RGD-peptide modified covalent organic frameworks for targeted cancer therapy

Abstract

The present disclosure provides nanoparticles having RGD-based peptides conjugated thereto. The nanoparticles may be formed one or more covalent organic frameworks (COFs) of the present disclosure. Also disclosed are methods of making the COFs and nanoparticles. The COFs have one or more binding partners and / or COF (following conjugation of the binding partner) may have an RGD-based peptide. The RGD- based peptide may be selected based on the desire to treat a specific cancer. COFs may have the following structure: Formula (I), where L is a linker group and Peptide is an RGD-based peptide.

Description

RGD-PEPTIDE MODIFIED COVALENT ORGANIC FRAMEWORKS FOR TARGETED CANCER THERAPYCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 567,358, filed on March 19, 2024, the disclosure of which is incorporated herein in its entirety by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted in .xml format and is hereby incorporated by reference in its entirety. Said .xml copy was created on March 19, 2025, is named “058636_00787_ST26.xml”, and is 6,064 bytes in size.BACKGROUND OF THE DISCLOSURE

[0003] Breast cancer remains the most commonly diagnosed cancer in women worldwide, leading to 685,000 deaths and 2.3 million new cases in 2020 alone (WHO Breast Cancer fact sheet). The alarming statistic about breast cancer conceals its diversity, as it comprises various subtypes with significant variations in treatment responses, each linked to specific molecular and histological characteristics. These types include (i) cancers positive for estrogen or progesterone receptors (ER / PR); (ii) those not expressing the human epidermal growth factor receptor 2 (HER2), which may or may not be ER / PR positive; and (iii) the triple-negative breast cancer (TNBC) subtype, characterized by the absence of both ER / PR expression and HER2 overexpression. Among these, TNBC presents a particular challenge, accounting for 15-17% of all breast cancers. TNBC is characterized by its cellular lack of differentiation, molecular heterogeneity, and heightened metastasis, which contribute to chemoresistance. The primary treatment for TNBC remains conventional cytotoxic systemic chemotherapy, with Doxorubicin (Dox) being one of the most frequently prescribed agents. However, the clinical efficacy of Dox is constrained by challenges like drug resistance, nonselective distribution, and poor penetration. Such limitations not only render its use inadequate but also lead to significant side effects, including cardiotoxicity, neuropathy, myalgia, immunosuppression, and myelosuppression.

[0004] A key objective is, therefore, to develop a methodology whereby specific tumors can be targeted, releasing chemotherapeutic agents exclusively within tumors while safeguarding the rest of the body from harm. Drug delivery systems based on nanoparticles (NPs) have shown numerous benefits in cancer treatment, including improvedpharmacokinetics, tumor targeting, moderate reduction in side effects, and drug resistance. Despite FDA-approved delivery systems such as PEGylated (DOXIL®, Caelyx®, and LipoDox®) and non-PEGylated (Myocet®) liposomes, tumors frequently reappear following chemotherapy primarily due to a lack of targeted delivery. Thus, drugs do not exhibit targeted release within cancerous cells, causing collateral damage. Therefore, more effective nanoparticle-based delivery systems are required.

[0005] A parallel area of research has been towards understanding tumor physiology in order to fight cancer more efficiently. Several overexpressed receptors on the surface of cancer cells have been identified and selected for receptor-mediated targeted delivery. The avPs integrin receptor, belonging to the integrin heterodimers family expressed on the cell surface, plays a critical role in cell-cell and cell-extracellular matrix adhesion. While it is present at minimal levels in resting endothelial cells, its expression surges in high- proliferating angiogenic endothelial cells, sites of inflammation, solid tumors, and leukemic cells. Notably, the overexpression of av[E integrin is evident in TNBC, positioning it as an optimal target for selective delivery. The avPs integrins are known to interact with the Arginyl-glycyl-aspartic acid (RGD) peptide motif, present in most extracellular matrix proteins. Based on their binding to the avPs integrin or to the avcomponent or other integrins, several linear and cyclic RGD peptides have been used to functionalize the surface of NPs for specific targeting of tumors, with several studies showing RGD-modified nanosystems enable active targeting of tumors.BRIEF SUMMARY OF THE DISCLOSURE

[0006] The present disclosure provides nanoparticles having RGD-based peptides conjugated thereto. The nanoparticles may comprise one or more covalent organic frameworks (COFs) of the present disclosure. Also disclosed are methods of making the COFs and nanoparticles.

[0007] In an aspect, the present disclosure provides imine-linked-covalent organic frameworks (COFs). The COFs may be referred to as nCOFs. The COFs may be formed from co-condensation of 2,6-diformylpyridine (DFP) comprising a reactive group (which may be referred to as DFP-R), and l,3,5-tris(4-aminophenyl)-benzene (TAB) monomers.

[0008] The COFs may have a reactive group suitable for conjugating a binding partner. Prior to conjugation to the COF, the binding partner comprises a reactive group complementary to the reactive group of the COF. That is, the reactive group of the COF and the reactive group of the binding partner are configured such that the binding partner can beconjugated to the COF via chemical reaction. The binding partner may comprise a linker group and an RGD-based peptide.

[0009] Each nanosheet is formed from the co-condensation of DFP-R monomers and TAB monomers. The monomers have the following structures (and are presented with their IUPAC names):5'-(4-aminophenyl)-[1 ,T:3',1"-terphenyl]-4,4"-diamine andpyridine-2,6-dicarbaldehyde substituted with RThe nanosheets may have the following structure:In various examples, at least one Xi is not -NH2.

[0010] In various examples, a binding partner is conjugated to the COF. Prior to conjugation, a binding partner may comprise a reactive group complementary to the reactive group of the COF, a linker group, and an RGD-based peptide. In various examples, the linker group is conjugated to the peptide via an amide bond formed from an acylation reaction between a side chain of the RGD-based peptide and a complementary reactive group of the linker group. For example, if the side chain is a lysine, reactive group could be an ester or carboxylic acid, which when reacted with the amine of the lysine, forms an amide (see below). In various embodiments, the linker group may comprise a polyethylene glycol (PEG) group or any other suitable linker (e.g., an aliphatic group). In various examples, prior to conjugation, a binding partner may have the following structure:Peptide where the azide is the reactive group, and the peptide is the RGD-based peptide. Following conjugation to the COF, the linker group may have the following structure:In such a structure, the linker group comprises the formed tnazole.

[0011] Prior to conjugation of the binding partner, the COF may comprise:where R is a reactive group. When R comprises an alkynyl group, the COF may have the following structure:

[0012] A binding partner and / or COF (following conjugation of the binding partner) may comprise an RGD-based peptide. The RGD-based peptide may be selected based on the desire to treat a specific cancer. For example, the RGD-based peptide may be a peptide that can interact (e.g., bind) with any one or more of the following integrins: av[33 integrin (breast cancer, melanoma, glioblastoma, ovarian cancer, prostate cancer), avp5 integrin (breast cancer), avps integrin (glioblastoma), asPi integrin (lung cancer), and avpe integrin (colorectal cancer, prostate cancer). Each of these examples highlights the potential of using RGD-based peptide-conjugated COFs in nanoparticles for targeted cancer therapy. By specifically binding to integrins overexpressed on the surface of cancer cells, these nanoparticles can deliver drugs more efficiently, reduce side effects by minimizing impact on healthy cells, and potentially improve the overall effectiveness of cancer treatment. In various examples, the RGD-based peptide is cyclic. In various other examples, the RGD-based peptide is linear. In various examples, the RGD-based peptide is conjugated to the linker group of the binding partner via a covalent bond formed between the linker group and a side chain of an amino acid residue of the RGD-based peptide. For example, the side chain, is the side chain of anucleophilic amino acid residue, such as, for example, cysteine, lysine, serine, ornithine, or the like. For example, an RGD-based peptide may have the following structure:or a pronated or deprotonated form thereof, where R is a side chain of an amino acid residue of the RGD-based peptide. In various examples, when R is the side chain of lysine, the RGD- based peptide has the following structure:or a protonated or deprotonated form thereof. The previously depicted structure depicts a cyclic form of RGKYK (SEQ ID NO: 1), where the sidechain nitrogen is conjugated to the linker group. In various examples, the RGD-based peptide may have one or more D-amino acid residues. In various examples, all the amino acid residues are L-isomers. In various examples, all the amino acid residues are D-isomers. In various examples, a portion of the amino acid residues are D-isomers and the remaining amino acid residues are L-amino acids. In various examples, two or more D-amino acid residues are sequential, in other examples, two or more D-amino acid residues are not sequential, and in additional other examples some of the D-amino acid residues are sequential and some of the D-amino acid residues are non-sequential. Additional examples of RGD-based peptides are c(RGDfK) (SEQ ID NO:2, where c denotes the peptide is cyclic and the lowercase F corresponds to D-phenylalanine), RGDSP (SEQ ID NO:3), and RGDT (SEQ ID NO:4). RGDSP (SEQ ID NO:3) and RGDT (SEQ ID NO:4) are linear peptides and may be conjugated to the linker group at the C- or N- terminus of the peptide (e.g., using RGDSP (SEQ ID NO:3) as an example:

[0013] In aspect, the present disclosure provides nanoparticles comprising COFs of the present disclosure. For example, a COF nanoparticle may comprise a plurality of COF nanosheets of the monomers. In embodiments, the COF nanoparticle comprises 10-25 COF nanosheets (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 COF nanosheets).

[0014] In an aspect, the present disclosure provides compositions. The composition may comprise COF nanoparticles incorporating cargo molecules.

[0015] The composition can comprise COF nanoparticles incorporating cargo molecules in a pharmaceutically acceptable carrier (e.g., carrier).

[0016] In an aspect, the present disclosure provides methods for treating an individual having or suspected of having cancer. The individual may be treated by administering a therapeutically effective amount of the nanoparticle loaded with one or more cargo molecules and having one or more binding partners conjugated thereto.

[0017] In an aspect, the disclosure provides kits. A kit may comprise pharmaceutical preparations comprising COF nanoparticles and printed material.BRIEF DESCRIPTION OF THE FIGURES

[0018] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0019] Figure 1 shows a) a schematic illustration of Alkyn-nCOF functionalization, drug loading, and release: (i) Conjugation of cRGD-PEG-N? peptides to Alkyn-nCOF via click chemistry, forming Alkyn-nCOF-cRGD; (ii) Doxorubicin (Dox) loading, creating Alkyn-nCOF-cRGD@Dox; (iii) pH-dependent Dox release mimicking tumor environment, b) Chemical structure and synthetic route of Alkyn-nCOF-cRGD obtained by copper-mediated cycloaddition of cRGD-PEG-Ns on the Alkyn-nCOF nanoparticle surface, c) XPS C is and N Is spectra of Alkyn-nCOF and Alkyn-nCOF-cRGD. d) Experimental and calculated PXRD patterns with Pawley fitting, Bragg positions, and differences of Alkyn-nCOF. e) Spacefilling representation of the stacked layers of the Alkyn-nCOF, where each color represents individual layers.

[0020] Figure 2 shows a) HRTEM (i, vi) and STEM (ii, vii) images of Alkyn-nCOF (top) and Alkyn-nCOF-cRGD (bottom), respectively, demonstrating an average particle diameter of approximately 60 nm. Particles were negatively stained with a 1% uranyl acetate substitute to enhance contrast and highlight the presence of peptides on the nanoparticle surface. High-resolution images of lattice fringes (iii, viii) and their reconstruction with corresponding interlayer spacing (d) approximately 0.3-0.4 nm (iv, ix), and selected area electron diffraction (SAED) patterns (v, x) for Alkyn-nCOF (top) and Alkyn-nCOF-cRGD (bottom), (xi) A space-filling model depicts three stacked layers of Alkyn-nCOF. b) Variabletemperature liquid-cell transmission electron microscopy (VT-LCTEM) experiment: Sequential LCTEM frames extracted from Movie 1 illustrating the in-situ heating of TAB and Alkyn-DFP precursor solution to 110 °C in the presence of acetic acid. The initial frame shows the COF precursor solution at 110 °C without nanostructures at 0 seconds, while the following frames (15-45 seconds) capture the changes in a selected region as the solution is heated. An average particle size of approximately 60 nm is achieved within 60 s at 110 °C, maintaining stability over prolonged periods at this temperature. Scale bar = 50 nm.

[0021] Figure 3 shows a) Fluorescence emission spectra of a diluted supernatant solution containing Dox before (red curve) and 24 h after the addition of Alkyn-nCOF-cRGD (black curve). Dox fluorescence intensity was used to derive the loading efficiency ( / = 488 nm, H2O, pH 7.4, 298 K). The experiment was performed in triplicate, b) Schematic diagram displaying three Dox molecules occupying a pore within Alkyn-nCOF. The nCOF pores areoccupied by Dox molecules depicted in orange and purple, while an additional Dox molecule, shown in salmon, adopts a sandwich-like conformation nestled between the layers of nCOF. c) In vitro Dox release from Alkyn-nCOF-cRGD at 37 °C in 10 mM PBS solution at pH 7.4 (black curve) or 5.4 (red curve), d) TEM images of Alkyn-nCOF-cRGD@Dox after 24 hours at pH 7.4 (black) and 5.4 (red).

[0022] Figure 4 shows a) Cell viability of MCF-7 and MDA-MB-231 after 48-hour treatment with Alkyn-nCOF (blue, left in each series) and Alkyn-nCOF-cRGD (green, right in each series) at concentrations up to 1 mg / mL. Error bars denote the standard deviations from triplicate measurements, b) Cytotoxic effects (ICso values) of free Dox (red, left in each series), Alkyn-nCOF@Dox (blue, middle in each series), and Alkyn-nCOF-cRGD@Dox (green, right in each series) on MCF-7 and MDA-MB-231 cells. Data represent the mean ± SD from three independent experiments. Statistical significance was assessed using Student's t-test, where ns denotes not significant, *p < 0.05, and **p < 0.01, when compared to the Dox-treated group, c) Western blot analysis illustrating Caspase-3 protein expression MDA- MB-231 cells treated for 24 hours with no additives (control, grey, left), free Dox (red, second from left), Alkyn-nCOF@Dox (blue, third from left), and Alkyn-nCOF-cRGD@Dox (green, right) at a Dox concentration of 1 pM. d) Quantitative analysis of normalized Caspase-3 expression relative to control in MDA-MB-231 cells treated for 24 hours with free Dox (red), Alkyn-nCOF@Dox (blue), and Alkyn-nCOF-cRGD@Dox (green) at a Dox concentration of 1 pM. Error bars denote standard deviations illustrating the variability within duplicate experiments.

[0023] Figure 5 shows a) High-resolution electron micrographs comparing cellular interactions in MCF-7 (upper panel) and MDA-MB-231 (lower panel) cells when exposed 4 hours to Alkyn-nCOF@Dox and Alkyn-nCOF-cRGD@Dox. White arrows pinpoint internalized nanoparticles (NPs) post a 4-hour incubation. Key identifiers: n = nucleus; mb = membrane; M = macropinosome; CCP = clathrin-coated pits; E = endosome; L = lysosome. b) Schematic representations detailing the mechanisms of macropinocytosis (left, blue) and a[3 integrin-mediated internalization (right, green). The journey of the compounds within the cells is depicted through sequential stages, from the formation of macropinosomes (left, blue) or clathrin-coated pits (right, green) to their eventual lysosomal degradation and pH-sensitive release into the cytoplasm. Corresponding numbers on the micrographs indicate these stages. c) Comparison of endocytotic vesicle size measured by TEM and % LDH release in breast cancer cell lines MCF-7 and MDA-MB-231 after a 24-hour treatment period. The cells, characterized by low (MCF-7) and high (MDA-MB-231) integrin levels, were exposed to aconcentration of [Dox] = 10 pM of Alkyn-nCOF@Dox (blue, middle of each series) and Alkyn-nCOF-cRGD@Dox (green, right in each series), with gray bars (left in each series) indicating the control cells without treatment. The y-axis on the right (red) represents the vesicle size in micrometers (pm), while the y-axis on the left (black) indicates the percentage of LDH release, a marker of cell membrane integrity. The red lines with corresponding data point markers provide a visual guide for interpreting changes in vesicle size across the different treatments and cell types. Error bars denote standard deviations. Statistical significance was assessed using Student's t-test, where *p <0.05; **p <0.01; ***p <0.001; ****p <0.0005;<0.0001 when compared to the control sample, d) Confocal laser scanning microscopy (CLSM) images displaying MCF-7 (top panel) and MDA-MB-231 cells (bottom panel) subjected to a 24-hour treatment with control (no additive), free Dox, Alkyn- nCOF@Dox, or Alkyn-nCOF-cRGD@Dox at a concentration of [Dox] = 10 pM. Dox fluorescence was captured with 488 nm excitation / 520 nm emission, while nuclear DAPI staining was visualized under 355 nm excitation and 450 nm emission, e) Flow cytometry analysis of Dox fluorescence within MCF-7 (left) and MDA-MB-231 (right) cells post 24- hour incubation with either no additive (control, gray), free Dox (red), Alkyn-nCOF@Dox (blue), or Alkyn-nCOF-cRGD@Dox (green) at a concentration of [Dox] = 1 pM. f) CLSM images of MDA-MB-231 cells after 4 hours of treatment with no additive (control), free Dox, Alkyn-nCOF@Dox, or Alkyn-nCOF-cRGD@Dox at [Dox] = 10 pM, and co-stained with markers for the plasma membrane, lysosomes, and mitochondria. All images include nuclear DAPI labeling. Channels used: DAPI (X ex — 400 nm), Dox ( ex — 488 nm), and red marker (Xex = 647 nm).

