WO2025101564A1 - Methods of preparing and using plodia interpunctella silk - Google Patents

Abstract

Described are methods of production of Plodia interpunctella (Pi) silk. The Pi silk is suitable for as a raw material for biomaterial fabrication.

Description

Methods of Preparing and Using Plodia interpunctella Silk

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/596,797, filed November 7, 2023, and U.S. Provisional Application No. 63/693,797, filed September 12, 2024. each of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED ESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under Grant No. R35 GM147041 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[0002] The Sequence Listing written in file T19231W0001_SeqListing.xml is 18 kilobytes in size, was created October 14, 2024. and is hereby incorporated by reference.

BACKGROUND

[0003] The need for materials in pharmaceutical, food packaging, biomedical, and industrial fields that reduce the use of non-renewable resources and biodegrade in the environment has brought interest in the use of biopolymers as material precursors. Silk is a natural polymer produced by a variety' of insects. Silk-based biomaterials, primarily derived from the silk fibroin (SF) protein of the Bombyx mori (Bm) mulberry silkworm, have been studied due to their advantageous mechanical properties, biocompatibility, and commercial availability for use in tissue engineering, disease models, and delivery of payloads with controlled release. The use of alternative silk sources, such as silk produced by the Antheraea yamamai silkworm and Trichonephila clavipes spider, in biomaterial fabrication has been considered in efforts to utilize other available biopolymers to expand the range of achievable properties in biomaterials. Use of alternative silks, however, is limited by insufficient silk production with controlled growth environments and inconsistent methods to generate materials such as films, particles, and hydrogels.

[0004] Silk is a protein fiber produced by insects with applications in medicine. Silk products include FDA-approved products like silk sutures and silk scaffolds. Utilization of silk in material fabrication is largely dominated by use of silk from the domesticated silkworm, Bombyx mori (Bm), stemming from its use in the textile industry. However, the use of a singular raw' material limits the variety of properties achievable in a material format and the availability of the material source because each organism that makes a silk fiber has a unique silk fibroin protein sequence (heavy and light chain, that originate from unique gene sequences) and a unique composition of the fibroin coating (varied proteins and compositions of proteins that make up the coating). Silks from species such as spiders, hornets, and other silkworms differ in protein properties which may translate into unique, advantageous biomaterial applications. However, challenges with the use of these proteins, such as scale of production, safe rearing protocols, and knowledge gaps in protein production, properties, and their translation to material formats, have limited their development in the biomaterial field when compared to the grow th of Bm.

[0005] Silks are a diverse class of natural protein fiber originating from a large distribution of silk-producing insects and arachnids. Silk fibers from different insects and arachnids are variable on multiple levels, including amino acid content, protein structure, protein sequence, protein composition, fiber formation, and physical properties. Environmental parameters such as temperature, humidity, and diet can also influence silk fiber protein composition, morphology’, elastic modulus, and ultimate tensile strength.

[0006] We describe Plodia interpunctella (Pi) as an alternative silk source. When novel materials are being created, analyzed for performance, or compared to current products, characterization of the raw material and reconstituted solutions used to form the biomaterial is important to understand the structure-function relationship between the two structural levels and propose modifications to material performance.

SUMMARY

[0007] For the development of alternative silk-based materials, we investigate a novel source of silk from the Plodia interpunctella (Pi) silkworm, a small agricultural pest that infests and damages food products via silk production that traps waste within stored goods. Described are methods to cultivate Pi in a lab setting with control over environmental parameters that impact silk production and properties (/.e., temperature, humidity, diet) to produce silk in scalable quantities. Pi has previously been investigated from an entomological standpoint, but largely lacks characterization and development on the fiber and material level. Described are methods for production of Pi silk or Pi silk fibroin (SF) suitable for as a raw material for biomaterial fabrication. Also described are methods for solubilization of Pi silk and/or Pi SF. Also described is the formation and characterization of /7 silk- and/or Pi SF silk-based particles and uses of Pi silk/SK as an alternative biopolymer-based encapsulation system. [0008] Described are methods of rearing Pi for production of Pi silk. The methods comprise growing Pi larvae at a controlled temperature and population density, with a controlled food (resource) supply. In some embodiments, the Pi larvae are grown at an average temperature about 24 °C. In some embodiments, the Pi larvae are grown at population density of about 0.72 larvae/ cm³. In some embodiments, the Pi larvae are provided about 0.05 to about 0.2 gram of diet per larvae.

[0009] In some embodiments, the diet provided to the larvae is supplemented, to improve larvae health, improve silk production, and/or to modily the produced silk.

[0010] Pi silk produced using the described methods can be collected and processed to provide an aqueous Pi silk solution. Further described are methods of producing the aqueous Pi silk solution. In some embodiments, the methods comprise: dissolving raw Pi silk in a salt solution, filtering the dissolved Pi silk, and dialyzing the filtered Pi silk.

[0011] The salt solution can be, but is not limited to, a LiBr solution, a Ca(NC>3)2 solution, a LiNOs solution, a Mg(NCh)2 solution, a MgCh solution, or a CaCh solution. In some embodiments, the salt solution comprises: 7-9.3 M LiBr. 8-9.3 M LiBr. 5-8 M Ca(NO3)2, 6-8 M Ca(NO3>2, 1-2 M L1NO3, 3-5 M MgCNOsty, 5-7 M MgCb, or 5-8 M CaCb. In some embodiments, the raw Pi silk is combined with the salt solution a silk: salt solution ratio (weight: volume) of about 1 :3 to about 1 :200. In some embodiments, the raw Pi silk in the salt solution is incubated at about 25 °C to about 100 °C for about 2 h to about 48 h.

[0012] The dissolved Pi silk is filtered to remove contaminants and increase stability of the isolate Pi silk. In some embodiments, filtering is performed by passing the dissolved Pi silk through a filter, such as a miracloth, have 22-25 pm pores or filter units such as PluriSelect pluriStrainer sets with 1, 5, 10, 15, 20, and 30 pm pores, made from Polyethylene terephthalate. [0013] In some embodiments, the filtered Pi silk is dialyze using a 3.5 kiloDalton (kDa) Molecular Weight Cutoff (MWCO) dialysis membrane. If larger MW Pi silk proteins are desired or if removal or other large proteins is desired, higher kDa MWCO dialysis membrane may be used. In some embodiments, dialysis is performed at a temperature of about 4 °C to about 26 °C. Dialysis can be performed against water, a salt solution, a buffered solution, or a solution containing another molecule, such as polyethylene glycol (PEG), polyethylene oxide, or acetone. In some embodiments, dialysis is performed against 2 or more solutions of decreasing salt concentration.

[0014] In some embodiments, the Pi silk is degummed. Degumming removes the outer coating from the Pi silk fiber, which provides for a more purified fiber with reduced concentrations of seroins, sericins, or other fiber coating proteins (e.g., mucins, P25-like protein, enzy mes). Degumming enriches the fiber for the heavy chain of silk fibroin. Degumming of the Pi silk can be performed using methods available in the art for degumming of silk. In some embodiments, the Pi silk is degummed by incubating the Pi silk in (a) water; (b) a solution containing an alkaline agent; (c) a solution containing a neutral soap solution; or (d) w ith an enzyme that degrades sericin; or a combination of one of more of (a)-(d).

[0015] In some embodiments, the described methods are used to rear genetically modified Pi. The described methods can also be used to form an aqueous Pi silk solution from silk obtained from genetically modified Pi larvae. In some embodiments, the genetically modified Pi contain a genetically modified silk fibroin heavy chain and/or fibroin light chain.

[0016] Also described are aqueous Pi silk solutions or aqueous Pi SF solutions made using any of the described methods.

[0017] In some embodiments, the aqueous Pi silk solutions contain solubilized proteins that include silk fibroin, sericins, seroins, mucins, or any other protein produced by the insect as part of the final silk fiber.

[0018] Also described is purified Pi silk or purified Pi SF made using any of the described methods.

[0019] In some embodiments, the Pi SF is chemically modified. Chemical modification can be used to attach a group or moiety to the Pi SF. The Pi SF can be chemically modified to attach a therapeutic agent (e.g.. a drug or biologically active molecule or API), a reactive group, a tracking molecule, an interaction modifier, or a moiety having affinity for another molecule. The Pi SF can be chemically modified to crosslink the Pi SF to itself or to another polymer.

[0020] The described aqueous Pi silk solutions and aqueous Pi SF solutions or the Pi silk or Pi SF can be utilized in formation of Pi silk-based biomaterials. A Pi silk-based biomaterial can be. but is not limited to. a sponge, a microparticle, a nanoparticle, a film, a hydrogel, an electrospun fiber, a porous silk fibroin material, an implant, or a scaffold. The biomaterial can be linked to, associated with, encapsulate, or provide a substrate for, one or more bioactive substances. The one or more bioactive substances can be a biologically active molecule, a cell, or a combination thereof.

[0021] In some embodiments, the /7 silk or Pi SF is combined with one or more additional polymers in the formation of the biomaterial.

[0022] The described Pi silk solutions and aqueous Pi SF solutions or the Pi silk or Pi SF can be used to form biomaterials for use in tissue regeneration, tissue engineering, wound repair, wound dressing, nerve regeneration, bone regeneration, a coating material for protection or modulation of growth, encapsulating a payload, or drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1. Predicted 2D structure in Pi SF proteins as derived from the EMBOSS 6.5.7 Prediction of secondary structure feature in Geneious Prime®. Lower Panel: Zoomed in prediction to show the variation in predicted secondary structures and the amino acids predicted to participate. Arrows represent regions of beta sheet structure while the cylinders represent alpha helix-like structure. Loops or turns are shown with a U-shaped arrow. The amino acid sequence for the portion of the Pi fibroin heavy chain (NCBI Reference Sequence XP 053613783.1) shown is provided in SEQ ID NO: 22.

[0024] FIG. 2. Formation of SF crystal structures through bound-water interactions.

[0025] FIG. 3. Images showing silk fiber morphology: A. and B. Representative images oiBm cocoon fibers. C. Web-like structures present on the interior ofBrn cocoons. D. and E. Representative SEM images of 77 sheet fibers. F. Web-like structures visible throughout Pi silk sheets.

[0026] FIG. 4. Graphs illustrating silk fiber thermal properties. A) TGA curves of Bm and Pi silk. Samples were run in triplicate, with data represented as an average with confidence intervals of 1 standard deviation. B. DSC curves of Bm and Pi silk. Temperature regions related to bound water (Tw), glass transitions (Tg), and material degradation (Td) are labeled on the plot. All scans were run at heating rates of 10°C/min. [0027] FIG. 5. Images and graphs illustrating Pi fiber mechanical property assessment. A. AFM surface image of Pi fiber (scan size: 700 nm); surface height is depicted in the relative height scale to the right of the image. B. Force map of A depicting elastic moduli ranges for each pixel; map moduli scale is depicted in the relative moduli scale to the right of the image. C. Distribution of elastic moduli values determined by the DMT model. The average elastic modulus was 208 MPa.

[0028] FIG. 6. AFM image of Pi fibers. A. AFM surface image of Pi fiber. Relative surface height of the fiber is depicted by the scale accompanying the image. B. 3D reconstruction of the fiber height profile show n in A.

[0029] FIG. 7. Representative AFM force curve DMT fit model.

[0030] FIG. 8. Silk protein weight in solution. A. MWD of Bm and Pi solutions determined by SDS-PAGE. B. Schematic depicting MWD in high and low degumming times for Bm and Pi solutions and potential polymer-polymer interactions.

[0031] FIG. 9A. Schematic depicting nanoparticle and microparticle fonnation via PVA phase separation using an aqueous Pi silk solution. [0032] FIG 9B. Schematic depicting silk particle formation with a bioactive molecule (upper panel). Images and graphs illustrating incorporation FITC-dextran as a model bioactive molecule and comparison between Bm and Pi particles.

[0033] FIG. 10. Schematic of generalized process flow for solubilization of Pi silk sheets. [0034] FIG. 11. Graphs illustration effect of rearing temperature on Pi silk production. (A) Head capsule size was measured, and silk mats were collected during larval stages of the life cycle. (B) Head capsule measurements characterized how rearing parameters affect the larvae growth cycle (n=4). Rearing parameter impacts on (C) fiber density (number of colored pixels, silk, to total area in a representative image) and (D) fiber diameter. For a given resource availability and population density, each temperature was statistically compared for fiber density and diameter. * p=0.025, ** p=0.01. n=3

[0035] FIG. 12. Mass of silk produced by Pi raised an (A) 24 °C, (B) 26°C, and (C) 30 °C and various resource availabilities and population densities. (A) ** p = 0.002, ** p = 0.0016; (B) * p = 0.048; (C) * p = 0.04, ** p = 0.0059, n=3. (D) Highest silk production for each temperature at 10 larvae/ g diet and 0.72 larvae/ mL, * p = 0.0391.

[0036] FIG. 13. Representative stress strain curve of a Pi silk mat obtained by measuring stress in response to strain at 1% strain per minute.

[0037] FIG. 14. Images of Pi fibers. A. AFM surface image of Pi fiber. Relative surface height of the fiber is depicted by the scale accompanying the image. B. SEM micrograph of a silk mat.

[0038] FIG. 15. SEM Images of Pi raw silk fibers and degummed Pi silk fibers.

[0039] FIG. 16. Graph illustrating molecular weight distribution of Pi silk solution generated through LiBr dissolution. Analyzed through gel electrophoresis (n=2).

[0040] FIG. 17. Image illustrating scheme for carbodiimide coupling with silk fibroin.

[0041] FIG. 18. Image illustrating scheme for cyanuric chloride activated coupling with tyrosine residues of silk fibroin.

[0042] FIG. 19. Image illustrating scheme for acylate grafting onto nucleophilic residues of silk fibroin, namely lysine. Downstream radical polymerization can be induced.

[0043] FIG. 20. Reaction scheme illustrating amine installation on tyrosine residues of silk fibroin through a diazonium coupling reaction.

[0044] FIG. 21. SEM images of isotropic porous Pi silk sponges (0.5 wt/v %).

[0045] FIG. 22. Fluorescence image of Pi silk microparticles (0.25 wt/v %) fabricated through PVA phase separation. Particle diameter ranges between 1-10 pm. [0046] FIG. 23. Fabrication and analysis of a i silk film (0.25 wt/v%). (A) SEM image of silk film cross section. (B) FTIR spectra and (C) relative protein content of Pi silk films compared to native silk fibers.

[0047] FIG. 24. SEM images of i fiber structures. (A) Non-degummed isotropic silk sheet. (B) Degummed isotropic silk sheet. (C) Non-degummed aligned fiber bundle. (D) Degummed aligned fiber bundle.

[0048] FIG. 25. Graph and images illustrating metabolic activity of NHLFs cultured on nondegummed (NDG) and degummed (DG) Pi silk sheets, normalized to tissue culture plastic.

[0049] FIG. 26. SEM and DLA Images illustrating Pi silk particle morphology, size, and polydispersity.

DETAILED DESCRIPTION

I. Definitions

[0050] The scope of the present invention is defined by the claims appended hereto and is not limited by certain embodiments described herein. Those skilled in the art, reading the present specification, will be aware of various modifications that may be equivalent to such described embodiments, or otherwise within the scope of the claims. In general, terms used herein are in accordance with their understood meaning in the art, unless clearly indicated otherwise. Explicit definitions of certain terms are provided below; meanings of these and other terms in particular instances throughout this specification will be clear to those skilled in the art from context. Additional definitions for the following and other terms are set forth throughout the specification. Patent and non-patent literature references cited within this specification, or relevant portions thereof, are incorporated herein by reference in their entireties.

[0051] It should be noted that, as used in this specification and the appended claims, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “an oligomer” includes a plurality of oligomers and the like. The conjunction "or" is to be interpreted in the inclusive sense, i.e., as equivalent to "and/or," unless the inclusive sense would be unreasonable in the context.

[0052] Use of "comprise," "comprises, " "comprising,” "contain," "contains," "containing," "include," "includes," and "including" are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary’ and explanatory only and are not restrictive of the teachings. [0053] In general, the term "about" indicates variation in a quantity' of a component of a composition not having any significant effect on the activity or stability of the composition. The term '‘about’’ means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. , the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 0 to 20%, 0 to 10%, 0 to 5%, or up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. All ranges are to be interpreted as encompassing the endpoints in the absence of express exclusions such as "not including the endpoints"; thus, for example, "within 10-15" includes the values 10 and 15. One skilled in the art will understand that the recited ranges include the end values, as whole numbers in between the end values, and where practical, rational numbers within the range (e g., the range 5-10 includes 5, 6, 7, 8, 9, and 10, and where practical, values such as 6.8, 9.35, etc.).

[0054] The terms “protein.” “polypeptide.” and “peptide,” used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides having modified peptide backbones. The term “domain” refers to any part of a protein or polypeptide having a particular function or structure.

II. Overview

[0055] Described are methods of producing Pi silk, compositions comprising the Pi silk, and methods of using the Pi silk.

[0056] The generation of Pi silk and SF solution is a tunable process and can be utilized to produce polymer solutions with varying properties as a function of growth conditions of the Pi, processing parameters, and purification or the Pi silk.

[0057] Silk fibers are primarily composed of fibroins and sericins, two classes of silk proteins attributed to mechanical strength and a glue-like coating, respectively, that are folded and fused into a double stranded structure through insect silk spinning. Silk fibroin (SF) is a structural protein composed of a heavy chain (-391 kDa) and light chain (-25 kDa). The silk fibroin heavy chain largely consists of amino acid repeats (GASX (SEQ ID NO: 1), ASAX (SEQ ID NO: 2), XGASA (SEQ ID NO: 3), GAGX (SEQ ID NO: 4), GASXASAX (SEQ ID NO: 5), ASAASA (SEQ ID NO: 6), ASAAGX (SEQ ID NO: 7), GAYGX (SEQ ID NO: 8), PVVIIEX (SEQ ID NO: 9), XVVVIX (SEQ ID NO: 10). VVIX (SEQ ID NO: 11). GAVGAX (SEQ ID NO: 12). XAAAAX (SEQ ID NO: 13)) that are primarily responsible for the formation of pl-sheet crystal structures in Pi silk.

[0058] A summary of silk-related terms, descriptions, and common titles referred to in this document are listed in Table 1.

Table 1. Summary of silk nomenclature frequently used in this document.

II. Methods of production of Pi silk

[0059] Described are methods for rearing (growing) Pi for production of Pi silk. The described methods provide for optimizing quantity and/or quality of wandering silk production while maintaining a sustainable colony in a controlled (e.g, laboratory or manufacturing/production) setting. Benefits in the utilization of Pi in biomaterials arise from its ability to be reared in a controlled setting with control over environmental parameters to minimize batch-to-batch variability in produced silk fibers. Pi larvae begin to lay silk, termed wandering silk, in scalable quantities in the 4th instar phase. This wandering silk accumulates in thin sheets that can be collected without interruption of insect pupation, enabling completion of the life cycle.

[0060] In some embodiment the Pi are grown at a temperature of about 24 °C. In some embodiments, the Pi are grown at a temperature of less than 26 °C. In some embodiments, the Pi are grown at a temperature of less than 25 °C. In some embodiments, the Pi are grown at a temperature of about 22 °C to about 26 °C, about 23 °C to about 25 °C, about 23.5 °C to about 24.5 °C, or about 24 °C. In some embodiments, the Pi are grown at a temperature of 24±2 °C, 24±1 °C, 24±0.5 °C, or 24 °C. In some embodiments, the Pi are grown at a temperature of 24±1 °C. In some embodiments, the Pi are grown at a temperature of 24±0.5 °C. In some embodiments, the Pi are grown at a temperature of 24 °C. In some embodiments, the Pi are grown at an average temperature of 24±2 °C, 24±1 °C, 24±0.5 °C or 24 °C. In some embodiments, the Pi are grown at an average temperature of 24±1 °C. In some embodiments, the Pi are grown at an average temperature of 24±0.5 °C. In some embodiments, the Pi are grown at an average temperature of 24 °C.

[0061] In some embodiments, the Pi are grown at a population density of about 0.72 larvae/cm³. In some embodiments, the Pi are grown at a population density of 0.72±0.2 larvae/cm³. 0.72±0.1 larvae/cm³, 0.72±0.05 larvae/cm³, 0.72±0.02 larvae/cm , or 0.72 larvae/cm³. In some embodiments, the Pi are grown at a population density of about 0.52 to about 0.92 larvae/cm³, about 0.62 to about 0.82 larvae/cm³, about 0.65 to about 0.8 larvae/cm³, 0.67 to about 0.79 larvae/cm³, about 0.70 to about 0.74 larvae/cm³, or about 0.71 to about 0.73 larvae/cm³.

