretreatment to demonstrate the proton sponge effect. Using calcein assay and modeling, pore formation for escape from endosomes was examined. JBNPs containing lysine-based JBNTs had significantly better endosomal escape, which appears to be due to the proton sponge effect (US Patent Publication No. 20220133893A1). Furthermore, JBNPs containing arginine-based JBNTs had significantly enhanced endosomal escape, which appears to be due to the pore formation effect for escape from early endosomes (Figures 12B and 12C).

[0184] Example 41: JBNPs of the present disclosure are highly biocompatible and have low cytotoxicity Human chondrocytes were seeded (5000 cells / well) and incubated overnight. Then, the cell viability of various vectors including JBNP was measured by Cell Counting Kit-8 (Sigma) assay. After 24 hours of co-incubation, the absorbance was obtained by a microplate reader. The JBNP of the present disclosure is less cytotoxic and more biocompatible than lipid nanoparticles, polymer nanoparticles, such as polyethyleneimine nanoparticles (PEI), lipid nanoparticles (LNP), and single-walled carbon nanotubes (SWNT) (Figure 9L).

[0185] Example 42: JBNPs of the present disclosure can be utilized for CRISPR delivery C28 / I2 cells were seeded in 24-well plates (5000 cells / well) and incubated overnight. Co-assembled JBNP-Cas9mRNA-eGFP and gRNA-ATTO550 dyes were directly introduced into the wells and transfected for 72 hours. FACS was then performed to quantify the co-delivery in cells (Figure 9T and Figure 9U). Co-delivery of Cas9mRNA-eGFP and gRNA-ATTO550 was imaged by scanning confocal laser microscopy (Figure 9W).

[0186] Results of Examples 10 to 42 An exemplary transferrin-targeted JBNP delivering mRNA to C28 / I2 cells is shown in Figure 7E. An exemplary targeting in vivo study is also shown in Figures 7B and 7C. Cartilage-targeted WYRGRL-ArgJBNP delivered siRNA with increased retention time in the knee joint.

[0187] Figures 8A-8G show that a small molecule drug (doxorubicin) can be delivered via JBNPs. Figures 8A and 8B show transfected cell monolayers being examined for apoptosis markers. Figures 8C and 8D show that spheroid formation is inhibited when treated with JBNP-DOX. Figures 8E-8G show delivery of JBNP-DOX to ovarian cancer spheroids. Figures 8H-8J show that a small molecule drug (doxorubicin) can be co-delivered with siRNA via JBNPs into SKOV-3 cell monolayers. Figures 8K-8O show co-delivery of DOX and siRNA into SKOV-3 ovarian cancer spheroids. Figures 8K and 8L are fluorescent images of DOX and siRNA-AF488 delivered via JBNPs. Figure 8M has a representative image of spheroids stained with caspase 3 / 7, an apoptosis marker. Figures 8N and 8O show FACS plots and quantification of apoptosis analysis.

[0188] FIG. 9A shows the biodistribution of siRNA delivered via ArgJBNP in BALB / cJ mice. FIG. 9B and 9C show the immunogenicity study of ArgJBNP-siRNA. FIG. 9D-9F shows the optimization of ArgJBNP-mRNA formulation based on delivery efficiency and cell viability. FIG. 9G and 9H show the response surface methodology (RSM) utilized to optimize ArgJBNP-mRNA formulation. FIG. 9I shows the uptake mechanism analysis of ArgJBNP-mRNA. FIG. 9J shows the 6-(p-toluidino)-2-naphthalene-6-sulfonic acid (TNS) assay to analyze the pKA of ArgJBNP-mRNA. FIG. 9K shows the UV-VIS analysis. FIG. 9L shows the results of the cell viability assay performed with ArgJBNP. FIG. 9M shows the gel shift assay of ArgJBNP-mRNA-gRNA. Figures 9N and 9O show flow cytometry analysis examining the delivery efficiency of ArgJBNP-mRNA compared to LysJBNP-mRNA and Lipofectamine® 2000-mRNA. Figures 9P-9S show that ArgJBNP-mRNA exhibits superior stability over 2 weeks (wk) at room temperature. Figures 9T and 9U show time-dependent delivery of Cas9mRNA and gRNA in C28 / I2 cells. Figure 9V shows time-dependent gene editing of RFP-HUVEC cells analyzed by flow cytometry. Figure 9W is a kinetic study of ArgJBNP-mediated delivery of Cas9eGFP mRNA and gRNA. Figure 9X is a 3D rendering image of delivery of gRNA and Cas9mRNA to C28 / I2 cells. In Figures 9Y and 9Z, ArgJBNP can deliver Cas9mRNA and gRNA to and edit chondrocytes of Ai14 mice. Additionally, Figures 10A-10D show the in vivo editing ability of ArgJBNP-CRISPR. Figure 10E shows a representative image of an H&E stained section of the liver after injection of ArgJBNP-CRISPR.