[0024] Figure 6 shows a) Biodistribution profile of non-drug-loaded nanoparticles in major organs (heart, liver, spleen, kidney) following intraperitoneal administration in healthy mice. Sacrifice and quantitative analysis were performed 24 hours post-administration of Alkyn-nCOF (blue bars, left in each series) and Alkyn-nCOF-cRGD (green bars, right in each series) nanoparticles. Dose: [NP] = 20 mg / kg, 200 pL, with three mice per group (n=3). The inset shows representative TEM images of liver sections from treated and control mice, illustrating the maintenance of normal liver architecture. Nd* = non detected, b) In vivo fluorescence imaging (IVIS® Spectrum) of tumor-bearing mice under anesthesia, depicting the distribution of Dox fluorescence 24 hours after treatment with PBS (control), free Dox, Alkyn-nCOF@Dox, or Alkyn-nCOF-cRGD@Dox. Dose: [Dox] = 5 mg / kg, [NP] = 10 mg / kg, 200 pL. Ex vivo fluorescent images using IVIS® Spectrum (c and d) and quantitative analysis of Dox radiance efficiency (e and f) of excised tumors (c and e) and vital organs,including liver, heart, kidney, and spleen (d and f) from the same groups of mice as in panel (b), showing fluorescence intensity indicative of Dox distribution within the tissues. Organs were dissected and analyzed via IVIS Spectrum for fluorescence imaging, 24 hours postinjection. Each bar represents one of the treatments: control (grey, left in each series), free Dox (red, second from the left in each series), Alkyn-nCOF@Dox (blue, third from left in each series), and Alkyn-nCOF-cRGD@Dox (green, right in each series). Values are mean ± SD (n = 3), ns: non-significant, * p < 0.05, ** p < 0.01 compared to the control sample.

[0025] Figure 7 shows a) Schematic of the treatment and evaluation timeline, b) Tumor volume progression in mice after administration of PBS (Control, n=l 1, gray), free Dox (n=15, red), Alkyn-nCOF@Dox (n=14, blue), and Alkyn-nCOF-cRGD@Dox (n=14, green), with a Dox dose of 5 mg / kg. c) Visual comparison of tumor in mice at the start (day 0) and after 20 days of treatment, d) Body weight tracking across different treatment groups throughout the 20-day treatment, e) IVIS® Spectrum fluorescence imaging depicting Dox distribution in anesthetized mice after 20-day treatment, f) Ex vivo images of excised tumors at the end of the treatment, with scale bars representing 1 cm. g) Post-treatment tumor weight analysis, h) IVIS® Spectrum fluorescence imaging of the harvested tumors, and i) quantification of Dox radiance efficiency among different treatment groups after treatment. (n=5) j) Ex vivo evaluation of Dox distribution in the liver, heart, spleen, and kidneys of tumor-bearing nude mice treated with PBS (control), Dox alone, Alkyn-nCOF@Dox, and Alkyn-nCOF-cRGD@Dox on day 20, including average ROI measurements. (n=5) Statistical significance was determined using one-way ANOVA with Tukey's post hoc test. Significance denoted as: *p <0.05; **p <0.01; ***p <0.001; ****p <0.0005.

[0026] Figure 8 shows alkyn-nCOF-cRGD tumor targeting and drug release: Integrin- mediated targeting of triple-negative breast cancer cells and acid-triggered Dox release, leading to targeted chemotherapy.

[0027] Figure 9 shows the chemical structure and synthetic route of Alkyn-nCOF obtained under micro wave irradiation.

[0028] Figure 10 shows stacked FTIR spectra of Alkyn-nCOF and its precursors, 4- ethynyl-l,6-diformyl pyridine (Alkyn-DFP) and l,3,5-tris(4-aminophenyl)benzene (TAB).

[0029] Figure 11 shows stacked FTIR spectra of Alkyn-nCOF and its precursors, 4- ethynyl-l,6-diformyl pyridine (Alkyn-DFP) and l,3,5-tris(4-aminophenyl)benzene (TAB) between 1800-500 cm-1.

[0030] Figure 12 shows stacked FTIR spectra of Alkyn-nCOF and Alkyn-nCOF- cRGD.

[0031] Figure 13 shows stacked13C solid-state NMR spectra of a) Alkyn-nCOF, b) cRGD-PEG-Ns, and c) Alkyn-nCOF-cRGD. The asterisks denote spinning sidebands.

[0032] Figure 14 shows stacked13C solid-state NMR spectra of a) Alkyn-nCOF, b) doxorubicin hydrochloride, and c) Alkyn-nCOF@Dox. The asterisks denote spinning sidebands.

[0033] Figure 15 shows XPS a) C Is and b) N Is spectra of cRGD-PEG-Ns.

[0034] Figure 16 shows PXRD patterns of Alkyn-nCOF at pH 7.4 (blue) versus 5.4(black) after 24 hours, with corresponding TEM images.

[0035] Figure 17 shows PXRD analysis of Alkyn-nCOF-cRGD.

[0036] Figure 18 shows PXRD analysis of Alkyn-nCOF@Dox (pink) and Alkyn- nCOF-cRGD @Dox (purple).

[0037] Figure 19 shows a) Transmission Electron Microscopy (TEM) image of Alkyn-nCOF nanoparticles, b) Particle size distribution of Alkyn-nCOF. The overlaid dashed lines represent Gaussian fit profiles, derived using the mean diameter calculated from the analysis of 500 individual particles.

[0038] Figure 20 shows TEM at different magnification (a, b, c) and STEM (d) images of Alkyn-nCOF.

[0039] Figure 21 shows a) Transmission Electron Microscopy (TEM) image of Alkyn-nCOF-cRGD nanoparticles, b) Particle size distribution of Alkyn-nCOF-cRGD. The overlaid dashed lines represent Gaussian fit profiles, derived using the mean diameter calculated from the analysis of 500 individual particles.

[0040] Figure 22 shows TEM images of Alkyn-nCOF-cRGD at different magnifications.

[0041] Figure 23 shows VT-LCTEM experiment: Sequential LCTEM frames extracted from Movie 1 illustrating the in-situ heating of TAB and Alkyn-DFP precursor solution to 110 °C in the presence of acetic acid. The initial frame shows the COF precursor solution at 110 °C without nanostructures (t = 0s), while the following frames capture the changes in a selected region as the solution is heated. An average particle size of approximately 60 nm is achieved within a minute at 110 °C, maintaining stability over prolonged periods at this temperature. Scale bar = 200 nm.

[0042] Figure 24 shows TEM images of Alkyn-nCOF@Dox (a,b) and Alkyn-nCOF- cRGD@Dox (c,d) at pH 7.4 (a,c, black) versus 5.4 (b,d, red) after 24 hours.

[0043] Figure 25 shows AFM images Alkyn-nCOF at different magnifications.

[0044] Figure 26 shows AFM images and height profiles of Alkyn-nCOF-cRGD.

[0045] Figure 27 shows a) Porosity analysis and b) pore size distribution of Alkyn- nCOF (blue), Alkyn-nCOF-cRGD (green), Alkyn-nCOF@Dox (pink) and Alkyn-nCOF- cRGD@Dox (purple). The activation of the sample was carried out at 125 °C for 24 hours under a high vacuum.

[0046] Figure 28 shows an absorption spectrum of Alkyn-nCOF. The experiment was performed in triplicate.

[0047] Figure 29 shows an absorption spectrum of Alkyn-nCOF-cRGD. The experiment was performed in triplicate.

[0048] Figure 30 shows a fluorescence emission spectrum of Alkyn-nCOF at a) / .e — 380 nm and b) / .«-= 488 nm (H2O, pH 7.4, 298 K). The experiment was performed in triplicate.

[0049] Figure 31 shows a fluorescence emission spectrum of Alkyn-nCOF (blue) and Alkyn-nCOF-cRGD (green). Inset: Fluorescence emission spectrum of free cRGD-PEG-Ns (black). ( / .e>= 380 nm, H2O, pH 7.4, 298 K). The experiment was performed in triplicate.

[0050] Figure 32 shows a fluorescence emission spectrum of Alkyn-nCOF-cRGD (green). ( / . ex = 488 nm, H2O, pH 7.4, 298 K). The experiment was performed in triplicate.

[0051] Figure 33 shows a fluorescence emission spectrum of Alkyn-nCOF@Dox at a) ex= 380 nm and488 nm (H2O, pH 7.4, 298 K). The experiment was performed in triplicate.

[0052] Figure 34 shows thermogravimetric analysis (TGA) of Alkyn-nCOF (blue, left in legend), Alkyn-nCOF-cRGD (green, second from left in legend), Alkyn-nCOF @Dox (pink, third from left in legend) and Alkyn-nCOF-cRGD@Dox (purple, right in legend). The thermal stability of the COF profile was recorded at a scan rate of 5 °C / min.

[0053] Figure 35 shows Zcta(Q-potcntial of Alkyn-nCOF (blue, left), Alkyn-nCOF- cRGD (green, second from left), Alkyn-nCOF@Dox (pink, third from left), and Alkyn- nCOF-cRGD@Dox (purple, right) at pH 7.4. The experiment was performed in triplicate.

[0054] Figure 36 shows the hydrodynamic diameter of Alkyn-nCOF (blue) and Alkyn-nCOF-cRGD (green) at pH 7.4. The experiment was performed in triplicate.

[0055] Figure 37 shows the hydrodynamic diameter of Alkyn-nCOF (blue) and Alkyn-nCOF@Dox (pink) at pH 7.4. The experiment was performed in triplicate.

[0056] Figure 38 shows the hydrodynamic diameter of Alkyn-nCOF-cRGD (green) and Alkyn-nCOF-cRGD@Dox (purple) at pH 7.4. The experiment was performed in triplicate.

[0057] Figure 39 shows the hydrodynamic diameter of a) Alkyn-nCOF (blue), b) Alkyn-nCOF-cRGD (green), c) Alkyn-nCOF@Dox (pink), and d) Alkyn-nCOF-cRGD@Dox (purple) at pH 7.4 and pH 5.4 (black). The experiment was performed in triplicate.

[0058] Figure 40 shows a calibration curve obtained by measuring the maximum fluorescence signal by OPA method at different cRGD-PEG-Ns concentrations (. / .max = 460 nm, H20, pH 7.4, 298 K).

[0059] Figure 41 shows fluorescence emission spectra of diluted supernatant solution before (blue curve) and 24 h after addition of Alkyn-nCOF (green curve) to determine the protein content covalently conjugated. Fluorescence intensity was used to deduce the conjugation efficiency. (ex= 360 nm, H2O, pH 7.4, 298 K). The experiment was performed in triplicate.

[0060] Figure 42 shows a calibration curve obtained by measuring the maximum fluorescence signal at different Dox concentrations ( max= 560 nm, H2O, pH 7.4, 298 K).

[0061] Figure 43 shows fluorescence emission spectra of diluted supernatant solution before (red curve) and after Alkyn-nCOF (black curve) was added. Fluorescence intensity was used to deduce the loading efficiency. ( / .«= 488 nm, H2O, pH 7.4, 298 K). Inset picture displays supernatant before (left) and after the addition of the NPs. The experiment was performed in triplicate.

[0062] Figure 44 shows dox release from Alkyn-nCOF in PBS at pH 7.4 (black) and at pH 5.4 (red). The % of drug release was measured using fluorescence emission.

[0063] Figure 45 shows comparative profile of MCF-7 vs. MDA-MB-231 breast cancer cells: The diagram contrasts the two cell lines, highlighting hormone receptor status, metastatic behavior, subtype, doxorubicin sensitivity, and integrin av[33 expression, reflecting their distinct prognostic and therapeutic challenges.

[0064] Figure 46 shows inhibition of a) MCF-7 and b) MDA-MB-231 cells after 48- hour incubation with Dox alone (red, left in legend), Alkyn-nCOF@Dox (blue, middle in legend), or Alkyn-nCOF-cRGD@Dox (green, right in legend) up to [Dox] = 100 pM. Error bars represent standard deviations of triplicate measurements.

[0065] Figure 47 shows quantitative analysis of Caspase-3 expression relative to control in MCF-7 cells treated for 24 hours with Dox, Alkyn-nCOF@Dox and Alkyn-nCOF- cRGD@Dox at a Dox concentration of 1 pM.

[0066] Figure 48 shows TEM images of MCF-7 cells.

[0067] Figure 49 shows TEM images of MCF-7 cells treated with Alkyn-nCOF@Dox ([Dox] = 10 pM) for 4 h at various magnifications. White arrows show nanoparticle uptake in cells and their transit in the cytoplasm.

[0068] Figure 50 shows TEM images of MCF-7 cells treated with Alkyn-nCOF@Dox ([Dox] = 10 pM) for 4 h at higher magnifications.

[0069] Figure 51 shows TEM images of MCF-7 cells treated with Alkyn-nCOF- cRGD@Dox ([Dox] = 10 pM) for 4 h at various magnifications. White arrows show nanoparticle uptake in cells and their transit in the cytoplasm.

[0070] Figure 52 shows TEM images of MCF-7 cells treated with Alkyn-nCOF- cRGD@Dox ([Dox] = 10 pM) for 4 h at higher magnifications.

[0071] Figure 53 shows TEM images of MDA-MB-231 cells.

[0072] Figure 54 shows TEM images of MDA-MB-231 cells treated with Alkyn- nCOF@Dox ([Dox] = 10 pM) for 4 h at various magnifications. White arrows show nanoparticle uptake in cells and their transit in the cytoplasm.

[0073] Figure 55 shows TEM images of MDA-MB-231 cells treated with Alkyn- nCOF@Dox ([Dox] = 10 pM) at higher magnifications.

[0074] Figure 56 shows TEM images of MDA-MB-231 cells treated with Alkyn- nCOF-cRGD@Dox ([Dox] = 10 pM) for 4 h at various magnifications. White arrows show nanoparticle uptake in cells through integrin-mediated endocytosis and their transit in the cytoplasm.

[0075] Figure 57 shows TEM images of MDA-MB-231 cells treated with Alkyn- nCOF-cRGD@Dox ([Dox] = 10 pM) for 4 h at higher magnification displaying uptake in cells through integrin-mediated endocytosis and their transit in the cytoplasm.

[0076] Figure 58 shows transmission electron microscopy (TEM) images and corresponding size distribution histograms of endocytotic vesicles in MCF-7 and MDA-MB- 231 breast cancer cells. Cells were treated with control (no treatment), Alkyn-nCOF@Dox, and Alkyn-nCOF-cRGD@Dox to assess nanoparticle internalization and vesicle formation. Control MCF-7 cells show an average vesicle size of 1.2 pm ± 0.4, while Alkyn-nCOF@Dox and Alkyn-nCOF-cRGD@Dox treatments result in average vesicle sizes of 2.3 pm ± 0.4 and 1.0 pm ± 0.3, respectively. For MDA-MB-231 cells, control vesicles measure 1.0 pm ± 0.3, with Alkyn-nCOF@Dox treatment increasing the average vesicle size to 1.8 pm ± 0.6 and Alkyn-nCOF-cRGD@Dox treatment decreasing it to 0.5 pm ± 0.2. The histograms reflect the distribution of vesicle sizes observed in the TEM images, indicating the effect of nanoparticle treatment on endocytic vesicle dynamics within these cell lines.

[0077] Figure 59 shows confocal images of MCF-7 cells incubated for 24 hours with no additives (control), Dox alone, Alkyn-nCOF@Dox or Alkyn-nCOF-cRGD@Dox ([Dox] = 10 pM). Nucleus were labeled using DAPI on all images. DAPI channel (Xex = 400 nm), Dox channel (Xex = 488 nm).

[0078] Figure 60 shows confocal images of MDA-MB-231 cells incubated for 24 hours with no additives (control), Dox alone, Alkyn-nCOF@Dox or Alkyn-nCOF- cRGD@Dox ([Dox] = 10 pM). Nucleus were labeled using DAPI on all images. DAPI channel (Xex = 400 nm), Dox channel (Xex = 488 nm).

[0079] Figure 61 shows comparative analysis of Dox uptake between MCF-7 and MDA-MB-231 breast cancer cell lines. The bar graph illustrates the relative quantity of Dox internalized by the two cell lines, with no additives (control, grey, left in each series), Alkyn- nCOF@Dox (blue, third from left in each series), Alkyn-nCOF-cRGD@Dox (green, right in each series), and doxorubicin (red, second from left in each series).

[0080] Figure 62 shows body weight of healthy animals at baseline (t=0) and after 24 hours. The control group is represented in grey (left in each series), while the groups treated with intraperitoneal injections of Alkyn-nCOF and Alkyn-nCOF-cRGD nanoparticles are shown in blue (middle in each series) and green (right in each series), respectively. Each bar corresponds to the average weight of three subjects (n=3), indicating no significant change in body weight post-treatment within the 24-hour observation period.

[0081] Figure 63 shows TEM images of the soupy solution of livers treated with a) no additives (control), b) Alkyn-nCOF, and c) Alkyn-nCOF-cRGD after homogenization. Livers of animals treated with Alkyn-nCOF and Alkyn-nCOF-cRGD display the presence of nanoparticles as dark materials.

[0082] Figure 64 shows ex vivo IVIS images of vital organs (liver, heart, kidney, spleen) depicting the distribution of Dox fluorescence 24 hours after treatment with PBS (control), free Dox, Alkyn-nCOF@Dox, or Alkyn-nCOF-cRGD@Dox. Dose: [Dox] = 5 mg / kg, [NP] = 10 mg / kg, 200 pL.

[0083] Figure 65 shows body weight of tumor-bearing mice at baseline (t=0) and after 24 hours. The control group is represented in grey (left in each series), while the groups treated with intraperitoneal injections of free Dox, Alkyn-nCOF and Alkyn-nCOF-cRGD nanoparticles are shown in red (second from left in each series), blue (third from left in each series) and green (right in each series), respectively. Each bar corresponds to the average weight of three subjects (n=3), indicating no significant change in body weight post-treatment within the 24-hour observation period.

[0084] Figure 66 shows ex vivo biodistribution and corresponding average ROI of Dox in tumor-bearing nude mice treated with PBS (control), Dox alone, Alkyn-nCOF@Dox, and Alkyn-nCOF-cRGD@Dox at the end of the treatment (20thday) of the liver. All data are presented as the mean ±SEM (n=5). * p <0.05; ** p <0.01 ; *** p <0.001.

[0085] Figure 67 shows ex vivo biodistribution and corresponding average ROI of Dox in tumor-bearing nude mice treated with PBS (control), Dox alone, Alkyn-nCOF@Dox, and Alkyn-nCOF-cRGD@Dox at the end of the treatment (20thday) of heart. All data are presented as the mean ±SEM (n=5). * p <0.05; ** p <0.01; *** p <0.001.