[0062] In some embodiments, the Pi are grown at a temperature of 24±1 °C at a population density of 0.72±0.2 larvae/cm³. In some embodiments, the Pi are grown at a temperature of 24±0.5 °C at a population density⁷ of 0.72±0. 1 larvae/cm³. In some embodiments, the Pi are grown at a temperature of 24±0.5 °C at a population density' of 0.72±0.05 larvae/cm³. In some embodiments, the Pi are grown at a temperature of about 24 °C at a population density of about 0.72 larvae/cm³. In some embodiments, the Pi are grown at a temperature of 24 °C at a population density of 0.72 larvae/cm³.

[0063] The Pi can be fed any suitable diet known in the art for growth of Pi. The diet can include, but is not limited to, a standardized wheat bran diet. An exemplary wheat bran diet can include, wheat bran, organic buckwheat honey, glycerin, deionized water, insect vitamin mix, and brewer's yeast (see Tables 6 or 10). Resource availability' (amount of diet provided per larva) can affect the quantity and/or quality of the produced raw silk. In some embodiments, the Pi are provided with about 1 gram of diet per 10 larvae (i.e., 10 larvae/g diet). In some embodiments, the Pi are provided with about 1 gram diet per 5 to 20 larvae, about 1 gram diet per 5 to 15 larvae, about I gram of diet per 7 to 13 larvae, about 1 gram of diet per 8 to 12 larvae, or about 1 gram of diet per 9 to 11 larvae. In some embodiments, the Pi are grown at a resource availability' of about 5 to about 20 larva per gram of diet, about 5 to about 15 larva per gram of diet, about 7 to about 13 larva per gram of diet, about 8 to about 12 larva per gram of diet, about 9 to about 11 larva per gram of diet, or about 10 larva per gram of diet. In some embodiments, the Pi are grown at a resource availability of 10 larva per gram of diet. In some embodiments, the Pi are grown at a resource availability of about 1 larva per 0.05-0.2 gram of diet, about 1 larva per 0.067-0.2 gram of diet, about 1 larva per 0.075-0. 15 gram of diet, about 1 larva per 0.08-0.125 gram of diet, about 1 larva per 0.09-0.11 gram of diet, or about 1 larva per 0.1 gram of diet. In some embodiments, the Pi are grown at a resource availability of about 1 larva per about 0. 1 gram of diet. In some embodiments, the Pi are grown at a resource availability of 1 larva per 0. 1 gram of diet (z.e., 10 larvae/g diet).

[0064] Dietary supplements can affect the conformational transition, thermal characteristics, mechanical attributes, and metallic ion composition of the produced silk. Thus, modification of diet can be used to modulate properties of the produced silk fibers. Silk fibers having different properties can be produced for different applications. In some embodiments, different dietary supplements can be added to the standard diet. Adding supplements to the standard diet provides for more accurate delivery to the feeding larv ae compared to conventional techniques involving the application of an aqueous solution of supplements onto leaves or insects. Additives or supplements can be combined with the standard Pi diet at any point in their life cycle.

[0065] In some embodiments, one or more amino acids are added to the standard diet. Addition of amino acids to the standard diet can induce a shift towards |3-sheet protein structures in the produced silk or SF. increase fiber strength of the produced silk or SF, or increase residues available for chemical modification in the produced silk or SF.

[0066] In some embodiments, metallic ions (e.g., Ca²⁺, Cu ² . K⁺, or Zn²⁺) are added to the standard diet. Addition of metallic ions to the standard diet can induce a shift towards (3-sheet protein structures and/or increased fiber strength.

[0067] In some embodiments, insect juvenile hormone is added to the standard diet. Addition of juvenile hormone to the standard diet can be used to prolong time in larval stage to increase silk production.

[0068] In some embodiments, nanoparticles (e.g., Cu or CaCCh) are added to the standard diet. Addition of nanoparticles to the standard diet can induce a shift towards [3-sheet protein structures and/or increased fiber strength.

[0069] In some embodiments, nanofibers or nanotubes (e.g., cellulose, carbon, or graphene) are added to the standard diet. Addition of nanofibers or nanotubes to the standard diet can induce a shift towards [3-sheet protein structures and/or increased fiber strength.

[0070] In some embodiments, plastics (e.g., polyethylene terephthalate (PET), low-density polyethylene (LDPE), high-density polyethylene (HDPE), polypropylene (PP), or polystyrene (PS)) are added to the standard diet. Addition of plastics to the standard diet can provide for alternative recycling or processing of plastics, chemical modification of plastics additives in silk.

[0071] In some embodiments, a dye can be added to the standard diet. Addition of dye to the standard diet can be used to produce colored silk for textiles, or fluorescence for optics or photonics applications.

[0072] In some embodiments, the Pi are grown at a temperature of 24±1 °C, at resource availability of about 5 to about 15 larva per gram of diet. In some embodiments, the Pi are grow n at a temperature of 24±0.5 °C, at resource availability of about 8 to about 12 larva per gram of diet. In some embodiments, the Pi are grown at a temperature of 24±0.5 °C, at resource availability of about 9 to about 11 larva per gram of diet. In some embodiments, the Pi are grown at a temperature of about 24 °C, at resource availability of about 10 larva per gram of diet. In some embodiments, the Pi are grown at a temperature of 24 °C, at resource availability of 10 larv a per gram of diet.

[0073] In some embodiments, the Pi are grown at a population density of 0.72±0.2 larvae/cm³ with a resource availability of about 5 to about 15 larva per gram of diet. In some embodiments, the Pi are grown at a population density of 0.72±0.1 larvae/cm³ w ith a resource availability of about 8 to about 12 larva per gram of diet. In some embodiments, the Pi are grown at a population density of 0.72±0.05 larvae/cm³ with a resource availability of about 9 to about 11 larva per gram of diet. In some embodiments, the Pi are grown at a population density of about 0.72 larvae/cm³ with a resource availability of about 10 larv a per gram of diet. In some embodiments, the Pi are grown at a population density of 0.72 larvae/cm³ with a resource availability of 10 larva per gram of diet.

[0074] In some embodiments, the Pi are grown at a temperature of 24± 1 °C and a population density of 0.72±0.2 larvae/cm³, with a resource availability of about 5 to about 15 larva per gram of diet. In some embodiments, the Pi are grown at a temperature of 24±0.5 °C and a population density of 0.72±0.1 larvae/cm³, with a resource availability of about 8 to about 12 larva per gram of diet. In some embodiments, the Pi are grown at a temperature of 24±0.5 °C and a population density of 0.72±0.05 larvae/cm³, with a resource availability of about 9 to about 11 larva per gram of diet.

[0075] In some embodiments, the Pi are grown at a temperature of about 24 °C and a population density of about 0.72 larvae/cm³. with a resource availability of about 10 larva per gram of diet. In some embodiments, the Pi are grown at a temperature of 24 °C and a population density of 0.72 larvae/cm³, with a resource availability of 10 larva per gram of diet.

[0076] The Pi can be grown in any container (or chamber) suitable for insect culture (e.g., insect rearing). In some embodiments, the container contains solid walls to facilitate collection of wandering silk. The containers can be, but are not limited to, plastic containers. Using the described methods, Pi enter the 4^th larval stage produce significant amounts of silk fibers while wandering around their environment (wandering silk). The wandering silk accumulates in sheets on the side of the rearing container. These silk sheets can be collected during Pi pupation, without interrupting the life cycle of the population. Adults can be allowed to emerge and can then be collected to produce eggs and start a new generation.

III. Manufacture of Aqueous Pi Silk Solutions

[0077] Described are methods of generating aqueous Pi silk solutions from raw Pi silk. The described methods comprise (a) dissolution of raw Pi silk to form dissolved Pi silk, (b) filtration of the dissolved Pi silk, and (c) dialysis of the filtered silk.

[0078] In some embodiments, dissolution of raw Pi silk comprises salt dissolution. In some embodiments, salt dissolution of Pi silk comprises contacting the Pi silk with a high concentration of salt. Salt dissolution of Pi silk comprises contacting the Pi silk with a salt solution, wherein the salt solution contains an appropriate concentration of the salt. The salt can be, but is not limited to, LiBr, Ca(NCh)2, LiNCh, Mg(NCh)2, MgCh, or CaCb. In some embodiments, concentration of the salt in the salt solution is about 1 molar (M) to about 10 M. In some embodiments, concentration of the salt in the salt solution is about 1 M, about 2 M, about 3 M, about 4 M, about 5 M, about 6 M, about 7 M, about 8 M, about 9 M, or about 10 M. In some embodiments, concentration of the salt in the salt solution is about 1 M to about 2

M. In some embodiments, concentration of the salt in the salt solution is about 3 M to about 5

M. In some embodiments, concentration of the salt in the salt solution is about 5 M to about 7

M. In some embodiments, concentration of the salt in the salt solution is about 5 M to about 8

M. In some embodiments, concentration of the salt in the salt solution is about 6 M to about 8

M. In some embodiments, concentration of the salt in the salt solution is about 7 M to about 9.3 M. In some embodiments, concentration of the salt in the salt solution is about 8 M to about 9.3 M. In some embodiments, concentration of the salt in the salt solution is about 9.3 M. An increase in the concentration of the salt can lead to a decrease in the dissolution time. A decrease in the concentration of the salt can lead to an increase in the dissolution time. [0079] In some embodiments, dissolution of raw Pi silk comprises incubating the raw Pi silk in a 7-9.3 M LiBr solution.

[0080] In some embodiments, dissolution of raw Pi silk comprises incubating the raw Pi silk in a 5-8 M Ca(NOs)2 solution.

[0081] In some embodiments, dissolution of raw Pi silk comprises incubating the raw Pi silk in a 6-8 M Ca(NO?)2 solution.

[0082] In some embodiments, dissolution of raw Pi silk comprises incubating the raw Pi silk in a 1-2 M LiNCh solution.

[0083] In some embodiments, dissolution of raw Pi silk comprises incubating the raw Pi silk in a 3-5 M Mg(NOs)2 solution.

[0084] In some embodiments, dissolution of raw Pi silk comprises incubating the raw Pi silk in a 5-7 M MgCb solution.

[0085] In some embodiments, dissolution of raw Pi silk comprises incubating the raw Pi silk in a 5-8 M CaCb solution.

[0086] In some embodiments, dissolution of raw Pi silk comprises incubating the raw Pi silk in the salt solution at a Pi silk to salt ratio of about 1 :3 to about 1 :200 grams of raw silk fibers or degummed silk fibers to mL of total solution. In some embodiments, dissolution of raw Pi silk comprises incubating the raw Pi silk in the salt solution at a Pi silk to salt ratio of about 1:3 to about 1 :50. In some embodiments, the Pi silk is incubated in the salt solution at a Pi silk to salt ratio of about 1 :3. In some embodiments, the Pi silk is incubated in the salt solution at a Pi silk to salt ratio of about 1 :4. In some embodiments, the Pi silk is incubated in the salt solution at a Pi silk to salt ratio of about 1:5. In some embodiments, the /’/ silk is incubated in the salt solution at a Pi silk to salt ratio of about 1 :6. In some embodiments, the Pi silk is incubated in the salt solution at a Pi silk to salt ratio of about 1 :7. In some embodiments, the Pi silk is incubated in the salt solution at a Pi silk to salt ratio of about 1 :8. In some embodiments, the Pi silk is incubated in the salt solution at a Pi silk to salt ratio of about 1:9. In some embodiments, the Pi silk is incubated in the salt solution at a Pi silk to salt ratio of about 1: 10. In some embodiments, the silk is incubated in the salt solution at aPi silk to salt ratio of about 1 : 15. In some embodiments, the /7 silk is incubated in the salt solution at a Pi silk to salt ratio of about 1 :20. In some embodiments, the Pi silk is incubated in the salt solution at a Pi silk to salt ratio of about 1:25. In some embodiments, the Pi silk is incubated in the salt solution at a Pi silk to salt ratio of about 1 :30. In some embodiments, the Pi silk is incubated in the salt solution at a Pi silk to salt ratio of about 1 :35. In some embodiments, the Pi silk is incubated in the salt solution at a Pi silk to salt ratio of about 1 :40. In some embodiments, the Pi silk is incubated in the salt solution at a Pi silk to salt ratio of about 1 :45. In some embodiments, the Pi silk is incubated in the salt solution at a Pi silk to salt ratio of about 1 :50. An increase the ratio of the Pi silk to salt solution can lead to a decrease in the dissolution time. A decrease the ratio of Pi silk to salt solution can lead to an increase in the dissolution time.

[0087] In some embodiments, dissolution of raw Pi silk comprises incubating the raw Pi silk in the salt solution at a temperature of about 25 °C to about 100 °C. In some embodiments, the Pi silk is incubated with the salt solution at about 60 °C to about 80 °C. In some embodiments, the Pi silk is incubated with the salt solution at about 25 °C. In some embodiments, the Pi silk is incubated with the salt solution at about 30 °C. In some embodiments, the /7 silk is incubated with the salt solution at about 35 °C. In some embodiments, the Pi silk is incubated with the salt solution at about 40 °C. In some embodiments, the Pi silk is incubated with the salt solution at about 45 °C. In some embodiments, the Pi silk is incubated with the salt solution at about

50 °C. In some embodiments, the Pi silk is incubated with the salt solution at about 55 °C. In some embodiments, the Pi silk is incubated with the salt solution at about 60 °C. In some embodiments, the Pi silk is incubated with the salt solution at about 65 °C. In some embodiments, the Pi silk is incubated with the

solution at about 70 °C. In some embodiments, the Pi silk is incubated with the salt solution at about 75 °C. In some embodiments, the Pi silk is incubated with the salt solution at about 80 °C. In some embodiments,

is incubated with the salt solution at about 85 °C. In some embodiments, the Pi silk is incubated with the salt solution at about 90 °C. In some embodiments, the Pi silk is incubated with the

solution at about 95 °C. In some embodiments, the Pi silk is incubated with the salt solution at about 100 °C. An increase the incubation temperature can lead to a decrease in the dissolution time. A decrease the incubation temperature can lead to an increase in the dissolution time.

[0088] In some embodiments, dissolution of raw Pi silk comprises incubating the raw Pi silk in the salt solution for about 2 hours to about 48 hours. In some embodiments, the Pi silk is incubated with the salt solution for about 2 hours to about 3 hours. In some embodiments, the Pi silk is incubated with the salt solution for about 4 hours. In some embodiments, the Pi silk is incubated with the salt solution for about 6 hours. In some embodiments, the Pi silk is incubated with the salt solution for about 8 hours. In some embodiments, the Pi silk is incubated with the salt solution for about 10 h. In some embodiments, the Pi silk is incubated with the salt solution for about 12 h. In some embodiments, the Pi silk is incubated with the salt solution for about 15 h. In some embodiments, the Pi silk is incubated with the salt solution for about 20 h. In some embodiments, the Pi silk is incubated with the salt solution for about 24 h to about 48 h. In some embodiments, the Pi silk is incubated with the salt solution for about 48 h. [0089] The salt solution containing the Pi silk can be mixed (e.g., stirred or agitated) during the dissolution step. Alternatively, the Pi silk can be incubated in the salt solution without mixing (e.g., stirring or agitating) during the dissolution step.

[0090] In some embodiments, dissolution of raw Pi silk comprises incubating the Pi silk in a 7-9.3 M LiBr solution at a silk:salt solution ratio of 1:30 to 1:50 at 25 °C to 100 °C for 2 to 3 h, either with or without mixing.

[0091] In some embodiments, dissolution of raw Pi silk comprises incubating the Pi silk in a 8-9.3 M LiBr solution at a silk:salt solution ratio of 1 :30 to 1 :50 at 25 °C to 100 °C for 2 to 24 h, either with or without mixing.

[0092] In some embodiments, dissolution of raw Pi silk comprises incubating the Pi silk in a 9.3 M LiBr solution at a silk:salt solution ratio of 1:4 at 60 °C for 2 to 3 h, either with or without mixing.

[0093] In some embodiments, dissolution of raw Pi silk comprises incubating the Pi silk in a 5-8 M Ca(NOs)2 solution at a silk: salt solution ratio of 1:30 to 1:50 at 25 °C to 100 °C for 48 h, either with or without mixing.

[0094] In some embodiments, dissolution of raw Pi silk comprises incubating the Pi silk in a 5-8 M Ca(NOs)2 solution at a silk:salt solution ratio of 1 :30 to 1 :50 at 60 °C to 80 °C for 48 h, either with or without mixing.

[0095] In some embodiments, dissolution of raw Pi silk comprises incubating the Pi silk in a 6-8 M Ca(NCh)2 solution at a silk: salt solution ratio of 1 :30 to 1:50 at 60 °C to 80 °C for 3 h, either with or without mixing.

[0096] In some embodiments, dissolution of raw Pi silk comprises incubating the Pi silk in a 6-8 M Ca(NO?)2 solution at a silk: salt solution ratio of 1 : 30 to 1 :50 at 20 °C to 26 °C for 24 h to 48 h, either with or without mixing.

[0097] In some embodiments, dissolution of raw Pi silk comprises incubating the Pi silk in a 1-2 M LiNOs solution at a silk:salt solution ratio of 1 :30 to 1:50 at 25 °C to 100 °C.

[0098] In some embodiments, dissolution of raw Pi silk comprises incubating the Pi silk in a 3-5 M Mg(NOs)2 solution at a silk: salt solution ratio of 1:30 to 1 :50 at 25 °C to 100 °C.

[0099] In some embodiments, dissolution of raw Pi silk comprises incubating the Pi silk in a 5-7 M MgCk solution at a silk: salt solution ratio of 1 :30 to 1 :50 at 25 °C to 100 °C.

[0100] In some embodiments, dissolution of raw Pi silk comprises incubating the Pi silk in a 5-8 M CaCk solution at a silk:salt solution ratio of 1 :30 to 1 :50 at 25 °C to 100 °C. [0101] Filtration of the dissolved Pi silk removes residual insect food, waste, and optionally other contaminants or impurities from the Pi silk. In some embodiments, filtration of the dissolved Pi silk comprises passing the dissolved Pi silk through a 22-25 pm pore filter. The 22-25 pm pore filter can be, but is not limited to, a 22-25 pm pore miracloth. Miracloth is a filtration material made of rayon-polyester with an acrylic binder. The dissolved Pi silk can be passed through the filter using a gravity filtration method, a vacuum filtration method, or another method typical in the art for passing a liquid through a filter.

[0102] In some embodiments, filtration of the dissolved Pi silk comprises passing the dissolved Pi silk through a 22-25 pm pore miracloth using a gravity filtration method.

[0103] In some embodiments, the filtration of the dissolved Pi silk comprises passing the dissolved Pi silk through a filter cascade from PluriSelect®, which could include the use of 1, 5, 10, 20, and/or 30 pm pore PET filters. The filters can be, but are not limited to filter membranes made from PET. The filters can be used in combination or individually. The dissolved Pi silk can be passed through the filter using a gravity filtration method, a vacuum filtration method, or another method typical in the art for passing a liquid through a filter.

[0104] Dialysis of the filtered Pi silk removes salt used in the dissolution step. Dialysis of the Pi silk can also increase stability the aqueous Pi silk solution product. In some embodiments, dialysis of the filtered Pi silk comprises dialyzing the filtered Pi silk using a dialysis membrane having a molecule weight cut-off that is lower than the desired size of the Pi silk fibroin in the aqueous Pi silk solution. In some embodiments, dialysis of the filtered Pi silk comprises dialyzing the filtered Pi silk using a 3.5 kDa dialysis membrane. Dialysis can be performed at about 4 °C to about 25 °C for about 4 to about 48 h.

[0105] In some embodiments, the filtered Pi silk is dialyzed against water (e.g., deionized water or ultrapure water (United States Pharmacopeia, resistivity of 18.2 MQ.cm at 25 °C, TOC < 10 ppb, and bacterial count <10 CFU/ml)), a salt solution, a buffered solution, or a PEG-containing solution (dialysis solution) of a combination thereof. The dialysis solution can be changed 2 or more times. In some embodiments, the dialysis solution can be changed 2, 3, 4, 5, 6, 7. 8, 9, 10, or more times.

