Semaglutide-loaded silk nanoparticles

Abstract

Disclosed herein are compositions comprising a population of silk fibroin nanoparticles having a polydispersity index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less, wherein a glucagon-like peptide-1 (GLP-1) compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures is embedded within the population of silk fibroin nanoparticles, wherein the population of silk fibroin nanoparticles is optionally surface treated with at least one of polyethylene glycol, human transferrin protein, the Fc receptor of IgG, or other novel surface modifiers including but not limited to peptides, proteins, or nanobodies. Methods of making and administering the compositions are described herein.

Description

PATENTAttorney Docket No. T002871 WO-2095.0707SEMAGLUTIDE-LOADED SILK NANOPARTICLESCLAIM TO PRIORITY

[0001] This application relates to, incorporates by reference for all purposes, and claims priority to United States Application Serial Number 63 / 713,431 filed on October 29, 2024, entitled “SEMAGLUTIDE-LOADED SILK NANOPARTICLES”.BACKGROUND

[0002] Oral drag delivery remains the most convenient, preferred, and patient-friendly route of administration due to its simplicity, ease of self-administration, and high patient compliance. However, many biological barriers within the human gastrointestinal (GI) tract significantly impede the successful oral transport and absorption of drags. These barriers include the acidic pH of the stomach, the diverse enzyme content in oral and stomach regions that can degrade drag molecules, and the mucus layer that lines the intestines, acting as a physical barrier to absorption.

[0003] Additionally, the intestinal epithelium is a complex barrier, composed of tightly joined cells that restrict the passage of substances to maintain selective permeability. Consequently, this complex environment results in poor bioavailability for both hydrophilic and hydrophobic drags, particularly affecting large or sensitive molecules like proteins and nucleic acids. Closed tight junctions of epithelial cells are impermeable to molecules larger than 11-15 angstroms, excluding most peptides and proteins from efficient oral uptake. To address this, peptide or protein formulations intended for oral delivery often incorporate permeation enhancers, which can present significant toxicity risks, especially for patients with compromised gastrointestinal health or co-morbidities.

[0004] Oral bioavailability of semaglutide remains very low, accompanied by considerable variability of up to 137%. Given these substantial barriers specifically associated with oral delivery, particularly for large or sensitive molecules like peptides, proteins, and nucleic acids, there is a pressing need to develop new delivery platforms capable of overcoming these limitations.

[0005] Current nanoparticle (NP) systems in drag delivery, including inorganic metal-based, lipid- based, and early polymer nanoparticles, continue to exhibit significant safety and functional limitations. Metal-based NPs can cause cytotoxicity, DNA damage, and potential long-term biological risks, while lipid-based NPs may induce oxidative stress damaging vital organs.Similarity, early polymers like poly(methyl methacrylate) and polystyrene are non-biodegradable and can provoke inflammation. In peptide drag delivery, current systems like liposomes, silica NPs, and polymeric NPs face challenges, including poor interactions with intestinal cells, limited mucus penetration, and low cell viability. Furthermore, these systems often only control release effectively at neutral pH, making them highly susceptible to rapid burst releases under acidic conditions such asPATENT Attorney Docket No. T002871 WO-2095.0707 those in the gastrointestinal tract. This burst release significantly elevates initial drug concentrations and potential toxicity. For example, a recent poly(lactic-co-glycolic acid)-poly(ethylene glycol) NP system was developed for semaglutide, which only sustained release up to 20 hours, with nearly 80% of the drug released within the first 15 minutes, highlighting a pronounced initial burst and thus increasing the risk severe side effects.SUMMARY

[0006] In some aspects, the techniques described herein relate to compositions including a population of silk fibroin nanoparticles having a polydispersity index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less, wherein a glucagon-like peptide-1 (GLP-1) compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures is embedded within the population of silk fibroin nanoparticles, wherein the population of silk fibroin nanoparticles is optionally surface treated with at least one of polyethylene glycol, human transferrin protein, the Fc receptor of IgG, or other novel surface modifiers including but not limited to peptides, proteins, or nanobodies.

[0007] In some aspects, the techniques described herein relate to a method including a) adding a silk solution dropwise into a volatile solvent that is miscible with water, thereby forming a precipitate-bearing solution, wherein the silk solution contains silk fibroin in an amount by weight of between 1% and 25%, including at least 1%, at least 2%, at least 3%, at least 5%, at least 6%, or at least 7% and at most 25%, at most 23%, at most 20%, at most 18%, at most 16%, at most 14%, at most 12%, or at most 10%, wherein a surface charge of the silk fibroin is optionally adjusted. The method further includes b) applying shear forces to the precipitate-bearing solution for a length of time sufficient to achieve evaporation of at least 95% of the volatile solvent, thereby producing a population of silk fibroin nanoparticles in water having a polydispersity index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less, wherein either the method further includes step c) or the silk solution comprises a glucagon-like peptide-1 (GLP-1) compound or other protein drug or peptide drug thereby embedding the GLP-1 compound or other protein drug or peptide drug within the silk fibroin nanoparticles, wherein step c) comprises suspending the silk fibroin nanoparticles and dissolving the glucagon-like peptide-1 (GLP-1) compound or other protein drug or peptide drug in a solvent thereby embedding the GLP-1 compound or other protein drug or peptide drug within the silk fibroin nanoparticles, and wherein the GLP-1 compound or other proteinPATENTAttorney Docket No. T002871 WO-2095.0707 drug or peptide drug includes but is not limited to insulin, monoclonal antibodies, and related structures.

[0008] In some aspects, the techniques described herein relate to a method including a) adding a silk solution dropwise into a volatile solvent that is miscible with water, thereby forming a precipitate-bearing solution, wherein the silk solution contains silk fibroin in an amount by weight of at least 1%, at least 2%, at least 3%, at least 5%, at least 6%, or at least 7%, and at most 25%, at most 23%, at most 20%, at most 18%, at most 16%, at most 14%, at most 12%, or at most 10%, wherein the precipitate-bearing solution includes the volatile solvent in an amount of at least 75% (v / v), wherein the silk solution comprises a glucagon-like peptide- 1 (GLP-1) compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures. The method further includes b) applying shear forces to the precipitate-bearing solution for a length of time sufficient to achieve evaporation of at least 95% of the volatile solvent, thereby producing a population of silk fibroin nanoparticles in water having a polydispersity index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less, wherein the presence of the GLP- 1 compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures in the silk solution results in the GLP-1 compound or other protein drag or peptide drag being embedded within the population of silk fibroin nanoparticles.

[0009] In some aspects, the techniques described herein relate to a method of administering the population of silk fibroin nanoparticles to a subject in need thereof.

[0010] These and other systems, methods, objects, features, and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description of the preferred embodiment and the drawings.

[0011] All documents mentioned herein are hereby incorporated in their entirety by reference. References to items in the singular should be understood to include items in the plural, and vice versa, unless explicitly stated otherwise or clear from the text. Grammatical conjunctions are intended to express any and all disjunctive and conjunctive combinations of conjoined clauses, sentences, words, and the like, unless otherwise stated or clear from the context.BRIEF DESCRIPTION OF THE FIGURES

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.PATENT Attorney Docket No. T002871 WO-2095.0707

[0013] The disclosure and the following detailed description of certain embodiments thereof may be understood by reference to the following figures:

[0014] Figs. 1A-1D depict physicochemical Properties of SNPs. Fig. 1 A depicts a schematic of SNP formation. Fig. IB depicts a diameter of SNPs, n=3. Fig. 1C depicts a Zeta potential of SNPs, n=2-3. Fig. ID depicts degradation of SNPs by enzymes after 24 hours, n=2. Error bars represent standard deviation. 2-way ANOVA with Dunnett’s multiple comparison test shows no significant differences.

[0015] Figs. 2A-2D depict drug release profile of semaglutide-loaded SNPs. Fig. 2A depicts a schematic showing drug coating on SNP. Fig. 2B depicts loading efficiency of semaglutide in CSNPs, n=3. Fig. 2C depicts in vitro release of semaglutide from CSNPs, n=3. Fig. 2D depicts in vitro release of semaglutide from CSNPs by percent, n=3. Error bars represent standard deviation.

[0016] Figs. 3A - 3H depict surface modification of SNPs. Fig. 3A depicts a scheme of SNPs and surface modifications. Fig. 3B depicts a scheme of PEGylation protocol using PEG-NHS to covalently attach PEG to surface of SNP. Fig. 3C depicts FTIR spectra of PEGylated SNPs vs native SNPs, n=3. Fig. 3D depicts a scheme of conjugated SNPs and controls. Fig. 3E depicts fluorescent NP aggregates after Tf conjugation compared with nonspecific binding of unconjugated SNPs incubated with the same primary and secondary antibodies, n=3. Fig. 3F depicts fluorescent NP aggregates after FcRn conjugation compared with nonspecific binding of unconjugated SNPs incubated with the same primary and secondary antibodies, n=3, scale bar = 100 pm. Fig. 3G depicts TF-Conj and, in Fig. 3H, FcRn-Conj SNP relative fluorescent units (RFUs) of the fluorescent secondary antibody attached to the primary antibody conjugated to the surface of the SNPs, compared to nonspecific binding in the “only antibody control” n = 3. All values were normalized against the blank SNPs. Error bars represent standard deviation. One-way ANOVA with Dunnett’s multiple comparisons test was used for statistical analysis.

[0017] Figs. 4A-4D depict SNP interactions with mucus. Fig. 4A depicts a scheme of mucus penetration testing. Fig. 4B depicts a library of SNPs tested for mucus penetration. Fig. 4C depicts penetration depth of SNPs in biosimilar mucus. Scale bars represent standard deviation. n>3. Fig. 4D depicts adhesion of SNPs to biosimilar mucus, n=3. One-way ANOVA with Tukey’s multiple comparisons test was used for statistical analysis.

[0018] Figs. 5A-5E depict interaction of SNPs with intestine epithelial cells. Fig. 5A depicts a schematic of SNP treatment to cells. Fig. 5B depicts TEER of Caco-2 monolayers after treatment with SNPs over time, n>4. Fig. 5C depicts tight junction staining of Caco-2 monolayer with no SNP treatment. Tight junction staining (ZO-1, red - tight junctions, FITC, green - SNPs) of Caco-2 monolayers after 5 hours of treatment (Fig. 5D) and 24 hours of treatment (Fig. 5E) with i) 130 nmPATENTAttorney Docket No. T002871 WO-2095.0707SNPs, ii) 65 nm SNPs, iii) EDA modified SNPs, iv) PEG SNPs, v) TF SNPs, and vi) FcRn SNPs. Scale bar = 50 pm.

[0019] Figs. 6A-6C depict exocytosis and transport of SNPs across cell monolayers. Fig. 6A depicts a schematic showing particle passage through cell monolayer. Fig. 6B depicts a Z slice of Caco-2 monolayer stained for ZO-1 (tight junctions, red), DAPI (blue), and SNPs (green) showing i) 130 nm SNPs, ii) EDA SNPs, and iii) Tf conj. SNPs at 24 hours. SNPs crossing cell monolayer. Circled areas show SNP crossing cell monolayer before crossing transwell membrane. Scale bar = 100 pm. Fig. 6C depicts fold change fluorescence measurements of SNPs transported to the basolateral side of Caco-2 monolayers at 8 and 24 hours after treatment, n=3. Error bars represent standard deviation. 2- way ANOVA with Tukey’s multiple comparisons were used for statistical analysis.

[0020] Fig. 7A and Fig. 7B depict MIN6 stimulation with SNPs. Fig. 7A depicts insulin secretion of MIN6 cells after treatment STZ and CSNPs, n=3. Error bars represent standard deviation. 2-way ANOVA with Dunnett’s multiple comparisons were used for statistical analysis. Fig. 7B depicts immunofluorescence images of MIN6 cells after STZ and i) control, no treatment, ii) free drug (4mM) treatment, and iii) CSNP treatment. Red, phalloidin = actin; green, FITC = SNPs. Scale bar = 200 pm.

[0021] Fig. 8 depicts encapsulation efficiency (%) of semaglutide-loaded SNPs.

[0022] Fig. 9 depicts Zeta potential measurements of native SNPs in PBS and water.

[0023] Fig. 10 depicts Zeta potential of PEGylated SNPs compared with native SNPs, n=3.

[0024] Fig. 11A and Fig. 1 IB depict immunofluorescence images of Caco-2 cells after 5 hours (Fig. 1 1 A) and 24 hours (Fig. 1 1 B) of treatment with 130 nm SNPs with staining for tight junctions (ZO- 1, red), DAPI (blue), and SNPs (FITC, green). Scale bar = 50 pm.DETAILED DESCRIPTION

[0025] Before the present disclosure is described in further detail, it is to be understood that the disclosure is not limited to the particular embodiments described. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The scope of the present disclosure will be limited only by the claims. As used herein, the singular forms "a", "an", and "the" include plural embodiments unless the context clearly dictates otherwise.

[0026] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; andPATENT Attorney Docket No. T002871 WO-2095.0707 (iv) the terms “about” and “approximately” are used as equivalents and may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

[0027] Oral drug delivery is highly preferred as a drug delivery route due to its non-invasive administration and strong patient compliance. The intestine offers a large absorptive surface and contains enterocytes and specialized M cells that promote drug uptake, making it an attractive route. However, several biological barriers limit its effectiveness. These include degradation in the acidic stomach, enzymatic breakdown in the intestine, and restricted permeability from tight epithelial junctions. Additionally, drugs with low solubility or stability often show poor oral bioavailability.

[0028] Nanoparticles have emerged as promising carriers for both small molecules and protein / peptide therapeutics to address these challenges. Their small size enables controlled release, improved intestinal adhesion, and potential enhancement in drug absorption. Moreover, nanoparticles can be fabricated from diverse materials and surface-modified to optimize physicochemical traits and boost bioavailability.

[0029] GI clearance is another major challenge for effective oral nanoparticle delivery, as it limits the time available for particles to interact with and cross the intestinal epithelium. Clearance mechanisms include the rapid turnover of the mucus layer, enzymatic degradation, peristaltic movement, and immune surveillance by gut-associated lymphoid tissue. These factors can lead to the premature removal or breakdown of nanoparticles before they reach their target site or release their payload. As a result, nanoparticles must be engineered to resist enzymatic degradation, evade immune detection, and penetrate the mucus barrier while maintaining sufficient residence time in the intestine. Strategies such as inert surface coatings (e.g., PEG), controlled particle size, and sustained- release formulations help extend nanoparticle retention in the GI tract and improve the likelihood of successful drug absorption.

[0030] Protein-based nanoparticles offer significant advantages for targeted drug delivery. They are metabolizable into amino acids by proteolytic enzymes, thereby reducing toxicity. Protein particles can be modified to target specific cells and tailored for desired surface charges, enhancing their therapeutic efficacy. Silk biomaterials, derived from Bombyx mori silkworm cocoons, have been effective in stabilizing, delivering and sustaining the release of a wide range of therapeutic agents such as growth factors, chemotherapy drugs, hormones, antibiotics, and peptides. Silk has the ability to foster the stability of peptides and proteins due to the unique block copolymer nature of the protein, with hydrophilic and hydrophobic domains, the inherent amphiphilic nature of the protein, and the limited water content in silk assembled structures - all providing stabilizing outcomes and protection of peptide and protein drugs from denaturation. Silk nanoparticles (SNPs) also stand outPATENTAttorney Docket No. T002871 WO-2095.0707 in this category due to their biocompatible and biodegradable nature, and their ability to be engineered with tunable hydrophobicity and surface charges to meet specific medical needs. These unique properties make SNPs a promising candidate in overcoming the limitations of earlier nanoparticle systems, leading to safer and more effective drug delivery strategies.

[0031] Semaglutide is a GLP-1 receptor agonist that has traditionally required injection due to its poor stability in the GI tract. Peptide drugs face numerous challenges when administered orally, including rapid degradation by intestinal enzymes, denaturation in acidic environments, and recognition by immune cells, all of which contribute to significantly reduced bioavailability. By loading semaglutide in silk nanoparticles, it is possible to shield the peptide from these harsh conditions and extend its therapeutic lifespan within the GI tract. In our system, SNPs demonstrated sustained release of semaglutide, in contrast to the rapid degradation profile observed when the drug was exposed to intestinal fluid or proteolytic enzymes alone. This prolonged release is critical for maintaining drug levels over time and enhancing the chances of absorption across the intestinal epithelium. Drug loading was achieved through a salt-bridge mechanism, which have previously been utilized to stabilize GLP-1 analogs to increase binding to its receptor. This interaction not only facilitates efficient embedding but may also help retain the drug within the particle matrix under physiological conditions, contributing to the overall stability and controlled release behavior of the formulation.

[0032] Herein, a nanotechnology-based strategy using SNPs to protect peptides and small molecules from enzymatic degradation enables controlled release in vitro. Surface modification reduces mucin interactions, enhancing mucus penetration and nanoparticle stability. By precisely tuning SNP properties like size, charge, and hydrophobicity, more effective traversal of these biological barriers is achieved and improved overall drug efficacy is realized. This strategy aims to significantly enhance the effectiveness of oral drug delivery systems.

[0033] The preserved bioactivity of semaglutide after nanoparticle loading is a critical finding, as drug formulation processes can sometimes result in partial or complete loss of function due to structural changes in the active compound. The comparable efficacy of SNP-loaded semaglutide to free drug suggests that the silk protein matrix provides a protective environment that maintains drug stability. This is particularly relevant for peptide-based therapeutics, which are highly susceptible to degradation in biological systems. We demonstrated that semaglutide retains its therapeutic activity following embedding within SNPs and remains effective in restoring insulin secretion in P-cell dysfunction models. In STZ-treated MIN6 cells, semaglutide-loaded SNPs significantly increased insulin secretion compared to untreated controls, with free drug restoring secretion to levels approaching those of healthy cells. The lack of effect in non-STZ-treated cells indicates that thePATENTAttorney Docket No. T002871 WO-2095.0707 observed benefits are specific to conditions of P-cell impairment and are not due to nonspecific stimulation of insulin release. Furthermore, immunofluorescence imaging confirmed interactions between cells and SNPs, supporting the hypothesis that these carriers can effectively associate with target cells. While the precise uptake mechanisms remain to be elucidated, SNP-cell engagement is a promising indicator for efficient drug delivery.

[0034] As described in the Examples below, semaglutide retains its therapeutic activity following embedding within SNPs and remains effective in restoring insulin secretion in P-cell dysfunction models. In STZ-treated MIN6 cells, semaglutide-loaded SNPs significantly increased insulin secretion compared to untreated controls, with free drug restoring secretion to levels approaching those of healthy cells. The lack of effect in non-STZ-treated cells indicates that the observed benefits are specific to conditions of P-cell impairment and are not due to nonspecific stimulation of insulin release. Furthermore, immunofluorescence imaging confirmed interactions between cells and SNPs, supporting the hypothesis that these carriers can effectively associate with target cells. SNP-cell engagement is a promising indicator for efficient drug delivery.