[0086] Figure 68 shows ex vivo biodistribution and corresponding average ROI of Dox in tumor-bearing nude mice treated with PBS (control), Dox alone, Alkyn-nCOF@Dox, and Alkyn-nCOF-cRGD@Dox at the end of the treatment (20thday) of the lung. All data are presented as the mean ±SEM (n=5). * p <0.05; ** p <0.01 ; *** p <0.001.

[0087] Figure 69 shows ex vivo biodistribution and corresponding average ROI of Dox in tumor-bearing nude mice treated with PBS (control), Dox alone, Alkyn-nCOF@Dox, and Alkyn-nCOF-cRGD@Dox at the end of the treatment (20thday) of the spleen. All data are presented as the mean i-SEM (n===5). * p <0.05; ** p <0.01; *** p <0.001.

[0088] Figure 70 shows ex vivo biodistribution and corresponding average ROI of Dox in tumor-bearing nude mice treated with PBS (control), Dox alone, Alkyn-nCOF@Dox, and Alkyn-nCOF-cRGD@Dox at the end of the treatment (20thday) of the spleen. AH data are presented as the mean ±SEM (n::::5). * p <0.05; ** p <0.01; *** p <0.001.DETAILED DESCRIPTION OF THE DISCLOSURE

[0089] Although claimed subject matter will be described in terms of certain embodiments, other embodiments, including embodiments that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the disclosure.

[0090] As used herein, unless otherwise indicated, “about”, “substantially”, or “the like”, when used in connection with a measurable variable (such as, for example, a parameter, an amount, a temporal duration, or the like) or a list of alternatives, is meant to encompass variations of and from the specified value including, but not limited to, those within experimental error (which can be determined by, e.g., a given data set, an art accepted standard, etc. and / or with, e.g., a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as, for example, variations of + / -10% or less, + / -5%or less, + / -!% or less, and + / -0. 1% or less of and from the specified value), insofar such variations in a variable and / or variations in the alternatives are appropriate to perform in the instant disclosure. As used herein, the term “about” may mean that the amount or value in question is the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error, or the like, or other factors known to those of skill in the art such that equivalent results or effects are obtained. In general, an amount, size, composition, parameter, or other quantity or characteristic, or alternative is “about” or “the like,” whether or not expressly stated to be such. It is understood that where “about,” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0091] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “0. 1% to 5%” should be interpreted to include not only the explicitly recited values of 0. 1% to 5%, but also, unless otherwise stated, include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5% to 1.1%; 0.5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, and other possible sub-ranges) within the indicated range. It is also understood (as presented above) that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about, it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0092] The term “treatment” as used herein refers to reduction in one or more symptoms or features associated with the presence of the particular condition being treated. Treatment does not necessarily mean complete cure or remission, nor does it preclude recurrence or relapses. For example, treatment in the present disclosure means reducing pain (e.g., decreasing pain sensitivity) or increasing pain sensitivity.

[0093] The term “therapeutically effective amount” as used herein refers to an amount of an agent sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. Treatment does not have to lead to complete cure, although it may. Treatment can mean alleviation of one or more of the symptoms or markers of the indication. The exact amount desired or required will vary depending on the particular compound or composition used, its mode of administration, patient specifics and the like. Appropriate effective amount can be determined by one of ordinary skill in the art informed by the instant disclosure using only routine experimentation. Treatment can be orientated symptomatically, for example, to suppress symptoms. It can be affected over a short period, over a medium term, or can be a long-term treatment, such as, for example within the context of a maintenance therapy. Treatment can be continuous or intermittent.

[0094] As used herein, unless otherwise stated, the term “group” refers to a chemical entity that is monovalent (i.e., has one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., has two or more termini that can be covalently bonded to other chemical species). The term “group” also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like). Illustrative examples of groups include:

[0095] The present disclosure provides nanoparticles having RGD-based peptides conjugated thereto. The nanoparticles may comprise one or more covalent organic frameworks (COFs) of the present disclosure. Also disclosed are methods of making the COFs and nanoparticles.

[0096] In an aspect, the present disclosure provides imine-linked-covalent organic frameworks (COFs). The COFs may be referred to as nCOFs. The COFs may be formed from co-condensation of 2,6-diformylpyridine (DFP) comprising a reactive group (which may be referred to as DFP-R), and l,3,5-tris(4-aminophenyl)-benzene (TAB) monomers.

[0097] The COFs may have a reactive group suitable for conjugating a binding partner. Prior to conjugation to the COF, the binding partner comprises a reactive group complementary to the reactive group of the COF. That is, the reactive group of the COF and the reactive group of the binding partner are configured such that the binding partner can be conjugated to the COF via chemical reaction. The binding partner may comprise a linker group and an RGD-based peptide.

[0098] Each nanosheet is formed from the co-condensation of DFP-R monomers and TAB monomers. The monomers have the following structures (and are presented with their IUPAC names):pyridine-2,6-dicarbaldehyde substituted with RThe nanosheets may have the following structure:In various examples, at least one Xi is not -NH2.

[0099] The COF may comprise various reactive groups to enable conjugation of a binding partner. Examples of reactive groups include an azide group, an alkynyl group, activated esters, carboxylate / carboxylic acid, halides, amines, hydroxides, and the like. For example, the reactive groups can be groups that are suitable for nucleophilic substitution or acylation. For example, the reactive group can be a nucleophile or an electrophile suitable for a substitution reaction. In various examples, the COF comprises an alkynyl group and the binding partner comprises an azide such the COF and the binding partner may be conjugated via a click reaction.

[0100] When the COF comprises a plurality of alkynyl groups, each nanosheet is formed from the co-condensation of DFP-R monomers and TAB monomers, where DFP-R is 4-(prop-2-yn-l-yloxy)pyridine-2,6-dicarbaldehyde. The monomers have the following structures (and are presented with their IUPAC names):and4-(prop-2-yn-1-yloxy)pyridine-2,6-dicarbaldehydeThe nanosheets may have the following structure:The nanosheets have a plurality ofgroups.In various examples, at least one Xi is not -NH2.

[0101] For example, a COF of the present disclosure comprises the following structure:

[0102] For example, a COF of the present disclosure comprises the following structure:

[0103] In various examples, a binding partner is conjugated to the COF. Prior to conjugation, a binding partner may comprise a reactive group complementary to the reactive group of the COF, a linker group, and an RGD-based peptide. In various examples, the linker group is conjugated to the peptide via an amide bond formed from an acylation reaction between a side chain of the RGD-based peptide and a complementary reactive group of the linker group. For example, if the side chain is a lysine, reactive group could be an ester orcarboxylic acid, which when reacted with the amine of the lysine, forms an amide (see below). In various embodiments, the linker group may comprise a polyethylene glycol (PEG) group or any other suitable linker (e.g., an aliphatic group). In various examples, prior to conjugation, a binding partner may have the following structure:Peptide where the azide is the reactive group, and the peptide is the RGD-based peptide. Following conjugation to the COF, the linker group may have the following structure:In such a structure, the linker group comprises the formed triazole.

[0104] Prior to conjugation of the binding partner, the COF may comprise:where R is a reactive group. When R comprises an alkynyl group, the COF may have the following structure:

[0105] A binding partner and / or COF (following conjugation of the binding partner) may comprise an RGD-based peptide. The RGD-based peptide may be selected based on the desire to treat a specific cancer. For example, the RGD-based peptide may be a peptide thatcan interact (e.g., bind) with any one or more of the following integrins: avPs integrin (breast cancer, melanoma, glioblastoma, ovarian cancer, prostate cancer), avp5 integrin (breast cancer), avPs integrin (glioblastoma), asPi integrin (lung cancer), and avPe integrin (colorectal cancer, prostate cancer). Each of these examples highlights the potential of using RGD-based peptide-conjugated COFs in nanoparticles for targeted cancer therapy. By specifically binding to integrins overexpressed on the surface of cancer cells, these nanoparticles can deliver drugs more efficiently, reduce side effects by minimizing impact on healthy cells, and potentially improve the overall effectiveness of cancer treatment. In various examples, the RGD-based peptide is cyclic. In various other examples, the RGD-based peptide is linear. In various examples, the RGD-based peptide is conjugated to the linker group of the binding partner via a covalent bond formed between the linker group and a side chain of an amino acid residue of the RGD-based peptide. For example, the side chain, is the side chain of a nucleophilic amino acid residue, such as, for example, cysteine, lysine, serine, ornithine, or the like. For example, an RGD-based peptide may have the following structure:or a pronated or deprotonated form thereof, where R is a side chain of an amino acid residue of the RGD-based peptide. In various examples, when R is the side chain of lysine, the RGD- based peptide has the following structure:or a protonated or deprotonated form thereof. The previously depicted structure depicts a cyclic form of RGKYK (SEQ ID NO: 1, which may be referred to as c(RGKYK) (SEQ ID NO: 1)), where the sidechain nitrogen is conjugated to the linker group. In various examples, the RGD-based peptide may have one or more D-amino acid residues. In various examples, all the amino acid residues are L-isomers. In various examples, all the amino acid residues are D-isomers. In various examples, a portion of the amino acid residues are D-isomers and the remaining amino acid residues are L-amino acids. In various examples, two or more D-amino acid residues are sequential, in other examples, two or more D-amino acid residues are not sequential, and in additional other examples some of the D-amino acid residues are sequential and some of the D-amino acid residues are non-sequential. Additional examples of RGD- based peptides are c(RGDfK) (SEQ ID NO:2, where c denotes the peptide is cyclic and the lowercase F corresponds to D-phenylalanine), RGDSP (SEQ ID NO:3), and RGDT (SEQ ID NO:4). RGDSP (SEQ ID NO:3) and RGDT (SEQ ID NO:4) are linear peptides and may be conjugated to the linker group at the C- or N-terminus of the peptide (e.g., using RGDSPIn various other examples, all the amino acid residues of SEQ ID NO:2 are the L-isomers.

[0106] In various examples, following conjugation of the binding partner to the COF, the COF may comprise:where L is a linker group and Peptide is an RGD-based peptide. For example, the COF may comprise:or a deprotonated or protonated form thereof. In various other examples, Peptide is SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0107] In aspect, the present disclosure provides nanoparticles comprising COFs of the present disclosure. For example, a COF nanoparticle may comprise a plurality of COF nanosheets of the monomers. In embodiments, the COF nanoparticle comprises 10-25 COF nanosheets (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 COF nanosheets).

[0108] The layers may be stacked in a staggered configuration. The COF nanosheets agglomerate such that a poly crystalline, spherical nanoparticle is formed. The COF network may have the structure presented in Figure 3.

[0109] The layers may be hcb (honeycomb) layers disposed in an abc sequence. The staggered configuration may result in the formation of hexagonal channels along the stacking axis. Cargo may be stored between layers. In various examples, the layers are crystalline and porous.

[0110] The COF nanoparticles may have various sizes. For example, the COF nanoparticles have a longest linear dimension (e.g., diameter) of 10-300 nm, including every 0.1 nm value and range therebetween. In various other examples, the COF nanoparticles have a longest linear dimension (e.g., diameter) of about 60 nm.[oni] The COF nanoparticles may be used as carriers for delivery of cargo molecules, such as compounds (e.g., small molecules). The COF nanoparticles may comprise one or more compounds that are the same or different. For example, the compounds may be therapeutic compounds, such as, for example, chemotherapeutic agents. The chemotherapeutic agents may have one or more aromatic groups. Examples of chemotherapeutic groups include, but are not limited to, doxorubicin, Pamufetinib, Pamufetinib, Mobocertinib, Famitinib, Crenolanib, Dovitinib, Cediranib, Donafenib, Sitravatinib, Infigratinib, Asciminib, Momelotinib, Ipatasertib, Capivasertib, Parsaclisib, Inavolisib, Buparlisib, Enzastaurin, Hypericin, Volasertib, Pelabresib, Abexinostat, Givinostat, Inavolisib, Vorasidenib, Navitoclax, Patidegib, Salinosporamide A, Senaparib, Fluzoparib, Veliparib, HC-1119, Proxalutamide, Nitroxolinc, Uproleselan sodium, Amcenestrant, Elacestrant, Giredestrant, RRx-001, Relacorilant, Belzutifan, Pimitespib, Xevinapant hydrochloride, Linrodostat, Epacadostat, Pevonedistat, Devimistat, Rose Bengal disodium, Acyclic retinoid, Anhydroicaritin, Napabucasin, Nirogacestat, cisplatin, paclitaxel, methotrexate, and the like, and combinations thereof.

[0112] The COF nanoparticles may release its cargo upon exposure to an external trigger. Although the COF nanoparticles may exhibit a slower, natural, release of protein cargo, exposure to an external trigger may increase the release of cargo. For example, the COF nanoparticles may release incorporated compounds upon contact with a medium where the pH is below 6 or below pH 5. For example, the pH may be 5 or lower.

[0113] In an aspect, the present disclosure provides compositions. The composition may comprise COF nanoparticles incorporating cargo molecules.

[0114] The composition can comprise COF nanoparticles incorporating cargo molecules in a pharmaceutically acceptable carrier (e.g., carrier). The carrier can be an aqueous carrier suitable for administration to individuals including humans. The carrier can be sterile. The carrier can be a physiological buffer. Examples of suitable carriers include sucrose, dextrose, saline, and / or a pH buffering element (such as, a buffering element that buffers to, for example, a pH from pH 5 to 9, from pH 6 to 8, (e.g., 6.5)) such as histidine, citrate, or phosphate. Additionally, pharmaceutically acceptable carriers may be determined in part by the composition being administered. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present disclosure. Additional, non-limiting examples of carriers include solutions, suspensions, and emulsions that are dissolved or suspended in a solvent before use, and the like. The composition may comprise one or more diluents. Examples of diluents, include, but are not limited to distilled water, physiological saline, vegetable oil, alcohol, dimethyl sulfoxide, and the like, and combinations thereof. Compositions may contain stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, and the like, and combinations thereof. Compositions may be sterilized or prepared by sterile procedure. A composition of the disclosure may also be formulated into a sterile solid preparation, for example, by freeze- drying, and may be used after sterilization or dissolution in sterile injectable water or other sterile diluent(s) immediately before use. Additional examples of pharmaceutically acceptable carriers include, but are not limited to, sugars, such as, for example, lactose, glucose, and sucrose; starches, such as, for example, com starch and potato starch; cellulose, including sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as, for example, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil, and soybean oil; glycols, such as, for example, propylene glycol; polyols, such as, for example glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as, for example, ethyl oleate and ethyl laurate; agar; buffering agents, such as, for example, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Additional non-limiting examples of pharmaceutically acceptable carriers can be found in: Remington: The Science and Practice of Pharmacy (2012) 22nd Edition, Philadelphia, PA. Lippincott Williams & Wilkins. For example, a composition comprises a plurality of COF nanoparticles incorporating one or more compounds, where the COFs have one or more binding partners conjugated thereto, anda sterile, suitable carrier for administration to individuals including humans — such as a physiological buffer such as sucrose, dextrose, saline, pH buffering (such as from pH 5 to 9, from pH 7 to 8, from pH 7.2 to 7.6, (e.g., 7.4)) element such as, for example, histidine, citrate, or phosphate. In various examples, the composition may be suitable for injection. Parenteral administration includes infusions and injections, such as, for example, intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous administration, and the like.

[0115] In an aspect, the present disclosure provides methods for treating an individual having or suspected of having cancer. The individual may be treated by administering a therapeutically effective amount of the nanoparticle loaded with one or more cargo molecules and having one or more binding partners conjugated thereto.

[0116] In one embodiment, the individual is a human or non-human mammal. Nonhuman animals include ungulates such as bovines. Additional non-limiting examples of non- human mammals include pigs, mice, rats, rabbits, cats, dogs, or other agricultural mammals, pet, or service animals, and the like.

[0117] Various cancers may be treated via a method of the present disclosure. For example, the cancer being treated may be associated with the RGD-based peptide conjugated to the COFs of the nanoparticle. For example, the cancer may be a solid cancer (e.g., tumor). In various examples, the cancer is breast cancer, melanoma, glioblastoma, lung cancer, ovarian cancer, colorectal cancer, or prostate cancer. In various examples, the cancer is associated with one or more of the following integrins: av[k integrin (breast cancer, melanoma, glioblastoma, ovarian cancer, prostate cancer), avps integrin (breast cancer), avps integrin (glioblastoma), asPi integrin (lung cancer), and avPe integrin (colorectal cancer, prostate cancer). For example, regarding otvPs and avPs integrins, these integrins are overexpressed in breast cancer and associated with tumor growth, angiogenesis, and metastasis. For example, regarding otvPs integrin, it is highly expressed in melanoma cells, facilitating their attachment and migration. For example, regarding otvPs and avPs integrins, these integrins are involved in the angiogenesis and invasiveness of glioblastoma, the most aggressive brain cancer. For example, regarding asPi integrin, this integrin is associated with the proliferation and survival of lung cancer cells. For example, regarding otvPs integrin, ovarian cancer cells frequently overexpress avPs integrin, which is involved in tumor growth and metastasis. For example, regarding avPe integrin, this integrin is overexpressed in colorectal cancer and is a marker of poor prognosis. For example, regarding avPs and avPe integrins, these integrins are associated with the metastasis of prostate cancer.

[0118] In various examples, the method comprises administering the composition or COF nanoparticles via non-oral routes, such as, for example, injection.

[0119] In an aspect, the disclosure provides kits. A kit may comprise pharmaceutical preparations comprising COF nanoparticles and printed material.In various examples, a kit comprises a closed or sealed package that contains the pharmaceutical preparation. In various examples, the package comprises one or more closed or sealed vials, bottles, blister (bubble) packs, or any other suitable packaging for the sale, or distribution, or use of the compounds and compositions comprising compounds of the present disclosure. The printed material may include printed information. The printed information may be provided on a label, or on a paper insert, or printed on the packaging material itself. The printed information may include information that identifies the compound in the package, the amounts and types of other active and / or inactive ingredients, and instructions for taking the composition, such as the number of doses to take over a given period of time, and / or information directed to a pharmacist and / or another health care provider, such as a physician, or a patient. The printed material may include an indication that the pharmaceutical composition and / or any other agent provided with it is for treatment of a subject. In various examples, the product includes a label describing the contents of the container and providing indications and / or instructions regarding use of the contents of the container to treat a subject. A kit may comprise a single dose or multiple doses.