[0106] In some embodiments, the filtered Pi silk is dialyzed against 2 or more solutions of decreasing salt concentration. The salt can be the same salt as used in the dissolution step or a different salt. The different salt can be, but is not limited to, NaCl or HEPES buffer.

[0107] In some embodiments, dialysis of the filtered Pi silk comprises dialyzing at 4 °C to 26 °C against 5-10 changes of ultrapure water dialysis solution over 48 h, with stirring, using a 3.5 kDa dialysis membrane. [0108] In some embodiments, dialysis of the filtered Pi silk comprises dialyzing against 5-10 NaCl solutions and/or HEPES buffers of decreasing concentrations for 48 hours at 4-26 °C using a 3.5 kDa dialysis membrane.

[0109] In some embodiments, dialysis of the filtered Pi silk comprises dialyzing against 5- 10 LiBr (or CafNCh ) solutions of decreasing concentrations for 48 hours at 4-26°C using a 3.5 kDa dialysis membrane.

[0110] In some embodiments, tangential flow filtration can be performed on the filtered Pi silk solubilized Pi silk proteins and salt solvent in lieu of or prior to dialysis.

[OHl] In some embodiments, the Pi silk is degummed. Degumming comprises removing sericins, and optionally other components, from the Pi silk fibroin. Degumming may be performed at any time in the purification of aqueous Pi silk solution of manufacture of a Pi silk material. In some embodiments, degumming is performed prior to dissolution (z.e., degumming of raw Pi silk). In some embodiments, degumming is performed after dissolution. In some embodiments, degumming is performed after filtration. In some embodiments, degumming is performed after dialysis.

[0112] In some embodiments, degumming Pi silk comprises:

(a) incubating the Pi silk in water;

(b) incubating the Pi silk in a solution containing an alkaline agent;

(c) incubating the Pi silk in a neutral soap solution;

(d) incubating the Pi silk with an enzyme that degrades sericin; or

(e) a combination of one or more of (a)-(d) (e.g, (b) and (c); (b) and (d); (c) and (d); or (b), (c), and (d)).

[0113] The Pi silk can be degummed by incubating in the water, in the alkaline agent solution, in the neutral soap solution, or with the enzyme for about 5 minutes to about 60 minutes. For combinations, steps can be performed sequentially (e.g, incubating the Pi silk with an alkaline agent solution followed by incubating of the alkaline agent solution treated Pi silk with neutral soap solution and/or an enzyme solution) or simultaneously (e.g, incubating the Pi silk with an alkaline solution containing a neutral soap and/or an enzyme). In some embodiments, degumming the Pi silk comprises incubating the silk in an alkaline solution containing a neutral soap. In some embodiments, degumming the Pi silk comprises incubating the silk in a Na2CO3Solution containing sodium lauryl sulfate. In some embodiments, degumming the Pi silk comprises incubating the silk in an alkaline solution containing a neutral soap and an enzyme.

[0114] The alkaline agent can be, but is not limited to, sodium carbonate (Na2CCh). [0115] The neutral soap can be, but is not limited to, sodium laury l sulfate.

[0116] The enzyme can be, but is not limited to, a protease. The protease can be, but is not limited to, papain, protease XIV, or collagenase.

[0117] Other degumming methods available in the art, such as methods to remove sericin from other types of silk, may also be used to degum the Pi silk.

[0118] In some embodiments, the Pi silk is degummed and the aqueous Pi silk solution comprises, consists essentially of, or consists of Pi silk fibroin in an aqueous solution. Degumming can be performed to remove at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the Pi sericin.

[0119] The aqueous Pi silk solution or aqueous Pi silk fibroin solution (if the Pi silk solution is degummed) can be further purified, concentrated, or size fractionated. Additional purification can be performed using methods available in the art for protein purification. Such purification methods include, but are not limited to, filtration, tangential flow filtration, ultrafiltration, and chromatography (e g., size exclusion, ion exchange, or affinity chromatography). Concentration of the Pi silk can be performed using methods available in the art for protein concentration. Such concentration methods include, but are not limited to, dialysis against a water absorbing polymer (e.g. , 10% (w/v) PEG (10,000 MW)), precipitation, filtration concentrators, and tangential flow filtration. Size fractionation of the Pi silk can be performed using methods available in the art for protein size fractionation. Such size fractionation methods include, but are not limited to, tangential flow filtration, dialysis against membranes having different molecule weight cut-off sized, and size exclusion chromatography .

[0120] Size fractionation can be performed to (a) isolate Pi silk or Pi silk fibroin having a molecule weight below a defined molecular weight; (b) isolate Pi silk or Pi silk fibroin having a molecule weight above a defined molecular weight; (c) isolate Pi silk or Pi silk fibroin having a molecule weight in a defined range; or (d) isolate Pi silk or Pi silk fibroin having a reduced poly dispersity.

IV. Aqueous Pi silk solutions

[0121] Also described are aqueous Pi silk solutions, and Pi silk and Pi SF made using the described methods. [0122] In some embodiments, the Pi SF generated using the described methods contain Pi silk fibroin having population sizes >230 kDa, about 174 kDa, about 162 kDa, about 150 kDa, about 76 kDa, about 26 kDa, and about 7 kDa as visualized by SDS-PAGE electrophoresis.

[0123] In some embodiments, the Pi SF generated using the described methods have a diameter of about 1 to about 2.5 pm. In some embodiments, the Pi SF generated using the described methods have a diameter of 1.3±0.46 pm, 2.1 ±0.5 pm, or 2. 14 ±0.46 pm.

[0124] In some embodiments, the Pi SF generated using the described methods have a diameter of 1.3 ±0.1 pm, 1.4 ±0.2 pm, 1.5 ±0.1, pm, 1.6 ±0.2 pm, or 1.9 ±0.3 pm.

[0125] In some embodiments, the Pi silk or Pi SF is modified. The Pi silk or Pi SF can be genetically modified or chemically modified. Modification of Pi SF can be used to alter a mechanical or biochemical property of the Pi SF, or to modify an interaction of the Pi SF.

[0126] The Pi SF can be chemically modified using methods available in the art for chemical modification of protein or Bombyx silk fibroin. Chemical modification includes, but is not limited to, covalently linking one or more groups or moieties to the

SF and crosslinking the Pi SF. The group or moiety linked to the Pi SF can be, but is not limited to, a therapeutic agent (e.g. a drug or bioactive molecule), a reactive group, a tracking molecule, an interaction modifier, or a moiety having affinity for another molecule. The moiety having affinity for another molecule includes, but is not limited to, an antibody binding domain, a ligand, or a ligand binding molecule (e g., a receptor, avidin, or streptavidin). The /7 SF can be crosslinked to itself or to another polymer.

[0127] Chemical modification typically modifies a reactive group in the Pi SF. Reactive groups include, carboxyl groups, amine groups, thiol groups, hydroxyl groups. Reactive groups present in Pi SF include, but are not limited to, the amino-terminal amino group, the carboxyterminal carboxyl group, the carboxyl groups of aspartate and glutamate, the amino groups of lysine and histidine, the thiol groups of cysteine and methionine, and the hydroxyl groups of tryptophan, tyrosine. In some embodiments, a Pi SF may also be modified at an asparagine, glutamine, or serine residues.

[0128] Chemical modification of Pi SF includes, but is not limited to, carbodiimide coupling, cyanuric chloride activated coupling, poly(methacrylate) grafting, and diazonium coupling.

V. Pi silk-based products and Pi silk-based biomaterials

[0129] Any of the aqueous Pi silk solutions (including Pi silk. Pi SF, modified Pi silk, or modified Pi SF) can be dried to form a dried Pi silk product. Thus, also described are Pi silk, Pi SF, modified Pi silk and modified Pi SF (collectively Pi silk products) made using the described methods.

[0130] Any of the aqueous Pi silk solutions or Pi silk products can be used to manufacture of a variety of silk-based products or silk-based biomaterials. Such silk-based products or silkbased biomaterials include, but are not limited to, sponges, microparticles, nanoparticles, films, hydrogels, electrospun fibers, porous silk fibroin materials, and scaffolds (e.g.. biocompatible scaffolds). In some embodiments, the Pi silk-based products or silk-based biomaterials are for use in biomedical, agricultural, cosmetic, or environmental applications. In some embodiments, the Pi silk-based products or silk-based biomaterials are for use in delivery of pharmaceutical agents. In some embodiments, the Pi silk-based products or silk-based biomaterials are for use in providing a scaffold for one or more pharmaceutical agents, including, but not limited to, drugs and/or cells. In some embodiments, the Pi silk-based products or silk-based biomaterials are for use as medical implants. Silk-based products can be applied to a variety of fields due to their ability to entrap cargo while maintaining stability', low immunogenicity, and the ability to biodegrade into byproducts harmless to human health or the environment.

[0131] A Pi silk sponge comprises a semicrystalline three-dimensional polymer matrix with interconnected pores throughout the structure. Porosity⁷, density⁷, cry stallinity⁷, compressive modulus, and mechanical strength of the porous sponge can be modified to match, mimic, or approximate natural tissues for tissue engineering, encapsulate payloads for delivery of payloads for medical or environmental applications, or achieve filtration of particulates for medical or environmental applications. In some embodiments, the three-dimensional structure and/or morphology of a Pi silk sponge can be modified through utilization of molds or patterns during production. A Pi silk sponge can be isotropic or anisotropic.

[0132] Pi silk microparticles and nanoparticles comprise semi-crystalline three-dimensional polymer matrices that are capable of encapsulation one or more cargo molecules (i.e., payload). In some embodiments, microparticles and nanoparticle are capable of release of the payload under appropriate conditions. Morphology⁷, size, polydispersity, crystallinity, and mechanical strength of a Pi microparticles or nanoparticle can be customized to the payload or use.

[0133] In some embodiments, Pi silk microparticles and nanoparticles (particles) are produced by (a) forming a Pi silk product solution comprising 1 - 10% Pi silk produce by weight in water; (b) blending the polymer solution with polyvinyl alcohol (PVA); (c) sonicating the solution to form the particles through hydrophobic collapse; (d) removing water; (e) removing PVA solvent through dissolution of the polymer-PVA film in water and centrifugation to collect the polymer particle pellet; and (f) re-distributing the particles in aqueous solution through sonication. Particle size can be modulated be varying the Pi silk product concentration, modulating sonication amplitude and time, modulating temperature during water evaporation from the polymer-PVA blend, and modulating the weight ratio of Pi silk product to PVA.

[0134] In some embodiments, Pi silk microparticles and nanoparticles can be used in vaccines. Such vaccines include, but are not limited to, mRNA vaccines.

[0135] A Pi silk film comprises a semicrystalline, continuous largely two-dimensional polymer matrix. Porosity, density, crystallinity. Young’s modulus, mechanical strength, and elongation or torsional properties of the Pi silk film can be modified to match, mimic, or approximate natural tissues. In some embodiments, a Pi silk film is produced by (a) forming a Pi silk product solution comprising 1-10% Pi silk product by weight in water; (b) placing the solution into a flat or patterned form; (c) removing water from the solution; and (d) inducing crystallinity of the Pi silk, thereby forming water-insoluble structure. Inducing crystallinity comprises washing with methanol, water vapor annealing, or autoclaving of the dried, insoluble film.

[0136] Pi silk films can be used in tissue regeneration, tissue engineering, wound repair, wound dressings, nerve regeneration, coating materials for protection or modulation of growth, or encapsulating payloads (including pharmaceutical agents).

[0137] A Pi silk hydrogel comprises a swollen, semicrystalline three-dimensional polymer matrix having the ability to retain or release fluids from the hydrogel. Swelling, permeability, degradation, compressive modulus, and mechanical strength of the hydrogel can be modified according to the intended used of the Pi silk hydrogel. In some embodiments, the Pi silk hydrogel can be made to match, mimic, or approximate a natural tissue. In some embodiments, the three-dimensional structure and/or morphology of a Pi silk hydrogel can be modified through utilization of molds or patterns during production. In some embodiments, a Pi silk hydrogel is produced by (a) forming a Pi silk product solution comprising 1-10% Pi silk product by weight in water; (b) crosslinking silk fibers of the Pi silk product; (c) placing the crosslinked silk fibers into a form or pattern; and (g) incubating the crosslinked silk fibers under conditions suitable for forming a gel. In some embodiments, crosslinking silk fibers comprises (a) sonicating the solution, (b) shifting the temperature (e.g., heating) of the solution, (c) shifting pH of the solution, (d) electrogelation, (e) applying high pressure to the solution, or (f) chemically crosslinking the silk fibers.

[0138] Pi silk hydrogels can be used, for example, for encapsulating payloads (including pharmaceutical agents), and filtering or entrapping of particulates. [0139] Pi silk electrospun fibers comprise semicrystalline three-dimensional polymer spun into micro- or nanoscale fibrous structures. Electrospun fibers can be spun into thin sheets, multi-layered sponges, or pattern molds to modify the three-dimensional structure of the product. Fiber diameter, porosity, crystallinity, surface roughness, and mechanical strength of the electrospun fiber structure can be modified according to a desire used. In some embodiments, Pi silk electrospun fibers can be made to match, mimic, or approximate natural tissues. Pi silk electrospun fibers can be isotropic or anisotropic.

[0140] In some embodiments, isotropic Pi silk electrospun fiber structures are produced by

(a) forming a Pi silk product solution comprising 1-10% Pi silk product by weight in water;

(b) mixing the solution with 5% wt/v PEO in water; (c) drawing the solution into a syringe or similar device equipped with, for example, a 16-gauge needle; (d) applying a voltage gradient between the needle and a collection surface; (e) spinning fibers onto the collection surface; (I) evaporating the water to form a water-insoluble structure; (g) removing the PEO; and (h) treating the spun fibers to induce crystallinity. Inducing crystallinity within the spun fibers includes: (i) washing with methanol, (ii) water vapor annealing, or (iii) autoclaving of a dried, insoluble fiber structure. In some embodiments the needle and collection surface are about 5 to about 20 cm apart. For anisotropic Pi silk electrospun fiber, the spun fibers are collected on a rotating mandrel.

[0141] In some embodiments, composite electrospun fibers can be achieved through dualsyringe electrospinning to generate overlapping layers of electrospun Pi silk fibers and an additional structural or bioactive polymer.

[0142] APi silk biocompatible scaffold comprises atwo- or three-dimensional material (<?.g., sponge, film, particle, hydrogel, or fiber) which is linked to, is associated with, or provides a substrate for one or more bioactive substances. The bioactive substance can be a biologically active molecule, a cell, or a combination thereof. The biologically active molecule can be, but is not limited to, an active pharmaceutical ingredient (API), a hormone, or a grow th factor. The cell can be, but is not limited to, a stem cell (e.g., an embry onic stem cell (ESC), adult stem cell (ASC), or an induced pluripotent stem cells (iPSCs)), an immune cell, a somatic cell, a fibroblast (e.g.. anormal human lung fibroblasts), an epithelial cell, or an endothelial cell. Such scaffolds have use in, for example, wound healing, tissue engineering, drug/biologic delivery, bone regeneration, and the like. Such scaffolds also have use in the manufacture of medical devices and implants. Pi silk biocompatible scaffolds can be formed in molds to have a desired shape or they may be 3D printed. [0143] Any of the Pi silk-based products or biomaterials can be combined with one or more additional structural of bioactive polymer in the formation of the silk-based product or biomaterial.

[0144] In another embodiment, composite silk hydrogels can be achieved through generation of a double-network hydrogel with Pi silk and an additional structural or bioactive polymer.

EXAMPLES

Example 1. Characterization of Pi silk: silk fiber structure and physical properties.

[0145] Interactions of specific functional groups on SF with water molecules influence silk fiber structure by induction or collapse of crystal structures. In silk spinning, the highly repetitive GAGAGS region of water-soluble SF undergoes rapid phase transitions to form antiparallel 0-sheet crystals (FIG. 2). Beta sheet structures form when water is removed from the protein backbone. The degree of hydration of the fiber impacts macromolecular stability of SF and the thermodynamics of phase transitions and cry stallization. Dehydration of fibers causes removal of bound water, transitioning protein structure from a water soluble, elastic state to a thermodynamically favored, glassy state. Many techniques can supply molecular information about silk structure including: 1) imaging by scanning and transmission electron microscopy’ (SEM and TEM), 2) cry stalline content with small and wide-angle X-ray scattering. 3) molecular structure through solid-state nuclear magnetic resonance (ss-NMR), and4) protein conformation with Fourier transform infrared spectroscopy (FTIR) and Raman.

[0146] The advantageous thermophysical properties of silk fibers are often attributed to crystalline units acting as crosslinks between silk proteins in the fiber. Differences in thermal properties — phase transition temperature, degradation rate, bound water content, and molecular mobility during glass transitions — between different silk fibers is influenced by' polymer chain mobility and evaluated through a combination of thermal gravimetric analy sis (TGA) w ith differential scanning calorimetry (DSC) or temperature- modulated DSC. Assessment of physical properties such as fiber elastic modulus, tensile strength, recovery, and adhesion can be accomplished on single fibers by dynamic mechanical analysis (DMA) and tensile testing, or in microscopic fiber regions through AFM and micro- and nanoindenters. Fiber mechanical properties are utilized to propose formation of materials with varying strength, elasticity, or extensibility, and are the most influential property in relation to silk biomaterial properties aside from crystallinity. [0147] (A) Characterization of silk-biomaterial precursors: protein solubility and protein solution properties. Treatment and processing of silk fibers into aqueous solution prior to material formation can influence biomaterial properties of the Pi silk.

[0148] The limitation in use of alternative silk source revolves in the dissolving step, as different silk fibers possess different protein structures that require different chemical interactions or have physical barriers that inhibit solubilization. An explanation for the success of Bm can be proposed through the Hofmeister Series (CO?² > SOi² > S2O3² > H2PO4 > F > Cl > Br > NO3 > I > C1O4 > SCN and N(CH₃)₄ ⁺ > NH₄ ⁺ > Cs⁺ > Rb⁺ > K⁺ > Na > Li⁺ > Ca²⁺ > Mg²⁺ > Zn²⁺ > Ba²⁺), which describes the distribution of salting in and salting out effects of ionic salts according to their properties of enhancing or weakening the hydrogen bonding network of water molecules. Divalent cations and poorly hydrated anions to the right of Na /CI tend to facilitate protein denaturation and unfolding — salting in — the desired effect for silk protein solubilization. Protein solubility is a function of salt type, concentration, and temperature, but is also dependent on attractive ion-backbone interactions with protein interfaces.

[0149] The molecular weight (MW) or molecular weight distribution (MWD) of proteins in solution determines the resulting solution properties. Generally speaking, higher MWD increases the degree of polymer entanglement, leading to increased strength, strain until rupture, and viscosity. Lower MWD has lower polymer entanglement and higher molecular mobility, resulting in less viscous solutions with lower strength and modulus, but typically flows easier. MWD in silk solutions is commonly described using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), with the use of tandem mass spectrometry, liquid chromatography tandem mass spectrometry , and NMR to identify and structurally characterize proteins. Impacts of MWD on bulk solution properties can be observed via rheological assessment of solution viscosity. The viscosity of silk solution varies as a function of concentration, salt content, and procedures employed to solubilize silk fibers due to modifying the MWD of the solution. MWD of silk solution influences properties relevant to potential biomaterial applications: injectability, flow, and polymer-polymer interactions that drive the formation and function of biomaterials.

[0150] (B) Silk biopolymers as encapsulation systems. Silk and silk fibroin can be used to form biomaterials to encapsulate, for example, drugs, bioactive molecules, or agricultural compounds (e.g., herbicides, insecticides, or fertilizers). Silk particles can be adapted to these various applications through modification of particle size and uniformity and entrapment of different payloads. Silk particles can be synthesized by inducing phase separation and P-sheet structure formation in SF, accomplished through a variety of methods, including (a) liquid-liquid phase separation, (b) microfluidic mixing, (c) emulsification, and (d) electro-spraying. Different parameters and methods of particle synthesis bring about different particle properties, which affect their biological fate and interactions with the environment or entrapped cargo. Common techniques used to evaluate particle properties are as follows: (a) morphology and size by brightfield imaging, SEM, and AFM, (b) polydispersity and zeta potential by dynamic light scattering (DLS), and (c) thermal properties by TGA and DSC. Encapsulation of cargo and particle properties and release behavior can be affected by the molecule encapsulated, the ratio of silk: cargo, and the environment in which the particles are formed or released. Release of pay loads is controlled by diffusion of the molecule into and out of the particle, swelling or degradation of the protein matrix, and/or the infdtration of solvent. SF crystallinity in particle systems is an important regulator of these mechanisms. Controlling the stability and distribution of encapsulated biomolecules within particles and the degradation of particles can be modified through synthesis methods.