[0035] Overall, oral delivery of peptide drugs remains a significant challenge due to instability of peptide and protein drugs in the gastrointestinal environment, where degradation by enzymes and poor permeability across the intestinal epithelium render drugs ineffective. While NPs offer a promising strategy to address these limitations, many synthetic NP systems raise concerns regarding biocompatibility and toxicity, and most protein-based NPs require harsh crosslinking to survive enzymatic conditions. SNPs present a compelling alternative, combining biocompatibility with structural resilience without the need for chemical crosslinking. They can penetrate mucus barriers, provide sustained release of embedded peptide drugs, and enhance epithelial permeability. Future work can further the understanding and optimization of these unique SNPs for oral drug delivery through the pursuit of cell-specific interactions along with in vivo validation studies.

[0036] Benefits of Using SNP

[0037] The GI tract presents a highly enzymatically active environment, particularly in the intestine, where proteolytic enzymes such as trypsin, pepsin, and pancreatin readily degrade protein-based materials. As a result, most protein nanoparticles are highly susceptible to enzymatic breakdown and require harsh chemical crosslinking with agents like glutaraldehyde to maintain structural integrity and prolong their stability during transit through the gut. However, SNPs offer a unique advantage due to the intrinsic stability of silk fibroin, as well as the stabilizing features of the silk due to the chemistry and structure. The P-sheet-rich structure and uniquely strong amino acid sequence of silk provides inherent resistance to proteolytic degradation, allowing silk nanoparticles to maintain their integrity in enzyme-rich conditions without the need for toxic or extensive chemical crosslinking.PATENTAttorney Docket No. T002871 WO-2095.0707This natural robustness makes silk an especially promising material for oral drug delivery applications where prolonged stability and sustained release are critical.

[0038] The inventors hypothesized that silk nanoparticles (SNPs) would resist degradation by physiological enzymes due to the GAGAGS amino acid sequence of silk fibroin and its ability to form stable 0-sheet-rich structures. These P-sheets create a tightly packed, crystalline network that limits enzymatic access and cleavage. As a result, SNPs are expected to remain structurally intact in the GI tract, supporting their potential for sustained drug release, protection of embedded drugs, and improved oral delivery stability. In some conditions, SNP samples increased in weight after exposure to enzymes. This may be due to slight heterogeneity of SNP samples but could also be indicative of the formation of an enzyme corona, where enzymes are attaching to the surface of nanoparticles.

[0039] Challenges Associated with Delivery Through the Gastrointestinal Tract

[0040] In the GI tract, NPs are rapidly coated by a dynamic layer of biomolecules, primarily proteins and enzymes, forming what is known as the protein or enzyme corona. This corona defines the nanoparticle’s biological identity and significantly influences its interaction with intestinal cells. Unlike the well-studied protein coronas formed in plasma after intravenous injection, the corona that forms on NPs during oral delivery is less understood, particularly due to the complexity and variability introduced by digestive fluids and food matrices.

[0041] Recent studies have shown that the protein corona formed during digestion can alter nanoparticle size, surface charge, and composition, which in turn affects cellular uptake and even induces morphological changes in intestinal epithelial cells. Notably, enzyme coronas have been shown to significantly reduce the uptake of synthetic polymeric nanoparticles by epithelial cells, potentially masking or sterically hindering targeting ligands that are meant to enhance specificity and absorption. As such, while targeting ligands remain a powerful tool for improving oral nanoparticle delivery, their efficacy may be compromised by corona formation in the GI tract, which could be occurring with transferrin-modified or FcRn-modified SNPs, hindering their targeting of receptors on enterocytes. The protein or enzyme corona fundamentally alters the surface characteristics of nanoparticles and plays a critical role in determining their interaction with intestinal cells and, ultimately, their ability to be taken up and transported across the epithelial barrier.

[0042] Role of Size of SNPs

[0043] Particle size is a critical parameter for effective drug delivery. The inventors theorized that sizes in the 50 nm to 200 nm range are particularly advantageous for drug delivery because they are small enough to penetrate biological barriers such as the mucus layer and cellular membranes yet large enough to avoid rapid clearance by renal filtration.PATENTAttorney Docket No. T002871 WO-2095.0707

[0044] Molecules can permeate the intestinal epithelium through two primary pathways: transcellular transport (through cells) and paracellular transport (between cells). Paracellular transport is advantageous because it avoids the risk of nanoparticle or drug degradation within the cell; however, it is limited by the size of tight junction openings, which typically allow only molecules up to 20 nm in diameter. Several factors influence the paracellular transport of nanoparticles, including size, charge, surface modifications, and the proteins that adsorb to the surface of the particle. While SNPs are too large to pass directly through tight junctions, they can enhance intestinal permeability by inducing tight junction rearrangement, allowing released drag to permeate the cell barrier. In vivo studies suggest that larger particles may be more effective in crossing epithelial barriers, particularly in pathological conditions such as cancer and inflammation, which can create a "leaky" barrier and facilitate the paracellular transport of larger molecules.

[0045] However, the inventors surprisingly discovered that it is likely that SNPs primarily traverse intestinal cell monolayers via intracellular mechanisms. Surface-modified SNPs may interact with cell membrane receptors and adhere to the surface rather than significantly disrupting tight junctions, indicating a complex interplay between particle properties and cellular transport mechanisms, as seen with the Tf-modified SNPs traversing to the basolateral compartment more readily than other SNPs.

[0046] In some embodiments, the population of silk nanoparticles may have an average particle diameter of between 20 nm and 170 nm and a PDI of 0.5 or less. The average particle diameter may be between 40 nm and 80 nm, between 50 nm and 75 nm, or between 55 nm and 70 nm. The average particle diameter may be at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least 50 nm, at least 55 nm, at least 60 nm, at least 65 nm, at least 100 nm, or at least 130 nm, or at least 150 nm. The average particle diameter may be at most 170 nm, at most 155 nm, at most 100 nm, at most 95 nm, at most 90 nm, at most 85 nm, at most 80 nm, at most 75 nm, at most 70 nm, or at most 65 nm. The average particle diameter may be 65 nm. Administering the population of silk fibroin nanoparticles to a subject or an intestinal model yields increased uptake compared with having a larger and / or smaller average particle diameter and / or a greater PDI.

[0047] Surface Modification and Charges of SNPs

[0048] Herein, the terms cationic SNP, EDA-coupled SNPs, and positively charged SNPs may all be used interchangeably refer to cationic SNPs generated as described in the Examples. The inventors unexpectedly discovered that surface modification of SNPs was highly relevant for modulating their interactions with biological systems, particularly mucus and cellular membranes, and the inventors discovered parameters to make cationic SNPs having similar size characteristics as non-cationic SNPs described herein, including use of 2% silk solution and a stir rate, which may be referred to elsewhere herein as a bath stir speed, of 800 rpm.PATENT Attorney Docket No. T002871 WO-2095.0707

[0049] To protect drugs from stomach acid, nanoparticles are often enterically coated or loaded into enteric capsules for site-specific release in the intestine. While silk fibroin has shown inherent resilience to extreme pH compared to other biopolymers, similar enteric encapsulation will likely be required for SNPs to effectively protect their cargos from stomach acid. Once SNPs reach the intestine, barriers such as mucosal layers must be traversed. Mucoadhesive systems, using polymers such as polylactic acid, poly(lactic-co-glycolic acid), and polyacrylic acid, help prolong GI retention and have been applied to drugs like docetaxel and insulin. Hydrophilic coatings like PVA and PEG also enhance mucus penetration and uptake. Chitosan and trimethyl chitosan, known for strong mucoadhesion and permeability enhancement, have been widely used for drug and gene delivery.

[0050] Mucus and Pore Penetration

[0051] As discussed above, once SNPs reach the intestine, barriers such as mucosal layers must be traversed.

[0052] The inventors hypothesized that surface modifications such as PEG would cause a neutrally charged surface on SNPs that would reduce interactions with mucins, forming the underpinning of a bio-inspired targeting approach and allowing nanoparticles to more effectively diffuse through the dense and adhesive mucus layer in the GI tract. As described in the Examples below, PEG coatings do cause SNPs to penetrate mucus barriers. This “stealth” effect also could minimize premature clearance and promote longer residence times at the mucosal surface. Additionally, targeting ligands like transferrin and FcRn can be conjugated to the nanoparticle surface to facilitate receptor- mediated uptake by epithelial cells. Transferrin receptors are expressed on the apical surface of intestinal cells and can mediate endocytosis and transport of iron across cell membranes, enabling more efficient transcellular transport of the nanoparticles and their cargo.

[0053] These surface modifications can enhance the permeability and retention of nanoparticles, supporting their use in challenging delivery environments such as the gut. It was discovered that attaching transferrin to the surface of the NPs caused a statistically significant increase in transport of SNPs across intestinal enterocytes, which could lead to more effective delivery of cargos. A skilled artisan would find it reasonable to believe that findings of SNP transport based on models lacking GLP-1 compound or other protein drug or peptide drug loading would be similar in SNPs with GLP- 1 compound or other protein drug or peptide drug loading.

[0054] In some embodiments, the population of silk nanoparticles may have a mucus penetration depth of between 150 pm and 5 mm in biosimilar mucus and exhibits cellular uptake in an intestinal model comprising Caco2 enterocyte monolayers. The mucus penetration depth may be at least 150 pm, at least 200 pm, at least 250 pm, at least 300 pm, at least 350 pm, at least 400 pm, at least 450PATENTAttorney Docket No. T002871 WG-2095.0707 pm, or at least 500 pm. The mucus penetration depth may be at most 5 mm, at most 4 mm, at most 3 mm, at most 2 mm, or at most 1 mm.

[0055] In some embodiments, at least a portion of a population of silk fibroin nanoparticles exhibits cellular uptake when introduced into a two-dimensional model of the intestinal epithelium comprising an enteroid-derived monolayer and a layer of biosimilar mucus. The cellular uptake may induce rearrangement of tight junctions within the two-dimensional model of the intestinal epithelium to form a rearranged model. An intestinal permeability of the rearranged model can be increased compared to a comparison intestinal permeability of a comparison two-dimensional model of the intestinal epithelium lacking administration of the population of silk fibroin nanoparticles but otherwise exposed to the same conditions as the rearranged model.

[0056] In some embodiments, wherein the population of silk fibroin nanoparticles with surface modification are taken up at higher rates than otherwise identical silk fibroin nanoparticles without surface modification in an intestinal model comprising Caco2 enterocyte monolayers.

[0057] Loading and Release of GLP-1 compound or other protein drug or peptide drug

[0058] Semaglutide, a peptide used in the treatment of type 2 diabetes, poses significant challenges for oral delivery due to its instability and tendency to cause a burst release, which can lead to undesirable side effects. Silk, however, offers a unique advantage in stabilizing protein and peptide therapeutics. Due to the porous nature of SNPs, sustained drug release is enabled.

[0059] In some embodiments, the population of silk fibroin nanoparticles has an overall active agent loading of between 0.1 pg active / mg SNP and 100 pg active / mg SNP. The loading may be at least 0.1 pg active / mg SNP, at least 1 pg active / mg SNP, at least 5 pg active / mg SNP, at least 10 pg active / mg SNP, at least 20 pg active / mg SNP, at least 30 pg active / mg SNP, at least 40 pg active / mg SNP, at least 50 pg active / mg SNP, at least 60 pg active / mg SNP, or at least 70 pg active / mg SNP. The loading may be at most 100 pg active / mg SNP, at most 90 pg active / mg SNP, at most 80 pg active / mg SNP, at most 70 pg active / mg SNP, at most 60 pg active / mg SNP, at most 50 pg active / mg SNP, at most 40 pg active / mg SNP, at most 30 pg active / mg SNP, or at most 20 pg active / mg SNP.

[0060] In other embodiments, the population of silk fibroin nanoparticles has GLP-1 compound or other protein drug or peptide drug loading of between 0.1 pg GLP-l / mg SNP and 100 pg GLP-l / mg SNP. The loading may be at least 0.1 pg GLP-l / mg SNP, at least 1 pg GLP-l / mg SNP, 5 pg GLP- l / mg SNP, at least 10 pg GLP-l / mg SNP, at least 20 pg GLP-l / mg SNP, at least 30 pg GLP-l / mg SNP, at least 40 pg GLP-l / mg SNP, at least 50 pg GLP-l / mg SNP, at least 60 pg GLP-l / mg SNP, or at least 70 pg GLP-l / mg SNP. The loading may be at most 100 pg GLP-l / mg SNP, at most 90 pg GLP-l / mg SNP, at most 80 pg GLP-l / mg SNP, at most 70 pg GLP-l / mg SNP, at most 60 pg GLP-PATENT Attorney Docket No. T002871 WO-2095.0707 1 / mg SNP, at most 50 jag GLP-l / mg SNP, at most 40 pg GLP-l / mg SNP, at most 30 pg GLP-l / mg SNP, or at most 20 pg GLP-l / mg SNP. The loading may be 10 pg GLP-l / mg SNP.

[0061] In some embodiments, the active agent may be loaded between 0.1% and 15% of the SNP by mass. The active agent may be at least 0.1% by weight, at least 0.5% by weight, at least 1% by weight, at least 3% by weight, at least 5% by weight, or at least 7% by weight. The active agent may be at most 15% weight, at most 13% by weight, or at most 11% by weight, at most 9% by weight, or at most 7% by weight.

[0062] Salt Bridges

[0063] As used herein, “salt bridge” refers to a combination of hydrogen bonding and ionic interactions. It is widely known that non-covalent interactions, a group including various ionic interactions, van der Waals forces, and it effects, are weak compared to covalent interactions. The inventors remarkably applied the use of this weak force to significantly improve loading of GLP-1 compounds or other proteins or peptides into SNPs as described below.

[0064] The use of salt bridges enhances electrostatic interactions between the GLP-1 compound or other protein drug or peptide drug and the SNPs. This may be reflected by a reduction in zeta potential. Semaglutide has a net negative charge and so do the native SNPs, so they electrostatically repel each other. By using ionic strength to alter the charge of SNPs to be more neutral, the drug is more likely to adhere.

[0065] Despite the porous nature of SNPs, native SNPs exhibited low encapsulation efficiency with ~1 pg of drug loaded onto 1 mg of SNPs. The inventors discovered that surprisingly, using phosphate-buffered saline (PBS) as a “salt bridge”, loading efficiency was increased to nearly 80% across all particle types, achieving an impressive enhancement to approximately 8 pg of drug per mg of nanoparticles. Surprisingly, the inventors also discovered that only a narrow range of concentrations of PBS provided the beneficial effects of the salt bridge. A skilled artisan will appreciate that salt solutions commonly aggregate SNPs; however, as noted in the Examples below, the use of 0.5X PBS increased loading.

[0066] Definitions

[0067] As used herein, embedded may mean adsorbed to at least one surface of the SNP, bonded to a surface of the SNP, incorporated into pores of SNPs. Embedded may also mean entrapped, loaded, coated, or otherwise distributed within or on the SNPs. The term “GLP-1” should be taken to mean a GLP-1 compound (e.g., semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide) or other protein drug or peptide drug, including but not limited to, insulin, monoclonal antibodies, or related structures. “GLP-1” may also mean a GLP-1 receptor agonist, a GLP-analogue, a GLP derivative, or a combination thereof, or a pharmaceutically acceptable salt, amide, or ester thereof. The GLP-1PATENTAttorney Docket No. T002871 WO-2095.0707 compound may also be a compound disclosed and / or claimed in International Patent Application Pub. Nos. WO 2011 / 080102 A2, WO 2011 / 080103 Al, WO 2012 / 080471 Al, WO 2014 / 005858 Al, WO 2014 / 177683 Al, WO 2019 / 038412 Al, each of which is incorporated herein in its entirety by reference for all purposes.

[0068] Dropvt •ise Addition

[0069] Dropwise addition may be a component of methods described herein. Dropwise addition may have a drop length of between 2 cm and 15 cm. The drop length may be at least 2 cm, at least 3 cm, at least 4 cm, at least 5 cm, at least 6 cm, at least 8 cm, or at least 10 cm. The drop length may be at most 15 cm, at most 13 cm, at most 10 cm, at most 8 cm, at most 7 cm, at most 6 cm, or at most 5 cm. Dropwise addition may have a drop rate of between 4 drops / min and 20 drops / min. The drop rate may be at least 4 drops / min, at least 6 drops / min, at least 8 drops / min, at least 9 drops / min, at least 10 drops / min, at least 11 drops / min, or at least 12 drops / min. The drop rate may be at most 20 drops / min, at most 18 drops / min, at most 16 drops / min, at most 14 drops / min, at most 12 drops / min, at most 10 drops / min, at most 9 drops / min, at most 8 drops / min, or at most 7 drops / min,

[0070] Methods of Making SNPs

[0071] In some embodiments, SNPs may be made by a method comprising steps a)-c). Step a) includes adding a silk solution containing silk fibroin dropwise into a volatile solvent that is miscible with water to form a precipitate-bearing solution. A surface charge of the silk fibroin may be adjusted. The silk solution may contain silk fibroin in an amount by weight of between 1% and 25%. The silk solution may contain silk fibroin in an amount by weight of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, or at least 25%.The silk solution may contain silk fibroin in an amount of at most 25%, at most 23%, at most 20%, at most 18%, at most 16%, at most 15%, at most 14%, at most 12%, at most 10%, or at most 5%.

[0072] Step b) includes applying shear forces to the precipitate-bearing solution for a length of time sufficient to achieve evaporation of at least 95% of the volatile solvent, resulting in SNPs in water. The SNPs may have a polydispersity index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less.

[0073] Step c) may include suspending the SNPs in a solvent with a glucagon-like peptide- 1 (GLP- 1) GLP-1 compound or other protein drug or peptide drug dissolved therein, thereby embedding the GLP-1 compound or other protein drag or peptide drag within the SNPs. The solvent may be a buffer solution, e.g., PBS. The PBS may be at a concentration between 0.1X and 2X. The concentration of PBS may be at least 0.15X, at least 0.2X, or at least 0.4X. The concentration of PBS may be at mostPATENTAttorney Docket No. T002871 WO-2095.07071.8X, at most 1.6X, at most 1.5X, at most 1.3X, at most 1.1X, at most IX, at most 0.8X, or at most 0.6X. The solvent may function as a salt bridge as described herein.

[0074] To generate cationic SNPs, silk fibroin with a modified or adjusted surface charge compared to native silk fibroin may be used in step a). The surface charge may be adjusted via carbodiimide coupling. Additional details may be found in the section Surface Modification of SNPs herein.

[0075] Additional details on synthesis and characterization of SNPs may be found in the Examples described herein. Nanoprecipitation of silk nanoparticles may be carried out as described in PCT / US2023 / 078662, which is herein incorporated by reference in its entirety.