[0120] The steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in an embodiment, the method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.

[0121] The following Statements provide various examples of the present disclosure.Statement 1. A nanoparticle comprising, one or more covalent organic frameworks (COFs), wherein each COF comprises one or more binding partners conjugated thereto, wherein each binding partner comprises a linker group conjugated to an RGD-based peptide, wherein the nanoparticle further comprises one or more compounds.Statement 2. A nanoparticle according to Statement 1, wherein the linker groups comprise a polyethylene glycol (PEG) group.Statement 3. A nanoparticle according to Statement 2, wherein the linker groups have the following structure:Statement 4. A nanoparticle according to Statement 1, wherein the binding partners are conjugated to the COF via a click reaction, acylation chemistry, or a substitution reaction.Statement 5. A nanoparticle according to Statement 1, wherein the binding partners are conjugated to the COF via a click reaction.Statement 6. A nanoparticle according to Statement 1, wherein the one or more RGD-based peptides are attached to the one or more linker groups via a sidechain of an amino acid residue of the one or more RGD-based peptides.Statement 7. A nanoparticle according to Statement 6, wherein the sidechain is the sidechain of a nucleophilic amino acid residue.Statement 8. A nanoparticle according to Statement 7, wherein the nucleophilic residue is cysteine, lysine, serine, or ornithine.Statement 9. A nanoparticle according to Statement 6, wherein the side chain is the side chain of an electrophilic amino acid residue.Statement 10. A nanoparticle according to Statement 9, wherein the electrophilic amino acid residue is aspartic acid or glutamic acid.Statement 11. A nanoparticle according to any one of the preceding Statements, wherein the RGD-based peptides are cyclic. In various other examples, the RGD-based peptides are linear.Statement 12. A nanoparticle according to Statement 11, wherein the RGD-based peptides have the following structure:or a protonated or deprotonated form thereof, wherein R is the side chain of the RGD-based peptide.Statement 13. A nanoparticle according to Statement 12, wherein the one or more RGD- based peptides have the following structure:or a protonated or deprotonated form thereof.Statement 14. A nanoparticle according to any one of Statements 1-11, wherein the RGD- based peptide is associated (e.g., capable of binding) with one or more of the following integrins: avPs integrin, avps integrin, avps integrin, asPi integrin, or avpe integrin. In various examples, the RGD-based peptide is c(RGDfK) (SEQ ID NO:2, where c denotes the peptide is cyclic and the lowercase F corresponds to D-phenylalanine), RGDSP (SEQ ID NO:3), or RGDT (SEQ ID NO:4).Statement 15. A nanoparticle according to any one of the preceding Statements, wherein the one or more compounds are therapeutic compounds.Statement 16. A nanoparticle according to Statement 15, wherein the therapeutic compounds are chemotherapeutic agents.Statement 17. A nanoparticle according to Statement 15, wherein the one or more compounds comprise one or more aromatic groups. Statement 18. A nanoparticle according to Statement 16, wherein the one or more compounds are doxorubicin, cisplatin, methotrexate, or paclitaxel, or any combination thereof.Statement 19. A nanoparticle according to any one of the preceding Statements, wherein the one or more COFs comprise the following structure:Statement 20. A nanoparticle according to Statement 18, wherein the one or more COFs comprise the following structure:Statement 21. A nanoparticle according to any one of the preceding Statements, wherein the nanoparticle comprises a COF comprising:where L is a linker group and Peptide is an RGD-based peptide (e.g., c(RGKYK) (SEQ ID NO: 1), c(RGDfK) (SEQ ID NO:2), RGDSP (SEQ ID NO:3), or RGDT (SEQ ID NO:4)), or a deprotonated or protonated form thereof.Statement 22. A covalent organic framework (COF), comprising the following structure:where L is a linker group and Peptide is an RGD-based peptide (e.g., c(RGKYK) (SEQ ID NO: 1), c(RGDfK) (SEQ ID NO:2), RGDSP (SEQ ID NO:3), or RGDT (SEQ ID NO:4)).Statement 23. A COF according to Statement 22, wherein the linker group comprises a polyethylene glycol (PEG) group.Statement 24. A COF according to Statement 23, wherein the linker group has the following structure:Statement 25. A COF according to any one of Statements 22 to 24, where the RGD-based peptide is attached to the linker group via a sidechain of an amino acid residue of the RGD- based peptide.Statement 26. A COF according to Statement 25, wherein the sidechain is the sidechain of a nucleophilic amino acid residue.Statement 27. A COF according to Statement 26, wherein the nucleophilic residue is cysteine, lysine, serine, or ornithine.Statement 28. A COF according to Statement 25, wherein the sidechain is the sidechain of an electrophilic amino acid residue.Statement 29. A COF according to Statement 28, wherein the electrophilic amino acid residue is aspartic acid or glutamic acid.Statement 30. A COF according to any one of Statements 22 to 29, wherein the RGD-based peptide is cyclic. In various other examples, the RGD-based peptide is linear.Statement 31. A COF according to Statement 30, wherein the RGD-based peptide has the following structure:or a protonated or deprotonated form thereof, wherein R is the sidechain of the RGD-basedpeptide, or c(RGKYK) (SEQ ID NO: 1), c(RGDfK) (SEQ ID NO:2), RGDSP (SEQ ID NON), or RGDT (SEQ ID NON).Statement 32. A COF according to Statement 31, wherein the RGD-based peptide has the following structure:or a protonated or deprotonated form thereof.Statement 33. A COF according to any one of Statements 22-32, wherein the RGD-based peptide is associated (e.g., capable of binding) with one or more of the following integrins: avPs integrin, avp5 integrin, avPs integrin, asPi integrin, or avPe integrin. In various examples, the RGD-based peptide is c(RGDfK) (SEQ ID NON), RGDSP (SEQ ID NO:3), or RGDT (SEQ ID NON).Statement 34. A covalent organic framework (COF), comprising the following structure:wherein R is a reactive group. Statement 35. A COF according to Statement 34, wherein the reactive group comprises an azide group, an alkynyl group, activated esters, carboxylate / carboxylic acid, halides, amines, hydroxides, and the like.Statement 36. A COF according to Statements 34 or 35, wherein the reactive group has the following structure:Statement 37. A composition comprising a nanoparticle according to any one of Statements 1 to 21 or a COF according to any one of Statements 22 to 36.Statement 38. A composition according to Statement 37 further comprising one or more pharmaceutically acceptable carriers.Statement 39. A method for treating a subject having or suspected of having cancer comprising administering to the subject a therapeutically effective amount of a nanoparticle according to any one of claims 1 to 21 or a composition according to claims 37 or 38.Statement 40. A method according to Statement 34, wherein the cancer is associated with one or more RGD-based peptides of the nanoparticle.Statement 41. A method according to Statements 39 or 40, wherein the cancer is a solid tumor.Statement 42. A method according to any one of Statements 39-41, wherein the cancer is breast cancer, melanoma, glioblastoma, lung cancer, ovarian cancer, colorectal cancer, or prostate cancer.Statement 43. A method according to any one of claims 39 to 42, wherein the cancer is breast cancer (e.g., triple -negative breast cancer).

[0122] The following example is presented to illustrate the present disclosure. It is not intended to be limiting.EXAMPLE

[0123] This example provides methods and COFs of the present disclosure.

[0124] Nanoscale imine-linked covalent organic frameworks (nCOFs) have shown tremendous potential as emerging chemotherapeutic delivery systems for cancer treatment This is due to their unique properties, including large surface area, tunable pore geometry, crystallinity, high flexibility in molecular architecture and functional design, and sensitivity to the acidic environment of tumors. Unlike amorphous organic polymers, nCOFs have a long-range ordered structure in which the organic building blocks are positionally controlledin two or three dimensions. This results in regular pores that facilitate drug loading and controlled release of prominent drugs such as Doxorubicin. Recently, we published the formation of nCOF by condensation of 2,6-diformylpyridine (DFP) with 1 ,3,5-tris(4- aminophenyl)-benzene (TAB) to form an imine-linked nCOF. The resulting nCOF was stable under physiological conditions but decayed in an acidic medium, an essential property of the microenvironment of most solid tumors, allowing site-specific drug release. The lack of existing RGD-conjugated COF-delivery systems and the chemical modifiability of our DFP- TAB nCOF to achieve active targeted delivery create promising opportunities for utilizing RGD-functionalized nCOFs in targeted cancer therapy.

[0125] We developed a pH-responsive nanoparticle (NP) drug delivery system using chemically modified nano-covalent organic frameworks (nCOFs) to target triple-negative breast cancer (TNBC) cells expressing high integrin levels. This system leverages the unique properties of nCOFs, which decompose in acidic environments, such as cancer cell vesicles, allowing the release of encapsulated chemotherapy drugs. An alkyne-enriched nCOF (Alkyn- nCOF) with an average size of 60 nm was synthesized and was subsequently surface- modified with cyclic RGD peptides through click chemistry, resulting in Alkyn-nCOF- cRGD. The RGD peptides, chosen for their affinity to the avPs integrin overexpressed on TNBC cell membranes, ensure targeted drug delivery. Both nanoparticles exhibited biocompatibility and stability under physiological conditions but disintegrated under acidic pH. NPs were loaded with Doxorubicin (Dox), a common chemotherapeutic agent for TNBC, achieving high loading capacities: 61% for Alkyn-nCOF and 42% for Alkyn-nCOF-cRGD. Testing on breast tumor cells displaying high (MDA-MB-231) and low (MCF-7) integrin expressions demonstrated that Alkyn-nCOF-cRGD significantly improves drug uptake and targeted cell death, owing to receptor-mediated endocytosis mechanism. In vivo studies in tumor (MDA-MB-231) mouse models further substantiated the superior therapeutic efficacy of Alkyn-nCOF-cRGD over both free Dox and Alkyn-nCOF, showcasing the benefits of receptor-mediated endocytosis and pH-triggered drug release. Our strategy, which integrates nCOF with targeted peptides, is a step forward in drug delivery, paving the way for nextgeneration personalized cancer therapy.

[0126] Described herein are alkyne -functionalized nCOF NPs (denoted Alkyn-nCOF) modified with cyclic RGD peptides as they have higher binding selectivity, are more conformationally stable, and are less susceptible to biodegradation than linear peptides. These NPs were then loaded with Dox, creating a system simultaneously targeting cancer cells and achieving controlled tumor-specific release of the drug used for the treatment of TNBC. TheAlkyn-nCOF NPs were synthesized with an average diameter of 60 nm. The alkynes on the surface of the NPs were covalently conjugated to pegylated cyclic RGD peptides with a terminal azide function (cRGD-PEG-Ns) by copper-mediated click chemistry (Figure la), and the resulting NPs are designated as Alkyn-nCOF-cRGD. Click chemistry reactions have been extensively used to prepare new bioconjugated structures of peptides and nanoparticles for biomedical and pharmaceutical applications. Because the triazole ring is strong, it reduces the susceptibility of the molecule to enzymatic degradation, reduction, hydrolysis, and oxidation, thus ensuring the metabolic stability and bioavailability of the bioconjugate in vivo. Successful conjugation of the pegylated cyclic RGD peptide to the surface of the Alkyn-nCOF NPs was demonstrated by XPS, solid-state NMR, FTIR, DLS, and TEM. Alkyn-nCOF and Alkyn-nCOF-cRGD showed excellent biocompatibility and stability at physiological pH and temperature conditions and successfully disintegrated completely at low pH. Subsequently, Alkyn-nCOF and Alkyn-nCOF-cRGD were loaded with Dox to a loading capacity of 61 % and 42 %, respectively, which are among the highest values reported for COFs (Table 2). Simultaneously, the pH-dependent drug release profile was observed to provide a platform for the release of Dox in the acidic environment of tumor tissues and enhance the therapeutic anticancer effect. The in vitro antitumor effect of Alkyn- nCOF@Dox and Alkyn-nCOF-cRGD@Dox was evaluated on breast tumor cells with high (MDA-MB-231) and low (MCF-7) integrin levels, demonstrating the effect of cRGD targeting on enhanced internalization of Dox through receptor-mediated endocytosis and targeted toxicity. The in vivo tumor targeting and therapeutic efficacy of Alkyn-nCOF-cRGD was significantly superior to free drug or peptide-free system (Alkyn-nCOF) in triplenegative breast tumors (MDA-MB-231), with no side effects observed, underscoring the enhanced tumor-targeted delivery capabilities of the cRGD-conjugated nanoparticles. This strategy reduces required drug doses, lowers treatment costs, and, most importantly, could prolong the survival of TNBC patients.

[0127] Results and discussion.

[0128] Porous imine-linked Alkyn-nCOF nanoparticles with an average diameter of ~60 nm were synthesized by co-condensation of 4-ethynyl-l,6-diformyl pyridine (Alkyn- DFP; 12 mg, 0.06 mmol) and l,3,5-tris(4-aminophenyl)-benzene (TAB; 21 mg, 0.06 mmol) in anhydrous 1,4-dioxane in the presence of acetic acid catalyst (0.5 mb, 3 M) under microwave irradiation at 110 °C for 120 minutes (Figure 9). The as-synthesized brownish gel was collected by centrifugation and washed with 1,4-dioxane and ethanol, followed by water. The Alkyn-nCOFs are highly dispersible in water, facilitating their use as a drug deliverysystem. To incorporate the active targeting function into the Alkyn-nCOF nanocarriers, conjugation of the azide-pegylated cyclic RGD peptide (cRGD-PEG-Nv MW Ik) was performed in a one-step procedure by copper-mediated click chemistry in water (Figure lb). Copper sulfate hexahydrate (3.2 mg, 0.02 mmol, 200 pL) and sodium ascorbate (12 mg, 0.06 mmol, 200 pL) were added to the Alkyn-nCOF solution (10 mg, 2 mb, pH 7). The resulting brown solution was stirred for 10 minutes at room temperature, then cRGD-PEG-Ns (10 mg, 100 pL) was added dropwise and stirred at room temperature overnight. The as-synthesized particles were collected by centrifugation, washed with water, and dialyzed for 48 hours. The obtained nanoparticles are referred to as Alkyn-nCOF-cRGD.

[0129] The chemical composition of Alkyn-nCOFs and Alkyn-nCOF-cRGD was investigated by Fourier-transform infrared (FTIR) spectroscopy, cross-polarization magicangle spinning nuclear magnetic resonance (CP / MAS NMR) and X-ray photoelectron spectroscopy (XPS).

[0130] The FTIR spectrum of Alkyn-nCOFs shows a weak stretching band at 1622 cm'1corresponding to the imine C=N bond, two broad and intense peaks at 1585 and 1345 cm'1attributed to C=N and C-N of the pyridines and a strong vibrational peak at 1515 cm'1corresponding to aromatic C=C bonds (Figures 10 and 11). Additionally, vibrational peaks corresponding to the C-O-CH2-C=C-H group of the Alkyn-DFP at 1443 cm'1are evident, consistent with C-0 stretching, and at 626 cm'1, corresponding to the C-H bending vibration of the terminal alkyne and the corresponding bond bending overtone at 1293 cm'1, originating from an out-of-plane overtone (harmonic) of the C=C-H bending. This overtone absorption appears at twice the fundamental frequency. The disappearance of the N-H bonding of the amine in the 3400-3200 cm'1range and the attenuation of the C=O signa at 1705 cm'1, (Figures 10 and 11) indicates complete consumption of the starting material.Following conjugation of the cyclic peptide cRGD-PEG with the Alkyn-nCOF nanoparticles, changes in the C-O-CH2-C=C-H group of the Alkyn-pyridines of the framework were observed (Figure 12). While the C-0 stretching vibration at 1442 cm'1remained unchanged, the terminal alkyne C-H bending vibration at 626 cm'1and the corresponding bond bending overtone at 1293 cm'1disappeared completely, while the other peaks became broader due to the presence of the peptide (Figure 12).

[0131] The CP / MAS NMR spectrum of Alkyn-nCOFs (Figure 13) shows chemical shifts from the aromatic carbons (100 to 150 ppm), carbons of the C=C alkyne (~ 78 ppm), -OCH2 (~ 57 ppm), and the imine (150 to 170 ppm). Absence of the aldehyde signal at ca. 200 ppm confirms the complete conversion of the monomers. The spectrum of cRGD-PEG-Ns shows chemical shifts of the aliphatic (10 to 80 ppm), aromatic (120 to 140 ppm), guanidine (~ 158 ppm), and carbonyl (165 to 180 ppm) carbons. The successful reaction between the cRGD-PEG peptide and the Alkyn-nCOFs was confirmed by the disappearance of the alkyne peaks at ~ 78 ppm and by the appearance of new peaks at ~ 70 ppm belonging to -OCH2 groups of the cyclic peptide. This latter peak is broad compared to those of the free peptide, indicating the successful coupling of cRGD-PEG to nCOF.

[0132] The chemical composition of Alkyn-nCOF and Alkyn-nCOF-cRGD was also confirmed by XPS. In the high-resolution C Is XPS spectra of Alkyn-nCOF, the three peaks with binding energies at (i) 284.6, (iii) 285.4, and (iii) 286.8 eV were assigned to (i) C=C / C-C, (ii) C=C, and (iii) carbon-oxygen / nitrogen bonds (C-0 in Alkyn-DFP / C-N=C in the imine bond and the pyridine), respectively (Figure 1c, Table 3). The N Is band was deconvoluted into two peaks at (i) 399.6 and (ii) 400.5 eV assigned to the nitrogen atoms in (i) the imine bond -N=C- and (ii) the N-pyridine, respectively (Figure 1c, Table 4).

[0133] High-resolution C Is XPS spectra of cRGD-PEG-Ns yielded four peaks: (i) C=C / C-C at 284.7 eV, (ii) N-C=O at 286.5 eV, and (iii) the side chain COOH along with the guanidinium at 287.6 eV (Figure 15, Table 3). In the high-resolution N Is XPS spectra, the peptide has amide N-C=O linkages that lead to photoemission at 399.9 eV (Figure 15 and Tables 4), along with a small peak to higher binding energies at 401.4 eV arising from the protonated guanidine group. The presence of the azide N=N“=N functional group is evident at 404.6 eV, corresponding to the central electron deficient N atom in the azido group.