[0151] (C) Evaluation of material and mechanical properties of Pi silk fibers and silk gland characteristics. Investigations into silk gland characteristics such as ionic and pH gradients can provide insight into fiber stability and solubility. Determination of silk fiber morphology, thermal properties, and elastic modulus can be used to establish standard property values that can be utilized downstream for material property evaluation. Fiber properties can be characterized using SEM, TGA and DSC, and AFM.

[0152] (D) Creation of Pi silk-based micro- and nanoparticles. Silk-based particles are a versatile biomaterial format in that they can entrap a variety of cargo for desired applications. Pi silk particles are synthesized using a liquid-liquid phase separation mechanism with polyvinyl alcohol (PVA). Encapsulation of fluorescein isothiocyanate (FITC) labeled dextran molecules is used as a model sy stem to study release behavior and properties of Pi silk particles. Neat silk particles and FITC-Dextran silk particle properties are evaluated with DLS, SEM, AFM, TGA, and DSC to investigate the impact of molecule inclusions.

[0153] (E) Structural characteristics of the Pi silk gland. Analysis of silk gland shape and pH gradients are evaluated using nano-CT to visualize gland structure in situ. pH microelectrodes are used to measure pH shifts from the posterior gland region to the anterior region. Pi larvae in the 4th instar, the larval stage in which they produce wandering silk (raw material for biomaterial applications), are stained in a buffered iodine solution to visualize internal soft tissue through diffusible iodine-based contrast-enhanced CT (diceCT). Measurement of gland pH shifts are performed using microelectrodes on silk glands dissected from 4th instar larva at three points within the posterior, middle, and anterior regions of the silk gland.

[0154] It was hypothesized that the gland of Pi would be much smaller than Bm due to relative insect size, but still maintain similar structural regions. Early work involved imaging isolated larval silk glands and histological sections of larva to assess insect anatomy and gland shape to later compare to CT data. Using nano-CT, a 3D reconstruction of 4th instar larva was generated, and structures of silk glands were segmented out in their natural position of the insect. CT reconstructions and segmented glands closely resembled those produced by histology and dissection, allowing identification of anatomical structures like fat bodies, salivary glands, and muscles and confirmation of silk gland shape and structure. The structure of the Pi silk gland was observed to be simpler than that of Bm. The Pi posterior region lacked folds or bended structures. However, it still possessed the primary folded structure in the middle region, which is attributed to the storage of silk dope and the region where sericins begin to form a coating around the fibroin core.

Example 2. Surface morphology and thermal properties of Pi fibers collected from insects reared at standard conditions.

[0155] To correctly compare the raw materials proposed and used for biomaterial fabrication, fibers from Pi silk sheets are analyzed. Fibers are imaged by SEM to measure long-axis fiber diameters and visualize any relevant structures or inclusions on the raw silk materials. Fiber samples are run on TGA and DSC to evaluate material transformations and degradation upon heating. TGA is completed prior to DSC to avoid degradation of material within the instrument. DSC curves are utilized to determine the glass transition, melting, and crystallization temperatures of the fibers in addition to estimation of crystalline content through cooling crystallization. Use of constant heating ramps (10 °C/min) across both techniques avoids shifts in temperatures and produced heats while enabling comparison to other studies in the literature. It is anticipated that Pi silk fibers will be smaller due to the relative size of the insects in addition to a lower glass transition point and crystalline content attributed to differences in estimated protein secondary structure. Bm’s large, uninterrupted P-sheet structures compared to Pi’s -sheet regions interrupted by a-helix structures result in a lower crystalline content that decreases thennal stability of Pi silk. [0156] Silk cocoon and sheet fibers for Pi and Bm silks were imaged by SEM (FIG. 3). Images were taken on samples from three fiber sources to measure fiber diameters and search for irregular material inclusions (FIG. 3A and D). Higher resolution images of Bm and Pi fibers were used to closely visualize and compare surface structure (FIG. 3B and E). The double-stranded structure of Bm fibers was also observed in Pi, with measured fiber diameters of 29.57 ± 2.03 pm (n=90) and 2.14 ± 0.46 pm (n=90) for Bm and Pi, respectively. Irregularly shaped, web-like structures connecting Pi fibers in the fiber sheets (FIG. 3D and F) in addition to debris from the insect was observed. These webbed structures could be similar to the web-like sericin coatings betw een Bm cocoon fibers or other globular proteins (FIG. 3C). Visualization of debris in the silk sheet suggested removal of these impurities in generation of a Pi silk solution, whether through physical cleaning, filtration, or surfactants.

[0157] TGA curves of three samples from Pi and Bm silks w ere generated to investigate material degradation and biological variability considerations before moving to DSC (FIG. 4A). Samples were normalized to weight after evaporation of absorbed water at 100 °C. Within 200-400 °C, both silks displayed rapid weight loss as the fibers began to degrade. Onset of degradation w as observed around 250°C and 180°C for Bm cocoon silk and Pi wandering silk, respectively. Initial heating scans in DSC showed bound water evaporation peaks (Tw) in all samples with no significant differences observed between samples (FIG. 4B). Glass transition regions (Tg) were approximated using similar methods by Mazzi et al. in studying shifts in various silk fibers pre- and post-degumming. Pi silk had a slightly lower glass transition region.

Example 3. Variability’ in elastic modulus of Pi fibers collected from insects reared at standard conditions.

[0158] Individual Pi and Bm fibers are analyzed through AFM to produce images and force spectroscopy curve maps. Sample measurements are conducted in QI-Advanced mode, a measurement mode on the AFM, due to its’ to ability to produce high-resolution force-map images while avoiding the introduction of lateral forces. Parameters of this mode are described in Table 2. Elastic modulus (also termed Young’s modulus) is detennined from force curve data using the Deryagin-Muller-Toporov (DMT) model of solids adhesion, an elasticity fit model included on in AFM processing software (FIG. 7). It is anticipated that Pi silk fibers will have a low er stiffness compared to Bm due to its interrupted P-sheet structures in its SF protein. Pi silk fibers may have higher elasticity due to the inclusion in a-helices between p-sheet regions.

Table 2. Parameters of QI- Advanced measurement mode

[0159] A representative image of aft fiber is shown in FIG. 6, where the double-stranded structure was observed with similar diameter to those measured by SEM. Exploration of mechanical property assessment is through QI- Advanced imaging mode of single fibers laid on glass slides. For accurate sample mapping, small regions of fibers are scanned. Generated force curves showed similarity to the DMT model in FIG. 7. with values of calculated elastic modulus mapped across the scanned surface in FIG. 5B and quantified in FIG. 5B. The average elastic modulus was 208 MPa, which was much smaller than Bm values reported in literature.

[0160] Potential challenges in AFM property measurements rise in the impact of hydration on silk fiber mechanical properties and the bottom substrate effect, in which soft samples appear stiffer than their natural properties. It is possible that variations in fiber modulus measurements can result from slight differences in humidity regardless of sample prep. One strategy to address this can be adapted from spider silk measurements, where samples are scanned or extended in water to enable measurements in consistent environmental conditions. Alternative bulk-material mechanical property analysis can be achieved through DMA. Multi-fiber aligned structures of larger diameter formed by larva can be used as a substitute and back-calculated to single fibers.

[0161] Differences in morphology, thermal stability, and mechanical properties have been observed betw een Pi fibers and Bm cocoon fibers. Though Pi silk degraded at a low er temperature and had lower stiffness.

Example 4. Rearing protocols for production of aqueous Pi silk fibroin solutions.

[0162] The generation of Pi silk solution has not been previously investigated.

[0163] (A) Solvent and temperature variables to generate Pi silk solution. Solubilization of Pi silk will first be investigated to identify requirements in processing. Dissolution of Pi silk was investigated through ionic salts present in the Hofmeister Series due to their ability to salt-in proteins. Use of ionic salts enables ease of manipulation of salt type, concentration, temperature, and silk: salt ratios to impact protein solubility by modifying pH and thermodynamics. A list of investigated parameters and their impact to the processing or final solution are shown in Table 3.

Table 3. Experimental design: Pi silk fiber solubilization parameters.

[0164] Methods used to solubilize Bm silk were not successful in solubilizing Pi silk. Bm degumming methods applied to Pi silk resulted in a hardened silk mat that was unable to be further processed or dissolved, preventing success in SF solution production. Additional degumming methods were than explored, including the use of neutral soap solutions, enzy mes, or simple water baths, and confirmation of sericin removal was determined visually by SEM, histological staining of degummed silk fibers, or by measuring weight loss from raw silk fibers to SF mat. The methods described for generation of Pi silk or Pi SF comprise the steps of: dissolution, dialysis, centrifugation, and purification.

[0165] Pi silk was first dissolved in concentrated LiBr at room temperature, filtered to remove residual insect food and waste, dialyzed against water for 48 hours at 4°C, and then refiltered to remove any remaining impurities or insoluble proteins before being stored at 4 °C. This method was successful in solubilizing Pi silk in a range of conditions (silk:solvent ratios: 1:20-1:200, LiBr concentration: 7-9.3 M). To increase maintenance of the protein stability in solution, other salts in the Hofmeister Series, such as CazNCh, or solvents were used.

[0166] Modifications of described methods can involve the use of co-solvent systems or dialysis treatments by varying dialysate, temperature, or time. Additionally, grinding silk fibers into fine particles can improve exposed surface area and aid in dissolution, similar to methods described by Loh et al (“Overview of milling techniques for improving the solubility of poorly water-soluble drugs.” Asian journal of pharmaceutical sciences 2015, 10 (4):255-274). Nanoparticles may be achieved through ground silk fibers by utilizing the "top-down" approach of nanomaterial manufacturing, which would involve the breakdown of fiber sheets to generate desired nanostructures.

[0167] (B) Solution properties. The MWD and purity of generated Pi silk solution was determined through SDS-PAGE. Solution viscosity was evaluated as a function of concentration, shear rate, and temperature using a cone-and-plate geometry on a rheometer. Experimental design conditions and hypothesized trends in solution viscosity are listed in Table 4. It is anticipated that increasing concentration of silk proteins in solution will increase the polymer entanglement, leading to a more viscous solution with higher flow resistance. Additionally, differences in [3-sheet content could result in shifts in shear-thickening behavior, as Bm SF precipitates out of solution upon formation of - sheets at high shear.

[0168] Using SDS-PAGE results, we observed differing MWD of proteins in solution between Bm and Pi methods (FIG. 8A). Bm solutions commonly exhibited a large broad distribution of SF fragments, which was observed. Pi silk solution displayed concentrated, individual protein peaks. These differences in MWD are likely attributed to the presence and absence of the silk degumming step. In degumming, Bm SF is purified from sericins, but also broken down into fragments. Bm MWD can be shifted to high-medium-low ranges by increasing the time of degumming from 30, 60, and 90, respectively. These initial differences in polymer chain length and protein distribution propose variations in polymer entanglement, w hich could lead to differences in solution viscosity and interactions of silk proteins in material formats, enabling different material properties (FIG. 8B).

[0169] If the resolution of distributed proteins in SDS-PAGE of Pi solutions is not optimal, this can be improved through use of high and low' percentage acrylamide gels to individually assess both ends of protein distribution. It is possible that the cone-and-plate geometry is not sensitive enough to detect changes in viscosity at low concentrations. To address this issue, use of a concentric cylinder geometry can be employed due to its larger shear surface.

Table 4. Experimental design and hypotheses.

[0170] Initial aqueous Pi silk solutions generated from the methods described above have successfully synthesized 3 material formats — films, sponges, and particles — exhibiting early success at being able to utilize Pi in biomaterial fabrication.

Example 5. Pi silk-based micro- and nanoparticles.

[0171] Silk-based encapsulation systems for controlled release of payloads have been investigated due to their low immunogenicity, stability⁷, and ability to biodegrade into byproducts harmless to human health or the environment. Water-insoluble silk particles can be generated through a variety of methods, but a simple, cost-effective method using PVA induces phase separation and formation of 0-sheet structures in the SF protein. This method also increases the potential of translatability, as it avoids the use of organic solvents. FITC- Dextran is utilized as a model cargo due to its commercial availability, ability⁷ to modify polymer chain lengths, and ability⁷ to be easily visualized within silk particles through fluorescent imaging. Parameters in the synthesis of silk-only and FITC- Dextran-silk particles can be modified to tune particle properties and influence the release behavior of FITC-Dextran from the protein matrix. Pi particles, with sizes ranges of <500 nm to about 10 pm are expected to be produced.

[0172] Silk particles are formed through phase separation by. Particle size is modulated by changing silk concentration relative to PVA concentration. However, alternative methods as sonication and temperature during film casting can also be used to modulate particle size. Particle morphology, size and polydispersity is assessed through SEM and DLS. Baseline thermal stability' and degradation temperatures is determined through TGA and DSC. Crystalline content of silk particles is estimated through the use of DSC or FTIR. Assessment of particle mechanical properties is analyzed by AFM. TGA, DSC, and AFM characterization can be performed on liquid samples. For FTIR analysis, silk particle solution is lyophilized prior to measurement.

[0173] Particle preparation through phase separation (hydrophobic collapse of SF) were applied to Pi silk solution to generate a small batch of 0.25% (w/v) particles. Imaged particle diameters ranged from 1 to 10 pm. Formation oiPi silk particles showed that this is an achievable material format, highlighting its feasibility for use in manufacture of Pi silkbased biomaterials.

[0174] In some experiments, the concentration of silk in the PVA solution was 3%. Increasing the concentration of silk can lead to increased particle size. Increasing the ratio of silk to PCT can also lead to increased particle size. Decreasing particle size can be accomplished with one or more rounds of sonication.

[0175] Particle populations can be filtered to reduce polydispersity.

[0176] Encapsulation of bioactive molecules. Encapsulation of FITC-Dextran is carried out with synthesized particles of one size distribution (10 pm or <500 nm) to study the ability of Pi particles to entrap and release desired payloads. Encapsulation of FITC- Dextran of MW 4 and 40 kDa is visualized via confocal microscopy to assess incorporation and distribution of FITC-Dextran in the particles. Encapsulation and loading efficiency are determined by Equations 1 and 2. mass of FITC-Dextran in particles

Encapsulation efficiency (^w/_w %) = ■ 100 mass input of FITC-Dextran (1)

_T mass of FITC-Dextran in particles

Loading efficiency (^w/_w%) = - _{mass of particles} ^P - MO (2)

[0177] It is anticipated that lower MW FITC-Dextran will be able to better infiltrate the silk particle, leading to higher encapsulation efficiency. The particles are also analyzed to determine whether encapsulation of FITC-Dextran molecules alters particle properties, such as by shifting polymer entanglement interactions relative to silk-only particles.

[0178] In other experiments, positively charged molecules or molecules with varying hydrophobicity are used in the encapsulation analyses.

Example 6. Pi sericulture and Silk Collection.

[0179] Insects were reared in containers modified for insect culture in a 16 h light: 8 h dark cycle at 24°C and 65±3.2% relative humidity. Insects were fed a standardized wheat bran diet. The life cycle of Pi silkworms was completed as follows: 130 mg of diet was placed in the container followed by 50 mg of Pi eggs. Insects were reared until the silkworms enter the 4th larval stage and began laying silk along the walls of the container. Silk sheets were collected while the insects were pupating; adults emerged and were then collected in a separate container to collect eggs and repeat the cycle. Adults were frozen for 24 hours for euthanasia before disposal.

[0180] (A) Pi Silk Solubilization. Pi silk sheets were cleared of loose insect debris. 50- 200 mg of Pi silk was dissolved in an 7-9.3 M LiBr solution at room temperature for 3 h with continuous mixing. Resulting solutions were filtered with Miracloth to remove food debris and insect waste before being transferred to 3.5 kDa MW dialysis tubing. Solutions were dialyzed against water for 48 h at 4°C before being re-filtered to remove any insoluble silk or remaining impurities. The concentration (w/v %) of Pi silk solutions were determined before storage at 4°C or use. Resulting solutions were further concentrated through dialysis against a 10% (w/v) PEG (10,000 MW) solution.

[0181] (B) Bm Silk Fibroin Extraction. Bm SF is described in detail by Rockwood et al. : 1) Degumming: 5 grams of Bm cocoons were cut into dime-sized pieces and boiled for 30 minutes in 2 liters of 0.02 M Na2CCh solution. The purified Bm SF mat was then rinsed in water and left to dry for a minimum of 48 h. 2) Dissolving: The Bm SF mat was dissolved in a 9.3 M LiBr salt solution at a ratio of 1 :4 (silk: salt solution) at 60°C for 4 h. 3) Dialysis: LiBr was removed from the solubilized silk solution by dialyzing against water for 48 h using a 3.5 kDa MW cutoff dialysis tubing. 4) Centrifugation: Aqueous Bm SF solution was removed from dialysis and centrifuged at 9,000 rpm for 20 minutes (4°C) three times to remove any insoluble proteins or impurities before the concentration (w/v %) of the solution is determined. Complete Bm SF solution was be stored at 4°C for up to 1 month or until use.

[0182] (C) Scanning Electron Microscopy. 7 mm biopsy punches of Bm cocoons and Pi silk sheets were secured to SEM sample stubs with carbon conductive tape. Samples were dried overnight in air and sputter-coated with 8 nm of Au prior to imaging on a Phenom Pure benchtop SEM. Long axis fiber measurements of Bm and Pi silk (n=90 for each) were completed in ImageJ analysis software across 3 different samples and 3 representative images from each sample.

[0183] (D) Thermal Gravimetric Analysis. 7 mm biopsy punches of Bm cocoons and Pi silk sheets were analyzed using TA Instruments TGA550. Samples were placed in platinum pans and ramped from ambient temperature to 800°C at 10 °C/min with air flow of 40 L/min and N2 flow of 60 L/min for an inert atmosphere. Data were analyzed using TRIOS software package. Weight % curves were normalized to sample weight after water evaporation at 100 °C. [0184] (E) Differential Scanning Calorimetry. 5-10 mg of Bm cocoons and Pi silk sheets were analyzed using TA Instrument DSC250. Samples were pressed into Al Tzero pans and ramped from 0 to 300 °C at 10 °C/min with air flow of 40 L/min and N2 flow of 60 L/min. Data were analyzed using TRIOS software package.

[0185] (F) Biological Atomic Force Microscopy. Single Pi fibers were isolated by placing 4^th instar larvae on glass microscope slides and allowing them to wander until silk fibers were visibly accumulated on the slide. Slides were dried in air overnight before imaging and scanning on a Bruker NanoWizard® 4 XP AFM. Imaging of Pi silk fibers was conducted in QI imaging mode wi th a qp-BioAC-Cl cantilever (NanoAndMore, spring constant: 0.3 Nm-1). Force spectroscopy of single fibers was performed in QI Advanced mode with a RTESPA-150 cantilever (Bruker, spring constant: 5 Nm-1). Image and force map data were processed in JPKSPM Data Processing software. Calculations of Young’s modulus was completed by fitting the linear region of the retract (adhesion) curve of force curves with the DMT model (spherical tip).

[0186] (G) Nano-Computed Tomography. Pi 4th instar larvae were fixed in phosphate buffer formalin overnight before the skin layer was nicked by needle pins and then returned to fixative for 48 h. Fixed larvae were submerged in Lugol’s iodine (5%) contrast solution for 1 week prior to scanning. Specimens were scanned using a GE Phoenix v|tome|x m 240 CT scanner with 180 kV transmission tube and diamond target with the voxel size, current, and voltage adjusted according to their size. The specimen presented in this document was scanned with a 5.34 pm voxel size, 250 pA current, and 70 kV voltage. 3D volume files were reconstructed with Datos|x CT software and later processed with VGStudio Max. Silk glands were segmented with VG Studio Max's segmentation tools and transported as .ply file for further use.