[0076] In an alternative embodiment, SNPs may be made by a similar method comprising steps a) and b). Step a) includes adding a silk solution containing silk fibroin and a GLP-1 compound or other protein drug or peptide drug dropwise into a volatile solvent that is miscible with water to form a precipitate-bearing solution. A surface charge of the silk fibroin may be adjusted. The silk solution may contain silk fibroin in an amount by weight of between 1% and 25%. The silk solution may contain silk fibroin in an amount by weight of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, or at least 25%. The silk solution may contain silk fibroin in an amount of at most 25%, at most 23%, at most 20%, at most 18%, at most 16%, at most 15%, at most 14%, at most 12%, at most 10%, or at most 5%.

[0077] Step b) includes applying shear forces to the precipitate-bearing solution for a length of time sufficient to achieve evaporation of at least 95% of the volatile solvent, resulting in SNPs in water. The presence of the GLP-1 compound or other protein drug or peptide drug in the silk solution results in the GLP-1 compound or other protein drug or peptide drug being embedded within the generated SNPs. The SNPs may have a polydispersity index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less.

[0078] In some embodiments, SNPs may be made by a method comprising steps a) and b), either further including step c) or including a GLP-1 compound or other protein drug or peptide drug within the silk solution. Step a) includes adding a silk solution dropwise into a volatile solvent that is miscible with water, thereby forming a precipitate-bearing solution, wherein the silk solution contains silk fibroin in an amount by weight of between 1% and 25%, including at least 1%, at least 2%, at least 3%, at least 5%, at least 6%, or at least 7%, and at most 25%, at most 23%, at most 20%, at most 18%, at most 16%, at most 14%, at most 12%, or at most 10%, wherein a surface charge of the silk fibroin is optionally adjusted. Step b) includes applying shear forces to the precipitatebearing solution for a length of time sufficient to achieve evaporation of at least 95% of the volatile solvent, thereby producing a population of silk fibroin nanoparticles in water having a polydispersityPATENTAttorney Docket No. T002871 WO-2095.0707 index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less. Step c) includes suspending the silk fibroin nanoparticles and dissolving the glucagondi ke peptide-1 (GLP- 1) compound or other protein drag or peptide drag in a solvent thereby embedding the GLP-1 compound or other protein drag or peptide drag within the silk fibroin nanoparticles. The GLP-1 compound or other protein drag or peptide drag includes but is not limited to insulin, monoclonal antibodies, and related structures in a concentration between 1 pg / mL and 100 mg / mL.

[0079] A skilled artisan will appreciate that a desired loading of active agent (GLP-1 compound or other protein drag or peptide drag) into SNPs may be achieved by selecting from a range of concentrations of active agents while making the SNPs. In other words, the concentration of active agent is not necessarily proportional to the resulting loading of active agent. For example, a skilled artisan will appreciate that a low concentration of active agent in solution when making the SNPs does not necessarily result in a low loading of active agent within the SNP. A skilled artisan will appreciate that certain factors (e.g., encapsulation efficiency, loading time, surface modification, stir rate, solvent, temperature, etc.) may affect the final loading amount. Once the active agent is loaded, the SNPs (and therefore the active agent loaded or embedded therein) may be concentrated or diluted to a desired concentration.

[0080] A skilled artisan will further appreciate that the specific method chosen of the two methods described herein may require different concentrations of active agent in solution for achieving a desired loading of active agent. For example, to achieve a desired loading, a concentration of active agent when the silk solution in step a) comprises the active agent may be different than a concentration when the method further comprises step c).

[0081] In some embodiments, the GLP-1 compound or other protein drug or peptide drag may be present in a solution used to form or load the SNPs at a concentration between 1 pg / mL and 100 mg / mL. The concentration may be at least 1 pg / mL, at least 5 pg / mL, at least 10 pg / mL, at least 20 pg / mL, at least 30 pg / mL, at least 40 pg / mL, at least 50 pg / mL, at least 60 pg / mL, at least 65 pg / mL, at least 70 pg / mL, at least 75 pg / mL, at least 80 pg / mL, at least 90 pg / mL, at least 100 pg / mL, at least 200 pg / mL, at least 300 pg / mL, at least 400 pg / mL, at least 500 pg / mL, at least 600 pg / mL, at least 700 pg / mL, at least 800 pg / mL, at least 900 pg / mL, at least 1 mg / mL, at least 10 mg / mL, at least 25 mg / mL, at least 50 mg / mL, or at least 75 mg / mL. The concentration may be at most 100 mg / mL, at most 50 mg / mL, at most 25 mg / mL, at most 10 mg / mL, at most 5 mg / mL, at most 1 mg / mL, at most 900 pg / mL, at most 800 pg / mL, at most 700 pg / mL, at most 600 pg / mL, at most 500 pg / mL, at most 400 pg / mL, at most 300 pg / mL, at most 200 pg / mL, at most 150 pg / mL, atPATENTAttorney Docket No. T002871 WO-2095.0707 most 125 pg / mL, or at most 100 pg / mL. The concentration may be 80 pg / mL. The concentration may be 250 pg / mL.

[0082] Loading of active agent on SNPs may be at a ratio of 0.1 pg active agent / mg SNPs to 100 pg active agent / mg SNPs, as described elsewhere herein.

[0083] The concentration of SNPs may be adjusted after making the SNPs to make various oral or nasal formulations including liquids, solutions, pastes, solids, aerosols, sprays, nebulized formulations, suspensions, syrups, slurries, emulsions, elixirs, gels, gel caps, beverages, and other pharmaceutically relevant formulations.

[0084] In the following embodiments, the statements apply to either of the two methods described herein unless stated otherwise. A skilled artisan will appreciate that the steps described herein may have minor variations that do not result in a different product. A skilled artisan may also recognize that selecting different concentrations of silk fibroin within the silk solution may be beneficial in some instances (e.g., controlling particle size, optimizing for GLP-1 compound or other protein drug or peptide drug or secondary active agent loading, or optimizing for surface modification).

[0085] In some embodiments, the applying of step b) may be performed by stirring or agitating the precipitate-bearing solution. The stirring or agitating may be performed with a magnetic stir bar or other method (e.g., sonication, vortexing, shaking, etc.). The stirring or agitating may be performed at a temperature between the freezing point of the precipitate-bearing solution and 60 °C.

[0086] In other embodiments, the silk solution may include a secondary active agent. Similar to the presence of the GLP-1 compound or other protein drug or peptide drug, the presence of the secondary active agent results in the secondary active agent being embedded within the population of silk nanoparticles. The embedding may be of the same mechanism or a different mechanism than the GLP-1 compound or other protein drug or peptide drug.

[0087] In some embodiments, the volatile solvent may be acetone, ether, an alcohol, or other solvents having comparable miscibility and / or boiling points, either alone or in combination with one another.

[0088] In other embodiments, the method may include a further step of crosslinking individual silk fibroin molecules within individual silk fibroin nanoparticles. Crosslinking may be achieved by adding an enzymatic crosslinker. The enzymatic crosslinker may be at least one of transglutaminase, laccase, tyrosinase or peroxidase, or a chemical crosslinker which is (e.g., glutaraldehyde, NHS / EDC, or related systems).

[0089] In some embodiments, the method may include a further step of modifying the surface of the silk fibroin nanoparticles within the population of silk fibroin nanoparticles. The term “modifying the surface” should be taken to mean modifying at least a portion of at least one surface of a silk fibroinPATENT Attorney Docket No. T002871 WO-2095.0707 nanoparticle within the population of silk fibroin nanoparticles. A skilled artisan will appreciate that different nanoparticles within the population of silk fibroin nanoparticles may have different degrees of surface modification without affecting the function of the population of silk fibroin nanoparticles. A skilled artisan will further appreciate that measuring surface modification of nanoparticles is a bulk measurement and may not reflect those differences with a high degree of accuracy.

[0090] Surface modifications may include covalent attachment of at least one of proteins, peptides, sugars, oligosaccharides, polysaccharides, other polymers, or nanobodies for transport through biological barriers and increasing delivery of therapeutics.

[0091] In some embodiments, the method may further include tuning features of the silk fibroin nanoparticle surfaces, optionally via the modification described in the previous paragraph. These features may include hydrophilicity, hydrophobicity, oleophilicity, oleophobicity, bioactive domains, and / or crystallinity. The tuning or surface modification may vary a degree of mucus adhesion and penetration as well as epithelial cell uptake and penetration. The method may include a further step of adjusting a surface charge of the population of silk fibroin nanoparticles.

[0092] The tuning may be achieved by affixing polyethylene glycol to the population of silk fibroin nanoparticles. The PEG may have a molecular weight of between 200 Da and 5 kDa. The molecular weight of the PEG may be at least 200 Da, at least 250 Da, at least 300 Da, at least 350 Da, at least 400 Da, at least 450 Da, or at least 500 Da. The molecular weight of the PEG may be at most 5 kDa, at most 4 kDa, at most 3.5 kDa, at most 3 kDa, a most 2.5 kDa, at most 2 kDa, at most 1.5 kDa, at most 1.25 kDa, at most 1.0 kDa, at most 0.8 kDa, at most 0.75 kDa, at most 0.6 kDa, or at most 0.5 kDa.

[0093] In other embodiments, an encapsulation efficiency of the GLP-1 compound or other protein drug or peptide drug may be between 10% and 90%. The encapsulation efficiency may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65% or at least 70%. The encapsulation efficiency may be at most 90%, at most 88%, at most 86%, at most 85%, at most 84%, at most 82%, or at most 80%.

[0094] Encapsulation efficiency may be calculated by dividing the amount of active agent (e.g., GLP-1 compound or other protein drug or peptide drug) incorporated into a carrier (e.g., SNP) by the amount of active agent that was originally added. Encapsulation efficiency may be calculated using Equation 1 below.Equation 1 : Encapsulation Efficiency amount of encapsulated active agent- — — — ; - x 100 = Encapsulation Efficiency (%) amount of added active agentPATENTAttorney Docket No. T002871 WO-2095.0707

[0095] Compositions

[0096] In some embodiments, compositions described herein include a population of silk nanoparticles having a polydispersity index (PDI) of 0.5 or less with a GLP-1 compound or other protein drug or peptide drug embedded within the population of silk nanoparticles. The PDI may be 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less. The silk nanoparticles may be surface treated (e.g., surface modified) with at least one of polyethylene glycol (PEG), human transferrin protein (Tf), the Fc receptor of IgG (FcRn), or other novel surface modifiers including but not limited to peptides, proteins, or nanobodies.

[0097] In other embodiments, a composition may be made by any of the methods described herein. The composition may be a hydrogel having the population of silk fibroin nanoparticles incorporated therein. The hydrogel may be a silk fibroin hydrogel.

[0098] Administration ofSNPs

[0099] In some embodiments, a method of administering the silk fibroin nanopaiticles described herein includes administering the population of silk fibroin nanoparticles to a subject in need thereof. The administration may be nasally or orally. A skilled artisan will appreciate that different methods or compositions may produce populations of silk nanoparticles which are better suited for one administration method over the other. Particle size, particle surface charge, loading, surface modification, or the PDI of the population of silk fibroin nanoparticles may be tuned to provide a desired mucoadhesion or cellular penetration and absorption upon oral or nasal administration.

[0100] Methods of Tuning Silk Nanoparticle Size Distribution

[0101] A method of tuning silk nanoparticle size distribution includes the following steps. The first step is selecting a predetermined molecular weight of silk fibroin and a predetermined concentration to produce a desired silk fibroin nanoparticle size distribution for inclusion in a silk solution. An optional next step is selecting at least one operational parameter including a shear force, a stir speed, a stir bar size, a temperature, a drop length, or a drop rate to produce the desired silk fibroin nanoparticle size distribution.

[0102] The silk solution includes a GLP-1 compound or other protein drug or peptide drug and is added dropwise into a volatile solvent that is miscible with water, thereby forming a precipitatebearing solution. The presence of the GLP-1 compound or other protein drug or peptide drug in the silk solution results in the GLP-1 compound or other protein drug or peptide drug being embedded within the population of silk fibroin nanoparticles as described herein. The GLP-1 compound or other protein drug or peptide drug is present in an amount by weight of between 1 ug / mL and 100 ug / mL. The silk solution may contain silk fibroin in an amount by weight of between 1% and 25%.PATENT Attorney Docket No. T002871 WO-2095.0707 The silk solution may contain silk fibroin in an amount by weight of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, or at least 25%.The silk solution may contain silk fibroin in an amount of at most 25%, at most 20%, at most 15%, at most 10%, or at most 5%. The precipitate-bearing solution includes the volatile solvent in an amount of at least 75% (v / v).

[0103] A shear force is then applied to the precipitate-bearing solution for a length of time sufficient to achieve evaporation of at least 95% of the volatile solvent. This produces a population of silk nanoparticles in water having the desired silk fibroin nanoparticle size distribution. The desired silk nanoparticle size distribution includes a PDI of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less.

[0104] In some embodiments, the applying may be performed by stirring or agitating at a stir speed selected to produce a population of silk fibroin nanoparticles of a predetermined size range. The stirring may be performed with a magnetic stir bar or other method (e.g., sonication, vortexing, or shaking). Similarly, the applying may be performed a temperature selected to produce a population of silk fibroin nanoparticles of a predetermined size range. The selected temperature may be between a freezing point of the precipitate-bearing solution and 60 °C.

[0105] In other embodiments, the silk solution may further comprise a secondary active agent. The presence of the secondary active agent may result in the secondary active agent being embedded within the population of silk fibroin nanoparticles.

[0106] In some embodiments, the volatile solvent may be acetone, ether, an alcohol, or other solvents having comparable miscibility and / or boiling points, either alone or in combination with one another.

[0107] In other embodiments, the method may include a further step of crosslinking individual silk fibroin molecules within individual silk fibroin nanoparticles. Crosslinking may be achieved by adding an enzymatic crosslinker. The enzymatic crosslinker may be at least one of transglutaminase, laccase, tyrosinase or peroxidase, or a chemical crosslinker which is (e.g., glutaraldehyde, NHS / EDC, or related systems).

[0108] In some embodiments, the method may include a further step of modifying the surface of the silk fibroin nanoparticles within the population of silk fibroin nanoparticles. The term “modifying the surface” should be taken to mean modifying at least a portion of at least one surface of a silk fibroin nanoparticle within the population of silk fibroin nanoparticles. A skilled artisan will appreciate that different nanoparticles within the population of silk fibroin nanoparticles may have different degrees of surface modification without affecting the function of the population of silk fibroin nanoparticles.PATENTAttorney Docket No. T002871 WG-2095.0707A skilled artisan will further appreciate that measuring surface modification of nanoparticles is a bulk measurement and may not reflect those differences with a high degree of accuracy.

[0109] Surface modifications may include covalent attachment of at least one of proteins, peptides, sugars, oligosaccharides, polysaccharides, other polymers, or nanobodies for transport through biological barriers and increasing delivery of therapeutics.

[0110] In some embodiments, the method may further include tuning features of the silk fibroin nanoparticle surfaces, optionally via the modification described in the previous paragraph. These features may include hydrophilicity, hydrophobicity, oleophilicity, oleophobicity, bioactive domains, and / or crystallinity. The tuning or surface modification may vary a degree of mucus adhesion and penetration as well as epithelial cell uptake and penetration. The method may include a further step of adjusting a surface charge of the population of silk fibroin nanoparticles.

[0111] The tuning may be achieved by affixing polyethylene glycol to the population of silk fibroin nanoparticles. The PEG may have a molecular weight of between 200 Da and 5 kDa. The molecular weight of the PEG may be at least 200 Da, at least 250 Da, at least 300 Da, at least 350 Da, at least 400 Da, at least 450 Da, or at least 500 Da. The molecular weight of the PEG may be at most 5 kDa, at most 4 kDa, at most 3.5 kDa, at most 3 kDa, a most 2.5 kDa, at most 2 kDa, at most 1.5 kDa, at most 1.25 kDa, at most 1.0 kDa, at most 0.8 kDa, at most 0.75 kDa, at most 0.6 kDa, or at most 0.5 kDa.

[0112] Approximately: as used herein, the term “approximately" or “about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0113] Composition: as used herein, may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc. In some embodiments, “composition” may refer to a combination of two or more entities for use in a single embodiment or as part of the same article. It is not required in all embodiments that the combination of entities result in physical admixture, that is, combination as separate co-entities of each of the components of the composition is possible; however many practitioners in the field may find it advantageous to prepare a composition that is an admixture of two or more of the ingredients in a pharmaceutically acceptable carrier, diluent, orPATENTAttorney Docket No. T002871 WO-2095.0707 excipient, making it possible to administer the component ingredients of the combination at the same time.

[0114] Improve, increase, or reduce: as used herein or grammatical equivalents thereof, indicate values that are relative to a baseline measurement, such as a measurement in a similar composition made according to previously known methods.

[0115] Substantially: as used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0116] It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as "comprising" certain elements are also contemplated as "consisting essentially of" and "consisting of" those elements. When two or more ranges for a particular value are recited, this disclosure contemplates all combinations of the upper and lower bounds of those ranges that are not explicitly recited. For example, recitation of a value of between 1 and 10 or between 2 and 9 also contemplates a value of between 1 and 9 or between 2 and 10.

[0117] As used herein, "silk fibroin" refers to silk fibroin protein whether produced by silkworm, spider, or other insect, or otherwise generated (Lucas et al., Adv. Protein Chem., 13: 107-242 (1958)). Any type of silk fibroin can be used in different embodiments described herein. Silk fibroin produced by silkworms, such as Bombyx mori, is the most common and represents an earth-friendly, renewable resource. For instance, silk fibroin used in a silk film may be attained by extracting sericin from the cocoons of B. mori. Organic silkworm cocoons are also commercially available. There are many different silks, however, including spider silk (e.g., obtained from Nephila clavipes), transgenic silks, genetically engineered silks, such as silks from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants, and variants thereof, that can be used. See, e.g., WO 97 / 08315 and U.S. Pat. No. 5,245,012, each of which is incorporated herein by reference in their entireties.PATENTAttorney Docket No. T002871 WO-2095.0707

[0118] According to various embodiments, a variety of functionalizing agents may be used and / or serve as a secondary active agent with the silk-containing embodiments described herein (e.g., silk nanoparticles, silk composition, etc.). It should be understood that the examples herein may recite one or a few silk-containing embodiments but are applicable to any silk-containing embodiment, as applicable. In some embodiments, a functionalizing agent may be any compound or molecule that facilitates the attachment to and / or development (e.g., growth) of one or more endothelial cells on a silk membrane. In some embodiments, a functionalizing agent may be any compound or molecule that facilitates the attachment and / or development (e.g., growth) of one or more megakaryocytes and / or hematopoietic progenitor cells on a silk matrix and / or silk membrane. In some embodiments, a functionalizing agent may be or comprise an agent suitable for facilitating the production of one or more of white blood cells and red blood cells.