[0134] After clicking the cRGD-PEG-Ns (Figure 1c and Tables 3-4), the N Is initially at 399.64 in Alkyn-nCOF (fwhm = 1.39) broadened (fwhm= 1.83) and shifted to slightly lower energy (399.5 eV) in Alkyn-nCOF-cRGD spectra. Moreover, the intensity of the N Is peak also increased (70%), indicating more N on the NP surface coming from the amide of the peptides. A weak contribution due to the presence of the -N= unit of the newly formed triazole rings and the charged guanidinium groups was identified as a shift and a broadness at ca. 401.05 eV (fwhm = 2.6 compared to 2.4 for Alkyn-nCOF) in addition to the N-pyridine contribution of the nCOF. Additionally, no azide was detected, confirming no peptides are non-covalently bound to the NP surface.

[0135] Furthermore, the C is region of Alkyn-nCOF-cRGD confirmed the successful peptide click and was deconvoluted into three peaks assigned to C=C / C-C (285.0 eV), C- O / C-N=C / N-C=O / triazole (286.6 eV), and the side chain COOH along with the guanidinium (288.3 eV) (Figure lb and Table 3). The peak at 285.0 eV assigned to C=C / C-C is more intense and broadened (fwhm = 1.44 compared with 1.15 for Alkyn-nCOF), clearlyindicating the presence of more C on the clicked surface due to new species / interaction components between the Alkyn-nCOF and the peptides on the surface. Significantly, no C=C units (or significantly diminished and adsorbed in the 284.5 eV peak) were observed, indicating a successful click on the surface of the nanoparticles. Moreover, the increase in cRGD-PEG surface concentration resulted in an increase in the intensity of C-O / C- N=C / N-C=O / triazole peak and the rise of a well-defined new peak corresponding to the COOH and guanidinium (288.3 eV). It is also important to note that no catalyst (CUSC>4-5H2O) residues were detected by XPS or ICP-MS, which could lead to unwanted toxicity.

[0136] Powder X-ray diffraction (PXRD) measurements were recorded to confirm the crystallinity of Alkyn-nCOF (Figure Id). The PXRD pattern shows the most intense diffraction peaks at 20 = 5.1° (110), 7.4° (101), 10.3° (220) / (211), indicating the long-range ordering in the framework. Following the principles of reticular chemistry, crystal structure models were constructed (Figure Id) based on the geometry of the building blocks, resulting in distorted hcb layers. A crystal structure model created in the P3 space group was geometrically optimized by energy minimization using a universal forcefield. The completed Pawley refinement supports the obtained unit cell with lattice parameters of a = b = 34.632 A, and c = 13.009 A, with three layers per unit cell and an interlayer spacing of "'4.3 A. The PXRD pattern of Alkyn-nCOF-cRGD shows the presence of peaks at 4.0°, 13.0°, and 24.6°. However, the intensity of these peaks decreased significantly compared to the pristine nCOF due to the presence of the peptide on the surface (Figure 17).

[0137] Lattice-resolution TEM images of Alkyn-nCOFs and Alkyn-nCOF-cRGD suggested that they are crystalline with consistent and continuous lattice fringes that extend throughout the COF (Figure 2a). The lattice spacings were 0.3-0.4 nm, corresponding to the interlayer distances. Furthermore, the selected area electron diffraction (SAED, Figure 2a) demonstrates the well-crystallized feature of Alkyn-nCOFs and Alkyn-nCOF-cRGD, which exhibited distinct electron diffraction spots, well matching to the t / 004 plane with -spacing of 0.32 nm (Figures 2a).

[0138] High-resolution transmission electron microscopy (HRTEM) images showed Alkyn-nCOFs and Alkyn-nCOF-cRGD as spheroidal morphology with an average diameter of ~60 nm (Table 1, Figures 2a, 19-22). After clicking the peptide, the morphology, size, and height of Alkyn-nCOF-cRGD remained unchanged compared with pristine nCOF because the peptide size is insignificant1.32 nm) (Table 1 and Figures 19-22). To verify the presenceof the peptide, samples were negatively stained with uranyl acetate substitute (1 %), which specifically stains the peptide, making it appear as dark spots in TEM; the differences between the Alkyn-nCOF and cRGD-conjugated nCOF are clearly evident. Small dark aggregates with an average size of 1-2 nm, corresponding to the peptides on the surface of the Alkyn-nCOF-cRGD, can be easily identified (Figure 2a). At the same time, the Alkyn-nCOFs do not exhibit these aggregates (Figure 2a).

[0139] Tapping mode AFM images provided additional evidence for the spheroidal morphology of the particles with a height of ~23 nm (Table 1, Figures 25-26). The mechanism of formation of Alkyn-nCOFs was directly monitored by in situ measurements using liquid cell TEM (Movie 1, Figures 2b and 23). The two linkers, Alkyn-DFP and TAB, dissolved in dioxane, were drop-casted onto a bottom chip. Once wetting of the liquid cell was confirmed, acetic acid was flowed into the cell and the liquid cell was heated to 110 °C (Movie 1, Figures 2b and 23: extracted frames from a movie). The formation of sheet-like structures was observed within a few seconds; then, nanosheets started to stack on top of each other, forming spherical particles, which explains why the diameter (60 nm) was larger than the height (23 nm).

[0140] The porosity of Alkyn-nCOFs was evaluated by measuring the adsorptiondesorption isotherms of nitrogen gas at 77 K (Figure 27). Alkyn-nCOFs particles exhibit type-II isotherms with Brunauer-Emmett-Teller (BET) surface areas of 140 m2g_ |. two narrow pore size distributions with average pore widths of 10.2 and 13.1 A, and a calculated pore volume of 0.17 cm3g-1(Figure 27 and Table 1). Due to the large size of the peptide, conjugation occurred only at the surface and not inside the pores, as confirmed by the analysis of BET. Indeed, a slight decrease in BET surface area (95 m2g_|) and pore volume (0.08 cm3g'1), but not in pore width (10. 1 / 13.1 A) were observed, confirming that the pores remained unaffected following clicking the peptide (Figure 27).

[0141] The electronic properties of Alkyn-nCOF and Alkyn-nCOF-cRGD were studied by absorption and fluorescence spectroscopy in water. The UV-vis absorption for an aqueous dispersion of Alkyn-nCOF and Alkyn-nCOF-cRGD exhibits two maxima at kmax = 283 and 366 nm (Figures 28 and 29). In addition, Alkyn-nCOF showed a broad emission band centered at a maximum of 468 nm when excited at 380 nm (Figure 30). The presence of the peptide on the surface of Alkyn-nCOF affected the emission of the material. After clicking the peptide, in addition to the broad emission peak of nCOF at 468 nm, an additional emission shoulder band centered at 438 nm corresponding to its emission was observed (Figure 31).

[0142] Thermogravimetric analysis (TGA) showed that Alkyn-nCOFs and Alkyn- nCOF-cRGD were stable up to 500 °C (Figure 34). Their chemical stability was also evaluated. The particles immersed in a PBS solution with a pH of 7.4 and kept at 37 °C for 24 hours maintained their shape, size, and crystallinity (Figure 16). Under acidic conditions (pH = 5.4), the particles decompose and lose their shape, size, and crystallinity, likely due to acid-catalyzed hydrolysis of the imine bonds (Figure 16).

[0143] The successful conjugation of the peptide to the nCOF surface was also confirmed through surface charge analysis at pH 7.4 (Figure 35). Upon clicking the peptide, the ^-potential of the Alkyn-nCOF significantly decreased from -6.7 ± 0.5 to -21.9 ± 1.1 mV. This decrease in ^-potential was attributed to the surface modification with cRGD-PEG cyclic peptides, providing additional evidence for the effective attachment of the peptide to the nCOF surface. A likely explanation lies in the preferential orientation of the anionic carboxyl groups (in the aspartic acid residue) and the cationic guanidine group (in arginine). As a consequence of the stronger negative charge and thus better interparticle repulsion, the average hydrodynamic diameter at pH 7.4 of Alkyn-nCOF-cRGD was 92.2 nm with a polydispersity (PDI) of 0.1 and no precipitation was observed overtime (Figures 16 and 39), in contrast to 220.0 nm with a PDI of 0.5 for Alkyn-nCOFs. At acidic pH, both systems decompose with a drastic decrease in particle size (Figures 16 and 39).

[0144] The weight % efficiency of peptide conjugation on nanoparticle surfaces reflects the proportion of conjugated peptides to the total nanoparticle weight. The amount of peptide on the nCOF surface was quantified using the o-phtalaldehyde (OPA) method, revealing a peptide conjugation efficiency of 54.2 wt% (Figure 41).

[0145] The properties of Alkyn-nCOF and Alkyn-nCOF-cRGD nanoparticles are summarized in Table 1. These bulk characterizations indicate the successful synthesis of nanoscale imine-linked Alkyn-nCOF nanoparticles. The presence of the alkyne functions was successfully conjugated with an azide -pegylated cyclic RGD peptide (cRGD-PEG-Ns) by copper-mediated click chemistry. This prompted us to use it for targeted delivery of Doxorubicin.

[0146] Table 1. Physicochemical characterization of the Alkyn-nCOF before and after clicking cRGD-PEG-Ns on the nanoparticle surface.

[0147] The anticancer drug Doxorubicin (Dox) was loaded in Alkyn-nCOF and Alkyn-nCOF-cRGD by impregnation at pH 7.4 and 37 °C for 24 hours (weight ratio Alkyn- nCOF: Dox = 1:5). Using fluorescence spectroscopy of the Dox-containing supernatant solution, the loading efficiency of Alkyn-nCOF and Alkyn-nCOF-cRGD was calculated to be 61 ± 4 wt %, and 42 ± 3 wt %, respectively (Figures 3a and 43), which are among the highest values compared with existing COF systems (Table 2 for comparison with reported COFs). For the same Dox concentration, an aqueous sample of free Dox shows greater fluorescence intensity than Alkyn-nCOF@Dox and Alkyn-nCOF-cRGD@Dox, indicating fluorescence quenching of Dox within the NPs (Figure 33). This quenching may be attributed to electronic interactions between the excited drug molecules and the nCOF pores or to self-quenching of the drug in the pores of the particle, where the effective concentration of the drug is relatively high. These results suggest that Dox is located within nCOF pores. The small dynamic radius and aromatic rings of Dox interact with Alkyn-nCOF through TT-TT conjugation. Additionally, -NH and -OH groups of Dox can form hydrogen bonds with the framework, providing strong physical adsorption forces for drug loading and retention. Indeed, a simulated annealing process performed with three Dox molecules per Alkyn-nCOF unit cell (corresponding to a loading of about 50 wt %) shows various preferential sorption sites (Figure 3b). Thus, Dox molecules fill the nCOF pores (orange- and purple molecules in Figure 3b). Interestingly, a sandwich conformation for a Dox molecule is also found between the nCOF layers (the salmon-colored molecule in Figure 3b). This behavior is possible due to the particular distorted hexagonal pore geometry of Alkyn-nCOF, allowing diffusion of Dox through the pores into the interlayer space. Van Der Waals interactions between nCOF layers and Dox molecules, which also involve alkyne groups, are present in all cases.

[0148] The13C CP / MAS NMR spectrum of Dox is well resolved (Figure 14), showing peaks derived from aliphatic (10 to 100 ppm), aromatic (110 to 165 ppm), and carbonyl (180 to 190 ppm) carbons. The successful loading of Alkyn-nCOF with Dox was confirmed by the presence of a distinct peak of the carbonyl carbons of Dox at ca. 187 ppm inaddition to the peaks of the Alkyn-nCOF (Figure 14). The presence of Dox molecules within the COFs was further confirmed by the sorption isotherm of the Alkyn-nCOF@Dox and Alkyn-nCOF-cRGD@Dox, which showed a decrease in BET surface area to 78 and 46 m2g_|. a pore size distribution with an average pore width of 11.7 A and a total pore volume of 0.045 cm3g-1, (Figure 27), indicating localization of Dox in the internal pores of the nCOFs. The PXRD peak intensity of the Alkyn-nCOF@Dox and Alkyn-nCOF-cRGD@Dox decreased significantly after Dox loading, indicating effective encapsulation of Dox in the nCOF pores (Figure 18).

[0149] The drug release behavior of Alkyn-nCOF@Dox and Alkyn-nCOF- cRGD@Dox was investigated using Dox fluorescence by mimicking the slightly acidic pH (~5.4) of the lysosomal compartments at 37 °C using the dialysis method in vitro. At pH 7.4, less than 10 % of the drug was released from Alkyn-nCOF and Alkyn-nCOF-RGD, demonstrating the efficient entrapment of Dox in the nCOF pores and the favorable structural and drug loading stabilities under physiological conditions (Figures 3c and 44). However, the rapid and complete release of Dox from Alkyn-nCOF and Alkyn-nCOF-cRGD was measured at a pH of 5.4 within 24 hours, confirming the expected pH sensitivity of the nanoparticles in acidic environments. At pH 7.4, no significant changes in the morphology of Alkyn- nCOF@Dox and Alkyn-nCOF-cRGD@Dox were observed within 24 hours, as shown from the TEM images and DLS measurements (Figures 3d, 24 and 39). At acidic pH, the system decomposes with a complete loss of morphology and a drastic decrease in particle size (Figures 3d and 24, and 39).

[0150] Taken together, these results indicate the nanoparticles we developed are desirable candidates for the specific release of Dox in an acidic environment of tumor tissues coupled with peptide-targeting properties.

[0151] Breast cancer is a complex and heterogeneous disease. Two representative cell lines corresponding to different breast cancer subtypes, with different aggressiveness, physiology, and treatment responses, were selected (Figure 45). MCF-7 cells originate from breast adenocarcinoma and typify as luminal breast cancers, characterized by a less aggressive nature. In contrast, MDA-MB-231 cells, known for their cancer stem-like features, fall under the category of triple-negative basal-type breast cancers. They are notorious for their aggressive behavior and pose a challenging prognosis due to higher instances of treatment resistance. The aggressive behavior of MDA-MB-231 cells is linked to their elevated expression of av[33 integrin. This integrin plays a crucial role in promoting tumor growth and invasiveness, contributing to these cancer cells enhanced migratory andmetastatic capabilities. However, otvPs integrins are weakly expressed on MCF-7. Therefore, MDA-MB-231 cells were used as integrin-positive cancer cells overexpressing otvPs integrin on the cell membrane. In contrast, MCF-7 cells were chosen as negative controls for lowly expressed avPs integrin. Based on the interaction between cyclic RGD peptides and a.\ integrins, we compared in vitro the ability of Alkyn-nCOF and Alkyn-nCOF-cRGD to internalize and release Dox in both cell lines. The biological activity of Alkyn-nCOF and Alkyn-nCOF-cRGD were compared in vitro and in vivo, with both systems loaded with the same amount of Dox (40 wt%).

[0152] To evaluate the biocompatibility of Alkyn-nCOF and Alkyn-nCOF-cRGD (no drug), in vitro viability studies were performed in MCF-7 and MDA-MB-231 cells (Figure 4a). Alkyn-nCOF and Alkyn-nCOF-cRGD did not elicit cytotoxic effects at concentrations up to 1 mg mb'1and after 48 hours of incubation. These in vitro results are indicative of biocompatibility.

[0153] We next evaluated the anticancer efficacy of three distinct Dox formulations - free Dox, Alkyn-nCOF@Dox, and Alkyn-nCOF-cRGD@Dox - on MCF-7 and MDA-MB-231 cells (Figures 4b and 46). In the case of MCF-7 cells, both free Dox (ICso = 0.52 pM±0.1) and Alkyn-nCOF@Dox (ICso = 0.44 pM±0.1) presented comparable ICso values. However, the Alkyn-nCOF-cRGD@Dox proved to be notably more potent (ICso = 0.28 pM±0.09, p < 0.05 compared to Dox). For the MDA-MB-231 cells, Alkyn-nCOF- cRGD@Dox again emerged as the most effective formulation (ICso = 0. 10 pM±0.03), showing significant superiority (p <0.01 compared to Dox) over both free Dox (ICso = 0.39 pM±0.09) and Alkyn-nCOF@Dox (ICso = 0.18 pM±0.1). The enhanced efficacy of Alkyn- nCOF-cRGD@Dox, especially pronounced in MDA-MB-231 cells, could be ascribed to its potential enhanced uptake through integrin otvPs receptor-mediated endocytosis resulting in augmented cytotoxicity of Dox due to its targeted delivery. This observation led us to further investigate the cell death and internalization mechanism of Dox, Alkyn-nCOF@Dox, and Alkyn-nCOF-cRGD@Dox in MDA-MB-231 compared to MCF-7 cells.

[0154] The Caspase family is essential in mediating apoptotic responses, with Caspase-3 being a key pro-apoptotic protease. To explore whether the cell death in MCF-7 and MDA-MB-231 cells treated with Alkyn-nCOF@Dox and Alkyn-nCOF-cRGD@Dox results from apoptosis via caspase cascade activation, we conducted Western blot experiments to measure Caspase-3 protein levels. These experiments compared the effects of these compounds to those induced by free Dox, as shown in Figure 4c. In MCF-7 cells, Caspase-3 was not detectable, which is consistent with previous findings indicating theabsence of Caspase-3 protein expression in these cells (Figure 47). In contrast, the Western Blot data for MDA-MB-231 cells, presented in Figure 4c-d, reveal a notable decrease in pro- Caspase-3 levels (32 KDa) following treatment with Dox, Alkyn-nCOF@Dox, and Alkyn- nCOF-cRGD@Dox. This decline points to apoptosis, likely driven by alterations in the caspase pathways, as a primary cell death mechanism in MDA-MB-231 cells after these treatments.