[0187] (H) Histological Preparation and Staining of Semi-thin Sections. Pi 4th instar larvae were fixed in paraformaldehyde 4% in phosphate buffered saline solution overnight before being cut into three sections (anterior, middle, posterior) and returned to fixative for 48 h. Samples were dehydrated through a series of ethanol solutions of increasing concentrations before being transferred to xylene. Dehydrated samples are left in a wax bath overnight before being embedded in wax. Samples were sectioned at 10 pm thickness and mounted on slides. Sections were deparaffinized and rehydrated before being stained with hematoxylin and eosin. Coverslips were secured to the stained samples. Stained sections were imaged on a Keyence BZ-X800 benchtop microscope. Masson’s trichome and fluorescent immunostaining (DAPI/Phalloidin) can also be used to visualize sample sections.

[0188] (I) Gel Electrophoresis. Pi silk solution and Bm SF solution MWDs were visualized with SDS-PAGE. 2 pg of solubilized silk protein per sample was loaded into a 4-12% polyacry lamide gel under reducing conditions and run in duplicate. 5 pL of broadspectrum ladder w as added to one well to provide a MW reference standard. The gel was run at 100 V for 5 minutes before the voltage was increased to 200 V for 20 minutes or until the ladder had extended the length of the gel. Gels were stained using a colloidal blue staining kit before being imaged on a LI-COR Odyssey Fc at 700 nm with a 30 second exposure time. MWDs of silk solutions were quantified by densiometric measurements along each lane with ImageJ gel analysis software.

[0189] (J) Pi Silk Particle Preparation. Pi and Bm silk particles were formed as described previously and as shown in FIG. 9. 0.25% (w/v) Pi silk solution was combined with a 5% (w/v) PVA stock solution in a 1 :4 w eight ratio for a total volume of 1 ml. The solution was then sonicated for 30 seconds at 25% amplitude before being cast in a petri dish and dried overnight. The dried film was removed from the dish and dissolved in DI water. The resulting solution was centrifuged and then pelleted particles were resuspended in ultrapure water and sonicated for 15 seconds at 15% amplitude. 40-50% encapsulation efficiency for FITC dextran was observed when using the two different sources of silk to form aqueous silk solutions. Results demonstrate the ability to entrap bioactive cargo in Pi particles at similar success rates as observed in Bm.

[0190] (K) Methods to process Pi silk sheets into aqueous solution. A depiction of the flow of steps is shown in FIG. 10.

[0191] (L) Degumming. Different methods for degumming the Pi silk were tried as follows:

(i) The Pi silk was boiled in DI water for 5, 10, 15, and 30 minutes. A 50-60% mass loss was observed and resulted in a hardened fiber mat that did not readily dissolve.

(ii) The Pi silk was boiled in 0.04 M Na2COs. The Pi silk fully dissolved in minutes, with no remaining mass. Thus, this method was not suitable for degumming Pi silk.

(iii) The Pi silk was boiled in 0.5 M CaCh. A 50% mass loss was observed and resulted in a hardened fiber mat that did not readily dissolve. (iv) The Pi silk was treated with 10-minute water washes at room temperature, 40 °C. or 60 °C. A 50-55% mass loss was observed and resulted in a partially stiff fiber mat that was difficult to dissolve.

[0192] (M) Dissolution of raw Pi silk sheets. Different methods for dissolution of Pi silk were tried as follows:

(i) Incubation of raw Pi silk sheets in 7-9.3 M LiBr at 60°C resulted in Pi silk sheets that turned dark brown/black and did not dissolve over the course of 3 days.

(ii) Incubation of raw Pi silk sheets in 0.5 M NaHCCh resulted in Pi silk sheets that were mostly dissolved, yielded a thick, muddy solution and that was not stable through dialysis or with time.

(iii) Incubation of raw Pi silk sheets in CaCh and CaCh-ethanol/methanol systems did not successful dissolve the Pi silk at a range of low to high concentrations and temperatures from room temperature to 60°C. Pi silk retained its structure and did not dissolve in CaCh or CaCh-ethanol/methanol.

(iv) Incubation of raw Pi silk sheets in 8-9.3 M LiBr at room temperature with mixing/shaking resulted in Pi silk dissolving in 2-3 hours. The resulting solutions was able to be filtered and transferred to dialysis. In some embodiments, the Pi silk was dissolved 8-9.3 M LiBr at room temperature with shaking for up to 1 day or longer. a) In some embodiments, ionic salts with higher salting-in capabilities, e.g., 6-8M Ca(NOs)2 are used. In some embodiments, the Pi silk sheets are dissolved at about 60°C to about 80°C water bath for about 3 hours. In some embodiments, the Pi silk sheets are dissolved room temperature for about 2 days.

[0193] (N) Filtration of Dissolved Pi silk. Different methods for filtering the postdissolutions and/or post-dialysis solutions to remove solid insect waste and impurities were tired as follows.

(i) Filtration using a metal mesh or sieve failed to remove all visible impurities and resulted in a solution in with the Pi silk crashed out of dialysis within a day.

(ii) Vacuum filtration, at 0.2 pm or 0.4 pm, removed all visible solids but resulted in a solution that was resistant to flow through membrane. Further vacuum filtration added undesirable shear/pushing forces to solution that did not aid in stability. (iii) Filtration using 70 pm and 40 pm cell filters/fme mesh strainers resulted in solutions that crashed out of dialysis within a day, likely caused by incomplete removal of impurities that destabilized solution during dialysis.

(iv) Filtration using 5 pm and 1 pm cell filters/fme mesh strainers resulted in clogging of the membrane in inhibition of flow.

(v) Dissolved Pi silk was filtered using a 22-25 pm Miracloth. The dissolved or dialyzed Pi silk was filtered using a 22-25 pm Miracloth using a gentle gravityfiltration method. The resulting solutions were clear of visible impurities and stable.

[0194] (M) Dialysis of filtered Pi silk. Different methods for dialyzing the filtered Pi silk are tried as follows:

(i) The filtered Pi silk was dialyzed against deionized water for about 48 h at room temperature using a 3.5 kDa dialysis membrane.

(ii) The filtered Pi silk was dialy zed against deionized w ater for about 48 h at about 4°C using a 3.5 kDa dialysis membrane.

(iv) The filtered Pi silk was dialyzed against NaCl solutions and HEPES buffers of decreasing concentrations using a 3.5 kDa dialysis membrane

(v) The filtered Pi silk was dialyzed against LiBr solutions of decreasing concentrations using a 3.5 kDa dialysis membrane.

Example 7. Plodia interpunctella growth conditions for silk production

[0195] Described are methods for growing Plodia interpunctella (Pi) and harvesting Pi silk. The descnbed methods, provide for improved production of silk and improve consistency in the produced silk. In some embodiments, the Pi are grown at about 24°C. The described methods also provide for optimization of resource availability (larvae/ gram diet), and population density (larvae/ mL). In some embodiments, the Pi are grown in a resource availability of 10 larvae/ gram food, and a population density of 0.72 larvae/cm³.

[0196] Pi produced significant amounts of silk fibers while wandering around their environment, indicating that Pi be reared indoors with a standardized diet, and the silk fibers can be collected as mats of silk fibers, generating relative abundance of silk material compared to what would be collected from the cocoon of this insect.

[0197] Growing the Pi at higher population densities led to production of silk fiber mats during the wandering stage of the larval lifecycle, providing for silk production and collection without collecting individual cocoons. [0198] (A) Diet. The Pi were reared on a standardized wheat bran diet similar to Silhacek et al. (Growth and development of the Indian meal moth, Plodia interpunctella (Lepidoptera: Phycitidae), under laboratory mass-rearing conditions." Annals of the Entomological Society of America. 1972; 65(5): 1084-7) in a 16 h light: 8 h dark cycle at the various conditions described in Table 5 and a relative humidity⁷ of 65 ± 3.2% as measured by a Govee-Hygrometer Thermometer. The artificial diet ingredient list and composition are shown in Table 6.

Table 5. Rearing parameters of Pi.

Table 6.

[0199] Standard rearing conditions prior to this set of experimentation was at 26°C and 70% relative humidity in plastic display boxes (tri-state plastics, 079-C, 7 7/16 x 5 5/16 x 3 3/4 inches) modified for insect rearing. A circular hole (diameter: 76 mm) was cut in the top of the lid where fine wire mesh was attached to cover the hole. In brief, Pi were reared within these boxes by first adding 130 g of standard wheat bran diet and then placing 50 mg of Pi eggs on top of the diet. The boxes were stored in the incubator at conditions as described above. After the adults emerged, they were placed within a state of quiescence by carbon dioxide and transferred to a mason jar fitted with a fine wire mesh at the top, which allows for eggs to pass through but not adults. Once eggs were collected, the adults are frozen for at least 24 hours for euthanasia and new boxes are created.

[0200] (B) Conditions for Pi Rearing. The Pi colony was maintained in custom cylindrical plastic containers (height: 9 cm, diameter: 6 cm) with a circular hole (diameter: 2.5 cm) cut in the bottom of the lid where fine wire mesh was attached to cover the hole. To set up each jar, eggs were collected and put in the respective incubator for their condition. Once hatched, the newly emerged larvae were counted and added to containers and immediately placed within their respective incubator (ThermoFisher Model 3900 Series). Each condition contained 3+ sets of replicate containers.

[0201] (C) Silk Mass Assessment. The Pi larvae were allowed to progress through their natural life cycles. Once they began pupation, the silk around the entire jar, both lid and side walls, was collected, except for a 2 mm margin around the base of the container to avoid additional mass caused by diet contamination. Additionally, once the silk was collected, any visible debris remaining on the mats (frass, head capsules, etc.) were removed to reduce or eliminate contamination. Cleaned silk mats were then placed in between aluminum foil and stored within a fume hood. The full set of samples analyzed is given in Table 7.

Table 7. Pi Rearing parameters.

[0202] (D) Silk Mat Analysis. To analyze the silk fiber mats, 8 mm biopsy punches were taken at different heights of the container to determine if variability existed within one silk mat. Images were taken at 10x magnification. Subsequently, DiameterJ was used to calculate the average silk fiber diameters for all conditions by selecting 4 random images from each condition and analyzing 3 random fibers from the image. Representative SEM images were also taken for each condition.

[0203] (E) Scanning Electron Microscopy (SEM). Sections of silk mats were excised using an 8 mm biopsy punch and added to an adhesive sticker placed on top of a ZEISS/LEO SEM Pin Stub Mount, 0 12.7 mm x 9 mm pin height. Samples were dried in a fume hood for 48 h and mounted on a Metallurgic Charge Reduction Sample Holder before being imaged at 5 kV on a Phenom Pure benchtop SEM.

[0204] (F) Developmental Time Analysis. To analyze the progression of developmental time, head capsule size was documented. Within each instar, the head capsule size remains constant. As the larvae develop to the next instar, they will shed their old head capsule and grow anew, larger one. Initial images were taken when the larvae were large enough to grab with feather light forceps. Images were taken by placing larvae next to a ruler and using a microscope. Measurements were taken every' 3 days, using 4 larvae from each jar. ImageJ software was used to measure the head capsule sizes.

[0205] (G) Fiber Density. To analyze the fiber density, 8 mm biopsy punches were taken at different heights of the container. Different heights account for variability' within one silk mat. Images of the biopsy' punches were taken using 10x magnification. Fiber density was analyzed on three clear images at each condition using the Histogram feature of the software ImageJ. The Histogram feature counts the pixels at each color condition. For a black and white image, the colored pixels are considered fibers with white pixels being open space. Therefore, fiber density was calculated by comparing the number of colored pixels to the total pixels in the image. Each fiber density' was for the same size image (966 x 725 pm).

[0206] (H) Fibroin-Heavy and Fibroin-Light Transcript Analysis. Total RNA was extracted from 12 silk glands from wandering phase Pi larvae (4 from each of the 3 replicate containers for each condition) using PureLink RNA Mini Kit. The silk glands were stored in RNAlater Stabilization Solution (Thermo Fisher) following manufacturers protocols after isolation until RNA extraction. cDNA was created from 1 pg total RNA extract for each group using Affini Script QPCR cDNA synthesis kit (Thermo Fisher). RT-qPCR was performed on 1 pL of cDNA using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) with Fibroin-Heavy and Fibroin-Light primers (Integrated DNA Technologies) as described in Table 8. As reference genes, the transcript levels of -actin (Pi_Bac3F and Pi_Bac3R) and ribosomal protein 7S (Pi_RPS7AF and Pi_RPS7AR) were quantified. All biological samples were run in triplicate. The RT-qPCR reactions were conducted in a Cl 000 Touch Thermal Cycler operated by the CFX96 Real-Time System (Bio-Rad, Hercules, CA) programmed for 35 cycles of 95 °C. 10 sec; 54°C, 30 sec; 95 °C, 10 sec. To confirm there was minimal genomic DNA contamination co-extracted during total RNA isolation, no reverse transcriptase control (NRT) reactions were made using the whole RNA extract from each sample. The relative gene expression was determined following the 2 ^{A< t} method as a standardized ratio to the geometric mean of the reference gene transcripts. To calculate the AACt all the conditions were normalized to the 30/20/0.72 fibroin-heavy expression value as represented by its relative expression being a value of 1 (Table 9).

Table 8. Reverse transcriptase primers utilized in manuscript.

Table 9. Fibroin -heavy' and -light chain relative gene expression

[0207] (I) Dynamic Mechanical Analysis ofSilkMat. Mechanical assessment of unprocessed Pi silk was conducted on 10 mm x 10 mm sections excised from silk mats that were collected from lids of our standard rearing boxes (tri-state plastics, 079-C, 7 7/16 x 5 5/16 x 3/4 inches) that were reared in the recommended conditions for silk production described herein (24 °C, resource availability of 10 larvae/ g diet, and population density of 0.72 larvae/ mL). The cross section of each silk mat was determined by SEM. Five (5) samples were collected from two different boxes and pooled for analysis. Dynamic mechanical analysis (DMA) was performed on an Anton Paar MCR 702e Rheometer (Anton Paar, Graz, Austria). Each sample was loaded into tensile clamps (upper clamp: U-SRF5; lower clamp: L-SRF5/LD) with Emery Cloth (3M) folded within each clamp and tightened to 10 centinewton/meter with a torsion screw driver. Anton Paar RheoCompass software was used to perform static tensile testing where sample was loaded and pre-stretched to a force of 0.01 N to remove any sag. The gap width was then recorded as the height of the sample between the clamps. Static testing was performed at room temperature and used to extend the samples from 0% to 50% of the original length at 1% per minute. The average Young’s modulus was calculated from the stress-strain curves at a stain range of 0.01-to-0.08. Ultimate tensile strength was calculated as the maximum point on the stress-strain curve.

[0208] (J) Atomic Force Microscopy (AFM). Imaging of single Pi silk fibers was conducted on a Bruker NanoWizard ® 4 XP AFM in the contact imaging mode with a NT_B30_v0030 cantilever (NanoAndMore, resonance frequency: 13 kHz, force constant: 0.2 Nm '). Fibers were isolated by placing a larva on glass microscope slides and allowing them to w ander until silk fibers were visibly accumulated on the slide. Single fibers were carefully stretched and secured over a glass microscope slide for imaging in air.

[0209] (K) Degumming Silk Fibers. Bombyx silk cocoons were degummed according to the established Rockwood et al. protocol. Pi silk mats were cleaned to remove frass, head capsules, and other waste on the surface of the silk fiber mat. Cleaned mats were degummed by boiling in deionized water for 10 minutes. The silk mats were then dried for 2 days in air before final weighing or assessment via SEM.

[0210] (L) Statistics. All statistics were performed in GraphPad Prism version 9.4.1 for windows, GraphPad Software, San Diego, California USA, www.graphpad.com. All statistics were performed by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test with an alpha value of 0.05, except for FIG. 12, which was analyzed as a one-way ANOVA. All graphs are shown with an average three or more biological replicates (n > 3) plus or minus a standard deviation (±SD).

[0211] (M) Temperature alters the rate of growth of Pi larvae. To leverage Pi for future applications, it is important to understand which laboratory rearing conditions yield consistent silk fibers while maintaining population viability. In FIG. 11, the relationships between grow th rates and fiber properties are compared between Pi populations with respect to feasible parameters for rearing in the laboratory, including temperatures (24-30 °C), resource availability (5, 10, 20 larvae/gram diet), and population density⁷ (0.36 vs. 0.72 larvae/ mL) by evaluating growth via head capsule size and overall silk production and fiber properties, as depicted in FIG. 11 A. First, we evaluated the impact of rearing temperature on the growth rates of Pi larvae, determining that the life cycle decreases from an average of 35 ±2 days to 24 ±2 days when temperature increases from 24 °C to 30 °C (FIG. 11B). Furthermore, the data indicate that the supplied diet (5, 10, and 20 larvae/ g diet) did not have an influence on the developmental rate. Thus, temperature, under the conditions we provided, had the greatest impact on physiological processes, including silk production.

[0212] (N) Impact of growth rate and temperature on silk fiber properties. When population density has reached an upper threshold at a given temperature, Pi larvae will leave the food source and start to crawl or wander around their environment. During this phase, they frequently spin silk fibers, leaving them in their path as they wander. This phenomenon causes the exposed surfaces of the insect rearing container to quickly become covered with silk fibers during the latter half of the growth cycle to form silk fiber mats. We analyzed the influence of rearing temperature on total silk fiber density FIG. 11C and silk fiber diameter FIG. 1 ID while holding the total rearing vessel volume constant. Examples of silk fiber mats collected prior to moth emergence are shown in FIG. 11A for all temperatures. Results show that fiber density remained statistically similar across all temperatures, resource availability, and population densities when analyzed by ImageJ (p > 0.4542).

[0213] Previous research has shown that Pi silk fibers have different diameters between wandering silk and pupal silk. We analyzed the diameter of the silk fibers in each mat (3 images per vessel) from 3 separate rearing vessels to obtain average silk fiber diameters FIG. I ID. Overall, trends show limited differences between observed fiber diameters, but some statistical differences were observed between the 24 °C and 26 °C groups, where 26°C fibers were statistically larger in diameter. The 26 °C samples contained lower amounts of total silk fiber production. [0214] The results demonstrate that wandering silk, spun over a longer period than conventional cocoon silk, can be produced in a consistent manner with fiber diameters in the range of 1-2 gm.

[0215] (O) Greater silk fiber production was achieved at 24 °C with higher population density and moderate resource availability’. Follow ing assessments in temperature change, we further explored the impacts of larval population density’ and resource availability’ on total silk fiber production FIG. 12. Results demonstrated that population density had a significant impact on i ’s production of silk fibers. As shown in FIG. 12A, statistical differences in total silk fiber production within a given temperature were found when comparing production by larvae at 24 °C with a resource availability of 5, 10 or 20 larvae/ g diet. When the resource availability was constant (10 larvae/ g diet), but the population density’ changed (0.36 vs. 0.72 larvae/ mL), total silk produced increased with increasing population density (p < 0.0001). This general trend was also present in the 26 °C and 30 °C conditions. Silk fiber production did not increase as dramatically within the 10 larvae/g diet (0.72 larvae/ mL) condition in the 26 °C group. Resource availability’ (larvae/g diet) also played a small role in the wandering silk behavior, where all temperature groups showed a general trend of increased silk fiber production as resource availability moved to the most restrictive state of 20 larvae/g diet.

[0216] Highest production of wandering silk fiber production was achieved at a resource availability of 10 larvae/ g diet and at a population density of 0.72 larvae/ mL across all temperatures. Keeping resource availability and population density constant, statistical differences in total silk fiber production were observed between 24 °C and 26 °C (p = 0.0391, FIG. 12D), where the highest silk mass achieved across all parameters occurred at 24 °C. Furthermore, our findings established 26 °C produces the least amount of wandering silk fibers. Silhacek et al. established optimal Pi rearing conditions at 28 °C. On a per lifecycle basis 24/10/0.72 (°C, larvae/g diet, larvae/mL) is the preferred choice for laboratory rearing.