[0189] Also, Figures 11A and 11B show the biodistribution of mCherry mRNA delivered via ArgJBNP. Figures 11C and 11D show protein corona analysis of ArgJBNP-mRNA. Figure 11C shows the protein corona by SDS-PAGE. Figure 11D is an LC / MS / MS analysis of the protein corona of ArgJBNP-mRNA.

[0190] Additionally, Figures 11E-11H examined the immunogenicity of ArgJBNP-mRNA. Figure 11E shows H&E stained sections of organs after repeated injections of ArgJBNP-scrambled mRNA. Figure 11F shows IgG and IgM immune responses. Figure 11G shows CBCs performed, including WBC, RBC, HGB and PLT counts. Figure 11H shows changes in body weight.

[0191] Figure 12A shows UV-VIS analysis of ArgJBNP at pH 5.2 and 7.4. Figure 12B shows endosomal escape of ArgJBNP at early endosome (Rab5) and late endosome (Rab7). Figure 12C also shows endosomal escape of ArgJBNP by using inhibitors.

[0192] Also, Figure 13A shows a cell viability assay performed on PEG-JBNP. PEGylation is to protect against non-specific cellular uptake (e.g., immune cells and other undesirable cells), and targeting peptides can be used with PEG to achieve active targeting. Figures 13B and 13C show the biodistribution of PEG-JBNP delivering siRNA to BALB / cJ mice.

[0193] Exemplary Figures 14A-14C show the combined PEG effect and targeting ability of WYRGRL-PEG-JBNP-mRNA-cy5.

[0194] The compositions, methods and articles can alternatively comprise, consist of, or consist essentially of any suitable materials, steps or ingredients disclosed herein. The compositions, methods and articles can additionally or alternatively be designed to be free, or substantially free, of any materials (or species), steps or ingredients that are not necessary to achieve the function or purpose of the compositions, methods and articles.

[0195] While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are not presently anticipated or contemplated may occur to applicant or others skilled in the art. Accordingly, the appended claims, as they may be submitted and amended, are intended to embrace all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. The Janus-based nanotube comprises at least one compound represented by formulas I to XII or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 During the ceremony, R １ is H or CH ３ And, R ２ is (CH ２ ), (CH ｊ CH ２ CH ２ O), or (CH ｋ CH ２ CH ２ NH), ｍ where j, k, and m are independently integers from 1 to 200, R ３ These are α-amino acids, β-amino acids, α-polypeptides, or β-polypeptides. L is a bond or linker group, T is a biologically active molecule or a targeted molecule. R ４ This is a coating material, a self-assembled nanomaterial.