[0155] The interaction between nanoparticles and cells is influenced by the nanoparticles' surface properties. Cell uptake primarily occurs through endocytosis, either through non-specific macropinocytosis or through a targeted approach using receptor- mediated endocytosis. When nanoparticles are equipped with specific ligands like the cRGD peptide, they induce targeted integrin-mediated endocytosis, mainly through the clathrin- mediated pathway. After internalization, nanoparticles generally move to endosomes and then to lysosomes for degradation, with behavior potentially varying based on factors like nanoparticle nature, ligands, and receptor expression levels of the cell. Therefore, we investigated the interaction and possible endocytic routes of Alkyn-nCOF@Dox and Alkyn- nCOF-cRGD@Dox in MCF-7 and MDA-MB-231 cells at the ultrastructural level using TEM. We qualitatively examined endocytosis pathways in normally functioning cells without using specific inhibitors, avoiding potential disruptions that inhibitors might introduce. Although various cytotoxicity studies have been conducted with drug-loaded COF nanoparticles, none have directly visualized their interactions with cell membranes and organelles.

[0156] In MCF-7 cells, large aggregates of both Alkyn-nCOF@Dox and Alkyn- nCOF-cRGD@Dox were detected near or under membrane protrusions and within large vacuoles in the cytoplasm. These aggregates appeared mainly in the perinuclear region, suggesting macropinocytosis as a likely pathway for NP internalization, with the macropinosomes’ endocytic vesicles varying in size and shape and sometimes reaching considerable diameters, 2.3 pM ±0.4 for Alkyn-nCOF@Dox and 1.0 pM ±0.3 for Alkyn- nCOF-cRGD@Dox (Figures 5a, b, 48-52 and 50) whereas MCF-7 control cells display vesicle size of 1.2 pM ±0.4. However, Alkyn-nCOF-cRGD@Dox treated MCF-7 cells display fewer particles directly interacting with the cell membrane. This observation hints at some level of receptor-mediated endocytosis, consistent with the low integrin levels in MCF- 7 cells (Figures 5a, b, 48-52). Vesicle size distributions in the cells are displayed in Figure 58.

[0157] In MDA-MB-231 cells, significant aggregates of Alkyn-nCOF@Dox are primarily detected near the membrane, where membrane ruffling is also observed. Theseaggregates gather in the perinuclear region within large vacuoles, resulting in increased cellular uptake through macropinocytosis (1.8 pM ±0.6 whereas MDA-MB-231 control cells 1.0 pM ±0.3). This uptake mode appears receptor-independent, similarly observed in MCF-7 cells (Figures 5a, b, and 53-57). MDA-MB-231 cells treated with Alkyn-nCOF-cRGD@Dox showed an increased number of smaller endocytic vesicles (0.5 pM ±0.2) containing fewer particle aggregates than those treated with non-targeted nanoparticles. The Alkyn-nCOF- cRGD@Dox particles mainly bind individually to the cell membrane, enhancing uptake into cancer cells through receptor-mediated endocytosis, such as clathrin-mediated pathways (Figures 5a, b, and 53-57). NP aggregates were observed in both clathrin-coated pits on the plasma membrane and internalized clathrin-coated vesicles, confirming this endocytic route. Alkyn-nCOF-cRGD@Dox -bound integrins are internalized and then transported to endosomes and lysosomes (Figures 5a, b and 53-57). The Alkyn-nCOF-cRGD@Dox nanoparticles adhere to the vesicle wall, unlike Alkyn-nCOF@Dox nanoparticles, which are evenly distributed inside the endosomes. Individual NPs that escaped the vesicles and present in the cytosol were also observed. Our findings emphasize that adding the cRGD peptide to the nanoparticle surface changes the uptake mechanism and cellular processing in MDAMB- 231 cells with high integrin levels. This alteration modifies the entry pathway, specifically directing it through receptor-mediated endocytosis, such as clathrin-mediated cycles.

[0158] The extracellular release of lactic acid dehydrogenase (LDH) is a well- established marker of cell membrane integrity. Elevated LDH levels in the extracellular environment typically indicate a breach in the cell membrane, which various factors, including the interaction with exogenous agents like nanoparticles, can cause. As LDH is predominantly localized within the cytoplasm, its detection outside the cell is a clear sign of membrane permeabilization or injury. To assess the extent of cell membrane damage in breast cancer cells with varying levels of integrin expression, we evaluated the release of LDH from MCF-7 and MDA-MB-231 cells post 24-hour treatment with Alkyn-nCOF@Dox and Alkyn-nCOF-cRGD@Dox, both at a Dox equivalent concentration of 10 pM (Figure 5c).

[0159] In MCF-7 cells, a significant increase in LDH release was noted upon treatment with Alkyn-nCOF@Dox. This increase was quantified at 73.4% ± 8.7, showing a statistically significant difference (p < 0.0001) compared to untreated control cells, which showed a 27.5% ±4.5 LDH release. In contrast, cells treated with Alkyn-nCOF-cRGD@Dox exhibited a moderate LDH release of 48.8% ± 8.3, which was still statistically significant (p < 0.05) compared to the control. This suggests that the larger vesicle size associated with Alkyn-nCOF@Dox treatment (~2.3 pM ± 0.2) could be a factor in the compromisedmembrane integrity. Conversely, when treated with Alkyn-nCOF-cRGD@Dox, the vesicle size was smaller (~1.0 pM ± 0.3), indicative of a degree of receptor-mediated endocytosis that may mitigate extensive membrane damage, as illustrated by the two vesicle size distributions (Figure 58).

[0160] In MDA-MB-231 cells, treatment with Alkyn-nCOF@Dox led to a markedly higher release of LDH (51.2% ± 6.7, p < 0.000001) in comparison to the control cells, which showed 11.4% ± 3.8 LDH release. This was significantly greater than the LDH release observed with Alkyn-nCOF-cRGD@Dox treatment, which was 29.5% ± 4.5 (p < 0.05 compared to control). This suggests that Alkyn-nCOF@Dox treatment causes considerable cell membrane damage. It can be correlated with TEM images, revealing larger endocytic vesicles averaging approximately 1.8 pm (±0.6) in size, suggesting that membrane distortion could lead to the formation of membrane defects and subsequent LDH release. In contrast, MDA-MB-231 cells treated with the targeted Alkyn-nCOF-cRGD@Dox presented smaller endocytic vesicles, around 0.5 pm (±0.2), containing fewer nanoparticle aggregates. This indicates less membrane distortion and, thus, a lower extent of LDH release than cells treated with non-targeted nanoparticles.

[0161] The cell's molecular characteristics profoundly influence the cellular response to nanoparticle treatment, as evidenced by the differential LDH release and vesicle size in MCF-7 and MDA-MB-231 cells. Moreover, the targeted approach of Alkyn-nCOF- cRGD@Dox appears to reduce non-specific cellular interactions, leading to less membrane damage. This suggests that receptor-targeted nanoparticles can offer a more refined treatment strategy that capitalizes on the unique molecular signatures of cancer cells, potentially leading to improved therapeutic outcomes with reduced collateral damage to the cells.

[0162] The intracellular internalization of free Dox, Alkyn-nCOF@Dox, and Alkyn- nCOF-cRGD@Dox was further detected by confocal laser scanning microscopy (CLSM) and flow cytometry. MDA-MB-231 and MCF-7 breast cancer cells were incubated with free Dox, Alkyn-nCOF@Dox, and Alkyn-nCOF-cRGD@Dox ([Dox] = 10 pM) for 24 hours. The Dox internalization was then visualized using CLSM at Dox excitation ( \ex = 488 nm / Xem = 520 nm) (Figures 5d and 59-60). The nucleus was also specifically stained using DAPI ( \ex = 360 nm / Xem = 460 nm). Free Dox was predominantly found in the nuclei of both cell types, where it intercalates with DNA strands, causing chromatin condensation and apoptosis.77'78In contrast, the fluorescence from Alkyn-nCOF@Dox and Alkyn-nCOF-cRGD@Dox were primarily in the cytoplasm, Alkyn-nCOF-cRGD@Dox exhibited greater fluorescenceintensity across all cell types within their cytoplasm compared to its non-targeted counterpart confirming efficient receptor-mediated endocytosis.

[0163] Flow cytometry was utilized to measure intracellular Doxorubicin concentrations in MDA-MB-231 and MCF-7 cells following a 24-hour treatment with free Dox, Alkyn-nCOF@Dox, and Alkyn-nCOF-cRGD@Dox ([Dox] = 1 pM, Figures 5e and 61). In MCF-7 cells exposed to free Dox and Alkyn-nCOF@Dox, Doxorubicin levels were 8 % and 10 % higher, respectively, than the control. However, cells treated with Alkyn- nCOF-cRGD@Dox exhibited a 65 % increase in Doxorubicin content. In comparison, MDA- MB-231 cells showed increases of 9 %, 20 %, and 97 % for free Dox, Alkyn-nCOF@Dox, and Alkyn-nCOF-cRGD@Dox treatments, respectively, against the control. Notably, the Alkyn-nCOF-cRGD@Dox treatment resulted in significantly elevated Doxorubicin levels, especially in MDA-MB-231, compared to free Dox and Alkyn-nCOF@Dox. This marked increase implies that the cRGD peptide effectively facilitates interaction with integrin av[E. enhancing the endocytosis of the targeted nanocarrier.

[0164] To determine the subcellular localization of the Dox formulations, MDA-MB- 231 cells were treated for 4 hours with free Dox, Alkyn-nCOF@Dox, or Alkyn-nCOF- cRGD@Dox and then stained with organelle -specific fluorescent markers (DAPI, LysoTracker™ 647 Deep Red, MitoTracker™ Deep Red FM, and CellMask™ 647 Deep Red to visualize nuclei, lysosomes and late endosomes, mitochondria and membrane, respectively) followed by CLSM imaging (Figure 5f). Four hours after treatment, free dox was colocalized with lysosomes, late endosomes, and nuclei in MDA-MB-231 cells (Figure 5f). Dox was shown to enter primarily through passive diffusion and endocytosis, potentially leading to lysosomal capture. Regardless of the uptake mechanism, its main target is the nucleus, where it interferes with DNA functionality. Alkyn-nCOF@Dox was also observed to colocalize with lysosomes and late endosomes but not with mitochondria or nuclei. Meanwhile, Alkyn-nCOF-cRGD@Dox co-localized with the cell membrane, validating receptor-mediated endocytosis, as well as lysosomes and late endosomes. (Figure 5f). We also detected increased fluorescence in the cytoplasm. This amplified cytoplasmic fluorescence can be partially attributed to the release of Dox from nanoparticles and partly due to the augmented number of internalized nanoparticles resulting from endosome fusion and lysosomal acidification (Figure 5f). These observations suggest that Alkyn-nCOF- cRGD@Dox's cellular internalization into the cytoplasm is facilitated by receptor-mediated endocytosis, triggering Dox release into the cytosol. These findings are consistent with the TEM, flow cytometry, CLSM results, and toxicity.

[0165] The therapeutic efficacy and targeting properties of Alkyn-nCOF-cRGD@Dox in vitro encouraged us to investigate its antitumor ability further in vivo in mice with triplenegative MDA-MB-231 breast tumors.

[0166] We initially investigated the immediate biodistribution of nanoparticles in athymic NU / J nude mice by assessing the percentage of the injected dose (ID) per gram of tissue. We administered both drug-free Alkyn-nCOF and Alkyn-nCOF-cRGD intraperitoneally (IP) at a concentration of 20 mg / kg body weight. This dosage is double that intended for subsequent toxicity studies (Figure 6a). 24 hours post-administration, the mice were euthanized, and pivotal organs were harvested. We specifically focused on the liver, spleen, and kidneys, anticipating them to be primary clearance organs that might exhibit cellular responses to nanoparticle accumulation. Additionally, the heart was scrutinized for any signs of cardiotoxicity. Following a thorough rinse with PBS, organs were homogenized and subsequently subjected to UV-VIS spectrometry for nanoparticle detection. Encouragingly, both Alkyn-nCOF and Alkyn-nCOF-cRGD were well-tolerated by the mice, with no discernible weight loss noted (Figure 62). Notably, approximately 11 % and 9 % of injected dose of Alkyn-nCOF and 15 % and 16 % of injected dose of Alkyn-nCOF-cRGD localized in the liver and the spleen, respectively, with no apparent accumulation in the heart or kidneys, implying effective circulatory clearance, in line with previous studies.82TEM imaging confirmed the presence of nanoparticles in the liver and spleen but also validated the absence of any structural alterations (Figures 6a inset, Figure 63).

[0167] To assess the tumor-targeting potential of Alkyn-nCOF-cRGD@Dox, we employed real-time in vivo fluorescence imaging (IVIS® Spectrum) and subsequent Dox radiance efficiency quantification. Athymic NU / J nude mice carrying subcutaneous MDA- MB-231 tumors (50-100 mm3) received IP injections of Alkyn-nCOF-cRGD@Dox, Alkyn- nCOF@Dox, Dox, or a saline solution ([Dox] = 5 mg / kg, [NP] = 10 mg / kg). Animals were anesthetized, and whole-body dorsal fluorescence images were captured 24 hours postinjection. Live animal imaging, as shown in Figure 6b, indicated an increase in fluorescence intensity only in the tumor of the animal treated with Alkyn-nCOF-cRGD@Dox, highlighting the targeted delivery facilitated by the cRGD conjugation.

[0168] Then, we euthanized the mice to harvest the tumors and vital organs, including the heart, liver, spleen, and kidney. We conducted a detailed examination of Dox's biodistribution by performing ex vivo IVIS® Spectrum imaging and quantifying Dox fluorescence. As shown in Figures 6c and 6e, a significant increase in fluorescence was noted in the tumors from mice treated with Alkyn-nCOF-cRGD@Dox. This contrasted themoderate fluorescence observed in the free Dox group, underscoring the enhanced tumor- targeted delivery capabilities of the cRGD-conjugated nanoparticles. We also analyzed the general biodistribution of the Dox treatments (Figures 6d and 6f). Organ-specific investigation indicated that the injected Alkyn-nCOF-cRGD was predominantly localized in the liver, with detectable fluorescence also present in the spleen and kidneys, as depicted in Figure 6d. The distribution pattern of Dox across the liver, spleen, and kidneys suggests efficient systemic clearance of the Alkyn-nCOF-cRGD that did not localize within the tumor.

[0169] These results underscore the precision targeting of Alkyn-nCOF-cRGD@Dox nanoparticles, their favorable tumor retention, and their effective systemic elimination. This promising profile motivates further investigation of their anti-tumor effects on MDA-MB-231 tumor-bearing mice.

[0170] Next, the anticancer therapeutic effect of Alkyn-nCOF-cRGD@Dox, Alkyn- nCOF@Dox, and free Dox was compared in an orthotopic tumor model. Athymic NU / J nude mice carrying subcutaneous MDA-MB-231 tumors (50-100 mm3) were IP injected every 2 days with Alkyn-nCOF-cRGD@Dox, Alkyn-nCOF@Dox, Dox, or saline solution as control ([Dox] = 5 mg. kg'1, [NP] = 10 mg. kg'1, 200 pL, Figure 7a). Tumor volume evolution shows that Alkyn-nCOF-cRGD@Dox treatment has significantly blocked the growth of tumors in mice (Figure 7b). A statistical analysis of these changes over the 20-day treatment period reveals that only the injections of Alkyn-nCOF-cRGD@Dox had a significant impact on slowing tumor growth (p <0.001) when compared to the control group (Figure 7b). In contrast, mice treated with Alkyn-nCOF@Dox and free Dox exhibited comparable and statistically insignificant levels of tumor suppression when compared to the control group. These results reflect the limited effectiveness of non-targeted chemotherapy.

[0171] Figure 7c depicts the progression of tumor size in mice across four treatment groups at the start (Day 0) and after 20 days. There was a noticeable increase in tumor size in the PBS and free Dox groups, accompanied by evident necrosis at Day 20. For the group treated with Alkyn-nCOF@Dox, the tumors enlarged significantly without any signs of necrosis. Remarkably, in the Alkyn-nCOF-cRGD@Dox group, the tumors had almost vanished by Day 20, highlighting the potential efficacy of this targeted treatment approach.

[0172] Moreover, body weight analysis shows a significant reduction in the weight of mice treated with free Dox. This indicates that free Dox has considerable side effects, resulting in weight loss even as the tumor continues to grow. In contrast, the Alkyn- nCOF@Dox, Alkyn-nCOF-cRGD@Dox, and saline-treated groups showed no significant changes in body weight compared to the initial weights at the start of treatment. These resultsdemonstrate that Alkyn-nCOF@Dox and Alkyn-nCOF-cRGD@Dox treatments have minimal side effects. (Figure 7d). After the 20-day treatment regimen, whole-body dorsal fluorescence imaging was performed, which explained the body weight results. Figure 7e illustrates a marked presence of off-target Dox fluorescence in the animals treated with free Dox. This explains the considerable weight loss in this group due to the free Dox systemic toxicity. This was in contrast to the groups treated with Alkyn-nCOF-cRGD@Dox or Alkyn- nCOF@Dox, which exhibited significantly reduced off-target fluorescence when compared to the control.

[0173] At the end of treatment, mice were sacrificed, and tumors and major organs were extracted. Tumor visualization and weight analysis show drastic differences between treatment groups (Figures 7f and g). The average tumor weight decreased by 67% in the free Dox group, 65% in the Alkyn-nCOF@Dox group, and a notable 95% reduction in the Alkyn- nCOF-cRGD@Dox group when compared to the control group (Figures 7g). These results demonstrate that incorporating the RGD peptide notably enhances targeted tumor therapy and subsequent drug delivery in vivo.

[0174] Further analysis was conducted using IVIS® Spectrum through ex vivo fluorescence imaging of tumors that did not vanish during the treatment and vital organs (heart, liver, spleen, lung, and kidney). The findings corroborated our initial observations. Notably, tumors from the Alkyn-nCOF-cRGD@Dox-treated mice displayed fluorescence signals that were markedly higher than those from the saline, free Dox, and Alkyn- nCOF@Dox groups (Figures 7h and 7i). To quantify, the Dox fluorescence in tumors treated with Alkyn-nCOF-cRGD@Dox was 16.5-fold higher than the saline group, 4.96-fold higher than the free Dox group, and 11.2-fold higher than the Alkyn-nCOF@Dox group (Figures 7h and 7i). Furthermore, a significant fluorescence indicative of Dox was detected in the heart, spleen, liver, and kidneys of mice treated with free Dox, showcasing the drug's nonselective distribution and its potential for unintended systemic toxicity (Figures 7j). In stark contrast, the organs harvested from mice treated with Alkyn-nCOF-cRGD@Dox or Alkyn- nCOF@Dox did not exhibit significant fluorescence when compared to the control (Figure 7j).