[0217] (P) Total silk fiber production per lifecycle vs. per year. An increase in temperature decreases the total length of a Pi lifecycle. Given that the lifecycle length is substantially shorter at 30 °C, total silk produced was greatest for conditions at 30°C, resource availability of 10 larvae/ g diet, and a population density of 0.72 larvae/mL. At 30°C. we achieved 15 total lifecycles per year. 10 cycles are completed per year at 24 °C. However, rearing Pi at higher temperatures over multiple generations diminishes the fitness and fecundity’ of the colonies and therefore diminish the production over multiple generations. In addition, increase numbers of life cycles has extra costs associates with increase food use, increase labor, increased energy costs. [0218] (Q) Silk fibroin gene expression impacted by rearing conditions. The fibroins in the silk mats are predominately responsible for mechanical properties such as elasticity and yield strength, with the majority of the mechanical properties being attributed to the fibroin-heavy protein. We analyzed silk fibroin -heavy and -light chain transcripts in Pi larvae at the early wandering stage. Results showed that transcript levels were highest in larvae reared at 24 °C and 30°C. P s fibroin-heavy transcript was highest at 30 °C.

[0219] (R) Pi silk fibers in advanced manufacturing and product development. Mechanical and structural characterization of the unprocessed silk fiber mats in FIG. 13. show that these materials have an average Young's Modulus and tensile strength of 10.6 ±3.8 MPa and 2.64 ±0.40 MPa, respectively, when measured under extension at 1% strain per minute as shown via a representative stress strain curve in FIG. 13 on an Anton Paar MCR 702 instrument equipped with a linear drive. Compared to previously studied silks which lie in the ‘natural fiber’ range described in Gibson et al. (Cellular Materials in Nature and Medicine. New York, NY: Cambridge University Press; 2010), Pi silk fiber mats reside in the soft tissue region, where they displayed mechanical properties that more closely resemble that of cartilage, which are significantly lower than other common silk producers. Furthermore, the Pi silk mats had higher average strain at the maximum stress (28.5 ±2.4%) compared to reported values for Bombyx (18 ±2%). However, the Pi spun silk fiber mats w ere non-uniform and randomly oriented. More order fibers may display different properties. Further investigation of the contribution of individual fibers via single fiber analysis using atomic force microscopy provides greater information on the sources of variability within these samples (FIG. 14). Analysis of a single fibers by AFM showed variation in fiber diameters (FIG. 146A), which could be visualized in the mat as raw Pi silk fibers using SEM (FIG. 14B).

[0220] The next step in analysis was the removal of sericin proteins from the silk fibers, through a process known as degumming. Pi silk was degummed in boiling deionized water. Raw and degummed Pi silk fibers are shown in FIG. 15. Recovery estimations show Pi raw silk w as around 45-55% fibroins.

Example 8. Growth conditions for Pi.

[0221] Pi were reared in a 16 h light: 8 h dark cycle at various temperatures 24 °C and 65% relative humidity in plastic display boxes (tri-state plastics. 079-C, 7.4375x5.3125x3.75 inches) modified for insect rearing. A circular hole (diameter: 76 mm) is cut in the top of the lid where fine wire mesh was super glued to cover the hole to allow- air flow within the container. The Pi w ere fed a standard diet as outlined in Table 10. Table 10. Ingredients in the standard diet used to rear Pi silkworms.

[0222] The maintenance of cultures was completed as follows: 130 mg of diet was placed in the rearing container prior to adding 50 mg of Pi eggs. Rearing boxes are then stored in the incubator at conditions as described above until the silkworms reach the 4^th larval stage and began laying silk along the walls of the container. Once substantial silk sheets were laid, the silk was collected while the insects were pupating and prior to the emergence of any adult moths before the container was returned to the incubator. Once the adults emerged, they were placed in a state of quiescence by carbon dioxide and transferred to a mason jar fitted with a fine wire mesh at the top, which allowed for eggs to pass through but not adults. Once eggs were collected, the adults were frozen for at least 24 hours for euthanasia and new boxes are created.

Example 9. Influence of environmental conditions on Pi silk production.

[0223] The rearing conditions above were modified to optimize the production of silk. To establish optimal growth conditions for production of Pi wandering silk sheets, three environmental parameters were investigated: temperature, population density, and resource availability. A list of investigated parameters is shown in Table 11. In these experiments, the Pi colony was maintained in custom cylindrical plastic containers (height: 9 cm, diameter: 6 cm) with a circular hole (diameter: 2.5 cm) cut in the bottom of the lid where fine wire mesh was super glued to cover the hole. The amount of diet in each container w as massed using a balance (Mettler Toledo XPE205). To set up each jar, eggs were collected and put in the respective incubator for their condition. Once hatched, the newly emerged larvae were counted and added to containers using a paint brush and immediately placed within their respective incubator. Each condition contained 3+ sets of replicate containers. Table 11. Rearing parameters for investigation into effects of environmental paraments on Pi silk production.

[0224] Various rearing strategies were examiner for their effects on total silk production and produced silk fiber properties. The mass of silk, diameter of the silk fibers, and silk fibroin gene expression were analyzed at each environmental condition. Results are summarized in Table 12.

[0225] The results indicated that population density had a significant impact on Pi silk production. Statistical differences in total silk fiber production within a given temperature were found when comparing production by larvae at 24 °C with a resource availability of 5, 10 or 20 larvae/g diet. At all temperatures, total silk production increased with increasing population density when resource availability was held constant. Resource availability played a small role in silk production, where all temperature groups showed a general trend of increased silk fiber production as resource availability moved to the most restrictive state (20 larvae/ g diet). However, trends also showed a decrease in fecundity of the insect population at temperatures at or greater than 30 °C. The highest silk fiber production was achieved at a resource availability' of 10 larvae/ g diet and at a population density of 0.72 larvae/ mL across all temperatures. Keeping resource availability and population density constant, statistical differences in total silk fiber production were observed between 24 and 26 °C (p = 0.0391), where the highest silk mass achieved across all parameters occurred at 24 °C. Overall, trends show limited differences between observed fiber diameters, but some statistical differences were observed between the 24 and 26 °C groups, where 26 °C fibers were statistically larger in diameter. To start to understand how gene expression and protein production are related as a function of environmental parameters, silk fibroin-heavy (Fib-H) and fibroin-light (Fib-L) chain transcripts, which are the two main fibroin proteins, were assessed. Transcript levels tended to be highest in larvae reared at temperatures outside of their optimal temperature range. Fib-H and Fib-L expression generally correlate with silk production, with higher silk fiber mass production and fibroin expression at 24 °C and 30 °C.

Table 12. Pi silk production over modified environmental conditions.

[0226] For Pi silk production, rearing conditions that yield consistent silk fibroin gene expression ratios and subsequent protein production is desired. We determined that rearing and production at 24 °C, 65% humidity, resource availability of 10 (larvae/ gram diet), and a population density of 0.72 (larvae/ rnL of container volume) is led to consistently high silk production. Further, rearing at 24 °C did not cause generational decline, thus providing for multigenerational production.

Example 10. Scale up of silk production for advanced manufacturing of raw material.

[0227] Scale up of Pi silk as a raw material can be achieved through modification of the rearing container in which they are cultured. As Pi lay silk on the walls of their container as they wander, increasing the accessible surface area is expected to correlate to an increase in the amount of silk that can be collected from each population. Modification of rearing container surface area can be achieved using methods available in the art, including, but not limited to, three-dimensional (3D) printing of polylactic acid (PLA). Design of 3D print inserts can be tailored to the dimensions of the rearing container and, upon implementation, be evaluated through 1) total mass of silk produced, 2) the quality of silk collected (thickness, density, cleanliness), 3) larval interaction with the insert, and 4) practical translation into a production setting. Separate 3D inserts can be designed with varying geometry, surface area, and materials to evaluate conditions that maximize silk production. Initial trials have shown that the larval life cycle is un-interrupted upon implementation of flat 3D inserts and can lead to an increase in total mass of silk fibers collected at the end of a Pi life cycle.

Example 11. Evaluation of silk fiber properties

[0228] (A) Protein Content and Gene Expression. To evaluate the influence of environmental factors and how they impact the fibroin components within the silk fibers, we evaluated the transcript levels of fibroin-heavy and fibroin-light genes. Total RNA was extracted from 12 silk glands from wandering phase Pi larvae (4 from each of the 3 replicate containers for each condition) using PureLink RNA Mini Kit. cDNA was created from 1 pg total RNA extract for each group using Affinity Script QPCR cDNA synthesis kit. RT-qPCR was performed on 1 pL of cDNA using SsoAdvanced Universal SYBR Green Supermix with Fibroin-Heavy and Fibroin-Light primers (T able 13). As reference genes, the transcript levels of -actin (77_Bac3 F and ft_Bac3R) and ribosomal protein 7S (F^>z_RPS7AF and / RPS7AR) were quantified. All biological samples were run in triplicate. The RT-qPCR reactions were conducted in a C 1000 Touch Thermal Cycler operated by the CFX96 Real-Time System programmed for 35 cycles of 95 °C, 10 sec; 54 °C, 30 sec; 95 °C, 10 sec. To confirm there was minimal genomic DNA contamination co-extracted during total RNA isolation, no reverse transcriptase control (NRT) reactions were made using the whole RNA extract from each sample. Relative gene expression (see Table 9) was determined following the 2 ^vv'^t method as a standardized ratio to the geometric mean of the reference gene transcripts. To calculate the AACt, all conditions were normalized to the 30/20/0.72 fibroin-heavy expression value as represented by its relative expression being a value of 1.

Table 13. Reverse transcriptase primers utilized for gene expression quantification.

[0229] (B) Morphology. Biopsy punches of Pi silk sheets taken from three separate insect populations were secured to scanning electron microscope (SEM) sample stubs with carbon conductive tape. Samples were dried overnight in air and sputter-coated with 8 nm of gold prior to imaging on a Phenom Pure benchtop SEM. Long axis and cross-sectional fiber measurements of non-degummed and degummed Pi silk (n=90 for each) were completed in ImageJ analysis software across 3 different samples and 3 representative images from each sample. Higher resolution images of single Pi fibers can be achieved through atomic force microscopy. Fibers were isolated by placing a larva on glass microscope slides and allowing them to wander until silk fibers were visibly accumulated on the slide. Slides were dried in air overnight before imaging and scanning on a Bruker NanoWizard® 4 XP AFM in QI imaging mode with a FESP-V2 cantilever (k = 2.8 Nm ' ). Image data was processed in JPKSPM Data Processing software.

[0230] (C) Mechanical Properties. Local and bulk scale mechanics of Pi silk fibers were measured using a Nanowizard 4XP ZEISS LSM 900 AFM and Anton Paar MCR 702e Rheometer. The cross-sectional area of fibers and aligned fiber bundles was determined using scanning electron microscopy prior to testing. Local scale measurements of single Pi fibers mechanical properties were completed through atomic force microscopy. Fibers were isolated by placing a larva on glass microscope slides and allowing them to wander until silk fibers were visibly accumulated on the slide. Force spectroscopy of single fibers was performed in QI Advanced mode with a FESP-V2 cantilever (k = 2.8 Nm ^x). Young's modulus (EAFM) and the adhesive force between the tip and the sample was extracted from each force curve in Bruker JPK SPM processing soft are. For bulk mechanics, aligned fiber bundles and silk fiber sheets were evaluated. Aligned fiber bundles were secured to a thin paper frame. Silk sheets were secured to thin paper sheets at their ends to prevent slipping in the instrument. The gauge length of all samples prior to tensile testing was 10 mm. The samples in the paper frames w ere inserted into the solid rectangular fixture (SRF) screw⁷ clamps of an Anton Paar MCT 702e Rheometer (Anton Paar, Graz, Austria) equipped with twin drive motors for dynamic mechanical analyzer (DMA) capabilities. The sample in the paper frame w as tightened in the clamps to 10 centinewton/meter with a torsion screwdriver. Once the sample w as secured, the side paper panels were cut with scissors. The mat samples were inserted into the clamps and tightened to 10 centinew ton/meter. Anton Paar RheoCompass software was used to pre-stretch the samples to 0.1 N and perform static extensional tests at 1 mm/min, and 10 mm/min strain rates. The Young’s modulus (EDMA), ultimate tensile strength (UTS), and break strain (Ebreak) w ere calculated from the resulting stress-strain curves. [0231] (D) Thermal Properties. Melting behavior of Pi silk was analyzed using TA Instruments TGA550. Samples (5-10 mg) were placed in platinum pans and ramped from ambient temperature to 800 °C at 10 °C/min with air flow of 40 L/min and N2 flow of 60 L/min. Mass loss curves are normalized to total sample mass after water evaporation at 100 °C and represented as an average curve ± standard deviation. Water content, Tonset, and Ta were determined using TA Instruments TRIOS analysis software. Standard mode DSC analysis of thermal transitions of Pi silk was completed using a TA Instrument DSC250. Samples (10 mg) were pressed into Al Tzero pans and ramped from 0 to 300 °C at 10 °C/min with a purge gas (N2) flow of 50 L/min. Temperature modulated DSC measurements were completed on a TA Instruments DSC2500. Modulation was performed on non-degummed and degummed silk samples sealed in Al Tzero pans at a modulation period of 60 seconds, temperature amplitude of 0.3 °C, and heating rate of 2 °C/min with a N2 flow of 50 L/min. Thermal transitions were analyzed using TA Instruments TRIOS analysis software.

[0232] (E) Crystalline Content. Quantification of the (3-sheet content and other secondary protein structures in silk fibers was performed using micro-attenuated total reflection Fourier transform infrared spectroscopy analysis. Spectra were collected with a Nicolet iS50 FTIR Spectrometer equipped with a zinc selenide crystal. Spectra was collected over 64 scans at a resolution of 4 cm ¹ over a 4,000-650 cm ¹ wavenumber range for non-degummed and degummed silk (n=6). Background spectra were collected at the same conditions to be subtracted from the sample spectra in OMNIC™ Spectra Software and then analyzed with Origin data analysis software. For analysis, deconvolution of the amide I region (1,590-1 ,720 cm ’j was performed to obtain relative amounts of respective secondary structures.

[0233] (F) Cell Interactions. Cytocompatibility assessments of P. interpunctella silk fibers are analyzed in vitro to assess cell-material interactions. Normal human lung fibroblasts (NHLF) are grown and metabolic activity is assessed. Non-degummed and degummed P. interpunctella silk samples are placed in 48-well plates and NHLFs are seeded at a density of about 30,000 cells/well. Cell metabolic activity is assessed by measuring the absorbance of wells stained with Alamar Blue at time points of 1 and 3 days. Cell phenotype is assessed at 1 and 3 days through immunostaining with DAPI (blue nuclei stain) and phalloidin (green actin filament stain).

Example 12. Manufacture of aqueous Pi silk solution.

[0234] Pi silk fibers differ in composition from other silks. For this reason, methods of providing aqueous silk solutions from, for example Bombyx, are not effective in providing Pi silk from raw silk materials. Described are methods of generating aqueous Pi silk solution from raw silk materials. The resulting solution is not a purified protein solution, but rather consists of all proteins present in the raw silk fibers directly spun by the insect. In general, the methods (1) dissolution, (2) filtration, and (3) dialysis.

[0235] (A) Dissolution. Solubilization of Pi silk is dependent on the processing method: salt type, salt concentration, silk: salt ratio, dissolution temperature, and solution mixing (Table 14). In general, the process begins by clearing Pi silk sheets of loose insect debris. A range of silk sheet mass, 50-200 mg, is then dissolved in a high molarity salt solution at a constant temperature until no solid silk mass is observed in the solution.

Table 14. Parameters modified to dissolve Pi silk into solution.

[0236] (B) Filtration. In general, the stability of the solution prior to dialysis was observed to be sensitive on the presence of solid, undissolved contaminants that remained in the solution. Unfiltered solutions, or solutions filtered with filter mesh >100 pm, placed on dialysis experienced crashing (precipitation) of solubilized polymers, likely due to de-stabilization of the system by the remaining impurities. The removal of these solids was achieved through filtration (e.g, vacuum or gravity filtration) at multiple mesh sizes until visible particulates were removed. Gravity filtration of solution using cloth or fine mesh strainers with mesh sizes <25 pm was sufficient to remove insoluble particulates prior to dialysis.

[0237] (C) Dialysis. Following filtration, the solution can be dialyzed to remove salt ions and stabilize the aqueous silk solution. In some embodiments, dialysis is completed at room temperature (25 °C) against ultrapure water for 48 h using a 3.5 kDa dialysis membrane, stirring the dialysate, and refreshing the dialysate six times within the 48-hour period (after 1 h, 4 h, the first night, the next morning, noon, and night, and the morning on the last day). Lowering the temperature at which dialysis occurs to 4 °C or dialysis against solutions of decreasing salt concentrations (LiBr, NaCl, HEPES buffer, or other salts used in dissolution) improved the stability- of solutions in dialysis. The resulting solutions could be further concentrated through dialysis against a 10% (w/v) PEG (10.000 MW) solution.

[0238] Pi silk generated using the described methods exhibited distinct protein populations of sizes >230 kDa, 174 kDa, 162 kDa, 150 kDa, 76 kDa, 26 kDa, and 7 kDa as visualized through gel electrophoresis (FIG. 16).

Example 13. Manufacture of aqueous Pi silk fibroin solution.

[0239] The described methods for producing aqueous Pi silk fibroin can be modified to enable scale up of production and fabrication of materials.

[0240] A Pi silk fibroin polymer solution in the range of 0. 1-10% by weight in water can be produced by (1) purification of silk fibroin through degumming of the native (raw) silk fiber; (2) dissolution of the degummed fibers through ionic salts; (3) removal of insoluble impurities through filtration (e.g., gravity filtration); (4) removal of residual salt ions through dialysis; and optionally (5) adjustment of polymer concentration. Polymer concentration can be lowered through dilution with water or increased with removal of excess water through evaporation or dewatering with polyethylene glycol (PEG) dialysis.

[0241] Methods for degumming of Pi silk are detailed in Table 15.

Table 15. Degumming methods of P/ silk.

[0242] Alternatively, a Pi silk fibroin polymer solution in the range of 0. 1-10% by weight in water can be produced by downstream purification of a Pi silk solution. Tangential flow filtration can be performed on a solution containing solubilized Pi silk proteins and salt solvent solution prior to dialysis. The solution can be desalted, fractionated, and concentrated in the laminar flow regime to produce the aqueous Pi silk fibroin solution product. Molecular weight cut-offs for size filtration of Pi silk fibroin-heavy chain and Pi silk fibroin-light chain are 390 kDa and 26 kDa. respectively. [0243] Alternatively, the production of Pi silk fibroin solution can be achieved through ion exchange or affinity chromatography.

Example 14. Manufacture of chemically modified Pi silk and silk fibroin solution.

[0244] (A) Chemical modification. Proteins can undergo chemical modifications in order to enable the formation of covalently crosslinked networks, impart sensing or controlled behavior, or to covalently connect bioactive signaling peptides/molecules. This is most often achieved by targeting reactive amino acid residues such as methionine, cystine, try ptophan, ty rosine, lysine, and histidine. In silk fibroin proteins, the molar abundance of these residues tends to be <10%. Pi silk fibroin heavy chain is known to contain ~0.3 mol% lysine, -2.6 mol% tyrosine, and -18.6 mol% serine based on the full-length sequence. These residues are present in sufficient quantity to allow' for the development of modified polymers and covalently crosslinked networks. These reactions either (a) target lysine residues, e.g., in a carbodiimide coupling reaction; (b) target tyrosine residues, e.g.. in a cyanuric chloride activated coupling reaction; or (iii) use a poly(methacrylate) grafting chemistry that can convert both lysine and serine residues. Diazonium coupling reactions targeting tyrosine residues can be leveraged to increase the number of reactive amine groups for more efficient poly(methacrylate) reactions. [0245] Carbodiimide coupling (EDC/NHS) (FIG. 17). This reaction is a standard method used to react primary' amines on the silk fibroin polymer with carboxylic acids, resulting in the formation of an amide bond. Primary' amines are found on the N and C termini of the silk proteins as well as in the side chains of lysine residues. The reaction can be used to react carboxylic acid on the silk polymer and primary amines found on the molecules is being reacted to the silk. Aspartic and glutamic acids are present in 0.29 mol% and 2.24 mol% in Pi silk fibroin heavy' chain, respectively. Reaction conditions to activate the acidic group in the silk fibrin chain include an aqueous mixture of l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a phosphate buffer at a slightly acidic pH. The desired amine bearing molecule to be covalently’ bound is then reacted.