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

5. The Janus-based nanotube is, A combination of compounds represented by formulas I to IV and compounds represented by formulas V to VIII, A combination of compounds represented by formulas I to IV and compounds represented by formulas IX to XIII, A combination of compounds represented by formulas V to VIII and compounds represented by formulas IX to XIII, A combination of compounds represented by formulas I to IV, compounds represented by formulas V to VIII, and compounds represented by formulas IX to XIII, or those combinations A self-assembled nanomaterial according to claim 1, comprising:

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

7. The acid-cleaving group is an N-acylhydrazone, a carbonate group, or an ester group. The aforementioned reducible disulfide linker is N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), or 4-(4'-acetylphenoxy)butanoic acid (AcBut), Val-Cit dipeptide, Phe-Lys dipeptide, α-methyl-substituted disulfide, modified cysteine ​​residue, or thiol-containing maytansinoid. The stimulus-responsive linker is a trans-cyclooctene linker or a thioether-containing linker, or The self-assembled nanomaterial according to claim 6, wherein the enzyme-cleavable linker is GPLGOAGQ (SEQ ID NO: 89), GDEVEAPKGC (SEQ ID NO: 90), citrulline-valine, glycosidase-cleavable linker, β-glucolonidase-cleavable linker, β-galactosidase-cleavable linker, phosphatase-cleavable linker, pyrophosphate-containing linker, dipeptide-containing linker, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Phe-Lys-PAABC (para-aminobenzylcarbamate), Val-Cit-PAABC-containing linker, Glu-Val-Cit-containing linker, or Vai-Ala-containing linker.

8. R ４ The self-assembled nanomaterial according to claim 1, wherein the coating material is selected from polymers, peptides, polypeptides, lipid-based materials, phosphate esters, or biomimetic membranes.

9. R ４ The self-assembled nanomaterial according to claim 1, wherein the material is selected from polyethylene glycol, chitosan, hyaluronic acid, poloxamer, polyvinyl alcohol, polysaccharide, neutral polyamino acid, negatively charged polyamino acid, phytokeratin, autopeptides, and antithrombotic peptides.

10. The self-assembled nanomaterial according to claim 1, wherein T is a targeting molecule selected from biologically active molecules, amphiphilic polymers, aptamers, peptides, cyclic peptides, proteins, polysaccharides, polyunsaturated fatty acids, and carbohydrates.

11. The self-assembled nanomaterial according to claim 1, wherein T is a targeting molecule, and the self-assembled nanomaterial further comprises a biologically active molecule attached to the self-assembled nanomaterial by covalent or non-covalent bonds.

12. The self-assembled nanomaterial according to claim 10, wherein the biologically active molecule is attached to the self-assembled nanomaterial by non-covalent bonds.

13. The self-assembled nanomaterial according to claim 10 or 11, wherein the biologically active molecule is at least partially encapsulated by the self-assembled nanomaterial.

14. The self-assembled nanomaterial according to claim 1, wherein the biologically active molecule includes nucleic acids, proteins, peptides, cyclic peptides, small molecule drugs, or combinations thereof.

15. The self-assembled nanomaterial according to claim 1, wherein the biologically active molecule includes miRNA, siRNA, mRNA, gRNA, crRNA, tracrRNA, tRNA, ssDNA, dsDNA, cDNA, or a combination thereof.

16. The self-assembled nanomaterial according to claim 1, wherein the Janus-based nanotubes are present in an amount of 0.1% to 99.9% by weight 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 nanotube in the self-assembled nanomaterial is 1 μg / mL to 1 g / mL.

18. The self-assembled nanomaterial according to claim 1, wherein the pH is 1 to 10.

19. The self-assembled nanomaterial according to claim 1, further comprising extracellular matrix (ECM) molecules.

20. The self-assembling nanomaterial according to claim 19, wherein the ECM molecule comprises hydroxyapatite, fibronectin, Matn1, Matn3, laminin, cartilage oligomeric matrix protein, collagen, elastin, vitronectin, fibrillin, perlecan, fibrinogen, osteonectin, tenascin, thrombospondin, intercellular adhesion molecules (ICAM1-5), integrin, proteoglycan, glycoprotein, or a combination thereof.

21. A pharmaceutical composition for injection comprising a self-assembled nanomaterial according to claim 1 and a pharmaceutically acceptable carrier.

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