[0175] Overall, the in vivo findings indicate that the targeted chemotherapy agent Alkyn-nCOF-cRGD@Dox, which utilizes the RGD peptide as a targeting agent, offers significantly improved treatment efficacy and specificity over conventional treatments with non-targeted Alkyn-nCOF@Dox and free Doxorubicin (Dox). The targeted approach not only more effectively suppresses tumor growth but also minimizes side effects, as evidencedby stable body weights and reduced systemic toxicity. These results underscore the promise of RGD-modified nCOF nanocarriers in achieving more precise and safer chemotherapy interventions.

[0176] A biocompatible, pH-responsive system for precise targeted drug delivery to breast cancer cells with high integrin levels was designed, which, until now, have not been present in previous therapies. The designed system features an alkyne-enriched nCOF functioning as a drug storage matrix, with its surface chemically tailored through click chemistry to present cyclic RGD peptides. These peptides have a specific affinity for the avPs integrin present on the membrane of TNBC cells, thereby facilitating targeted drug delivery to the affected cells. The synthesized nanoparticles have one of the highest loading capacities for Dox currently available in COFs, which can be selectively released in the tumors’ acidic environment via particle decay. The cyclic RGD peptide units enhance the uptake of the nanoparticles by cancer cells in vitro and in vivo via integrin receptor-mediated endocytosis. In cancer cells, the release of anticancer drug was triggered by the intrinsic acidity of endo / lysosomes, resulting in significantly enhanced inhibition of triple-negative breast cancer cells in vivo. The preparation of nCOFs with targeting peptide ligands via click chemistry for targeted cancer therapy with vastly reduced side effects opens a window for developing versatile, multifunctional drug delivery systems with great potential for cancer therapy and other pathologies.

[0177] All reagents and starting materials were purchased from Sigma-Aldrich and used without further purification. Deionized water was used from Millipore Gradient Milli-Q water purification system. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 (E. Merck). The plates were inspected under UV light. Column chromatography was performed on silica gel 60F (Merck 9385, 0.040-0.063 mm). Infrared spectra were recorded on an Agilent Technologies Cary 600 Series FTIR Spectrometer using the ATR mode. The samples’ PXRD patterns were recorded using an X-ray Panalytical Empyrean diffractometer. N2 adsorption-desorption isotherms were obtained at 77 K using Micromeritics ASAP 2020 surface area analyzer. High-resolution transmission electron microscopy (HRTEM) images were obtained using a Talos F200X Scanning / Transmission Electron Microscope (STEM) with a lattice-fringe resolution of 0.14 nm at an accelerating voltage of 200 kV equipped with CETA 16M camera. The high-resolution images of periodic structures were analyzed using TIA software. The topography of the self-templated samples was analyzed by dynamic atomic force microscopy (5500 Atomic Force Microscope; Keysight Technologies Inc., Santa Rosa, CA). We acquired topography, phase, and amplitude scans simultaneously. Siliconcantilevers (NanosensorsTM, Neuchatel, Switzerland) with resonant frequencies of 250-300 kHz and force constants of 100-130 Nm'1were used. The set point value was kept at 2.5V. AFM scans were collected at 1024 points / lines with a scan speed of 0.20 at a fixed scan angle of 0°. Scan artifacts were minimized by acquiring a typical scan at an angle of 90° under identical image acquisition parameters. We used GwyddionTM free software (version 2.47), an SPM data visualization and analysis tool for postprocessing the AFM scans. UV-visible absorption spectra were recorded with an Agilent Technologies Cary 5000 Series UV-Vis- NIR Spectrophotometer in water at room temperature (298 K). Solutions were examined in 1 cm spectrofluorimetric quartz cells. The experimental error of the wavelength values was estimated to be ~1 nm. Emission spectra in water at room temperature were recorded on a Perkin Elmer LS55 Fluorescence Spectrometer using an excitation wavelength of 488 nm, corresponding to the wavelength that absorbs maximally. Dynamic light scattering (DLS) measurements were performed on a Malvern Zetasizer NanoSeries to obtain the size and C,- potential of the nanoparticles. Magic Angle Spinning (MAS) solid-state NMR experiments were carried out on a Bruker Avance-HD 600 MHz spectrometer operating at a static field of 14. 1 T using a 4.0 mm MAS probe. Dry samples were packed into 4.0 mm zirconia rotors and were spun at a MAS frequency of 14 kHz.3H-13C CP / MAS experiments were performed using a standard linearly ramped cross-polarization pulse sequence.13C chemical shifts were externally referenced to the adamantane CH2 signal at 38.48 ppm. All the NMR data were processed using TopSpin software. Thermogravimetric analysis (TGA) was performed on TA SDT Q600. The copper content of the nCOF was analyzed using the Inductively coupled plasma mass spectrometry (ICP-MS) on the Agilent 7800 series instrument after digesting the nCOF in nitric acid. Copper concentrations were determined by the external calibration method using copper calibration standard solutions in the 1-100 ppm range trace metal grade HNO3. The digested nCOF samples were diluted in milliQ water to a final co 100 to 200 ppm concentration measurements with no gas and helium mode. For The ICP-MS instrument control Agilent Mass Hunter software was used under the following conditions: RF power 1550 W, nebulizer gas flow 1.03 L / min, auxiliary gas flow 0.90 L / min, and plasma gas flow 15 L / min. Data were processed using Agilent Mass Hunter software. Magic Angle Spinning (MAS) solid-state NMR experiments were carried out on a Bruker Avance-HD 600 MHz spectrometer operating at a static field of 14.1 T using a 4.0 mm MAS probe. Dry samples were packed into 4.0 mm zirconia rotors and were spun at a MAS frequency of 14 kHz. H-13C CP / MAS experiments were performed using a standard linearly ramped cross-polarization pulse sequence.13C chemical shifts were externally referenced to the adamantane CH2 signal at 38.48 ppm. All the NMR data were processed using TopSpin software. Phase contrast and fluorescence images were observed on an Olympus FV1000MPE confocal scanning microscope. Flow cytometry analyses were performed on Attune NxT Flow cytometer.

[0178] Table 2. Reported COFs for Dox Delivery.

[0179] Synthesis of 4-ethynyl-l,6-diformyl pyridine (Alkyn-DFP). 4-ethynyl-l,6- diformyl pyridine synthesis was synthesized according to the published procedure with no modifications.

[0180] Synthesis of Alkyn-nCOF. Alkyn-nCOF was synthesized by condensation of4-ethynyl-l,6-diformyl pyridine (Alkyn-DFP, 12 mg, 0.06 mmol, 1 equiv.) and 1 ,3,5-tris(4- aminophenyl)benzene (TAB, 21 mg, 0.06 mmol, 1 equiv.) in anhydrous 1,4-dioxane (3 mb) with an acetic acid catalyst (0.5 mb, 3M) sealed in a 10 mb glass tube and heated undermicrowave irradiation to 110 °C for 120 minutes. The as-synthesized brownish gel was collected by fdtration and washed with 1,4-dioxane and ethanol, followed by water.

[0181] Synthesis of Alkyn-nCOF-cRGD. The cRGD-PEG-Ns (MW Ik) copper- catalyzed cycloadditions to the Alkyn-nCOF surface were carried out in water. Copper(II) sulfate (3.2 mg, 0.02 mmol, 200 pL) and sodium ascorbate (12 mg, 0.06 mmol, 200 pL) were added to the aqueous nCOF solution (10 mg, 2 mb). The resulting brown solution was stirred (10 min) at room temperature, then the cRGD-PEG-Ns azido reactant (10 mg, 100 pL) was added dropwise and stirred overnight. The solution was then cleaned with H2O several times by centrifugation and finally by dialysis for 48 hours to remove unreacted species.

[0182] Dox loading in Alkyn-nCOF and Alkyn-nCOF-cRGD. The anticancer drug doxorubicin (Dox) was loaded into Alkyn-nCOF or Alkyn-nCOF-cRGD by a simple impregnation method. Alkyn-nCOF or Alkyn-nCOF-cRGD (10 mg) was suspended in an aqueous Dox solution at pH 7 ([Dox] = 0.4 mM, 10 mb, nCOF: Dox ratio = 1:5). The solution was placed under magnetic stirring for 24 hours. The solution was then cleaned with H2O several times by centrifugation and finally by dialysis for 48 hours to remove unloaded Dox molecules. The Dox-loaded Alkyn-nCOF or Alkyn-nCOF-cRGD were denoted Alkyn- nCOF@Dox and Alkyn-nCOF-cRGD@Dox, respectively.

[0183] In vitro Dox release. The kinetic of Dox release from Alkyn-nCOF@Dox and Alkyn-nCOF-cRGD@Dox was measured by dialysis. Alkyn-nCOF@Dox or Alkyn-nCOF- cRGD@Dox (5 mb, 1.0 mg / mL) were placed into dialysis bags (MWCO: 3500 Da) and incubated at 37 °C in PBS solutions (30 mb) at pH 5.4, or 7.4 under gentle stirring. At regular intervals, solutions were centrifuged, and supernatants were collected. The kinetic curves of released Dox from Alkyn-nCOF or Alkyn-nCOF-cRGD were plotted over time; samples were run in triplicate.

[0184] For Fourier Transform infrared (FTIR) spectroscopy see Figures 10 to 14.

[0185] X-ray photoelectron (XPS) spectroscopy. X-ray photoelectron spectroscopy (XPS) analysis was performed to analyze the elemental composition and understand the interactions between the nCOF surface and the peptide. XPS experiments were carried out on a Kratos Axis Ultra DLD spectrometer under a base pressure of ~ 2x 10-10mbar. A monochromated Al Ka X-ray source (1486.69 eV) irradiated samples at room temperature. XPS spectra were recorded from an analysis area of 700 pm x 300 pm. High-resolution XPS data of core levels were obtained with an energy resolution of 0.05 eV. For consistency, XPSmeasurements were calibrated to Cis (~ 285 eV). Data were analyzed using CasaXPS package with Shirley background subtraction (see Figure 15).

[0186] Table 3. Binding energy maxima, FHWM, A (eV) and % for C Is spectrum deconvolution curves of Alkyn-nCOF, cRGD-PEG-N? and Alkyn-nCOF-cRGD.

[0187] Table 4. Binding energy maxima, FHWM, A (eV) and % for N Is spectrum deconvolution curves of Alkyn-nCOF, cRGD-PEG-N? and Alkyn-nCOF-cRGD.

[0188] Powder X-ray diffraction (PXRD). Powder X-ray diffraction (PXRD) measurements were carried out to confirm the crystalline nature of the framework (see Figures 16 to 18).

[0189] High-resolution transmission electron microscopy (HRTEM). High-resolution transmission electron microscopy (HRTEM) images were obtained using a Talos F200X Scanning / Transmission Electron Microscope with a lattice -fringe resolution of 0.14 nm at an accelerating voltage of 200 kV equipped with a CETA 16M camera. The samples were prepared on holey carbon film mounted on a copper grid. A drop of diluted particle solution was spotted on the grid and dried overnight at room temperature (298 K). The obtained images of periodic structures were analyzed using TIA software. All the relevant areas were marked using bright field imaging mode at spot size 3, and the marked areas were also scanned using the STEM-HDAAF mode at spot size 9 for imaging and spot size 6 for conducting the STEM-EDAX. The STEM mode helps provide the elemental composition as it works on the principle of mass determination. Such measurements can be performed at low electron dose by collecting the high-angle dark-field signal using an annular detector. This mode is generally used to image the elements with different masses, with the heavier mass element appearing brighter. The samples were scanned at spot size 9 and with a screen current of 60 pA. The data was analyzed using Velox analytical software (see Figures 19 to 22).

[0190] In-Situ Variable-Temperature Liquid-Cell Transmission Electron Microscopy (VT-LCTEM). The in-situ variable-temperature liquid-cell transmission electron microscopy (VT-LCTEM) experiments were performed using a Protochips Inc. (Morrisville, NC) Poseidon Select liquid-cell TEM holder with on-chip heating capability. A pair of Protochips Poseidon Heating Chips SiNx membrane windows (50 pm x 400 pm and 550 pm x 20 pm lateral window dimensions, respectively) with 50 nm gold spacer were plasma cleaned for 60s. The chips were loaded on the holder so the windows of the two chips were crossed, so the viewing area was a 50 pm x 20 pm rectangle. This was done to avoid the bulging of the windows. A drop (about 2 pl) of the solution was loaded onto the bottom chip before placing the top chip and sealing the chip setup. The integrity of the windows was visually inspected for any cracks / leaks. The window integrity and O-ring seal was further checked by pumping the holder down to vacuum (5.4.1 O'6mbar) in a “pre-pump” vacuum chamber. The holder was inserted into the TEM to begin the in-situ observations and initiate the on-chip liquid heating (See Figures 23 and 24).

[0191] The two linkers, Alkyn-DFP and TAB, dissolved in dioxane, were drop-casted onto a bottom chip. Once wetting of the liquid cell was confirmed acetic acid was flowed into the cell, and the liquid cell was heated to 110 °C. The formation of sheet-like particles was observed within a few seconds.

[0192] AFM data can be seen in Figures 25 and 26.

[0193] N2 adsorption-desorption experiments. Low-pressure gas adsorption measurements were performed on 3 -Flex Surface Characterization Analyzer (Micromeritics) at relative pressures up to 1 atm. The cryogenic temperatures were controlled using liquid nitrogen baths at 77 K. The apparent surface area was determined from N2 adsorption isotherm collected at 77 K by applying the Brunauer-Emmet-Teller (BET) model between P / Po values of 0.0005 and 0.05 for microporous COFs.

[0194] In atypical experiment, 20 to 30 mg of homogenous polycrystalline samples are transferred (dry) to a 6-mm large bulb glass sample cell and are evacuated at room temperature using a turbo molecular vacuum pump and then gradually heated to 125 °C, held for 24 h and cooled to room temperature (see Figure 27).

[0195] UV-vis data can be seen in Figures 28 and 29.

[0196] Fluorescence spectroscopy data can be seen in Figures 30 to 33.

[0197] TGA analysis can be seen in Figure 34.

[0198] Dynamic light scattering (DLS). DLS measurements were carried out on a Zetasizer Nano-ZS (Malvern Instruments) to determine the Zcta(Q-potcntial as well as the hydrodynamic size of the particles. All samples were analyzed at room temperature in 10 mM phosphate-buffered saline (PBS). Each experiment was performed in triplicate (see Figures 35 to 39).

[0199] Quantification of cRGD-PEG-Ns conjugated on the surface of the Alkyn- nCOF using the OPA method. The O-phthalaldehyde (OPA) method formed a fluorescent product after a reaction with a primary amine. OPA reacts to the primary amines and lysinesidechains of proteins in die presence of a thiol, usually 2-mercaptoethanoh under alkaline conditions and gives a fluorescence signal at 436-475 nm after excitation at 330-390 nm. The fluorescence signal is proportional to the amount of protein in the sample, allowing a sample to be quantified by comparing it to a standard curve of known protein content. 100 pL of the sample was added to 1 mL of OPA reagent, and fluorescence measurement was recorded precisely 2 minutes after addition.

[0200] To avoid contamination or overlap between species’ signals, the amount of peptide conjugated was calculated using the supernatant containing the peptide not conjugated in the NP surface at t=0 and t=24 hours.

[0201] The efficiency of peptide conjugation on nanoparticle surfaces is expressed in weight % and represents the percentage of the total nanoparticle weight that corresponds to the weight of the conjugated peptides. It indicates how much of the nanoparticles' surface is covered with the desired peptides (see Figures 40 and 41).

[0202] Quantification of Dox loaded in Alkyn-nCOF and Alkyn-nCOF-cRGD using fluorescence spectroscopy. To avoid contamination, the overlap between species’ signals, and possible quenching of the Dox fluorescence due to its pore confinement, the Dox loading was calculated using the supernatant containing the Dox that was not loaded in the porous structure by fluorescence spectroscopy based on comparison to a calibration curve of the Dox standard solution (Zex = 488 nm) (see Figures 42 and 43).

[0203] pH-dependent Dox release experiments. The effect of pH on the release of Dox from Alkyn-nCOF and Alkyn-nCOF-cRGD was monitored over time in water buffered with PBS (10 mM) at 37 °C and pH = 7.4 and 5.4. The pH of the solutions was adjusted using a 1 M HCl(aq) solution. At regular intervals, solutions were centrifuged, and supernatants were collected. The solution fluorescence intensity was measured compared to a calibration curve (Figure 42).

[0204] In vitro biological studies. Cell culture. Human breast cancer cell lines adenocarcinoma MCF-7 (ATCC HTB-22™) and MDA-MB-231 (ATCC HTB-26) cell lines were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin / streptomycin at 5 % CO2 and 37 °C (see Figure 45).

[0205] In vitro cell viability. Cell viability was assessed using CellTiter-Blue® Cell Viability assay (CTB, Promega). The assay measures the metabolic reduction of a non- fluorescent compound, resazurin, into a fluorescent product, resofurin, in living cells. As non- viable cells rapidly lose their metabolic activity, the amount of the resofurin product can beused to estimate the number of viable cells following treatment. Once produced, resofurin is released from living cells into the surrounding medium. Thus, the fluorescence intensity of the medium is proportional to the number of viable cells present.

[0206] 96-well plates were seeded with MCF-7 or MDA-MB-231 (~5,000 cells per well in 100 pL of DMEM) and incubated at 37 °C for 24 hours. The medium was removed and replaced with fresh medium (control) or various concentrations of test compounds and incubated at 37 °C for 48 hours. After that, cells were incubated with 80 pL DMEM and 20 pL of CTB per well for 6 hours at 37 °C. The fluorescence of the resofurin product (Xex / em 560 / 620) was measured. Untreated wells were used as control.

[0207] The percentage of cell viability was calculated using the following formula:Viability (%) = [(Ftreated - Fbl ank) I (Fcontrol - Fblan k)] X 100All assays were conducted in triplicate, and the mean ICso ± standard deviation was determined.