[0246] Cyanuric chloride activated coupling (FIG. 18). This reaction can be used to couple molecules to the ty rosine residues of silk fibroin. The reaction requires a target molecule that has either an amino functional group or a nucleophilic hydroxyl group. This molecule can then be conjugated to cyanuric chloride and reacted with the tyrosine residues on the fibroin polymer in basic conditions. The reaction conditions are varied from the carbodiimide reaction which progresses under slightly acidic conditions. [0247] Poly (methacrylate) grafting (FIG. 19). Acrylate monomers can be attached to nucleophilic amino acids of silk fibroin. The acrylate monomers can the undergo radical polymerization downstream. Lysine and serine residues can be modified acrylate monomers.

[0248] Diazonium coupling (FIG. 20). Diazonium reactions with silk utilize an electrophilic aromatic substitution reaction between ty rosine side chains and the diazonium salt. This leads to the production of azobenzene derivatives. This method can be used to install small molecules such as amines (shown in FIG. 20), sulfonic acids, carboxylic acids, ketones, etc.

Example 15. Assessment of solution properties or characteristics.

[0249] (A) Molecular weight range and fractions. The molecular weight distribution (MWD) and amino acid composition of the polymer solution product will vary depending on the processing steps or modifications to the silk used to generate the solution. MWD can be quantified with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on silk, SF, and chemically modified silk and SF solutions. MWD can be assessed using methods available in the art for MWD. In some embodiments, Pi silk MWD is determined using polyacrylamide gel (e g., SDS-PAGE) analysis. High and low percentage acrylamide gels can be used to assess high- or low-end distributions of solutions at higher resolution. Other methods of MW determination include, but are not limited to, gel permeation chromatography, size exclusion chromatography, light scattering, viscosity detection, and mass spectrometry.

[0250] (B) Amino acid composition. Amino acid composition can be quantified with reversephase high-performance liquid chromatography following hydrolyzation of the polymers in solution with HC1.

[0251] (C) Rheology. The viscosity, yield point, crystallization behavior, and melting behavior of silk, SF, and chemically modified silk and SF solutions can be evaluated with an Anton Paar MCR 702e Rheometer. The behavior of polymer solutions will vary⁷ as a function of polymer concentration, temperature, and applied shear stress to the system. The viscosity correlates to the entanglement of polymers in solution under deformation and is the ratio of shear stress to shear rate. The yield point is determined as the lowest shear stress required to deform a sample’s structure and cause it to flow. Crystallization or the formation of solid precipitate is expected at high shear as the polymer system will display shear thickening behavior prior to precipitating out of solution. Crystallization and gelling behavior occur as the sample is heated or cooled and depend on the strength of polymer binding interactions in response to temperature fluctuations. Example 16. Manufacture of silk-based products.

[0252] Pi silk products can be used to manufacture a range of silk-based products. Such silkbased products include, but are not limited to, sponges, microparticles, nanoparticles, films, hydrogels, and electrospun fibers, each of which can be formed using Pi silk solution, Pi silk fibroin solution, or chemically modified Pi silk or Pi silk fibroin solution.

[0253] (A) Sponges. Pi silk, silk fibroin, and chemically modified silk and silk fibroin sponges comprise of a semicrystalline three-dimensional polymer (native or chemically modified silk or silk fibroin) matrix with interconnected pores throughout the structure. Porosity, density, crystallinity, compressive modulus, and mechanical strength of the porous sponge can be modified to match, mimic, or approximate natural tissues for tissue engineering, encapsulate payloads for delivery of payloads for medical or environmental applications, or achieve filtration of particulates for medical or environmental applications.

[0254] Isotropic porous Pi sponges are produced by (1) forming a silk, silk fibroin, or chemically modified silk or silk fibroin solution; (2) forming a Pi polymer solution comprising 1-10% by weight in water; (3) placing the polymer solution into a form, pattern, or mold; (4) removing water from the polymer through lyophilization; and (5) contacting or treating the polymer to induce crystallinity and form a water-insoluble porous structure. Isotropic porous Pi sponges are shown in FIG. 21.

[0255] Anisotropic porous Pi sponges are produced by (1) forming a silk, silk fibroin, or chemically modified silk or silk fibroin solution: (2) forming a Pi polymer solution comprising 1 -10% by weight in water; (3) placing the polymer solution into a metal mold; (4) directionally freeze-templating the polymer by placing dry’ ice and ethanol (50-100%) on one side of the metal mold; (5) removing water from the polymer through lyophilization; and (6) contacting or treating the polymer to induce crystallinity and form a water-insoluble porous structure. Methods to induce crystallinity within the sponge include washing with methanol, water vapor annealing, or autoclaving of the dried, insoluble sponge. Encapsulation of active payloads or formation of composite porous sponges with additional polymers can be achieved through mixing the additional payload or polymer with the silk, silk fibroin, or chemically modified silk and silk fibroin solution prior to placing the solution into a mold.

[0256] (B) Micro- and nanoparticles . Pi silk, silk fibroin, and chemically modified silk and silk fibroin micro- and nanoparticles comprise of a semi-crystalline three-dimensional polymer (native or chemically modified silk or silk fibroin) matrix that is capable of encapsulation and stabilization of diverse payloads for release capabilities. Morphology, size, polydispersity, crystallinity, mechanical strength of the Pi silk polymer particle can be modified to encapsulate particulates for delivery of payloads for medical or environmental applications or stabilize bioactive molecules. Micro- and nanoscale Pi particles are produced by (1) forming a silk, silk fibroin, or chemically modified silk or silk fibroin solution; (2) forming a Pi polymer solution comprising 1-10% by weight in water; (3) blending the polymer solution with polyvinyl alcohol (PVA) in various weight ratios (control of 1 :4 silk to PVA); (4) hydrophobic collapse of polymer particles through sonication; (5) removing the water content in the silk polymer-PVA solution through evaporation; (6) removal of PVA solvent through dissolution of the polymer- PVA film in water and centrifugation to collect the polymer particle pellet; (7) re-distribution of polymer particles in aqueous solution through sonication. Pi Silk particles made using these methods are shown in FIG. 22. Methods to modulate particle size include varying silk concentration, sonication amplitude and time, the temperature of water evaporation from the polymer-PVA blend, and the weight ratio of silk polymer to PVA. Passive encapsulation of active payloads or formation of composite porous sponges with additional polymers can be achieved through mixing the additional payload or polymer with the silk, silk fibroin, or chemically modified silk and silk fibroin solution prior to mixing the polymer solution with PVA.

[0257] (C) Films. Pi silk, silk fibroin, and chemically modified silk and silk fibroin films comprise of a semicrystalline, continuous two-dimensional polymer (native or chemically modified silk or silk fibroin) matrix. Porosity, density, crystallinity, Young’s modulus, mechanical strength, and elongation or torsional properties of the polymer film can be modified to match, mimic, or approximate natural tissues for tissue engineering, adhere to skin or other natural tissues for wound tissue repair, coat natural materials for protection or modulation of growth, encapsulate payloads for delivery of payloads for medical or environmental applications, and achieve filtration or entrapment of particulates for medical or environmental applications. Thin Pi silk polymer films are produced by (1) forming a silk, silk fibroin, or chemically modified silk or silk fibroin solution; (2) forming a Pi polymer solution comprising 1-10% by weight in water; (3) placing the polymer solution into a flat or patterned form; (4) removing water from the polymer through evaporation; and (5) contacting or treating the polymer to induce crystallinity and form a water-insoluble structure. A Pi silk film is shown in FIG. 23. Methods to induce crystallinity within the film include washing with methanol, water vapor annealing, or autoclaving of the dried, insoluble film. Encapsulation of active payloads or formation of composite films with additional polymers can be achieved through mixing the additional payload or polymer with the silk, silk fibroin, or chemically modified silk and silk fibroin solution prior to placing the solution into a form. [0258] (D) Hydrogels. Pi silk, silk fibroin, and chemically modified silk and silk fibroin hydrogels comprise a swollen, semicrystalline three-dimensional polymer (native or chemically modified silk or silk fibroin) matrix with the ability to retain or release fluids from the structure. Swelling, permeability, degradation, compressive modulus, and mechanical strength of the polymer hydrogel can be modified to match, mimic, or approximate natural tissues for tissue engineering, encapsulate payloads for delivery of payloads for medical, food, and environmental applications, or filtration of particulates for medical or environmental applications. The three-dimensional structure and morphology of the hydrogel product can be modified through utilization of molds or patterns within production. Pi hydrogels are produced by (1) forming a silk, silk fibroin, or chemically modified silk or silk fibroin solution; (2) forming a Pi polymer solution comprising 1-10% by weight in water; (3) contacting or treating the polymer solution to induce crosslinking within the structure; (4) placing the treated polymer solution into a form or pattern; and (5) incubation of the polymer solution to allow gelation. Physical crosslinking of the polymer can be achieved by (a) sonication, (b) temperature shifts, (c) pH shifts, (d) electrogelation, and (e) high pressure treatments. Chemical crosslinking of the polymer can be achieved through chemical modification of the polymer solution and addition of chemical cross-linking agents. Encapsulation of active payloads or formation of composite hydrogels with additional polymers can be achieved through mixing the additional payload or polymer with the silk, silk fibroin, or chemically modified silk or silk fibroin solution prior inducing gelation and placing the solution into a mold. In another embodiment, composite silk hydrogels can be achieved through generation of a double-network hydrogel with Pi silk and an additional structural or bioactive polymer.

[0259] (E) Electrospun fibers. Pi silk, silk fibroin, and chemically modified silk and silk fibroin electrospun fibers comprise of a semicrystalline three-dimensional polymer (native or chemically modified silk or silk fibroin) spun into micro- or nanoscale fibrous structures. Electrospun fibers can be spun into thin sheets, multi-layered sponges, or pattern molds to modify the three-dimensional structure of the product. Fiber diameter, porosity, crystallinity, surface roughness, and mechanical strength of the electrospun fiber structure can be modified to match, mimic, or approximate natural tissues for tissue engineering, encapsulate payloads for delivery of payloads for medical or environmental applications, or achieve filtration of particulates for medical or environmental applications.

[0260] Isotropic Pi electrospun silk fiber structures are produced by (1) forming a silk, silk fibroin, or chemically modified silk or silk fibroin solution; (2) forming a Pi polymer solution comprising 1-10% by weight in water; (3) mixing the Pi polymer solution with 5% wt/v PEO in water; (4) drawing the silk polymer-PEO solution into a 10-20 mL syringe equipped with a 16-gauge needle; (5) applying a voltage gradient between the needle and collection surface (between 5-20 cm apart); (6) spinning fibers onto the collection surface at desired thickness; (7) contacting the polymer to form a water-insoluble structure; (8) removal of PEO through contact with water; (9) treatment of the electrospun fiber structure to induce cry stallinity.

[0261] Anisotropic Pi electrospun silk fiber structures are produced by spinning fibers onto a collection surface on a rotating mandrel.

[0262] Methods to induce crystallinity within the spun fibers include washing with methanol, water vapor annealing, or autoclaving of the dried, insoluble fiber structure. Functionalization of electrospun fibers can be achieved through encapsulation of active pay loads or formation of composites with additional polymers through mixing the additional payload or polymer with the silk, silk fibroin, or chemically modified silk and silk fibroin solution prior to loading into the syringe.

[0263] In some embodiments, composite electrospun fibers can be achieved through dualsyringe electrospinning to generate overlapping layers of electrospun Pi silk fibers and an additional structural or bioactive polymer.

Example 17. Manufacture of a Pi silk films.

[0264] Water-insoluble Pi films can be formed through evaporation of a silk polymer or silk polymer blend solution onto a flat or patterned mold. Insolubility can be achieved through posttreatment of the dried film, leading to the generation of water-insoluble thin film in a simple, cost-effective method with increased translatability to medical applications as it avoids the use of organic solvents. This process can be scaled up or down as needed for product supply or solution sourcing. This method can be utilized to form films from z silk solution, Pi silk fibroin solution, chemically modified Pi silk and silk fibroin solution, and Pi silk/silk fibroin polymer blends. Film thickness, size, and mechanical properties can be modulated by changing fabrication parameters such as silk concentration, mold size or pattern, temperature of film casting, and post-treatment methods to better fit proposed applications.

[0265] Encapsulation of active payloads and formation of polymer blend fdms. A wide range of molecules and polymers can be encapsulated or distributed within silk film products due to the complexity of the silk protein matrix. Model drugs, small peptides, or other bioactive agents of interest varying in hydrophobicity, structure, size, and biocompatibility can be passively encapsulated by or blended with the silk polymer by combining the desired concentration of payload or polymer with silk solution prior to film casting. Exemplary molecules that can be encapsulated in Pi silk films include, but are not limited to. therapeutic agents, decellularized extracellular matrix, chitosan, polycaprolactone collagen, alginate, cellulose, hemoglobin, nucleic acids (e.g., antisense oligonucleotides, siRNA), anti-microbial drugs (e.g, crystal violet, rifampin, gentamicin, octominin, saflufenacil, salicylaldehyde, sericin extracts), dyes, magnetic tracers, and agricultural compounds (e.g, metal ions (e.g., for use in seed coatings), lambda cyhalothrin, ivermectin, commercial pesticide granules).

Example 18. Evaluation of silk polymer film properties.

[0266] (A) Morphology. Pi silk films are freeze-fractured before securing samples to SEM sample stubs with carbon conductive tape. Samples are dried overnight in air prior to imaging on a Phenom Pure benchtop SEM. Film thickness and surface defects or patterns are observed with measurements completed in ImageJ analysis software across 3 different samples and 3 representative images from each sample. Pi silk film surface features can be assessed with atomic force microscopy. Samples are secured to glass slides or in 25 mm imaging plates for imaging in water or PBS. Film structures are imaged and scanned on a Bruker NanoWizard® 4 XP AFM in QI imaging mode. Image data is processed in JPKSPM Data Processing software. [0267] (B) Crystalline Content. Quantification of the (3-sheet content and other secondary protein structures in Pi silk films is performed using micro-attenuated total reflection Fourier transform infrared spectroscopy (microATR FTIR) analysis. Spectra are collected with a Nicolet iS50 FTIR Spectrometer, equipped with a micro attenuated total reflections (microATR) germanium crystal and MCT/A detector. Measurements consist of 128 scans with a resolution of 4 cm ¹ over wavenumbers ranging 4,000-650

Background spectra are collected using the same conditions and subtracted from each sample spectra. For analysis, the amide I region is deconvoluted to obtain relative amounts of respective secondary structures. Measurements are completed for neat-silk films, polymer blend films, and films containing or coated with active or therapeutic agents.

[0268] (C) Mechanical Properties. The Young's modulus, ultimate tensile strength, shear modulus, and maximum torque of Pi silk films are measured using an Anton Paar MCR 702e Rheometer. The width and thickness of film samples are measured with digital calipers and scanning electron microscopy prior to testing, respectively. 10 mm x 10 mm samples are cut from cast films and placed into tensile clamps, tightened to 10 cN/m with a torsion screwdriver, and pre-stretched to 0.01 N to remove any sag in the sample. The gap width is recorded as the height of the film sample between the clamps. Anton Paar RheoCompass software is utilized to perform tensile and torsion testing. Microscale measurements of Pi silk film mechanical properties are assessed with atomic force microscopy. Samples are secured to glass slides or in 25 mm imaging plates for imaging in water or PBS. Force spectroscopy is performed on film surfaces in QI Advanced mode on a Bruker NanoWizard® 4 XP AFM. Force map data is processed in JPKSPM Data Processing software. Calculations of elastic modulus are completed by fitting the linear region of the force curves. Measurements are completed for neat-silk films, polymer blend films, and films containing or coated with active or therapeutic agents. Tests can be completed in air, in water, in PBS. or other liquids that are relevant to the desired application.

[0269] (D) Thermal Properties. Biopsy punches of Pi silk films are analyzed using TA Instruments TGA550. Samples are placed into platinum pans and ramped from ambient temperature to 800 °C at 10 °C/min with air flow of 40 L/min and N2 flow of 60 L/min. Mass % curves are normalized to sample mass after total water evaporation at 100 °C. Biopsy punches of Pi silk films are analyzed using TA Instrument DSC250. Samples are sealed into Al Tzero pans and ramped from 0 to 300 °C at 10 °C/min with a purge gas (N2) flow of 50 L/min. Data are analyzed using TRIOS software package to determine material phase transition regions, temperatures of glass transition, melting, and degradation, and crystalline content. Measurements will be completed for neat-silk films, polymer blend films, and films containing or coated with active or therapeutic agents.

[0270] (E) Cell interactions. Cytocompatibility assessments of /h silk films are completed in vitro to assess how processing parameters improve cell-material interactions. Silk films are formed from Pi silk solution. Pi silk fibroin solution, chemically modified silk or silk fibroin solution, polymer blend solutions, and therapeutic/active agent-silk composite solutions. Bioactivity of the silk film product is evaluated in vitro with cell types relevant to the proposed application (immortalized cell lines, primary cells, and stem cells relevant to the target tissue or environment). Cell metabolic activity, proliferation and viability, migration, and phenotype are assessed with standard assays, staining, and imaging. Cell interactions are assessed over 1 week of culture with specific assessments performed at 1, 3, 5, and 7 days of culture. If relevant to the application, longer time periods are assessed. Samples are imaged with a Zeiss LSM 980 confocal microscope or Keyence BZ-X800 microscope.

Example 19. Manufacture of a three-dimensional porous Pi silk sponges.

[0271] Water-insoluble isotropic and anisotropic Pi sponges can be formed through removal of water from frozen polymer solutions contained in a flat or patterned mold. Insolubility can be achieved through post-treatment of the dried sponge, leading to the generation of w ater- insoluble three-dimensional porous structures in a simple, cost-effective method with increased translatability- to medical applications as it avoids the use of organic solvents. Active agents or additional polymers of interest can be combined with the silk polymer solution prior to freezing. This method is utilized to form sponges from z silk solution, Pi silk fibroin solution, chemically modified Pi silk and silk fibroin solution, and Pi silk/silk fibroin polymer blends. Sponge porosity, thickness, size, and mechanical properties can be modulated by changing fabrication parameters such as silk concentration, mold size or pattern, temperature of polymer freezing, and post-treatment methods to better fit proposed applications.

[0272] (A) Encapsulation of active payloads and formation of polymer blend sponges. A wide range of molecules and polymers can be encapsulated or distributed within silk sponge products due to the complexity of the silk protein matrix. Model drugs, small peptides, or other bioactive agents of interest varying in hydrophobicity, structure, size, and biocompatibility can be passively encapsulated by or blended with the silk polymer by combining the desired concentration of payload or polymer with silk solution prior to freezing. This method is utilized to form composite sponge products generated from Pi silk solution. Pi silk fibroin solution, chemically modified Pi silk and silk fibroin solution, and Pi silk/silk fibroin polymer blends. [0273] (B) Evaluation q/'Pi sponge system properties.

[0274] (i) Morphology- and Structure. Pi silk sponge morphology-, porosity-, and internal structure are visualized through scanning electron microscopy. Samples will be secured to SEM sample stubs with carbon conductive tape and dried overnight in air prior to imaging on a Phenom Pure benchtop SEM. Sponge thickness, uniformity-, and structural integrity are observed and measurements of porosity- are completed in ImageJ analysis software. Measurements are completed for neat-silk sponges, polymer blend sponges, and sponges containing active or therapeutic agents.

[0275] Pi silk sponge morphology and internal structure can also be assessed through histological sectioning and staining, in addition to the study of cell infiltration into the 3D structure. Samples are fixed in phosphate buffered formalin overnight before dehydration through a series of ethanol solutions of increasing concentrations. Samples are cleared in xylene before being placed in a wax bath overnight. Sponges are embedded in wax molds, sectioned at 10 pm thickness, and mounted on slides for analysis. Sections are deparaffinized and rehydrated before being stained with hematoxylin and eosin, Masson’s trichome, or fluorescent immunostaining (DAPI/Phalloidin) to visualize sample sections and cell infiltration. Coverslips are secured to the stained samples before imaging on a Keyence BZ- X800 benchtop microscope.

[0276] (ii) Crystalline Content. Quantification of the (3-sheet content and other secondary protein structures in silk sponges is performed using micro-attenuated total reflection Fourier transform infrared spectroscopy (microATR FTIR) analysis. Spectra are collected with a Nicolet iS50 FTIR Spectrometer, equipped with a micro attenuated total reflections (microATR) germanium crystal and MCT/A detector. Measurements will consist of 128 scans with a resolution of 4 cm ¹ over wavenumbers ranging 4,000-650

Background spectra are collected using the same conditions and subtracted from each sample spectra. For analysis, the amide I region is deconvoluted to obtain relative amounts of respective secondary structures. Measurements are completed for neat-silk sponges, polymer blend sponges, and sponges containing active or therapeutic agents.