[0119] In some embodiments, a functionalizing agent may be or comprise a cell attachment mediator and / or an extracellular matrix protein, for example: collagen (e.g., collagen type I, collagen type III, collagen type IV, or collagen type VI), elastin, fibronectin, vitronectin, laminin, fibrinogen, von Willebrand factor, proteoglycans, decorin, perlecan, nidogen, hyaluronan, and / or peptides containing known integrin binding domains (e.g., “RGD” integrin binding sequence, or variations thereof), that are known to affect cellular attachment.

[0120] In some embodiments, a functionalizing agent may be any soluble molecule produced by endothelial cells. Non-limiting examples include fibroblast growth factor-1 (FGF1) and vascular endothelial growth factors (VEGF).

[0121] According to some embodiments, a plurality of functionalizing agents may be used. For example, in some embodiments wherein production of platelets is desired, provided compositions may comprise the use of laminin, fibronectin and / or fibrinogen, and type IV collagen in order to facilitate the attachment and growth of endothelial cells on a silk membrane (e.g., a porous silk membrane) and / or attachment of megakaryocytes to a silk matrix.

[0122] In some embodiments, a functionalizing agent may be embedded or otherwise associated with a silk membrane, silk nanoparticle, silk particle, and / or silk matrix such that at least a portion of the functionalizing agent is surrounded by a silk membrane and / or silk matrix as contrasted to a functionalizing agent simply being positioned along the surface of a silk nanoparticle, silk particle, silk membrane and / or silk matrix. In some embodiments, a functionalizing agent is distributed along and / or incorporated in substantially the entire surface area of a silk membrane / silk wall. In some embodiments, a functionalizing agent is distributed and / or incorporated only at one or more discrete portions of a silk nanoparticle, silk particle, silk membrane / wall and / or silk matrix. In somePATENTAttorney Docket No. T002871 WO-2095.0707 embodiments, a functionalizing agent is distributed in and / or along at least one of the lumen-facing side of a silk wall and the matrix-facing side of a silk wall.

[0123] According to various embodiments, any application-appropriate amount of one or more functionalizing agents may be used. In some embodiments, the amount of an individual functionalizing agent may be between about 1 pg / mL and 1,000 pg / mL (e.g., between about 2 pg / mL and 1,000 pg / mL, 5 pg / mL and 1,000 pg / mL, 10 pg / mL and 1,000 pg / mL, 10 pg / mL and 500 pg / mL, 10 pg / mL and 100 pg / mL). In some embodiments, the amount of an individual functionalizing agent may be at least 1 pg / mL (e.g., at least 5 pg / mL, 10 pg / mL, 15 pg / mL, 20 pg / mL, 25 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, or 900 pg / mL). In some embodiments, the amount of an individual functionalizing agent is at most 1,000 pg / mL (e.g., 900 pg / mL, 800 pg / mL, 700 pg / mL, 600 pg / mL, 500 pg / mL, 400 pg / mL, 300 pg / mL, 200 pg / mL, 100 pg / mL, 90 pg / mL, 80 pg / mL, 70 pg / mL, 60 pg / mL, 50 pg / mL, 40 pg / mL, 30 pg / mL, 20 pg / mL, 10 pg / mL, or 5 pg / mL).

[0124] In some aspects, the composition comprises one or more sensing agents, such as a sensing dye. The sensing agents / sensing dyes are environmentally sensitive and produce a measurable response to one or more environmental factors. In some aspects, the environmentally- sensitive agent or dye may be present in the composition in an effective amount to alter the composition from a first chemical-physical state to a second chemical-physical state in response to an environmental parameter (e.g., a change in pH, light intensity or exposure, temperature, pressure or strain, voltage, physiological parameter of a subject, and / or concentration of chemical species in the surrounding environment) or an externally applied stimulus (e.g., optical interrogation, acoustic interrogation, and / or applied heat). In some cases, the sensing dye is present to provide one optical appearance under one given set of environmental conditions and a second, different optical appearance under a different given set of environmental conditions. Suitable concentrations for the sensing agents described herein can be the concentrations for the colorants and additives described elsewhere herein. A person having ordinary skill in the chemical sensing arts can determine a concentration that is appropriate for use in a sensing application of the inks described herein.

[0125] In some aspects, the first and second chemical-physical state may be a physical property of the composition, such as a mechanical property, a chemical property, an acoustical property, an electrical property, a magnetic property, an optical property, a thermal property, a radiological property, or an organoleptic property. Exemplary sensing dyes or agents include, but are not limited to, a pH sensitive agent, a thermal sensitive agent, a pressure or strain sensitive agent, a light sensitive agent, or a potentiometric agent.PATENTAttorney Docket No. T002871 WG-2095.0707

[0126] Exemplary pH sensitive dyes or agents include, but are not limited to, cresol red, methyl violet, crystal violet, ethyl violet, malachite green, methyl green, 2-(p- dimethylaminophenylazo)pyridine, paramethyl red, metanil yellow, 4-phenylazodiphenylamine, thymol blue, metacresol purple, orange IV, 4-o-Tolylazo-o-toluindine, quinaldine red, 2,4- dinitrophenol, erythrosine disodium salt, benzopurpurine 4B, N,N-dimethyl-p-(m-tolylazo) aniline, p-dimethylaminoazobenene, 4,4'-bis(2-amino- 1 -naphthylazo)-2,2'-stilbenedisulfonic acid, tetrabromophenolphthalein ethyl ester, bromophenol blue, Congo red, methyl orange, ethyl orange, 4-(4-dimethylamino-l-naphylazo)-3-methoxybenzenesulfonic acid, bromocresol green, resazurin, 4- phenylazo-l-napthylamine, ethyl red 2-(l-dimethylaminophenyazo) pyridine, 4-(p- ethoxyphenylazo)-m-phenylene-diamine monohydrochloride, resorcin blue, alizarin red S, methyl red, propyl red, bromocresol purple, chlorophenol red, p-nitrophenol, alizarin, 2-(2,4- dinitrophenylazo)-l-napthol-3,6-disulfonic acid, bromothymol blue, 6,8-dinitro-lH-quinazoline-2,4- dione, brilliant yellow, phenol red, neutral red, m-nitrophenol, cresol red, turmeric, metacresol purple, 4,4'-bis(3-amino-l-naphthylazo)-2,2'-stilbenedisulfonic acid, thymol blue, p-naphtholbenzein, phenolphthalein, o-cresolphthalein, ethyl bis(2,4-dimethylphenyl) ethanoate, thymolphthalein, nitrazine yellow, alizarin yellow R, alizarin, p-(2,4-dihydroxyphenylazo) benzenesulfonic acid, 5,5'- indigodisulfonic acid, 2,4,6-trinitrotoluene, 1,3,5-trinitrobenzene, and clayton yellow.

[0127] Exemplary light responsive dyes or agents include, but are not limited to, photochromic compounds or agents, such as triaryhnethanes, stilbenes, azastilbenes, nitrones, fulgides, spiropyrans, napthopyrans, spiro-oxazines, quinones, derivatives, and combinations thereof.

[0128] Exemplary potentiometric dyes include, but are not limited to, substituted amiononaphthylehenylpridinium (ANEP) dyes, such as di-4-ANEPPS, di-8-ANEPPS, and N-(4- Sulfobutyl)-4-(6-(4-(Dibutylamino)phenyl)hexatrienyl)Pyridinium (RH237).

[0129] Exemplary temperature sensitive dyes or agents include, but are not limited to, thermochromic compounds or agents, such as thermochromic liquid crystals, leuco dyes, fluoran dyes, octadecylphosphonic acid.

[0130] Exemplary pressure or strain sensitive dyes or agents include, but are not limited to, spiropyran compounds and agents.

[0131] Exemplary chemi-sensitive dyes or agents include, but are not limited to, antibodies such as immunoglobulin G (IgG) which may change color from blue to red in response to bacterial contamination.

[0132] In some aspects, the compositions comprise one or more additive, dopant, or biologically active agent suitable for a desired intended purpose. In some aspects, the additive or dopant may be present in the composition in an amount effective to impart an optical or organoleptic property to thePATENTAttorney Docket No. T002871 WO-2095.0707 composition. Exemplary additives or dopants that impart optical or organoleptic properties include, but are not limited to, dyes / pigments, flavorants, aroma compounds, granular or fibrous fillers.

[0133] Additionally or alternatively, the additive, dopant, or biologically active agent may be present in the composition in an amount effective to "functionalize" the composition to impart a desired mechanical property or added functionality to the composition. Exemplary additive, dopants, or biologically active agent that impart the desired mechanical property or added functionality include, but are not limited to: environmentally sensitive / sensing dyes: active biomolecules; conductive or metallic particles; micro and nanofibers (e.g., silk nanofibers for reinforcement, carbon nanofibers); nanotubes; inorganic particles (e.g., hydroxyapatite, tricalcium phosphate, bioglasses); drugs (e.g., antibiotics, small molecules, or low molecular weight organic compounds); proteins and fragments or complexes thereof (e.g., enzymes, antigens, antibodies, and antigen-binding fragments thereof); DNA / RNA (e.g., siRNA, miRNA, mRNA); cells and fractions thereof (viruses and viral particles; prokaryotic cells such as bacteria; eukaryotic cells such as mammalian cells and plant cells; fungi).

[0134] In some aspects, the additive or dopant comprises a flavoring agent or flavorant.

[0135] Exemplary flavorants include ester flavorants, amino acid flavorants, nucleic acid flavorants, organic acid flavorants, and inorganic acid flavorants, such as, but not limited to, diacetyl, acetyl propionyl, acetoin, isoamyl acetate, benzaldehyde, cinnamaldehyde, ethyl propionate, methyl anthranilate, limonene, ethyl decadienoate, allyl hexanoate, ethyl maltol, ethylvanillin, methyl salicylate, manzanate, glutamic acid salts, glycine salts, guanylic acids salts, inosinic acid salts, acetic acid, ascorbic acid, citric acid, fumaric acid, lactic acid, malic acid, phosphoric acid, tartaric acid, derivatives, and mixtures thereof.

[0136] In some aspects, the additive or dopant comprises an aroma compound. Exemplary aroma compounds include ester aroma compounds, terpene aroma compounds, cyclic terpenes, and aromatic aroma compounds, such as, but not limited to, geranyl acetate, methyl formate, methyl acetate, methyl propionate, methyl butyrate, ethyl acetate, ethyl butyrate, isoamyl acetate, pentyl butyrate, pentyl pentanoate, octyl acetate, benzyl acetate, methyl anthranilate, myrcene, geraniol, nerol, citral, citronellal, cironellol, linalool, nerolidol, limonene, camphor, menthol, carone, terpineol, alpha-ionone, thujone, eucalyptol, benzaldehyde, eugenol, cinnamaldehyde, ethyl maltol, vanillin, anisole, anethole, estragole, thymol.

[0137] In some aspects, the additive or dopant comprises a colorant, such as a dye or pigment. In some aspects, the dye or pigment imparts a color or grayscale to the composition. The colorant can be different than the sensing agents and / or sensing dyes below. Any organic and / or inorganic pigments and dyes can be included in the inks. Exemplary pigments suitable for use in the presentPATENTAttorney Docket No. T002871 WG-2095.0707 disclosure include International Color Index or C.I. Pigment Black Numbers 1, 7, 11, and 31, C.I. Pigment Blue Numbers 15, 15:1, 15:2, 15:3, 15:4, 15:6, 16, 27, 29, 61 and 62, C.I. Pigment Green Numbers 7, 17, 18 and 36, C.I. Pigment Orange Numbers 5, 13, 16, 34 and 36, C.I. Pigment Violet Numbers 3, 19, 23 and 27, C.I. Pigment Red Numbers 3, 17, 22, 23, 48: 1, 48:2, 57:1, 81 :1, 81:2, 81:3, 81:5, 101, 114, 122, 144, 146, 170, 176, 179, 181, 185, 188, 202, 206, 207, 210 and 249, C.I. Pigment Yellow Numbers 1. 2, 3, 12, 13, 14, 17, 42, 65, 73, 74, 75, 83, 30, 93, 109, 1 10, 128, 138, 139, 147, 142, 151, 154 and 180, D&C Red No. 7, D&C Red No. 6 and D&C Red No. 34, carbon black pigment (such as Regal 330, Cabot Corporation), quinacridone pigments (Quinacridone Magenta (228-0122), available from Sun Chemical Corporation, Fort Lee, N.J.), diarylide yellow pigment (such as AAOT Yellow (274- 1788) available from Sun Chemical Corporation); and phthalocyanine blue pigment (such as Blue 15:3 (294-1298) available from Sun Chemical Corporation). The classes of dyes suitable for use in present invention can be selected from acid dyes, natural dyes, direct dyes (either cationic or anionic), basic dyes, and reactive dyes. The acid dyes, also regarded as anionic dyes, are soluble in water and mainly insoluble in organic solvents and are selected, from yellow acid dyes, orange acid dyes, red acid dyes, violet acid dyes, blue acid dyes, green acid dyes, and black acid dyes. European Patent 0745651, incorporated herein by reference, describes a number of acid dyes that are suitable for use in the present disclosure. Exemplary yellow acid dyes include Acid Yellow 1 International Color Index or C.I. 10316); Acid Yellow 7 (C.I. 56295); Acid Yellow 17 (C.I. 18965); Acid Yellow 23 (C.I. 19140); Acid Yellow 29 (C.I. 18900); Acid Yellow 36 (C.I. 13065); Acid Yellow 42 (C.I. 22910); Acid Yellow 73 (C.I. 45350); Acid Yellow 99 (C.I. 13908); Acid Yellow 194; and Food Yellow 3 (C.I. 15985). Exemplary orange acid dyes include Acid Orange 1 (C.I. 13090 / 1); Acid Orange 10 (C.I. 16230); Acid Orange 20 (C.I. 14603); Acid Orange 76 (C.I. 18870); Acid Orange 142; Food Orange 2 (C.I. 15980); and Orange B.

[0138] Exemplary red acid dyes include Acid Red 1 (C.I. 18050); Acid Red 4 (C.I. 14710); Acid Red 18 (C.I. 16255); Acid Red 26 (C.I. 16150); Acid Red 27 (C.I. 16185); Acid Red 51 (C.I. 45430, available from BASF Corporation, Mt. Olive, N.J.); Acid Red 52 (C.I. 45100); Acid Red 73 (C.I. 27290); Acid Red 87 (C.I. 45380); Acid Red 94 (C.I. 45440) Acid Red 194; and Food Red 1 (C.I. 14700). Exemplary violet acid dyes include Acid Violet 7 (C.I. 18055): and Acid Violet 49 (C.I. 42640). Exemplary blue acid dyes include Acid Blue 1 (C.I. 42045); Acid Blue 9 (C.I. 42090); Acid Blue 22 (C.I. 42755); Acid Blue 74 (C.I. 73015); Acid Blue 93 (C.I. 42780); and Acid Blue 158A (C.I. 15050). Exemplary green acid dyes include Acid Green 1 (C.I. 10028); Acid Green 3 (C.I. 42085); Acid Green 5 (C.I. 42095); Acid Green 26 (C.I. 44025); and Food Green 3 (C.I. 42053). Exemplary black acid dyes include Acid Black 1 (C.I. 20470); Acid Black 194 (Basantol® X80, available from BASF Corporation, an azo / 1 :2 CR-complex.PATENT Attorney Docket No. T002871 WO-2095.0707

[0139] Exemplary direct dyes for use in the present disclosure include Direct Blue 86 (C.I. 74180); Direct Blue 199; Direct Black 168; Direct Red 253; and Direct Yellow 107 / 132 (C.I. Not Assigned).

[0140] Exemplary natural dyes for use in the present disclosure include Alkanet (C.I.75520,75530); Annatto (C.I. 75120); Carotene (C.I. 75130); Chestnut; Cochineal (C.I.75470); Cutch (C.I. 75250, 75260); Divi-Divi; Fustic (C.I. 75240); Brazilin (C.I. 75280); Logwood (C.I. 75200); Osage Orange (C.I. 75660); Paprika; Quercitron (C.I. 75720); Saffron (C.I. 75100); Sandal Wood (C.I. 75510, 75540, 75550, 75560); Sumac; and Turmeric (C.I. 75300). Exemplary reactive dyes for use in the present disclosure include Reactive Yellow 37 (monoazo dye); Reactive Black 31 (diazo dye); Reactive Blue 77 (phthalo cyanine dye); and Reactive Red 180 and Reactive Red 108 dyes. Suitable also are the colorants described in The Printing Ink Manual (5th ed., Leach et al. eds. (2007), pages 289-299). Other organic and inorganic pigments and dyes and combinations thereof can be used to achieve the colors desired.

[0141] In addition to or in place of visible colorants, compositions provided herein can contain ETV fluorophores that are excited in the ETV range and emit light at a higher wavelength (typically 400 nm and above). Examples of ETV fluorophores include but are not limited to materials from the coumarin, benzoxazole, rhodamine, napthalimide, perylene, benzanthrones, benzoxanthones or benzothia-xanthones families. The addition of a UV fluorophore (such as an optical brightener for instance) can help maintain maximum visible light transmission. The amount of colorant, when present, generally is between 0.05% to 5% or between 0.1% and 1% based on the weight of the composition.

[0142] For non-white compositions, the amount of pigment / dye generally is present in an amount of from at or about 0.1 wt% to at or about 20 wt% based on the weight of the composition. In some applications, a non-white ink can include 15 wt% or less pigment / dye, or 10 wt% or less pigment / dye or 5 wt% pigment / dye, or 1 wt% pigment / dye based on the weight of the composition. In some applications, a non-white ink can include 1 wt% to 10 wt%, or 5 wt% to 15 wt%, or 10 wt% to 20 wt% pigment / dye based on the weight of the composition. In some applications, a non-white ink can contain an amount of dye / pigment that is 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt% or 20 wt% based on the weight of the composition.

[0143] For white compositions, the amount of white pigment generally is present in an amount of from at or about 1 wt% to at or about 60 wt% based on the weight of the composition. In some applications, greater than 60 wt% white pigment can be present. Preferred white pigments include titanium dioxide (anatase and rutile), zinc oxide, lithopone (calcined coprecipitate of barium sulfate and zinc sulfide), zinc sulfide, blanc fixe and alumina hydrate and combinations thereof, althoughPATENT Attorney Docket No. T002871 WO-2095.0707 any of these can be combined with calcium carbonate. In some applications, a white ink can include 60 wt% or less white pigment, 55 wt% or less white pigment, 50 wt% white pigment, 45 wt% white pigment, 40 wt% white pigment, 35 wt% white pigment, 30 wt% white pigment, 25 wt% white pigment, 20 wt% white pigment, 15 wt% white pigment, or 10 wt% white pigment, based on the weight of the composition. In some applications, a white ink can include 5 wt% to 60 wt%, 5 wt% to 55 wt%, 10 wt% to 50 wt%, 10 wt% to 25 wt%, 25 wt% to 50 wt%, 5 wt% to 15 wt%, or 40 wt% to 60 wt% white pigment based on the weight of the composition. In some applications, a non-white ink can an amount of dye / pigment that is 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 1 1 wt%, 12 wt%, 13 wt%, 14 wt%, 15%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%,24 wt%, 25%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35%,36 wt%, 37 wt%, 38 wt%, 39 wt%, 40 wt%, 41 wt%, 42 wt%, 43 wt%, 44 wt%, 45%, 46 wt%, 47 wt%, 48 wt%, 49 wt%, 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55%, 56 wt%, 57 wt%, 58 wt%,59 wt% or 60 wt% based on the weight of the composition.