[0208] In vitro cell toxicity. The ICso values were determined from a plot of percentage cell inhibition against drug concentration (in pM). All assays were conducted in triplicate, and the mean ICso ± standard deviation was determined. The experiment was performed in triplicate (see Figure 46).

[0209] Western blotting. MCF-7 or MDA-MB-231 cells were seeded at 80% confluency in 6-well plates in complete DMEM and incubated for 24 hours. Then, cells were incubated for 24 hours with no additives (control), Dox alone, Alkyn-nCOF@Dox, or Alkyn- nCOF-cRGD@Dox ([Dox] = 1 pM). Then, for each experiment, the cells were collected and washed with PBS. Cells were lysed in 200 pL ofNP40 lysis buffer (150 mM NaCl, 1.0% NP- 40, 50 mM Tris-Cl (pH 8.0)) containing protease inhibitor cocktail (Roche # 11697498001). We then separated 30 pg of protein lysate by 10% Mini-PROTEAN® TGX™ precast polyacrylamide gels (Biorad # 4561033)and transferred the proteins to nitrocellulose membranes. The membranes were probed using the following antibodies: caspase-3 primary antibody (abeam #abl84787), GAPDH primary antibody (Santa Cruz # sc-32233), Antirabbit IgG (Cell Signaling #7074), and Goat Anti-Mouse IgG (Cell Signaling #91196) . The protein signals were detected using Clarity Western ECL Substrate (Biorad # 1705060 ) and ChemiDoc imaging systems (Biorad). Western blot bands were quantified using Fiji-Image J software (see Figure 47).

[0210] Intracellular distribution of Alkyn-nCOF@Dox or Alkyn-nCOF-cRGD@Dox using TEM. For TEM analysis, MCF-7 and MDA-MB-231 cells were seeded in T75 flasks in complete DMEM and incubated for 4 hours with cell-medium alone (control), Alkyn- nCOF@Dox or Alkyn-nCOF-cRGD@Dox ([Dox] = 10 pM) in DMEM. After harvesting, cell pellets were washed twice with PBS. The cells were cryo-fixed within a few milliseconds at a pressure of 2000 bar under liquid nitrogen using a high-pressure freezer (Leica Microsystems, Germany). After freezing, the sample pod was released automatically into a liquid nitrogen bath. While still in liquid nitrogen, the sample carrier was separated from the specimen pod using precooled fine-tipped tweezers and transferred to the cryo-transfer storage box for the flat specimen carrier, where the samples were stored in preparation for freeze substitution. Freeze substitution was performed using an automatic freeze substitution (AFS) unit (Leica EM AFS2, Heerbrugg, Switzerland) in a 10 mL solution of cold, dry absolute acetone (v / v) containing 1 % osmium tetroxide (w / v), 0.5 % uranyl acetate (w / v) and 5 % distilled water (v / v). The AFS unit was slowly warmed from -90 °C to 0 °C (2 °C / hour), with the temperature being held at both -60 °C and -30 °C for 8 hours. Samples were transferred to room temperature in a closed container to prevent condensation, rinsed with absolute acetone (3 x 5 minutes), and infiltrated with 30, 60, and 100 % Epon resin for 3 hours each. Epon was exchanged, and individual samples were embedded in 1 mL Eppendorf® lids for 24 hours at 60 °C. Finally, the samples were sectioned with an ultramicrotome at room temperature using a diamond knife, and the ultrathin sections were examined under TEM (Talos F200X STEM). The experiment was performed in triplicate (see Figures 48 to 58).

[0211] LDH release experiment. To assess the ability of Alkyn-nCOF@Dox, or Alkyn-nCOF-cRGD@Dox to disrupt the plasma membrane of the cancer cells the detection of lactate dehydrogenase (LDH) release from treated MCF-7 and MDA-MB-231 cells was performed using LDH-Glo™ Cytotoxicity Assay (Promega #J2380). cells were incubated for 24 hours with no additives (control), Dox alone, Alkyn-nCOF@Dox, or Alkyn-nCOF- cRGD@Dox ([Dox] = 1 pM). After 24-hours incubation, 5 pL of the medium was diluted in 50 pL of LDH storage buffer and transferred into a new 96 well plates, and then, 50 pL of LDH detection reagent was added. Luminescence was recorded After 30 min of incubation using a Cytation 5 multimode reader (Biotek).

[0212] In vitro internalization study by confocal microscopy. MCF-7 or MDA-MB- 231 cells were seeded on sterile coverslips in complete DMEM and incubated for 24 hours. Then, cells were incubated for 24 hours with no additives (control), Dox alone, Alkyn-nCOF@Dox, or Alkyn-nCOF-cRGD@Dox ([Dox] = 10 pM). Then, for each experiment, the cells were washed with PBS, fixed with paraformaldehyde solution (3.7 %) for 10 minutes, and washed thrice with PBS. The coverslips were then fixed onto a microscope slide with mounting media. Samples were analyzed using confocal microscopy. Each sample was assayed in duplicate, and the experiment was repeated 3 times (see Figures 59 and 60).

[0213] Flow cytometry analysis. MCF-7 or MDA-MB-231 cells were grown in petri dishes to a density of 100,000 cells per mL in DMEM for 24 hours. Cells were incubated for 4 hours with no additives (control), Alkyn-nCOF@Dox or Alkyn-nCOF-cRGD@Dox ([Dox] = 1 pM). Dox uptake was measured with an Attune Nxt flow cytometer (Thermo Fisher) using BL1 channel (Blue (488nm) laser and 530 / 30nm filter). The experiment was performed in triplicate (see Figure 61).

[0214] In vitro organelle co-localization study by confocal microscopy. MDA-MB- 231 cells were seeded on sterile coverslips in complete DMEM and incubated for 24 h. Cells were incubated for 4 hours with no additives (control), Dox alone, Alkyn-nCOF@Dox, or Alkyn-nCOF-cRGD@Dox ([Dox] = 10 pM). The cells were stained with organelles markers to understand the internalization of the different Dox compositions. Cells were incubated for 30 min either with LysoTracker™ 647 Deep Red (labeling lysosomes and endosomes), MitoTracker™ Deep Red FM (labeling mitochondria), and CellMask™ 647 Deep Red (labeling membrane), followed by three cycles of PBS washing. Then, for each experiment, the cells were fixed with formaldehyde solution (3.7 %) for 10 min, followed by washing thrice with PBS. The cells were kept for 5 min in the PBS during washing cycles. ProLong Live Antifade Reagent containing DAPI to label the nucleus was added to suppress photobleaching and preserve the fluorescent signals. The coverslips were then fixed onto a microscope slide. The intracellular internalization of the different Dox compositions was observed using confocal microscopy (Olympus FV1000MPE) measuring the fluorescence signal of the Dox (with excitation / emission of 488 / 560 nm) in cells as well as the fluorescence emission from the 4 organelle markers labeling the plasmic membrane, the lysosomes, the mitochondria (with excitation / emission of 647 / 668 nm) as well as the nucleus (with excitation / emission of 400 / 461 nm).

[0215] In vivo biological studies. All animal experiments were conducted in accordance with the policies of the New York University Institutional Animal Care and Use Committee (IACUC). Athymic NU / J nude mice (4-6 weeks old, about 20 g) were fed with normal conditions of 12 h light and dark cycles and given access to food and water adlibitum. All animal experimentation was approved by the Institutional Animal Care and Use Committee of NYU AD.

[0216] Biodistribution study on healthy animals. The mice (n=3) were randomized into 3 groups and treated with 0.1 mU of saline (control), Alkyn-nCOF, and Alkyn-nCOF- cRGD at a [NP] = 20 mg / kg dose (200 pU) via intraperitoneal injection. Mice were sacrificed 24 hours post-injection, and major organs, including liver, kidney, heart, and spleen, were harvested. Each organ was rinsed with PBS and immersed in lysing buffer, and the tissues were homogenized using a PowerEyzer 24 Homogenizer. 100 pl of the soupy solution was loaded in 12 wells in 96-well plate with clear bottoms, and the UV-vis intensity was measured using a microplate reader and compared to untreated (control) animals (see Figures 62 and 63).

[0217] Tumor model establishment. The MDA-MB-231 cancer model was employed as an example of aggressive triple -negative breast cancer to evaluate the therapeutic effect of the different Dox formulations. 5* 106MDA-MB-231 cells in 200 pE of DMEM medium were injected subcutaneously into the right axillary region of nude mice. The mice were utilized in subsequent experiments after the tumor size had reached approximately 75-100 mm 3.

[0218] Biodistribution study on tumor-bearing mice. To quantify the amount of Dox in tissues, tumor-bearing mice (n = 3) were divided into three groups at random and injected with 0.2 mb of saline (control), Alkyn-nCOF@Dox, and Alkyn-nCOF-cRGD@Dox ([Dox] = 10 mg.kg'1, [NP] = 20 mg.kg'1, 200 pL) via intraperitoneal injection.

[0219] The Dox signal was tracked in live animals using an IVIS Spectrum optical imaging system (PerkinElmer / Revvity). In vivo, real-time fluorescence imaging analysis was used to qualitatively evaluate the tissue distribution of Dox generated by analyzing fluorescent, whole-body dorsal images of the mice using IVIS at the end of the treatment. The anesthetized mice were positioned on the IVIS heated stage (37°C) in a left lateral decubitus orientation. The fluorescent scans were performed 24h post i.p. injection using IVIS Spectrum imaging system in vivo imaging system and employing a set of excitation and emission filters centered around excitation and emission peaks of Dox (470nm / 560nm). Images were analyzed using Living Image software employing a spectral unmixing approach to distinguish the specific fluorescent signal emitted by Dox from the inherent fluorescence background. To accomplish this, the autofluorescence background was quantified from tumors in untreated control mice.

[0220] After 24 post-injection, animals were sacrificed, and the tumors and major organs, including the heart, liver, spleen, and kidney, were harvested for ex vivo imaging on the IVIS system. Each organ was rinsed with PBS, and the fluorescence intensity was detected. Dox fluorescent images were taken for all organs and analyzed with Living Image software. Fluorescence values are represented as a percentage of total fluorescence. Treatment groups were compared for differences in Dox accumulation via one-way ANOVA with post hoc Tukey HSD using GraphPad PRISM software (see Figures 64 and 65).

[0221] In vivo antitumor efficacy. To assess the antitumor effect, tumor-bearing mice were randomly divided into four groups (Control n=l 1, Doxorubicin n=15, Alkyn- nCOF@Dox and Alkyn-nCOF-cRGD@Dox n=14) and injected with 0.1 mb of saline, Dox alone ([Dox] = 5 mg.kg'1), Alkyn-nCOF@Dox and Alkyn-nCOF-cRGD@Dox ([Dox] = 5 mg.kg'1, [NP] = 10 mg.kg'1, 200 pL) via intraperitoneal injection every 2 days for 20 days. Animal weight and tumor volume was measured before each injection. Tumor size was monitored via caliper measurement every 2 days, and the tumor volume was estimated using the Eq Q):V=0.5 x length x (width)2(1)On day 20, the mice were sacrificed, and the tumor mass and main organs were harvested, weighed, and photographed.

[0222] In vivo biodistribution study using IVIS Spectrum. Following the methodology described in section 5.3, the quantification of Dox signal on day 20 was conducted utilizing the IVIS Spectrum optical imaging system (PerkinElmer / Revvity). Subsequently, in vivo fluorescence imaging was employed to qualitatively assess the tissue distribution of Dox at the conclusion of the 20-day treatment period, involving intraperitoneal injections every 2 days. Fluorescent imaging took place 24 hours after the last intraperitoneal injection utilizing the IVIS Spectrum in vivo imaging system and the same combination of filters. Anesthetized mice were positioned on a heated plate (37°C) in a ventral decubitus orientation within the IVIS Spectrum chamber, allowing for fluorescence imaging with a dorsal view. The fluorescent signals were then analyzed using a similar spectral unmixing approach.

[0223] After 20 days of treatment, all the tumor-bearing mice were sacrificed, and the tumor and major organs, including the heart, liver, spleen, lung, and kidney, were harvested for ex vivo imaging on the IVIS Spectrum system. Each organ was rinsed with PBS, and thefluorescence intensity was detected. Dox fluorescent images were taken for all organs and analyzed with Living Image software. Fluorescence values are represented as a percentage of total fluorescence. Treatment groups were compared for differences in Dox accumulation via one-way ANO VA with post hoc Tukey HSD using GraphPad PRISM software (see Figures 66 to 70).

[0224] Statistical Analysis. All statistical analysis was performed with GraphPad PRISM 8. All data are expressed as mean ±SD. Data were analyzed using one-way ANOVA with post hoc Tukey tests SPSS (IBM, SPSS Statistics, version 23, USA). * p <0.05; ** p <0.01; *** p <0.001.

[0225] Although the present disclosure has been described with respect to one or more particular embodiments and / or examples, it will be understood that other embodiments and / or examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

CLAIMS:

1. A nanoparticle comprising, one or more covalent organic frameworks (COFs), wherein each COF comprises one or more binding partners conjugated thereto, wherein each binding partner comprises a linker group conjugated to an RGD-based peptide, wherein the nanoparticle further comprises one or more compounds.

2. The nanoparticle according to claim 1, wherein the linker groups comprise a polyethylene glycol (PEG) group.

3. The nanoparticle according to claim 2, wherein the linker groups have the following structure:

4. The nanoparticle according to claim 1, wherein the one or more RGD-based peptides are attached to the one or more linker groups via a sidechain of an amino acid residue of the one or more RGD-based peptides.

5. The nanoparticle according to claim 4, wherein the sidechain is the sidechain of a nucleophilic amino acid residue.

6. The nanoparticle according to claim 5, wherein the nucleophilic residue is cysteine, lysine, serine, or ornithine.

7. The nanoparticle according to claim 4, wherein the sidechain is the sidechain of an electrophilic amino acid residue.

8. The nanoparticle according to claim 7, wherein the electrophilic amino acid residue is aspartic acid or glutamic acid.

9. The nanoparticle according to claim 1, wherein the RGD-based peptides are chosen from c(RGKYK) (SEQ ID NO: 1), c(RGDfK) (SEQ ID NO:2), RGDSP (SEQ ID NO:3), and RGDT (SEQ ID NON).

10. The nanoparticle according to claim 9, wherein the RGD-based peptides have the following structure:or a protonated or deprotonated form thereof, wherein R is the side chain of the RGD-based peptide.

11. The nanoparticle according to claim 10, wherein the one or more RGD-based peptides have the following structure:or a protonated or deprotonated form thereof.

12. The nanoparticle according to claim 1, wherein the RGD-based peptide is associated with one or more of the following integrins: avPs integrin, avPs integrin, avPs integrin, asPi integrin, or avPe integrin.

13. The nanoparticle according to claim 1, wherein the one or more compounds are therapeutic compounds.

14. The nanoparticle according to claim 13, wherein the therapeutic compounds are chemotherapeutic agents.

15. The nanoparticle according claim 13, wherein the one or more compounds comprise one or more aromatic groups.

16. The nanoparticle according to claim 14, wherein the one or more compounds are doxorubicin, cisplatin, methotrexate, or paclitaxel.

17. The nanoparticle according to claim 1, wherein the one or more COFs comprise the following structure:

18. The nanoparticle according to claim 17, wherein the one or more COFs comprise the following structure:

19. The nanoparticle according to claim 1, wherein the nanoparticle comprises a COF comprising the following structure:where L is a linker group and Peptide is an RGD-based peptide (e.g., c(RGKYK) (SEQ IDNO: 1), c(RGDfK) (SEQ ID NO:2), RGDSP (SEQ ID NO:3), or RGDT (SEQ ID NO:4)), or a deprotonated or protonated form thereof.

20. A covalent organic framework (COF), comprising the following structure:wherein L is a linker group, and Peptide is an RGD-based peptide.

21. The COF according to claim 20, wherein the linker group comprises a polyethylene glycol (PEG) group.

22. The COF according to claim 19, wherein the linker group has the following structure:

23. The COF according to claim 20, wherein the RGD-based peptide is attached to the linker group via a sidechain of an amino acid residue of the RGD-based peptide.

24. The COF according to claim 23, wherein the sidechain is the sidechain of a nucleophilic amino acid residue.

25. The COF according to claim 24, wherein the nucleophilic residue is cysteine, lysine, serine, or ornithine.

26. The COF according to claim 23, wherein the sidechain is the sidechain of an electrophilic amino acid residue.

27. The COF according to claim 26, wherein the electrophilic amino acid residue is aspartic acid or glutamic acid.

28. The COF according to claim 20, wherein the RGD-based peptide are chosen from c(RGKYK) (SEQ ID NO: 1), c(RGDfK) (SEQ ID NO:2), RGDSP (SEQ ID NO:3), and RGDT (SEQ ID NON).

29. The COF according to claim 28, wherein the RGD-based peptide has the following structure:or a protonated or deprotonated form thereof, wherein R is the side chain of the RGD-based peptide.

30. The COF according to claim 29, wherein the RGD-based peptide has the following structure:or a protonated or deprotonated form thereof.

31. The COF according to claim 20, wherein the RGD-based peptide is associated with one or more of the following integrins: avPs integrin, avPs integrin, avPs integrin, asPi integrin, or avPe integrin.

32. A covalent organic framework (COF), comprising:wherein R is a reactive group.

33. The COF according to claim 32, wherein the reactive group comprises an azide group, an alkynyl group, activated esters, carboxylate / carboxylic acid, halides, amines, hydroxides, and the like.

34. The COF according to claim 32, wherein the reactive group has the following structure:

35. A composition comprising a nanoparticle according to claim 1.

36. The composition according to claim 32, further comprising one or more pharmaceutically acceptable carriers.

37. A method for treating a subject having or suspected of having cancer comprising administering to the subject a therapeutically effective amount of a nanoparticle according to claim 1.

38. The method according to claim 37, wherein the cancer is associated with one or more RGD-based peptides of the nanoparticle.

39. The method according to claim 37, wherein the cancer is a solid tumor.

40. The method according to claim 37, wherein the cancer is breast cancer, melanoma, glioblastoma, lung cancer, ovarian cancer, colorectal cancer, or prostate cancer.

41. The method according to claim 37, wherein the cancer is breast cancer.

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