[0144] In some aspects, the additive or dopant comprises a conductive additive. Exemplary conductive additives include, but are not limited to graphite, graphite powder, carbon nanotubes, and metallic particles or nanoparticles, such as gold nanoparticles. In some aspects, the conductive additive is biocompatible and non-toxic.

[0145] In some aspects, the secondary active agent is a biologically active agent. The term “biologically active agent" as used herein refers to any molecule which exerts at least one biological effect in vivo. For example, the biologically active agent can be a therapeutic agent to treat or prevent a disease state or condition in a subject. Biologically active agents include, without limitation, organic molecules, inorganic materials, proteins, peptides, nucleic acids (e.g., genes, gene fragments, gene regulatory sequences, and antisense molecules), nucleoproteins, polysaccharides, glycoproteins, and lipoproteins. Classes of biologically active compounds that can be incorporated into the composition provided herein include, without limitation, anticancer agents, antibiotics, analgesics, anti-inflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, anticonvulsants, hormones, muscle relaxants, antispasmodics, ophthalmic agents, prostaglandins, antidepressants, anti-psychotic substances, trophic factors, osteoinductive proteins, growth factors, and vaccines.

[0146] The term “active agent” or “secondary active agent” may also be used herein to refer to a biological sample (e.g., a sample of tissue or fluid, such as for instance blood) or a component thereof, and / or to a biologically active entity or compound, and / or to a structurally or functionally labile entity.PATENTAttorney Docket No. T002871 WO-2095.0707

[0147] Exemplary secondary active agents include, but are not limited to, therapeutic agents, diagnostic agents (e.g., contrast agents), and any combinations thereof. In some embodiments, the active agent present in a silk matrix (e.g., a silk microsphere), composition, or the like can include a labile active agent, e.g., an agent that can undergo chemical, physical, or biological change, degradation and / or deactivation after exposure to a specified condition, e.g., high temperatures, high humidity, light exposure, and any combinations thereof. In some embodiments, the active agent present in the silk matrix (e.g., a silk microsphere), composition, or the like can include a temperature-sensitive active agent, e.g., an active agent that will lose at least about 30% or more of its original activity or bioactivity, upon exposure to a temperature of at least about 10 °C. or above, including at least about 15 °C. or above, at least about room temperature or above, or at least about body temperature (e.g., about 37 °C.) or above.

[0148] The secondary active agent can be generally present in the silk matrix (e.g., a silk microsphere), composition, or the like in an amount of about 0.01% (w / w) to about 70% (w / w), about 0.1% (w / w) to about 50% (w / w), or about 1% (w / w) to about 30% (w / w). The active agent can be present on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like and / or encapsulated and dispersed in the silk matrix (e.g., a silk microsphere), composition, or the like homogeneously, heterogeneously, or in a gradient. In some embodiments, the active agent can be added into the silk solution, which is then subjected to the methods described herein for preparing a silk matrix (e.g., a silk microsphere), composition, or the like. In some embodiments, the active agent can be coated on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like. In some embodiments, the active agent can be loaded in a silk matrix (e.g., a silk microsphere), composition, or the like by incubating the silk microsphere in a solution of the active agent for a period of time, during which an amount of the active agent can diffuse into the silk matrix (e.g., a silk microsphere), composition, or the like, and thus distribute within the silk matrix (e.g., a silk microsphere), composition, or the like.

[0149] In some aspects, the secondary active agent is a therapeutic agent. As used herein, the term “therapeutic agent” means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes. As used herein, the term “therapeutic agent” includes a “drug” or a “vaccine.” This term includes externally and internally administered topical, localized and systemic human and animal pharmaceuticals, treatments, remedies, nutraceuticals, cosmeceuticals, biologicals, devices, diagnostics and contraceptives, including preparations useful in clinical and veterinary screening, prevention, prophylaxis, healing, wellness, detection, imaging, diagnosis, therapy, surgery, monitoring, cosmetics, prosthetics, forensics and the like. This term can also be used in reference to agriceutical,PATENTAttorney Docket No. T002871 WO-2095.0707 workplace, military, industrial and environmental therapeutics or remedies comprising selected molecules or selected nucleic acid sequences capable of recognizing cellular receptors, membrane receptors, hormone receptors, therapeutic receptors, microbes, viruses or selected targets comprising or capable of contacting plants, animals and / or humans. This term can also specifically include nucleic acids and compounds comprising nucleic acids that produce a therapeutic effect, for example deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleic acid analogues (e.g., locked nucleic acid (LNA), peptide nucleic acid (PNA), xeno nucleic acid (XNA)), or mixtures or combinations thereof, including, for example, DNA nanoplexes, siRNA, microRNA, shRNA, aptamers, ribozymes, decoy nucleic acids, antisense nucleic acids, RNA activators, and the like. Generally, any therapeutic agent can be included in the composition provided herein.

[0150] The term “therapeutic agent’’ also includes an agent that is capable of providing a local or systemic biological, physiological, or therapeutic effect in the biological system to which it is applied. For example, the therapeutic agent can act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell attachment, and enhance bone growth, among other functions. Other suitable therapeutic agents can include anti-viral agents, hormones, antibodies, or therapeutic proteins. Other therapeutic agents include prodrugs, which are agents that are not biologically active when administered but upon administration to a subject are converted to biologically active agents through metabolism or some other mechanism. Additionally, a silk-based drug delivery composition can contain one therapeutic agent or combinations of two or more therapeutic agents.

[0151] A therapeutic agent can include a wide variety of different compounds, including chemical compounds and mixtures of chemical compounds, e.g., small organic or inorganic molecules; saccharides; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; antibodies and antigen binding fragments thereof; nucleic acids; nucleic acid analogs and derivatives; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof. In some aspects, the therapeutic agent is a small molecule.

[0152] The term “bioactivity,” as used herein in reference to an active agent, generally refers to the ability of an active agent to interact with a biological target and / or to produce an effect on a biological target. For example, bioactivity can include, without limitation, elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological target. The biological target can be a molecule or a cell. For example, a bioactivity can refer to the ability of an active agent to modulate the effect / activity of an enzyme, block a receptor, stimulate a receptor, modulate thePATENT Attorney Docket No. T002871 WO-2095.0707 expression level of one or more genes, modulate cell proliferation, modulate cell division, modulate cell morphology, or any combination thereof. In some instances, a bioactivity can refer to the ability of a compound to produce a toxic effect in a cell. Exemplary cellular responses include, but are not limited to, lysis, apoptosis, growth inhibition, and growth promotion; production, secretion, and surface expression of a protein or other molecule of interest by the cell; membrane surface molecule activation including receptor activation; transmembrane ion transports; transcriptional regulations; changes in viability of the cell; changes in cell morphology; changes in presence or expression of an intracellular component of the cell; changes in gene expression or transcripts; changes in the activity of an enzyme produced within the cell; and changes in the presence or expression of a ligand and / or receptor (e.g., protein expression and / or binding activity). Methods for assaying different cellular responses are well known to one of skill in the art, e.g., western blot for determining changes in presence or expression of an endogenous protein of the cell, or microscopy for monitoring the cell morphology in response to the active agent, or FISH and / or qPCR for the detection and quantification of changes in nucleic acids. Bioactivity can be determined in some embodiments, for example, by assaying a cellular response.

[0153] In reference to an antibody, the term “bioactivity” includes, but is not limited to, epitope or antigen binding affinity, the in vivo and / or in vitro stability of the antibody, the immunogenic properties of the antibody, e.g., when administered to a human subject, and / or the ability to neutralize or antagonize the bioactivity of a target molecule in vivo or in vitro. The aforementioned properties or characteristics can be observed or measured using art-recognized techniques including, but not limited to, scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence ELISA, competitive ELISA, SPR analysis including, but not limited to, SPR analysis using a BIAcore biosensor, in vitro and in vivo neutralization assays (see, for example, International Publication No. WO 2006 / 062685), receptor binding, and immunohistochemistry with tissue sections from different sources including human, primate, or any other source as needed. In reference to an immunogen, the “bioactivity” includes immunogenicity, the definition of which is discussed in detail later. In reference to a vims, the “bioactivity” includes infectivity, the definition of which is discussed in detail later. In reference to a contrast agent, e.g., a dye, the “bioactivity” refers to the ability of a contrast agent when administered to a subject to enhance the contrast of structures or fluids within the subject’s body. The bioactivity of a contrast agent also includes, but is not limited to, its ability to interact with a biological environment and / or influence the response of another molecule under certain conditions.

[0154] As used herein, the term “small molecule” can refer to compounds that are “natural productlike,” however, the term “small molecule” is not limited to “natural product-like” compounds.PATENTAttorney Docket No. T002871 WO-2095.0707Rather, a small molecule is typically characterized in that it contains several carbon — carbon bonds and has a molecular weight of less than 5000 Daltons (5 kDa), preferably less than 3 kDa, still more preferably less than 2 kDa, and most preferably less than 1 kDa. In some cases, it is preferred that a small molecule has a molecular weight equal to or less than 700 Daltons.

[0155] Exemplary therapeutic agents include, but are not limited to, those found in Harrison’ s Principles of Internal Medicine, 13th Edition, Eds. T.R. Harrison et al. McGraw-Hill N.Y., NY; Physicians’ Desk Reference, 50th Edition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8th Edition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, ETSP XII NF XVII, 1990, the complete contents of all of which are incorporated herein by reference.

[0156] Therapeutic agents include the herein disclosed categories and specific examples. It is not intended that the category be limited by the specific examples. Those of ordinary skill in the art will recognize also numerous other compounds that fall within the categories and that are useful according to the present disclosure. Examples include a radiosensitizer, a steroid, a xanthine, a beta- 2-agonist bronchodilator, an anti-inflammatory agent, an analgesic agent, a calcium antagonist, an angiotensin-converting enzyme inhibitors, a beta-blocker, a centrally active alpha- agonist, an alpha- 1 -antagonist, an anticholinergic / antispasmodic agent, a vasopressin analogue, an antiarrhythmic agent, an antiparkinsonian agent, an antiangina / antihypertensive agent, an anticoagulant agent, an antiplatelet agent, a sedative, an anxiolytic agent, a peptidic agent, a biopolymeric agent, an antineoplastic agent, a laxative, an antidiarrheal agent, an antimicrobial agent, an antifungal agent, a vaccine, a protein, or a nucleic acid. In a further aspect, the pharmaceutically active agent can be coumarin, albumin, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives: xanthines such as theophylline and doxophylline; beta-2- agonist bronchodilators such as salbutamol, fenterol, clenbuterol, bambuterol, and salmeterol; antiinflammatory agents, including antiasthmatic anti-inflammatory agents, antiarthritis antiinflammatory agents, and non-steroidal anti-inflammatory agents, examples of which include but are not limited to sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesulide, naproxen, acetaminophen, ibuprofen, ketoprofen and piroxicam; analgesic agents such as salicylates; calcium channel blockers such as nifedipine, amlodipine, and nicardipine; angiotensin converting enzyme inhibitors such as captopril, benazepril hydrochloride, fosinopril sodium, trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride, and moexipril hydrochloride; beta-blockers (i.e., beta adrenergic blocking agents) such as sotalol hydrochloride, timolol maleate, esmolol hydrochloride, carteolol, propanolol hydrochloride,PATENTAttorney Docket No. T002871 WG-2095.0707 betaxolol hydrochloride, penbutolol sulfate, metoprolol tartrate, metoprolol succinate, acebutolol hydrochloride, atenolol, pindolol, and bisoprolol fumarate; centrally active alpha-2-agonists such as clonidine; alpha- 1 -antagonists such as doxazosin and prazosin; anticholinergic / antispasmodic agents such as dicyclomine hydrochloride, scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate, and oxybutynin; vasopressin analogues such as vasopressin and desmopressin; antiarrhythmic agents such as quinidine, lidocaine, tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamil hydrochloride, propafenone hydrochloride, flecainide acetate, procainamide hydrochloride, moricizine hydrochloride, and disopyramide phosphate; antiparkinsonian agents, such as dopamine, L-Dopa / Carbidopa, selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine, and bromocriptine; antiangina agents and antihypertensive agents such as isosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol and verapamil; anticoagulant and antiplatelet agents such as coumadin, warfarin, acetylsalicylic acid, and ticlopidine; sedatives such as benzodiazepines and barbiturates; anxiolytic agents such as lorazepam, bromazepam, and diazepam; peptidic and biopolymeric agents such as calcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporin, insulin, somatostatin, protirelin, interferon, desmopressin, somatotropin, thymopentin, pidotimod, erythropoietin, interleukins, melatonin, granulocyte / macrophage-CSF, heparin, and GLP-1 and / or GIP receptor agonists (e.g., semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide); antineoplastic agents such as etoposide, etoposide phosphate, cyclophosphamide, methotrexate, 5-fluorouracil, vincristine, doxorubicin, cisplatin, hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase, altretamine, mitotane, and procarbazine hydrochloride; laxatives such as senna concentrate, casanthranol, bisacodyl, and sodium picosulphate; antidiarrheal agents such as difenoxin hydrochloride, loperamide hydrochloride, furazolidone, diphenoxylate hydrochloride, and microorganisms; vaccines such as bacterial and viral vaccines; antimicrobial agents such as penicillins, cephalosporins, and macrolides, antifungal agents such as imidazolic and triazolic derivatives; and nucleic acids such as DNA sequences encoding for biological proteins, and antisense oligonucleotides.

[0157] Anti-cancer agents include alkylating agents, platinum agents, antimetabolites, topoisomerase inhibitors, antitumor antibiotics, antimitotic agents, aromatase inhibitors, thymidylate synthase inhibitors, DNA antagonists, farnesyltransferase inhibitors, pump inhibitors, histone acetyltransferase inhibitors, metalloproteinase inhibitors, ribonucleoside reductase inhibitors, TNF alpha agonists / antagonists, endothelin A receptor antagonists, retinoic acid receptor agonists, immuno-modulators, hormonal and antihormonal agents, photodynamic agents, and tyrosine kinase inhibitors.PATENT Attorney Docket No. T002871 WO-2095.0707

[0158] Antibiotics include aminoglycosides (e.g., gentamicin, tobramycin, netilmicin, streptomycin, amikacin, neomycin), bacitracin, carbapenems (e.g., imipenem / cilastatin), cephalosporins, colistin, methenamine, monobactams (e.g., aztreonam), penicillins (e.g., penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, and vancomycin; and bacteriostatic agents such as chloramphenicol, clindamycin, macrolides (e.g., erythromycin, azithromycin, clarithromycin), lincomycin, nitrofurantoin, sulfonamides, tetracyclines (e.g., tetracycline, doxycycline, minocycline, demeclocycline), and trimethoprim. Also included are metronidazole, fluoroquinolones, and rifampin.

[0159] Enzyme inhibitors are substances which inhibit an enzymatic reaction. Examples of enzyme inhibitors include edrophonium chloride, N-methylphysostigmine, neostigmine bromide, physostigmine sulfate, tacrine, 1 -hydroxymaleate, iodotubercidin, p-bromotetranisole, 10-(alpha- diethylaminopropionyl)-phenothiazine hydrochloride, calmidazolium chloride, hemicholinium-3,3,5- dinitrocatechol, diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II, 3- phenylpropargylamine, N°-monomethyl-L-arginine acetate, carbidopa, 3-hydroxybenzylhydrazine, hydralazine, clorgyline, deprenyl, hydroxylamine, iproniazid phosphate, 6-MeO-tetrahydro-9H- pyrido-indole, nialamide, pargyline, quinacrine, semicarbazide, tranylcypromine, N,N- diethylaminoethyl-2,2-diphenylvalerate hydrochloride, 3-isobutyl-l-methylxanthine, papaverine, indomethacin, 2-cyclooctyl-2-hydroxyethylamine hydrochloride, 2,3-dichloro-a-methylbenzylamine (DCMB), 8,9-dichloro-2,3,4,5-tetrahydro-lH-2-benzazepine hydrochloride, p-aminoglutethimide, p- aminoglutethimide tartrate, 3-iodotyrosine, alpha-methyltyrosine, acetazolamide, dichlorphenamide, 6-hydroxy-2-benzothiazolesulfonamide, and allopurinol.

[0160] Antihistamines include pyrilamine, chlorpheniramine, and tetrahydrozoline, among others.

[0161] Anti-inflammatory agents include corticosteroids, nonsteroidal anti-inflammatory drugs (e.g., aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, and fenamates), acetaminophen, phenacetin, gold salts, chloroquine, D-Penicillamine, methotrexate colchicine, allopurinol, probenecid, and sulfinpyrazone.

[0162] Muscle relaxants include mephenesin, methocarbamol, cyclobenzaprine hydrochloride, trihexyphenidyl hydrochloride, levodopa / carbidopa, and biperiden.

[0163] Anti-spasmodics include atropine, scopolamine, oxyphenonium, and papaverine.

[0164] Analgesics include aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, fenamates, acetaminophen, phenacetin, morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids (e.g., codeine sulfate, fentanyl citrate, hydrocodone bitartrate, loperamide, morphine sulfate, noscapine, norcodeine, normorphine, thebaine, nor-binaltorphimine,PATENT Attorney Docket No. T002871 WO-2095.0707 buprenorphine, chlornaltrexamine, funaltrexamine, nalbuphine, nalorphine, naloxone, naloxonazine, naltrexone, and naltrindole), procaine, lidocaine, tetracaine and dibucaine. Ophthalmic agents include sodium fluorescein, rose bengal, methacholine, adrenaline, cocaine, atropine, alphachymotrypsin, hyaluronidase, betaxolol, pilocarpine, timolol, timolol salts, and combinations thereof.

[0165] Prostaglandins are art recognized and are a class of naturally occurring chemically related long-chain hydroxy fatty acids that have a variety of biological effects.

[0166] Anti -depressants are substances capable of preventing or relieving depression.

[0167] Examples of anti-depressants include imipramine, amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazid.

[0168] Trophic factors are factors whose continued presence improves the viability or longevity of a cell trophic factors include, without limitation, platelet-derived growth factor (PDGP), neutrophilactivating protein, monocyte chemoattractant protein, macrophage- inflammatory protein, platelet factor, platelet basic protein, and melanoma growth stimulating activity; epidermal growth factor, transforming growth factor (alpha), fibroblast growth factor, platelet- derived endothelial cell growth factor, insulin-like growth factor, glial derived growth neurotrophic factor, ciliary neurotrophic factor, nerve growth factor, bone growth / cartilage-inducing factor (alpha and beta), bone morphogenetic proteins, interleukins (e.g., interleukin inhibitors or interleukin receptors, including interleukin 1 through interleukin 10), interferons (e.g., interferon alpha, beta and gamma), hematopoietic factors, including erythropoietin, granulocyte colony stimulating factor, macrophage colony stimulating factor and granulocyte-macrophage colony stimulating factor; tumor necrosis factors, and transforming growth factors (beta), including beta-1, beta-2, beta-3, inhibin, and activin.

[0169] Hormones include estrogens (e.g., estradiol, estrone, estriol, diethylstilbestrol, quinestrol, chlorotrianisene, ethinyl estradiol, mestranol), anti-estrogens (e.g., clomiphene, tamoxifen), progestins (e.g., medroxyprogesterone, norethindrone, hydroxyprogesterone, norgestrel), antiprogestin (mifepristone), androgens (e.g., testosterone cypionate, fluoxymesterone, danazol, testolactone), anti-androgens (e.g., cyproterone acetate, flutamide), thyroid hormones (e.g., triiodothyronne, thyroxine, propylthiouracil, methimazole, and iodixode), and pituitary hormones (e.g., corticotropin, somatotropin, oxytocin, and vasopressin). Hormones are commonly employed in hormone replacement therapy and / or for purposes of birth control. Steroid hormones, such as prednisone, are also used as immunosuppressants and anti-inflammatories. In some aspects, the additive is an agent that stimulates tissue formation, and / or healing and regrowth of natural tissues, and any combinations thereof. Agents that increase formation of new tissues and / or stimulates healing or regrowth of native tissue at the site of injection can include, but are not limited to, fibroblast growth factor (FGF), transforming growth factor-beta (TGF-beta, platelet-derived growthPATENTAttorney Docket No. T002871 WO-2095.0707 factor (PDGF), epidermal growth factors (EGFs), connective tissue activated peptides (CTAPs), osteogenic factors including bone morphogenic proteins, heparin, angiotensin II (A-II) and fragments thereof, insulin-like growth factors, tumor necrosis factors, interleukins, colony stimulating factors, erythropoietin, nerve growth factors, interferons, biologically active analogs, fragments, and derivatives of such growth factors, and any combinations thereof.

[0170] In some aspects, the silk composition can further comprise at least one additional material for soft tissue augmentation, e.g., dermal filler materials, including, but not limited to, poly(methyl methacrylate) microspheres, hydroxyapatite, poly(L-lactic acid), collagen, elastin, and glycosaminoglycans, hyaluronic acid, commercial dermal filler products such as BOTOX® (from Allergan), DYSPORT®, COSMODERM®, EVOLENCE®, RADIESSE®,RESTYLANE®, JUVEDERM® (from Allergan), SCULPTRA®, PERLANE®, and CAPTIQEIE®, and any combinations thereof.

[0171] In some aspects, the additive is a wound healing agent. As used herein, a “wound healing agent" is a compound or composition that actively promotes wound healing process.

[0172] Exemplary wound healing agents include, but are not limited to dexpanthenol: growth factors; enzymes; hormones; povidon-iodide; fatty acids; anti-inflammatory agents; antibiotics; antimicrobials; antiseptics; cytokines; thrombin; analgesics; opioids; aminoxyls; furoxans; nitrosothiols; nitrates and anthocyanins; nucleosides, such as adenosine; and nucleotides, such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP); neurotransmitter / neuromodulators, such as acetylcholine and 5-hydroxytryptamine (serotonin / 5-HT); histamine and catecholamines, such as adrenalin and noradrenalin; lipid molecules, such as 5-sphingosine-l -phosphate and lysophosphatidic acid; amino acids, such as arginine and lysine; peptides such as the bradykinins, substance P and calcium gene-related peptide (CGRP); nitric oxide; and any combinations thereof.

[0173] In certain aspects, the active agents provided herein are immunogens. In one aspect, the immunogen is a vaccine. Most vaccines are sensitive to environmental conditions under which they are stored and / or transported. For example, freezing may increase reactogenicity (e.g., capability of causing an immunological reaction) and / or loss of potency for some vaccines (e.g., HepB, and DTaP / IPV / FQB), or cause hairline cracks in the container, leading to contamination. Further, some vaccines (e.g., BCG, Varicella, and MMR) are sensitive to heat. Many vaccines (e.g., BCG, MMR, Varicella, Meningococcal C Conjugate, and most DTaP-containing vaccines) are light sensitive. See, e.g., Galazka et al., Thermostability of vaccines, in Global Programme for Vaccines & Immunization (World Health Organization, Geneva, 1998); Peetermans et al., Stability of freeze-dried rubella vims vaccine (Cendehill strain) at various temperatures, J. Biological Standardization 179 (1973). Thus,PATENT Attorney Docket No. T002871 WO-2095.0707 the compositions and methods provided herein also provide for stabilization of vaccines regardless of the cold chain and / or other environmental conditions.

[0174] In some aspects, the additive is a cell, e.g., a biological cell. Cells useful for incorporation into the composition can come from any source, e.g., mammalian, insect, plant, etc. In some aspects, the cell can be a human, rat or mouse cell. In general, cells to be used with the compositions provided herein can be any types of cells. In general, the cells should be viable when encapsulated within compositions. In some aspects, cells that can be used with the composition include, but are not limited to, mammalian cells (e.g. human cells, primate cells, mammalian cells, rodent cells, etc.), avian cells, fish cells, insect cells, plant cells, fungal cells, spore cells, bacterial cells, and hybrid cells. In some aspects, exemplary cells that can be used with the compositions include platelets, activated platelets, stem cells, totipotent cells, pluripotent cells, and / or embryonic stem cells. In some aspects, exemplary cells that can be encapsulated within compositions include, but are not limited to, primary cells and / or cell lines from any tissue. For example, cardiomyocytes, myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes, embryonic stem cells, adult stem cells, hematopoietic stem cells, hematopoietic cells (e.g. monocytes, neutrophils, macrophages, etc.), ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts, neurons, sperm cells, egg cells, liver cells, epithelial cells from lung, epithelial cells from gut, epithelial cells from intestine, liver, epithelial cells from skin, etc., and / or hybrids thereof, can be included in the silk / platelet compositions disclosed herein. Those skilled in the art will recognize that the cells listed herein represent an exemplary, not comprehensive, list of cells. Cells can be obtained from donors (allogenic) or from recipients (autologous). Cells can be obtained, as a non-limiting example, by biopsy or other surgical means known to those skilled in the art.

[0175] In some aspects, the cell can be a genetically modified cell. A cell can be genetically modified to express and secrete a desired compound, e.g. a bioactive agent, a growth factor, differentiation factor, cytokines, and the like. Methods of genetically modifying cells for expressing and secreting compounds of interest are known in the art and easily adaptable by one of skill in the art.

[0176] Differentiated cells that have been reprogrammed into stem cells can also be used.

[0177] For example, human skin cells reprogrammed into embryonic stem cells by the transduction of Oct3 / 4, Sox2, c-Myc and Klf4 (Junying Yu, et. ah, Science, 2007, 318, 1917-1920 and Takahashi K. et. al, Cell, 2007, 131, 1-12).

[0178] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules”.PATENTAttorney Docket No. T002871 WO-2095.0707

[0179] As used herein, “about”, “approximately”, “substantially”, and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus > 10% of the particular term.

[0180] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0181] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0182] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0183] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the abovedescribed elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0184] While the invention has been illustrated and described in detail in the foregoing drawings and description, the same is to be considered as illustrative and not restrictive in character, it beingPATENTAttorney Docket No. T002871 WO-2095.0707 understood that only illustrative embodiments thereof have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. For example, any of the features or functions of any of the embodiments disclosed herein may be incorporated into any of the other embodiments disclosed herein.

[0185] The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The following examples do not in any way limit the invention.

[0186] EXAMPLES

[0187] Example 1: Synthesis, Characterization, and Loading ofSNPs

[0188] All chemicals were purchased from Sigma- Aldrich (Burlington, MA). Silk cocoons were purchased from Tajima Shoji Co., Japan. Semaglutide was purchased from MedChemExpress (FITC-tagged, Monmouth Junction, NJ). Transwells with 0.4 pm pore size were purchased from Corning (Corning, NY), and 8 um pore size transwells were purchased from Millipore Sigma (Burlington, MA).

[0189] Silk fibroin was isolated from Bombyx mori cocoons as previously described (See Xiao L, Lu G, Lu Q, Kaplan DL. Direct Formation of Silk Nanoparticles for Drug Delivery. ACS Biomater Sci Eng. 2016;2(l l):2050-7). Briefly, B. mori cocoons were boiled in a 0.02 M sodium carbonate solution for 30 minutes to remove sericin. The extracted silk fibroin was then dried for 12 hours in a chemical hood before being dissolved in a 9.3 M lithium bromide solution at 60 °C for 4 hours, yielding a 20% w / v solution. This solution was dialyzed against distilled water using Pierce Slide-a- Lyzer cassettes, MWCO 3,500 Da (ThermoFisher Scientific, Rockford, IL) to remove the lithium bromide. The solution was centrifuged (9,000 rpm, 4 °C, 20 min cycle), and a final concentration of the aqueous silk fibroin was acquired ~6-8% w / v.

[0190] Nanoprecipitated silk nanopaiticles were prepared as previously described in PCT / US2023 / 078662. Briefly, a 5-6% w / v silk solution was added dropwise to acetone to generate a solution that is >75% v / v acetone, while stirring. The nanoparticle solution was stirred for >3 hours to evaporate the acetone. Once evaporated, the nanoparticle solution was sonicated with a Branson Ultrasonic Cell Disruptor (Brookfield, CT) for 30 seconds at 30% amplitude to yield particles of ~65 and 130 nm, confirmed using dynamic light scattering (DLS).

[0191] To generate cationic SNPs, silk was carbodiimide-coupled with ethylenediamine (EDA) using l-ethyl-3-(3-(dimethylamino)propyl) carbodiimide / A-hydroxy succinimide (EDC / NHS)PATENT Attorney Docket No. T002871 WO-2095.0707 bioconjugation chemistry. EDC and NHS were weighed, and 0.2 M 2-(A-morpholino)ethanesulfonic acid (MES) buffer (pH 6) was added to reach a final reaction concentration of 0.05 M as previously described. Silk, MES buffer, EDC, NHS, and EDA were combined and stirred at 200 rpm for 18 hours at RT. The silk solution was dialyzed for 72 h to remove the unbound EDA and other byproducts, followed by centrifuging (9,000 rpm, 20 min, 4 °C) to remove silk debris. The nanoprecipitation protocol was then carried out using 2% cationic silk directly after chemical modification by concentrating to the appropriate concentration with an acetone bath stir speed of 800 rpm to yield positively charged SNPs with a diameter of 130 nm.

[0192] In this study, we formulated SNPs of two distinct sizes (130 nm and 65 nm) and generated 130 nm positively charged SNPs by modifying them with EDA (Figs. IB and 1C). While proteinbased nanoparticles typically require chemical crosslinking with agents like glutaraldehyde to resist enzymatic degradation, silk nanoparticles exhibit inherent resistance to enzymatic breakdown, showing slow degradation after 24-hour exposure to proteolytic enzymes typically found in the GI tract, with no significant differences in weight after enzyme treatment (Fig. ID). This finding aligns with previous studies showing that enzymes like Protease XIV and chymotrypsin are much more effective at degrading silk fibroin than other physiological enzymes. Typically, smaller nanoparticles degrade faster than larger ones due to their higher surface area to volume ratio. However, there are a higher number of nanoparticles in the solution of small particles, which may be protecting them from this phenomenon.

[0193] To label the SNPs, fluorescein isothiocyanate (FITC) was dissolved in dimethyl sulfoxide (DMSO) to achieve a 10 mg / mL solution. Per 100 mg of silk solution needed for the nanoprecipitation process, 1 mg of FITC was added (e.g., for 4 mL of a 5% w / v silk solution, 200 pL of the FITC stock solution was added to add 2 mg of FITC to 200 mg of silk). After adding the FITC to the silk solution and ensuring homogeneous mixing, the nanoprecipitation process was conducted. Any unbound FITC was dialyzed and / or ultracentrifuged at 60,000 rpm, 4 °C, for 30 min (Beckman Coulter Optima Max TL with TLA-110 rotor, Brea, CA) from the SNPs with several wash cycles until the supernatants revealed no leached or unbound FITC using a Varioskan LUX Multimode Microplate Reader (491 nm excitation and 516 nm emission, Varioskan ThermoFisher, Waltham, MA).

[0194] The size and ^-potential of the SNPs were measured by using a ZetaPALS DLS machine (Brookhaven Instruments, Holtzville, NY). Similarly to previous studies, size measurements were taken 3 times on the DLS machine for each sample (technical replicates) and then averaged for each batch of SNPs dissolved from a fresh batch of silk (biological replicates).PATENT Attorney Docket No. T002871 WO-2095.0707

[0195] To measure degradation of the SNPs, 10 mg of SNPs were incubated in 1.0 mg / ml protease XIV37’38, 0.0875 mg / ml chymotrypsin, 0.143 mg / ml trypsin, and 0.1 mg / ml pepsin at 37°C for 24 hours. Particles were then centrifuged (3x, 60,000 rpm, 30 min, 4 °C) and the dry weight of the pellet was recorded. The dry weight of untreated SNPs before and after incubation was also recorded.

[0196] To coat semaglutide on SNPs using adsorption (CSNPs), 30 mg SNPs and 300 pg semaglutide were stirred in a 3.7 mL solution overnight at 4°C. The following day, the SNP- semaglutide mixture was ultracentrifuged (3x, 60,000 rpm, 30 min, 4 °C) to remove unbound drug. Supernatants were saved each time for the quantification of loading. For the PBS formulation, particles and drug were stirred in half deionized water and half IX PBS. Of note, salt solutions aggregate SNPs, which is undesirable when performing an adsorption reaction, so a lower concentration of buffer was chosen to act as a salt bridge for the semaglutide.

[0197] For the in vitro release profile, 5 mg of semaglutide-loaded SNPs suspended in PBS was pipetted into transwells with 0.4 pm pore size, which were suspended in 1 mL of phosphate buffer pH 7.4 at 37 °C. The release media was saved for quantification at each time point and replaced with fresh media. These experiments were performed with technical replicates per experiment, with separate experiments (new batch of the SNPs derived from freshly degummed silk), using three conditions: the 130 nm and 65 nm SNPs from native silk and 130 nm EDA modified SNPs. The amount of semaglutide in the release samples was quantified with a plate reader (494 nm excitation and 519 nm emission).

[0198] Semaglutide, a peptide used in the treatment of type 2 diabetes, poses significant challenges for oral delivery due to its instability and tendency to cause a burst release, which can lead to undesirable side effects. Silk, however, offers a unique advantage in stabilizing protein and peptide therapeutics. Due to the porous nature of SNPs, sustained drug release is enabled, and we attempted to load 10 pg of semaglutide per mg of SNPs via adsorption (Fig. 2A). Initially, native SNPs exhibited low encapsulation efficiency with ~1 pg of drag loaded onto a mg of SNPs, but by using phosphate-buffered saline (PBS) as a salt bridge, loading efficiency was increased to nearly 80% across all particle types, achieving approximately 8 pg of drug per mg of nanoparticles (Fig. 2B and Fig. 8). This improvement is likely driven by enhanced electrostatic interactions between the drag and the SNPs (Fig. 9). The resulting release profiles extend beyond 30 days, with EDA-modified SNPs showing faster release kinetics (Figs. 2C and 2D), although such profiles may differ under intestinal conditions.

[0199] Example 2: Surface ModificationPATENT Attorney Docket No. T002871 WO-2095.0707

[0200] Particles were PEGylated by incubating precipitated particles with m-dPEG4-NHS ester in a 1:3 ratio of SNPs to PEG with stirring for 16 hours. FTIR spectroscopy was performed using JASCO FTIR 6200 spectrometer (JASCO, Oklahoma City, OK) and curves were normalized using MATLAB (Natick, MA).

[0201] Transferrin (Tf) and Fc fragment of the IgG receptor (FcRn) conjugation was conducted similarly to previously reported methods of attaching ligands to SNPs. EDC and NHS were weighed, and 0.05 M MES buffer (pH 6) was added similarly to previously described protocols. Briefly, 6.2 mg of EDC and 2 mg of NHS were dissolved in 0.05 M MES buffer (pH 6) and brought to 1.2 mL with 2.5 mg of SNPs. Then, 25 pg of either FcRn (R&D Systems 8639FC050, Minneapolis, MN) or Tf (SigmaAldrich T8158) was added to the MES, EDC / NHS, and SNP mixture and stirred at 200 rpm for 18 h. The next day, this mixture was ultracentrifuged (3x, 60,000 rpm, 30 min, 4 °C) to pellet the particles. The pellet was resuspended in DI water and spun down 2 more times to wash. The particles were then resuspended in 3 mL of DI water, containing 0.2% blocking buffer (lOx, Abeam, ab210904, Cambridge, UK) and incubated with either Transferrin antibody (ThermoFisher MAI-20106), or Human FcRn Antibody (R&D Systems MAB8639100) and incubated for 1 h shaking at RT. SNPs were then ultracentrifuged (3x, 60,000 rpm, 30 min, 4 °C) to wash unbound primary antibody, and then incubated with 0.01 mg / mL secondary antibody AlexaFluor 488 (Thermofisher A-l 1029) for 1 h shaking at RT. Blank SNPs were also incubated in 0.2% blocking buffer, primary, and secondary antibodies as a control for nonspecific binding of the antibody to the SNPs. The fluorescence of the particle aggregates was read using a plate reader (495 nm excitation, 519 emission), and the particles were imaged on a Keyence all-in-one fluorescent microscope.

[0202] We explored three types of surface modifications: 1) a neutral, hydrophilic polymer (polyethylene glycol, PEG), which is utilized to traverse the mucus membrane found in the intestine, 2) the targeting ligand transferrin (Tf), and 3) the Fc fragment of the IgG receptor (FcRn) (Fig. 3A), both of which are used to target receptors on enterocytes and for favorable mucus interactions. PEG was conjugated to the SNPs using an NHS ester reaction (Fig. 3B), and successful surface attachment was confirmed via FTIR spectroscopy (Fig. 3C) and zeta potential (Fig. 10). Transferrin and FcRn were conjugated using EDC / NHS chemistry, leveraging methods previously employed for antibody attachment. Immunocytochemistry confirmed successful ligand conjugation through both fluorescence imaging (Fig. 3E) and quantitative fluorescence measurements (Figs. 3G and 3H) with statistically significant differences between conjugated SNPs and appropriate antibody controls included for comparison (Fig. 3D).

[0203] Example 3: Biosimilar Mucus (BSM) Preparation and Mucoadhesion StudyPATENTAttorney Docket No. T002871 WO-2095.0707

[0204] BSM was prepared according to a published protocol. Polysorbate 80 (0.163% w / v), phosphatidylcholine (0.18% w / v), and cholesterol (0.36% w / v) were dissolved in a solution of 137 mM NaCl, 10 mM 2-(A-morpholino)ethanesulfonic acid (MES), 1.3 mM CaCh, and 1.0 mM MgSCb, pH 6.5. A second solution of BSA (3.1% w / v), mucin from porcine stomach (5% w / v), and polyacrylic acid (0.9% w / v) was prepared in the same buffer excluding NaCl. The solutions were mixed in a 1:9 ratio to obtain the final volume. The pH was adjusted to 6.5, and the solution and seeded onto glass 96 well plates with a thickness of 1000 um. The solution was then equilibrated at 4 °C overnight. Following the equilibration step, the SNPs were seeded on the BSM at 0.1 mg / mL and incubated at 37 °C for 24 h. To wash, PBS was added to each well followed by agitation at 25 °C for 5 min. The plate was then centrifuged at 5,000 rpm for 5 min and the supernatant carefully removed. This was repeated for a total of three washes. Samples were then read using a plate reader (491 nm excitation, 516 emission) to measure SNP adhesion to biosimilar mucus. Penetration depth was measured using confocal imaging using z stacks using Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) with Z-series capability.

[0205] A major challenge for orally administered drug carriers is traversing the mucus layer of the GI tract. Effective strategies include either penetrating this barrier by employing small, neutral particles or by adhering to the mucus to ensure sustained delivery. We assessed both the penetration and adhesion properties of our SNP library in a biosimilar mucus model (Fig. 4A and 4B). Smaller SNPs and PEGylated variants showed statistically superior penetration into the biosimilar mucus layer (Fig. 4C), with all particle types capable of penetrating layers approximately 200 microns thick or more. Adhesion studies revealed that EDA-modified SNPs had the highest degree of mucus binding, whereas PEGylated SNPs exhibited minimal adhesion (Fig. 4D), likely due to its hydrophilic nature and neutral charge.

[0206] Example 4: SNP Treatment of Cell-based Models, Transport Studies, and Bioactivity of Semagiutide

[0207] Caco2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% antibiotic-antimycotic (Sigma- Aldrich) and 10% fetal bovine serum. Cells were seeded in 24 well plates at a density of 10,000 cells / well on Coming Transwell permeable polycarbonate membrane inserts (8-micron pore size) and grown for 2 weeks with media changes every 2 days. FITC labeled SNPs were suspended in media at 100 pg / ml and 200 pl of particle suspension was added to the top of the transwell and incubated for 24 hours. At 2, 5, 8, and 24 hours after treatment, Trans-epithelial electrical resistance (TEER, EVOM World Precision Instruments, Sarasota, FL) measurements were taken and normalized to TEER measurements before treatment.PATENTAttorney Docket No. T002871 WO-2095.0707

[0208] At 5 and 24 hours, samples were fixed with 4% paraformaldehyde for 10 minutes at room temperature and was then washed with PBS. Permeabilization buffer (Triton X in PBS) was added to the wells for 10 minutes and washed. Samples were stained with ZO-1 antibody (ThermoFisher 61- 7300) for tight junction visualization at 1:200 dilution overnight at 4°C and then washed 3 times with PBS. A secondary antibody (tagged with AlexaFluor 594, Thermofisher A32740) was added to the wells at 1:400 dilution, and 4',6-diamidino-2-phenylindole (DAPI; Invitrogen) was added to the wells at 1 pg / mL and incubated for 30 minutes at room temperature. The samples were then washed and imaged on a Leica SP8 confocal microscope with Z-series capability. Images were assembled with Leica confocal software (ver. 2.61, Leica).

[0209] To measure the transport of nanoparticles through cells, SNPs were used to treat cells like in section 2.8. After 8 or 24 hours, the SNP -containing media from the apical compartment was removed, and the monolayer was washed 3 times. Fresh media was added and left to incubate for another 24 hours. The media from the basolateral compartments was taken and read for FITC fluorescence using a plate reader (495 nm excitation, 519 emission). SNP fluorescence was normalized to the fluorescence of each particle type using a standard curve of SNPs in media due to differing fluorescence intensities. Fig. 1 1 shows immunofluorescence images of Caco-2 cells after A) 5 hours and B) 24 hours of treatment with 130 nm SNPs with staining for tight junctions.

[0210] MIN6 mouse insulinoma cells (Sigma) were cultured using DMEM, 15% FBS, P- mercaptoethanol, and Penicillin / Streptomycin. To study insulin secretion after treatment with SNPs, cells were seeded onto 24-well plates at 30,000 cells / well and allowed to recover for 1 day. Cells were then treated with ImM streptozotocin (STZ) to induce a diabetes phenotype. Cells were treated with low-glucose Krebs-Ringer bicarbonate buffer for 1 hour, then treated with semaglutide-loaded SNPs (130 nm, PBS formulation) for 24 hours. Fresh media was added for an additional 24 hours, and then this media was taken and analyzed using the Alpco Ltd Chemi Rodent Insulin ELISA (ALPCO, Salem, NH). Cells were fixed using 4% paraformaldehyde, stained with phalloidin (Invitrogen, Alexa Fluor 594) and imaged on a Keyence all-in-one fluorescent microscope.

[0211] The effects of SNPs on opening of tight junctions (measured by reduction in TEER) and the transport across intestinal monolayers was evaluated through Caco-2 monolayers (Fig 5A). As shown, incubation of SNPs on the apical side of Caco-2 monolayers led to a reduction in TEER (Fig 5B). Before incubation with SNPs, staining for ZO-1 tight junction proteins showed a continuous ring appearance between adjacent cells (Fig 5C), while post incubation with SNPs, ZO-1 staining appeared discontinuous, indicating the opening of cell tight junctions after 5 hours (Fig 5D). To confirm only tight junctions were disrupted and cell death was not induced, DAPI staining wasPATENTAttorney Docket No. T002871 WG-2095.0707 utilized (Fig 10). After 24 hours, the continuous staining for ZO-1 was mostly restored (Fig 5E), with minimal loss.

[0212] Transport or uptake of SNPs across Caco-2 monolayers were investigated by examining the fluorescent signal from SNPs in the basolateral compartment of Caco-2 cells grown on transwells (Fig 6A). Confocal images show some particles passing through cell monolayers (Fig 6A). After 8 hours, little exocytosis of SNPs was shown to the apical compartment, but this increased for EDA modified and Tf-conjugated SNPs at the 24-hour time point (Fig 6B). At 24 hours, Tf-conjugated SNPs showed the highest fluorescence signal in the basolateral compartment, significantly more than both native and EDA-modified SNPs (Fig 6C).

[0213] Caco-2 monolayers alone exhibit minimal nanoparticle transport compared with more complex intestine model, reflecting their limited capacity for transcytosis. This limitation arises because these models lack M cells, which play a critical role in facilitating translocation of particles across the intestinal epithelium. Incorporating Raji-B cells into the culture induces M-cell characteristics, significantly enhancing transport. M cells possess high endocytic activity and are well-suited for translocating drugs and nanoparticles across the epithelial barrier. While the Caco-2 monolayer represents a stringent in vitro barrier and thus a worst-case scenario for transport, in vivo conditions are more permissive due to the natural presence of M cells and additional uptake pathways, likely resulting in improved nanoparticle translocation.

[0214] To determine whether semaglutide retained its bioactivity after loading into SNPs, we evaluated its efficacy in MIN6 pancreatic P-cell line. Previous research has shown that semaglutide can recover insulin secretion of diseased pancreatic cells. Cells were assessed with and without STZ treatment to model P-cell dysfunction. In STZ-treated cells, free semaglutide significantly restored insulin secretion to nearly healthy control levels. Treatment with semaglutide -loaded SNPs (130 nm, PBS formulation) also resulted in a statistically significant increase in insulin secretion compared to untreated diabetic controls (Fig 7A), confirming that the drug maintained therapeutic activity following nanoparticle embedding. No differences in insulin secretion were observed among treatment groups in non-STZ-treated cells, indicating that neither free drug nor SNP formulations affected insulin output in healthy P-cells. Immunofluorescence imaging revealed clear interactions between cells and SNPs (Fig 7Biii), with visible nanoparticle association at the cell surface. These observations support that the formulation allows cellular engagement while preserving drug efficacy.

[0215] Example 5: Statistical Analysis

[0216] All data are expressed as mean ± standard deviation. GraphPad Prism (GraphPad Software, La Jolla, CA) was utilized to carry out one and two-way analysis of variance (ANOVA) with Dunnett’s and Tukey’s multiple comparisons tests: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), andPATENTAttorney Docket No. T002871 WG-2095.0707 p < 0.0001 (****). For each experiment, unless otherwise stated, nanoparticles were evaluated in at least triplicate, where different batches of silk were dissolved to generate the nanoparticles for each “biological” replicate. Meaning, each “n” was evaluated by using a separate batch of SNPs. Each biological replicate also had technical replicates.

[0217] EQUIVALENTS AND SCOPE

[0218] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combinations (or subcombinations) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.

[0219] In addition to the features described above and elsewhere herein, the present disclosure also includes the following clauses:

[0220] Clause 1. A composition comprising a population of silk fibroin nanoparticles having a polydispersity index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less, wherein a glucagon-like peptide- 1 (GLP-1) compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures is embedded within the population of silk fibroin nanoparticles, wherein the population of silk fibroin nanoparticles is optionally surface treated with at least one of polyethylene glycol, human transferrin protein, the Fc receptor of IgG, or other novel surface modifiers including but not limited to peptides, proteins, or nanobodies.

[0221] Clause 2. A method comprising: a) adding a silk solution dropwise into a volatile solvent that is miscible with water, thereby forming a precipitate-bearing solution, wherein the silk solution contains silk fibroin in an amount by weight of between 1% and 25%, including at least 1%, at least 2%, at least 3%, at least 5%, at least 6%, or at least 7% and at most 25%, at most 23%, at most 20%, at most 18%, at most 16%, at most 14%, at most 12%, or at most 10%, wherein a surface charge of the silk fibroin is optionally adjusted; b) applying shear forces to the precipitate-bearing solution for a length of time sufficient to achieve evaporation of at least 95% of the volatile solvent, thereby producing a population of silk fibroin nanoparticles in water having a polydispersity index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less. 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less, wherein either the method further includes step c) or the silk solution comprises a glucagon-like peptide-1 (GLP-1) compound or other protein drug or peptide drug thereby embedding the GLP-1 compound or otherPATENTAttorney Docket No. T002871 WO-2095.0707 protein drug or peptide drug within the silk fibroin nanoparticles, wherein step c) comprises suspending the silk fibroin nanoparticles and dissolving the glucagon-like peptide-1 (GLP-1) compound or other protein drug or peptide drug in a solvent thereby embedding the GLP-1 compound or other protein drug or peptide drug within the silk fibroin nanoparticles, and wherein the GLP-1 compound or other protein drag or peptide drag includes but is not limited to insulin, monoclonal antibodies, and related structures in a concentration between 1 pg / mL and 100 mg / mL.

[0222] Clause 3. The method of the immediately preceding clause, wherein the solvent comprises a concentration of ions sufficient to enhance electrostatic interactions of the population of silk nanoparticles and the GLP-1 compound or other protein drug or peptide drag including but not limited to insulin, monoclonal antibodies, and related structures (e.g., a salt bridge).

[0223] Clause 4. The method of any one of the two immediately preceding clauses, wherein the solvent is a buffer solution.

[0224] Clause 5. The method of the immediately preceding clause, wherein the buffer solution is PBS at a concentration between 0.1 and 2X PBS.

[0225] Clause 6. The method of any one of clause 2 to the immediately preceding clause, the method comprising step c).

[0226] Clause 7. A method comprising: a) adding a silk solution dropwise into a volatile solvent that is miscible with water, thereby forming a precipitate-bearing solution, wherein the silk solution contains silk fibroin in an amount by weight of at least 1%, at least 2%, at least 3%, at least 5%, at least 6%, or at least 7%, and at most 25%, at most 23%, at most 20%, at most 18%, at most 16%, at most 14%, at most 12%, or at most 10%, wherein the precipitate-bearing solution includes the volatile solvent in an amount of at least 75% (v / v), wherein the silk solution comprises a glucagon- like peptide-1 (GLP-1) compound or other protein drag or peptide drag including but not limited to insulin, monoclonal antibodies, and related structures in a concentration between 1 pg / mL and 100 mg / mL; b) applying shear forces to the precipitate-bearing solution for a length of time sufficient to achieve evaporation of at least 95% of the volatile solvent, thereby producing a population of silk fibroin nanoparticles in water having a polydispersity index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less, wherein the presence of the GLP-1 compound or other protein drug or peptide drag including but not limited to insulin, monoclonal antibodies, and related structures in the silk solution results in the GLP-1 compound or other protein drag or peptide drag being embedded within the population of silk fibroin nanoparticles.

[0227] Clause 8. The method of any one of clauses 2 to 7, wherein the applying of step b) is performed by stirring or agitating the precipitate-bearing solution.PATENTAttorney Docket No. T002871 WO-2095.0707

[0228] Clause 9. The method of clause 8, wherein the stirring or agitating is performed with a magnetic stir bar, sonication, vortexing, and / or shaking).

[0229] Clause 10. The method of clause 8, wherein the stirring or agitating is performed at a temperature of between a freezing point of the precipitate-bearing solution and 60 °C.

[0230] Clause 11. The method of any one of the preceding clauses, wherein the silk solution comprises a secondary active agent, wherein the presence of the secondary active agent in the silk solution results in the secondary active agent being embedded within the population of silk fibroin nanoparticles.

[0231] Clause 12. The method of any one of the preceding clauses, wherein the volatile solvent is acetone, ether, an alcohol, or other solvents having comparable miscibility and / or boiling points.

[0232] Clause 13. The method of any one of the preceding clauses, the method further comprising sonicating the population of silk fibroin nanoparticles.

[0233] Clause 14. The method of any one of the preceding clauses, the method further comprising crosslinking individual silk fibroin molecules within individual silk fibroin nanoparticles.

[0234] Clause 15. The method of the immediately preceding clause, wherein the crosslinking is achieved by adding an enzymatic crosslinker to the population of silk fibroin nanoparticles, wherein the enzymatic crosslinker is optionally transglutaminase, laccase, tyrosinase or peroxidase, or chemical crosslinker which is optionally glutaraldehyde, NHS / EDC, or related systems.

[0235] Clause 16. The method of any one of the preceding clauses, the method further comprising tuning features of the silk fibroin nanoparticle surfaces including but not limited to hydrophilicity, hydrophobicity, bioactive domains, and crystallinity.

[0236] Clause 17. The method of any one of the preceding clauses, the method further comprising surface modifying the population of silk fibroin nanoparticles.

[0237] Clause 18. The method of the immediately preceding clause, the surface modifying comprising covalently or electrostatically affixing polyethylene glycol to the population of silk fibroin nanoparticles.

[0238] Clause 19. The method of clause 17, the surface modifying comprising covalently or electrostatically affixing transferrin protein to the population of silk fibroin nanoparticles.

[0239] Clause 20. The method of clause 17, the surface modifying comprising covalently or electrostatically affixing FcRn nanobodies to the population of silk fibroin nanoparticles.

[0240] Clause 21. The method of clause 17, the surface modifying comprising carbodiimide coupling.PATENTAttorney Docket No. T002871 WO-2095.0707

[0241] Clause 22. The method of clause 17 to the immediately preceding clause, wherein the surface modifications vary a degree of mucus adhesion and penetration as well as epithelial cell uptake and penetration.

[0242] Clause 23. The method of clause 17, wherein the surface modifications include covalent attachment of at least one of proteins, peptides, sugars, oligosaccharides, polysaccharides, other polymers, or nanobodies for transport through biological barriers and increasing delivery of therapeutics.

[0243] Clause 24. The method of any one of clauses 17 to the immediately preceding clause, wherein a surface charge of the population of silk fibroin nanopaiticles with surface modifying is different from the population of silk fibroin nanoparticles lacking surface modifying.

[0244] Clause 25. The method of clause 17, wherein the polyethylene glycol has a molecular weight of between 200 Da and 5 kDa, including but not limited to, at most 5 kDa, at most 4 kDa, at most 3.5 kDa, at most 3 kDa, a most 2.5 kDa, at most 2 kDa, at most 1.5 kDa, at most 1.25 kDa, at most 1.0 kDa, at most 0.8 kDa, at most 0.75 kDa, at most 0.6 kDa, or at most 0.5 kDa and at least 200 Da, at least 250 Da, or at least 500 Da.

[0245] Clause 26. The method of any one of the preceding clauses, the method further comprising adjusting a surface charge of the population of silk fibroin nanoparticles.

[0246] Clause 27. The method of any one of the preceding clauses, wherein an encapsulation efficiency of the GLP-1 compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures is between 10% and 90%, including but not limited to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, wherein encapsulation efficiency is measured by stirring together a known amount of GLP-1 compound or other protein drug or peptide drug with a known amount of SNPs overnight in solution, centrifuging the solution to remove unembedded GLP-1 compound or other protein drug or peptide drug into a supernatant, and quantifying the unembedded GLP-1 compound or other protein drug or peptide drug within the supernatant.

[0247] Clause 28. A composition made by the method of any one of the preceding clauses.

[0248] Clause 29. The composition of the immediately preceding clause, wherein the composition is a hydrogel having the population of silk fibroin nanoparticles incorporated therein.

[0249] Clause 30. The composition of the immediately preceding clause, wherein the hydrogel is a silk fibroin hydrogel.PATENTAttorney Docket No. T002871 WO-2095.0707

[0250] Clause 31. A method of administering the silk fibroin nanoparticles of any one of the preceding clauses comprising administering the population of silk fibroin nanoparticles to a subject in need thereof.

[0251] Clause 32. The method of the immediately preceding clause, wherein the silk fibroin nanoparticles are administered orally.

[0252] Clause 33. The method of clause 31, wherein the silk fibroin nanoparticles are administered nasally.

[0253] Clause 34. A method of tuning silk nanoparticle size distribution, the method comprising: selecting, for inclusion in a silk solution, a predetermined molecular weight of silk fibroin and a predetermined concentration to produce a desired silk fibroin nanoparticle size distribution; optionally selecting, for inclusion in the method, at least one operational parameter including a shear force, a stir speed, a stir bar size, a temperature, a drop length, or a drop rate to produce the desired silk fibroin nanoparticle size distribution; adding the silk solution dropwise into a volatile solvent that is miscible with water, thereby forming a precipitate-bearing solution, wherein the precipitatebearing solution includes the volatile solvent in an amount of at least 75% (v / v), wherein the silk solution includes a glucagon-like peptide- 1 (GLP-1) compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures in a concentration between 1 iig / mL and 100 mg / mL; applying a shear force to the precipitate-bearing solution, the applying continuing for a length of time sufficient to achieve evaporation of at least 95% of the volatile solvent, thereby producing a population of silk fibroin nanoparticles in water having the desired silk fibroin nanoparticle size distribution, wherein the desired silk fibroin nanoparticle size distribution includes a poly dispersity index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less, wherein the presence of the GLP-1 compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures in the silk solution results in the GLP-1 compound or other protein drug or peptide drag including but not limited to insulin, monoclonal antibodies, and related structures being embedded within the population of silk fibroin nanoparticles.

[0254] Clause 35. The method of clause 34, wherein the silk solution contains silk fibroin in an amount by weight of at least 1%, at least 2%, at least 3%, at least 5%, at least 6%, or at least 7%, and at most 25%, at most 23%, at most 20%, at most 18%, at most 16%, at most 14%, at most 12%, or at most 10%.PATENTAttorney Docket No. T002871 WO-2095.0707

[0255] Clause 36. The method of clause 34, wherein the applying is performed by stirring or agitating at a stir speed selected to produce a population of silk fibroin nanoparticles of a predetermined size range.

[0256] Clause 37. The method of clause 34, wherein the applying is performed at a temperature selected to produce a population of silk fibroin nanoparticles of a predetermined size range.

[0257] Clause 38. The method of clause 34, wherein the selected temperature is between a freezing point of the precipitate-bearing solution and 60 °C.

[0258] Clause 39. The method of clause 34, wherein the dropwise addition is at a drop length of at least 6 cm, at least 7 cm, or at least 8 cm.

[0259] Clause 40. The method of clause 34, wherein the dropwise addition is at a drop length of at most 8 cm, at most 7 cm, or at most 6 cm.

[0260] Clause 41 . The method of clause 34, wherein the dropwise addition is at a drop rate of between 8 drops / min and 10 drops / min, including but not limited to at least 8 drops / minute or at least 9 drops / min and at most 10 drops / min or at most 9 drops / min.

[0261] Clause 42. The method of clause 34, wherein the stirring is performed with a magnetic stir bar, sonication, vortexing, shaking, or a combination thereof.

[0262] Clause 43. The method of any one of clauses 34 to the immediately preceding clause, wherein the silk solution comprises a secondary active agent, wherein the presence of the secondary active agent in the silk solution results in the secondary active agent being embedded within the population of silk fibroin nanoparticles.

[0263] Clause 44. The method of any one of clauses 34 to the immediately preceding clause, wherein the volatile solvent is acetone, ether, an alcohol, or other solvents having comparable miscibility and / or boiling points.

[0264] Clause 45. The method of any one of clauses 34 to the immediately preceding clause, the method further comprising sonicating the population of silk fibroin nanoparticles.

[0265] Clause 46. The method of any one of clauses 34 to the immediately preceding clause, the method further comprising crosslinking individual silk fibroin molecules within individual silk fibroin nanopaiticles.

[0266] Clause 47. The method of the immediately preceding clause, wherein the crosslinking is achieved by adding an enzymatic crosslinker to the population of silk fibroin nanoparticles, wherein the enzymatic crosslinker is optionally transglutaminase, laccase, tyrosinase, or peroxidase, or chemical crosslinker which is optionally glutaraldehyde, NHS / EDC, or related systems.

[0267] Clause 48. The method of any one of clauses 34 to the immediately preceding clause, the method further comprising surface modifying the population of silk fibroin nanoparticles.PATENTAttorney Docket No. T002871 WO-2095.0707

[0268] Clause 49. The method of the immediately preceding clause, the surface modifying comprising covalently or electrostatically affixing at least one of polyethylene glycol, transferrin protein, or FcRn nanobodies to the population of silk fibroin nanoparticles.

[0269] Clause 50. The method of clause 48, the surface modifying comprising covalently or electrostatically affixing transferrin protein to the population of silk fibroin nanoparticles.

[0270] Clause 51. The method of clause 48, the surface modifying comprising covalently or electrostatically affixing FcRn nanobodies to the population of silk fibroin nanoparticles.

[0271] Clause 52. The method of clause 48, the surface modifying comprising covalently or electrostatically affixing polyethylene glycol to the population of silk fibroin nanoparticles.

[0272] Clause 53. The method of any one of clauses 48 to the immediately preceding clause, wherein the surface charge of the population of silk fibroin nanoparticles is different from a comparison population of silk fibroin nanoparticles that has not been surface modified.

[0273] Clause 54. The method of any one of clauses 34 to the immediately preceding clause, wherein the encapsulation efficiency of the GLP- 1 compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures is at least 10%, with the preferred embodiment of at 90%, including but not limited to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

[0274] Clause 55. The method of any of clauses 1 to 27 and claim 34 to the preceding clause wherein the GLP-1 compound or other protein drug or peptide drug is embedded via distribution throughout the population of silk fibroin nanoparticles.

[0275] Clause 56. The method of any of clauses 1 to 27 and claim 34 to the preceding clause wherein the GLP-1 compound or other protein drug or peptide drug is embedded via adsorption onto at least one surface of the silk fibroin nanoparticles and / or incorporation into at least one pore of the silk fibroin nanoparticle.

[0276] Clause 57. A composition made by the method of any one of clause 34 to the preceding clause.

[0277] Clause 58. The composition of the immediately preceding clause, wherein the composition is a hydrogel having the population of silk fibroin nanoparticles embedded therein.

[0278] Clause 59. The composition of the immediately preceding clause, wherein the hydrogel is a silk fibroin hydrogel.

[0279] Clause 60. The composition or method of any one of the preceding clauses, wherein the population of silk fibroin nanoparticles has an average diameter below a predetermined size threshold of 500 nm.PATENTAttorney Docket No. T002871 WO-2095.0707

[0280] Clause 61. The composition or method of any one of the preceding clauses, wherein the population of silk fibroin nanoparticles has a mucus penetration depth of between 150 pm and 5 mm, wherein the mucus penetration depth is a measurement of vertical movement through a biosimilar mucus under force of gravity following placement atop the biosimilar mucus and incubation for a penetration length of time of between 12 hours and 48 hours.

[0281] Clause 62. The composition or method of any one of the preceding clauses, wherein at least a portion of the population of silk fibroin nanoparticles exhibits cellular uptake when introduced into a two-dimensional model of the intestinal epithelium comprising an enteroid-derived monolayer and a layer of biosimilar mucus.

[0282] Clause 63. The composition or method of the immediately preceding clause, wherein the cellular uptake induces rearrangement of tight junctions within the two-dimensional model of the intestinal epithelium to form a rearranged model.

[0283] Clause 64. The composition or method of the immediately preceding clause, wherein an intestinal permeability of the rearranged model is increased compared to a comparison intestinal permeability of a comparison two-dimensional model of the intestinal epithelium lacking administration of the population of silk fibroin nanoparticles but otherwise exposed to the same conditions as the rearranged model.

[0284] Clause 65. The composition or method of any one of the preceding clauses, wherein at least a portion of the population of silk fibroin nanoparticles is capable of cellular uptake in an intestinal model comprising Caco2 enterocyte monolayers.

[0285] Clause 66. The composition or method of any one of the preceding clauses, wherein the population of silk fibroin nanoparticles with surface modification are taken up at higher rates than otherwise identical silk fibroin nanoparticles without surface modification in an intestinal model comprising Caco2 enterocyte monolayers.

[0286] Clause 67. The composition or method of any one of the preceding clauses, wherein the population of silk fibroin nanoparticles has an average particle diameter of between 20 nm and 170 nm, including between 50 nm and 75 nm, between 55 and 70 nm, or between 110 nm and 150 nm, optionally wherein the average particle diameter is 65 nm or 130 nm, and a PDI of 0.5 or less, wherein administering the population of silk fibroin nanoparticles to a subject or an intestinal model yields increased uptake compared with having a larger and / or smaller average particle diameter and / or a greater PDI.

[0287] Clause 68. The composition or method of any one of the preceding clauses, wherein the population of silk fibroin nanoparticles has a mucus penetration depth of between 150 pm and 5 mmPATENTAttorney Docket No. T002871 WG-2095.0707 in biosimilar mucus and exhibits cellular uptake in an intestinal model comprising Caco2 enterocyte monolayers.

[0288] Clause 69. The composition or method of any one of the preceding clauses, wherein the GLP-1 compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures is the GLP-1 compound.

[0289] Clause 70. The composition or method of any one of the preceding clauses, wherein the GLP-1 compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures is a GLP-1 receptor agonist, a GLP-analogue, a GLP derivative, or a combination thereof, or a pharmaceutically acceptable salt, amide, or ester thereof.

[0290] Clause 71. The composition or method of any one of clauses 1 to 68, wherein the GLP-1 compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures is a GLP-1 receptor agonist or a pharmaceutically acceptable salt, amide, or ester thereof.

[0291] Clause 72. The composition or method of any one of clauses 1 to 68, wherein the GLP-1 compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures is a GLP-1 analogue or a pharmaceutically acceptable salt, amide, or ester thereof.

[0292] Clause 73. The composition or method of any one of clauses 1 to 68, wherein the GLP-1 compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures is a GLP-1 derivative or a pharmaceutically acceptable salt, amide, or ester thereof.

[0293] Clause 74. The composition or method of any one of clauses 1 to 68, wherein the GLP-1 compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures is semaglutide or a pharmaceutically acceptable salt, amide, or ester thereof.

[0294] Clause 75. The composition or method of any one of clauses 1 to 68, wherein the GLP-1 compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures is a compound disclosed and / or claimed in International Patent Application Pub. Nos. WO 2011 / 080102 A2, WO 2011 / 080103 Al, WO 2012 / 080471 Al, WO 2014 / 005858 Al, WO 2014 / 177683 Al, WO 2019 / 038412 Al, each of which is incorporated herein in its entirety by reference for all purposes.

[0295] Clause 76. The composition or method of any one of the preceding clauses, wherein a particle size, a particle surface charge, a loading, a surface modification, or the PDI of the populationPATENTAttorney Docket No. T002871 WO-2095.0707 of silk fibroin nanoparticles are tuned to provide a desired mucoadhesion upon oral or nasal administration.

[0296] Clause 77. The composition or method of any one of the preceding clauses, wherein a particle size, a particle surface charge, a loading, a surface modification, or the PDI of the population of silk fibroin nanoparticles are tuned to provide a desired cellular penetration and absorption upon oral or nasal administration.

[0297] Clause 78. The composition or method of any one of the preceding clauses, wherein the population of silk fibroin nanoparticles has an overall active agent loading of between 0.1 pg active / mg SNP and 100 pg active / mg SNP, including at least 0.1 active / mg SNP, at least 1 active / mg SNP, at least 5 pg active / mg SNP, at least 10 pg active / mg SNP, at least 20 pg active / mg SNP, at least 30 pg active / mg SNP, at least 40 pg active / mg SNP, at least 50 pg active / mg SNP, at least 60 pg active / mg SNP, or at least 70 pg active / mg SNP, and at most 100 pg active / mg SNP, at most 90 pg active / mg SNP, at most 80 pg active / mg SNP, at most 70 pg active / mg SNP, at most 60 pg active / mg SNP, at most 50 pg active / mg SNP, at most 40 pg active / mg SNP, at most 30 pg active / mg SNP, or at most 20 pg active / mg SNP.

[0298] Clause 79. The composition or method of any one of the preceding clauses, wherein the population of silk fibroin nanoparticles has GLP-1 loading of between 0.1 pg GLP-l / mg SNP and 100 pg GLP-l / mg SNP, including at least 0.1 GLP-l / mg SNP, at least 1 GLP-l / mg SNP, at least 5 pg GLP-l / mg SNP, at least 10 pg GLP-l / mg SNP, at least 20 pg GLP-l / mg SNP, at least 30 pg GLP-l / mg SNP, at least 40 pg GLP-l / mg SNP, at least 50 pg GLP-l / mg SNP, at least 60 pg GLP- l / mg SNP, or at least 70 pg GLP-l / mg SNP and at most 100 pg GLP-l / mg SNP, at most 90 pg GLP-l / mg SNP , at most 80 pg GLP-l / mg SNP, at most 70 pg GLP-l / mg SNP, at most 60 pg GLP- l / mg SNP, at most 50 pg GLP-l / mg SNP, at most 40 pg GLP-l / mg SNP, at most 30 pg GLP-l / mg SNP, or at most 20 pg GLP-l / mg SNP.

[0299] The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

PATENTAttorney Docket No. T002871 WO-2095.0707CLAIMSWhat is claimed is:

1. A composition comprising a population of silk fibroin nanoparticles having a polydispersity index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less, wherein a glucagon-like peptide- 1 (GLP-1) compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures is embedded within the population of silk fibroin nanoparticles, wherein the population of silk fibroin nanoparticles is optionally surface treated with at least one of polyethylene glycol, human transferrin protein, the Fc receptor of IgG, or other novel surface modifiers including but not limited to peptides, proteins, or nanobodies.

2. A method comprising: a) adding a silk solution dropwise into a volatile solvent that is miscible with water, thereby forming a precipitate-bearing solution, wherein the silk solution contains silk fibroin in an amount by weight of between 1% and 25%, including at least 1%, at least 2%, at least 3%, at least 5%, at least 6%, or at least 7% and at most 25%, at most 23%, at most 20%, at most 18%, at most 16%, at most 14%, at most 12%, or at most 10%, wherein a surface charge of the silk fibroin is optionally adjusted; b) applying shear forces to the precipitate-bearing solution for a length of time sufficient to achieve evaporation of at least 95% of the volatile solvent, thereby producing a population of silk fibroin nanoparticles in water having a polydispersity index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less, wherein either the method further includes step c) or the silk solution comprises a glucagon- like peptide-1 (GLP-1) compound or other protein drug or peptide drug thereby embedding the GLP- 1 compound or other protein drug or peptide drug within the silk fibroin nanoparticles, wherein step c) comprises suspending the silk fibroin nanoparticles and dissolving the glucagon-like peptide-1 (GLP-1) compound or other protein drug or peptide drug in a solvent thereby embedding the GLP-1 compound or other protein drug or peptide drug within the silk fibroin nanoparticles, and wherein the GLP-1 compound or other protein drug or peptide drug includes but is not limited to insulin, monoclonal antibodies, and related structures.

3. The method of the immediately preceding claim, wherein the solvent comprises a concentration of ions sufficient to enhance electrostatic interactions of the population of silkPATENTAttorney Docket No. T002871 WO-2095.0707 nanoparticles and the GLP-1 compound or other protein drug or peptide drug including but not limited to insulin, monoclonal antibodies, and related structures (e.g., a salt bridge).

4. The method of any one of the two immediately preceding claims, wherein the solvent is a buffer solution.

5. The method of any one of claim 2 to the immediately preceding claim further comprising step c).

6. A method comprising: a) adding a silk solution dropwise into a volatile solvent that is miscible with water, thereby forming a precipitate-bearing solution, wherein the silk solution contains silk fibroin in an amount by weight of at least 1%, at least 2%, at least 3%, at least 5%, at least 6%, or at least 7%, and at most 25%, at most 23%, at most 20%, at most 18%, at most 16%, at most 14%, at most 12%, or at most 10%, wherein the precipitate-bearing solution includes the volatile solvent in an amount of at least 75% (v / v), wherein the silk solution comprises a glucagon-like peptide-1 (GLP-1) compound or other protein drag or peptide drag including but not limited to insulin, monoclonal antibodies, and related structures; b) applying shear forces to the precipitate-bearing solution for a length of time sufficient to achieve evaporation of at least 95% of the volatile solvent, thereby producing a population of silk fibroin nanoparticles in water having a polydispersity index (PDI) of 0.5 or less, including but not limited to, a PDI of 0.450 or less, 0.40 or less, 0.35 or less, 0.325 or less, 0.30 or less, 0.275 or less, 0.250 or less, 0.225 or less, or 0.200 or less, wherein the presence of the GLP-1 compound or other protein drag or peptide drag including but not limited to insulin, monoclonal antibodies, and related structures in the silk solution results in the GLP-1 compound or other protein drag or peptide drag being embedded within the population of silk fibroin nanoparticles.

7. The method of any one of claims 2 to 76, wherein the applying of step b) is performed by stirring or agitating the precipitate-bearing solution.

8. The method of any one of the preceding claims, wherein the silk solution comprises a secondary active agent, wherein the presence of the secondary active agent in the silk solution results in the secondary active agent being embedded within the population of silk fibroin nanoparticles.

9. The method of any one of the preceding claims, wherein the volatile solvent is acetone, ether, an alcohol, or other solvents having comparable miscibility and / or boiling points.PATENT Attorney Docket No. T002871 WO-2095.070710. The method of any one of the preceding claims, the method further comprising sonicating the population of silk fibroin nanoparticles.

11. The method of any one of the preceding claims, the method further comprising crosslinking individual silk fibroin molecules within individual silk fibroin nanoparticles.

12. The method of any one of the preceding claims, the method further comprising tuning features of the silk fibroin nanoparticle surfaces including but not limited to hydrophilicity, hydrophobicity, bioactive domains, and crystallinity.

13. The method of any one of the preceding claims, the method further comprising surface modifying the population of silk fibroin nanoparticles.

14. The method of the immediately preceding claim, the surface modifying comprising covalently or electrostatically affixing polyethylene glycol to the population of silk fibroin nanoparticles.

15. The method of claim 14, the surface modifying comprising covalently or electrostatically affixing transferrin protein to the population of silk fibroin nanoparticles.

16. The method of claim 14, the surface modifying comprising covalently or electrostatically affixing FcRn nanobodies to the population of silk fibroin nanoparticles.

17. The method of claim 14, the surface modifying comprising carbodiimide coupling.

18. The method of claim 14 to the immediately preceding claiml3, wherein the surface modifications vary a degree of mucus adhesion and penetration as well as epithelial cell uptake and penetration.

19. The composition or method of any one of the preceding claims, wherein the population of silk fibroin nanoparticles has a mucus penetration depth of between 150 pm and 5 mm, wherein the mucus penetration depth is a measurement of vertical movement through a biosimilar mucus under force of gravity following placement atop the biosimilar mucus and incubation for a penetration length of time of between 12 hours and 48 hours.

20. A method of administering the silk fibroin nanoparticles of any one of the preceding claims comprising administering the population of silk fibroin nanoparticles to a subject in need thereof.

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