[0277] (iii) Mechanical Properties. The Young’s modulus and ultimate tensile strength of isotropic and anisotropic Pi silk sponges are measured using an Anton Paar MCR 702e Rheometer. Dried or hydrated sponge samples are evaluated depending on application relevancy. The width and thickness of sponge samples are measured with digital calipers and scanning electron microscopy prior to testing, respectively. Sponges are placed into tensile clamps, tightened with a torsion screwdriver, and pre-stretched to 0.01 N to remove any sag in the sample. The gap width is recorded as the height of the sponge sample between the clamps. Anton Paar RheoCompass software is utilized to perform tensile testing through static extension, amplitude sweeps, frequency sweeps, hysteresis tests, and/or fatigue tests if relevant to the current application.

[0278] (iv) Thermal Properties. Biopsy punches of dried Pi silk sponges are analyzed using TA Instruments TGA550. Samples are placed into platinum pans and ramped from ambient temperature to 800 °C at 10 °C/min with air flow of 40 L/min and N2 flow of 60 L/min. Mass % curves are normalized to sample mass after total water evaporation at 100 °C. Biopsy punches of dried Pi silk sponges are analyzed using TA Instrument DSC250. Samples are sealed into Al Tzero pans and ramped from 0 to 300 °C at 10 °C/min with a purge gas (N2) flow of 50 L/min. Data are analyzed using TRIOS software package to determine material phase transition regions, temperatures of glass transition, melting, and degradation, and cry stalline content. Measurements are completed for neat-silk sponges, polymer blend sponges, and sponges containing active or therapeutic agents.

[0279] (v) Cell interactions. Cytocompatibility assessments of Pi silk sponges are completed in vitro to assess how processing parameters improve cell-material interactions. Silk sponges are formed from Pi silk solution, Pi silk fibroin solution, chemically modified silk or silk fibroin solution, polymer blend solutions, and therapeutic/active agent-silk composite solutions. Bioactivity of the silk sponge product is evaluated in vitro with cell types relevant to the proposed application (immortalized cell lines, primary cells, and stem cells relevant to the target tissue or environment). Cell metabolic activity, proliferation and viability, and phenoty pe are assessed with standard assays, staining, and imaging. Infiltration of cells into the three- dimensional porous structure are visualized through standard histological techniques. Cell interactions are assessed over 1 week of culture with specific assessments performed at 1, 3, 5, and 7 days of culture. If relevant to the application, longer time periods are assessed. Samples are imaged with a Zeiss LSM 980 confocal microscope or Keyence BZ-X800 microscope.

Example 20. Manufacture of Pi silk particles.

[0280] Silk particles are formed through phase separation and induction of the hydrophobic collapse of silk proteins in solution. This is achieved through combination of PVA and /7 silk, leading to the generation of water-insoluble silk particles in a simple, cost-effective method with increased translatability' to medical applications as it avoids the use of organic solvents. This method is utilized to form particles from Pi silk solution, Pi silk fibroin solution, chemically modified Pi silk and silk fibroin solution, and Pi silk/silk fibroin polymer blends. Particle size can be modulated by changing fabrication parameters such as silk concentration relative to PVA concentration, sonication amplitude, and temperature of film casting to better fit proposed applications.

[0281] (A) Encapsulation of active payloads. A wide range of molecules can be encapsulated or entrapped in silk particle systems due to the complexity' of the silk protein matrix within the particle. Molecules are passively encapsulated by combining the desired concentration with silk solution prior to combination with PVA. The encapsulation efficiency can be determined by dividing the mass of loaded molecules into the particles divided by the initial mass input. Loading efficiency is determined by dividing the mass of loaded molecules into the particles by the mass of silk particles. Additionally, Pi silk fibroin contains a unique sequence of reactive residues to previously used silk fibroin particle systems that can be leveraged to form ionic interactions between the silk particle systems and drug payload. This provides a means to modulate the pharmacokinetics of drug pay loads. These methods are utilized to form encapsulation systems in particles generated from Pi silk solution, Pi SF solution, chemically modified Pi silk and SF solution, and /7 silk/SF polymer blend solution.

[0282] (B) Evaluation of Pi silk particle system properties. [0283] (i) Morphology and size. Pi silk particle morphology and size are visualized through scanning electron microscopy. 10 pL of Pi particle suspension is pipetted onto carbon conductive tape secured to SEM sample stubs and dried overnight in air. Sample stubs are sputter-coated with 8 nm of Au prior to imaging on a Phenom Pure benchtop SEM. Particle size is measured using ImageJ analysis software. Measurements can be determined for neatsilk particle systems, polymer blend particle systems, and particles encapsulating active or therapeutic agents. The particle size and poly dispersity of silk particle formulations in ultrapure water are determined at 25 °C by dynamic light scattering using a Zetasizer Nano-ZS Malvern Instrument. All measurements are conducted with refractive indexes of 1.33 for water and 1.60 for silk protein.

[0284] (ii) Mechanical Properties. Measurements of Pi silk particles’ mechanical properties are assessed using atomic force microscopy. Aliquots of particle solutions are pipetted onto mica substrates and dried in air prior to testing. Force spectroscopy is performed on film surfaces in QI Advanced mode on a Bruker NanoWizard® 4 XP AFM. Force map data is processed in JPKSPM Data Processing software. Calculations of elastic modulus are completed by fitting the linear region of the force curves. Measurements can be determined for neat-silk particle systems, polymer blend particle systems, and particles encapsulating active or therapeutic agents.

[0285] (iii) Thermal Properties. Pi particles suspended in ultrapure water are analyzed using TA Instruments TGA550. Samples are pipetted into platinum pans and held at 70 °C to allow drying before ramping to 800 °C at 10 °C/min with air flow of 40 E/min and N2 flow of 60 L/min. Mass % curves are normalized to sample mass after total water evaporation at 100 °C. Pi silk particles suspended in ultrapure water are analyzed using TA Instrument DSC250. Samples are pipetted into Al Tzero pans and covered with a hermetically sealed lid before being ramped from 0 to 300 °C at 10 °C/min with a purge gas (N2) flow of 50 L/min. Data are analyzed using TRIOS software package to determine material phase transition regions, temperatures of glass transition, melting, and degradation, and crystalline content. Measurements can be determined for neat-silk particle systems, polymer blend particle systems, and particles encapsulating active or therapeutic agents.

[0286] (iv) Crystalline Content. Quantification of the |B-sheet content and other secondary protein structures in silk particles is performed using micro-attenuated total reflection Fourier transform infrared spectroscopy (microATR FTIR) analysis. Particle suspensions are lyophilized at -80 °C and 0.320 mbar for 3 days prior to analysis. Spectra are collected with a Nicolet iS50 FTIR Spectrometer, equipped with a micro attenuated total reflections (microATR) germanium crystal and MCT/A detector. Measurements include 128 scans with a resolution of 4 cm ¹ over wavenumbers ranging 4.000-650 cm Background spectra are collected using the same conditions and subtracted from each sample spectra. For analysis, the amide I region is deconvoluted to obtain relative amounts of respective secondary structures. Measurements can be determined for neat-silk particle systems, polymer blend particle systems, and particles encapsulating active or therapeutic agents.

[0287] (v) Cell Interactions. Cytocompatibility assessments of Pi silk micro- and nanoparticles are performed in vitro to assess how processing parameters affect cell-material interactions. Silk particles are formed from z silk solution, Pi silk fibroin solution, chemically modified silk or silk fibroin solution, polymer blend solutions, or therapeutic/active agent-silk composite solutions. Bioactivity of the silk particle product is evaluated in vitro with cell types relevant to the proposed application (e.g, immortalized cell lines, primary cells, or stem cells relevant to the target tissue or environment). Cell metabolic activity, proliferation and viability, and phenoty pe are assessed with standard assays, staining, and imaging. Cell interactions are assessed over 1 week of culture with specific assessments performed at 1, 3. 5, and 7 days of culture. If relevant to the application, longer time periods are assessed. Samples are imaged with a Zeiss LSM 980 confocal microscope or Keyence BZ-X800 microscope.

Example 21. Pi Silk Uses and Devices.

[0288] (A) Medical device.'bio implants. Medical devices that undergo implantation can often trigger adverse immune responses, complicating their integration and functionality within the body. To overcome these challenges, Pi silk fibroin, which is biocompatible, can be utilized as a coating for these devices. The biocompatibility of Pi silk fibroin minimizes the risk of immune reactions, promoting better acceptance and integration of the implants. In addition to its potential use as a biocompatible coating, Pi silk fibroin can be used as a biodegradable material for various medical applications. Its mechanical properties and biodegradability make it a candidate for suturing material and screws.

[0289] (B) 3D printing. A common limitation of traditional silk fibroin-based bioink formulations is their tendency to plasticize under shear forces ty pically encountered during 3D printing, preventing the proper formation of structures. This issue arises due to the extensive (3-sheet regions present in. e.g, Bombyx, silk fibroin proteins. Pi silk contains lower (3-sheet content and features a more variable repeating structure, making it less sensitive to shear forces. This characteristic could allow Pi silk to maintain its integrity during the 3D printing process, leading to more reliable and consistent outcomes.

[0290] In addition to its reduced sensitivity to shear forces. Pi silk has several other properties that make it suitable for 3D printing applications. Its mechanical properties, such as elasticity and strength, differ compared with previously reported silk fibroin, providing novel properties in designing bioinks for various biomedical applications. Its unique protein composition and structure can be exploited to produce bioinks with tailored properties, enabling the fabrication of scaffolds with specific mechanical and biological characteristics. This adaptability' is particularly advantageous for applications requiring precise control over the microarchitecture of printed constructs, such as in the development of vascularized tissues or organs.

Example 22. Modified functional properties of Pi fibers through purification of silk fibroin. [0291] Upon degumming and removal of sericins. we observed a 45-55% mass loss. Degumming also resulted in the removal of web-like structures in the Pi silk sheets as determined visually (FIG. 24). We further investigated the mechanical properties of nondegummed and degummed aligned fiber bundles. Such fiber bundles are produced naturally by the Pi larvae as they drop down on a silk fiber from the top of their rearing containers to their food source and travel back up. These aligned structures resembled a solid, floss-like fiber consisting of many individual silk fibers (FIG. 24), in contrast to the isotropic, random orientation of silk fibers in the silk sheets. Fiber bundles exhibit similar mass loss and modified morphology upon degumming (FIG. 24). Results from these structural property assessments are summarized in Table 16. Lastly, we assessed the cell-material interactions of ft silk fiber sheets with normal human lung fibroblasts (NHLF) analyze Pi bioactivity' as a raw material for biomaterial applications.

[0292] Degumming of Pi silk fibers did not significantly shift protein structure, though there was a slight increase in crystallinity between non-degummed and degummed Pi silk as estimated by P-sheet content through FTIR. Non-degummed Pi silk exhibited a higher midpoint degradation temperature (Td) than degummed samples (p<0.01). The decrease in degradation temperature observed in degummed samples may be due to shorter polymers resulting from the degumming processes in which the polymer chain experienced thermal stress that began to degrade or shorten the polymer, modifying the molecular mass distribution of the sample. This modification of polymer chain length through degumming can be applied further to biomaterial products, wherein increasing or decreasing the degumming time of the silk can tune the molecular mass distribution of silk fibroin and resulting material properties. Nondegummed Pi silk exhibited melting behavior around 247° C prior to degradation and athermal transition between 150-170° C that we hypothesize is within the glass transition region. Degummed Pi silk did not have an observable melting point or glass transition, which could result from the heat treatment during degumming. Degumming can modify the structure, stability’, or interactions between the proteins in Pi silk fibers, causing a more gradual transition of polymer chain disentanglement or crystallization.

Table 16. Properties of Pi fiber sheets and bundles. NDG = non-degummed. DG = degummed

[0293] We performed bulk tensile mechanical assessments on non-degummed and degummed silk sheets and aligned fiber bundles to determine the impact of anisotropy on Pi silk. Isotropic silk sheets demonstrated an elastic response at low strain (<0.05 mm/mm) and a plastic, mostly brittle response at high strains (>0.2 mm/mm). Non-degummed and degummed fiber bundles all exhibited a linear, elastic response at low strains (<0.02) and exhibited a strainstiffening response at higher strains. Since these fiber bundles were highly aligned, they could withstand much larger forces than those of the isotropic sheets, almost an order of magnitude difference in stress. This anisotropy contributed to an even load spread across the entire material during extension rather than the rearrangement of random fibers from the sheets that caused premature breaking. We observed a decrease in the elastic response of the degummed materials due to the removal of the web-like structures from the surface of the fibers. Degummed sheets produced larger mechanical property values for Young’s modulus (20-44 MPa) and UTS (8-18 MPa). There were large statistically significant differences in Young’s modulus of the non-degummed (536 ± 127 MPa) versus degummed (3052 ± 965 MPa) fiber bundles, with around a 5-fold change in values. While there were differences when comparing strain rates, these overall values are not statistically significant, demonstrating that the aligned fiber bundles were less dependent on strain rate than the isotropic sheets. Similar trends existed for the UTS results where there are no statistical differences in non-degummed and degummed aligned fiber bundles when comparing different strain rates. Overall, the effect of degumming had the most significant impact on the extensional properties of Pi aligned fiber bundles. This demonstrates that mechanical properties of Pi materials have some tunability from the raw material format.

[0294] The mechanical property similarities between non-degummed and degummed Pi fibers provides the framework for future applications for Pi biomaterials. Degummed Pi silk has potential as a replacement to synthetic polymer sources in biomaterials and medicine due to its equivalent range in tensile modulus (2 - 4 GPa) and tensile strength (50 - 100 MPa). Degummed and non-degummed Pi fibers fall within the range of tensile strength and modulus of current materials demonstrating its suitability as an alternative material for tendon regeneration applications. Pi aligned fiber bundles closely matched the mechanical properties of highly aligned tissue types such as tendon and ligaments, suggesting utility in aligned soft tissue applications such as tendon and ligament engineering. The structure of aligned Pi fiber bundles also matched the structural hierarchy of the native collagen subunits found in tendon and the stress-strain curves of Pi aligned fiber bundles match fracturing found in the plastic regime of native tissue.

[0295] It is anticipated that degumming Pi silk will improve the bioactivity and reduce immunogenicity of silk sheets due to the removal of sericin proteins. The metabolic activity of cells on both samples (n=7) remained relatively constant over the investigated culture time (FIG. 25). However, a slight decrease in cell adhesion to the silk sheet surface was observed in both non-degummed and degummed cultures after 3 days (FIG. 25). The bioactivity of Pi silk may be shifted through the processing steps use to fabricate Pi silk into biomaterial formats. Additionally, cell-material interactions may be enhanced through the addition of extracellular matrix proteins (e.g, collagen, fibronectin). In this study, cells appeared to initially adhere to the surface of the silk fiber sheet. The cells then migrated through the gaps and layers of fibers within the sheet over longer time periods. Migration into and through the Pi sheet could prove useful in tissue engineering applications to act as a scaffold or in filtration applications where large particulates are filtered out and smaller molecules transport through the sheet. Example 23. Encapsulation of a bioactive agent in a Pi silk particle

[0296] The ability of Pi silk particles to entrap bioactive agents was evaluated by analyzing kinetic entrapment of hemoglobin within a silk particle matrix. Pi silk particles were fabricated through phase separation with PVA with tunable particle diameter and morphology. Hemoglobin was incorporated into the silk particles during fabrication by combining the silk solution with hemoglobin at a desired concentration prior to the addition of PVA and sonication. Encapsulation of hemoglobin in silk particles was visualized through fluorescent imaging following incubation with hemoglobin-FITC conjugated antibody. Particle morphology, size, and polydispersity were assessed using SEM and DLS (FIG. 26). Pi silk particles (0.2 wt/v%) loaded with hemoglobin (0. 1 mg/mL) having particle diameter ranges between 0.200 and 1 pm were observed.

Example 24. Cytocompatibility o/ Pi silk films.

[0297] Cell-material interactions upon the addition of a bioactive component (e.g, porcine decellularized extracellular matrix (dECM)) to both silk and silk fibroin films are analyzed to assess composite film systems tailored to wound healing applications. It is anticipated that Pi silk fibroin films will induce a lower immune response due to lack of interactions with sericin proteins. Addition of dECM is anticipated to increase cell proliferation on silk and silk fibroin films. Biopolymer films are formed from 3% Pi silk solution or purified Pi SF solution. Porcine dECM is added to Pi silk and silk fibroin solution to obtain a concentration of 0.5 mg/mL. Films are cast a petri dish or PDMS mold and allowed to dry overnight prior to autoclaving to induce crystallinity and sterilize the product. Normal human lung fibroblasts are seeded onto films for study of cell metabolic activity and proliferation over a week of culture. Cell metabolic activity is assessed with an Alamar Blue assay. Activity is assessed by measuring the absorbance of wells stained with Alamar Blue with a spectrophotometer at time points of 1, 3, and 7 days. Cell viability is measured by using a live-dead assay. Calcein and propidium iodide are used to differentiate between live (fluoresce green) and dead (fluoresce red) cells and used to quantify cell death at time points of 1 , 3, and 7 days. Cell phenotype is assessed at each time point through immunostaining with DAPI (blue nuclei stain) and phalloidin (green actin filament stain). Samples will be fixed and stained with primary' and secondary antibodies and imaged with a Zeiss LSM 980 confocal microscope or Keyence BZ-X800 microscope.

Claims

Claims:

1. A method of rearing Plodia interpunctella (Pi) for production of Pi silk comprising: growing Pi larvae at an average temperature about 22 °C to about 26 °C, at population density of about 0.52 to about 0.921arvae/cm³.

2. The method of claim 1 , wherein the Pi larvae are grown at an average temperature of

24±1 °C.

3. The method of claim 2, w herein the Pi larvae are grow n at an average temperature of 24±0.5 °C.

4. The method of any one of claims 1-3, wherein the Pi larvae are grown at a population density of 0.72±0.2 larvae/cm³.

5. The method of claim 4, wherein the Pi larvae are grown at a population density of 0.72±0. 1 larvae/cm³.

6. The method of claim 5, wherein the Pi larvae are grown at a population density of 0.72±0.05 larvae/cm’.

7. The method of any one of claims 1-6, wherein the Pi larvae are provided about 0.05 to about 0.2 gram of diet per larvae.

8. The method of claim 7, w herein the Pi larvae are provided about 0.067 to about 0.2 gram of diet per larvae; about 0.07 to about 0. 15 gram of diet per larvae; about 0.08 to about 0. 125 gram of diet per larvae; or about 0.09 to about 0. 1 Igram of diet per larvae.

9. The method of claim 8, w herein the Pi larvae are provided about 1 larva per 0.1 gram of diet.

10. The method of any one of claims 7-9, wherein the diet comprises: w heat bran, organic buckwheat honey, glycerin, water, an insect vitamin mix, and brewer's yeast.

11. The method of any one of claims 7-9, wherein the diet further comprises one or more of: one or more amino acids; one or more metallic ions; one or more insect juvenile hormones; one or more nanoparticles, nanofibers, or nanotubes; one or more plastics, or a dye.

12. A method of producing raw Pi silk comprising: rearing Pi larvae according to the method of any one of claims 1-11 and collecting Pi silk mats from walls of a container in which the Pi larvae are reared.

13. A method of producing an aqueous Pi silk solution comprising:

(a) dissolving raw Pi silk in a salt solution;

(b) filtering the dissolved Pi silk; and

(c) dialyzing the filtered Pi silk.

14. The method of claim 13, wherein the salt comprises: LiBr, Ca(NO3)2, LiNCh, Mg(NO₃)2, MgCh. or CaCl₂.

15. The method of claim 14, wherein the salt solution comprises: 7-9.3 M LiBr, 8-9.3 M LiBr, 5-8 M Ca(NO₃)₂, 6-8 M Ca(NO₃)₂, 1-2 M LiNO₃, 3-5 M Mg(NO₃)₂, 5-7 M MgCh, or 5-8 M CaCh.

16. The method of any one of claims 13-15, wherein dissolving the raw Pi silk comprises incubating the raw Pi silk in the salt solution at a raw Pi silk to salt solution ratio (grams of raw fiber or degummed fiber to mL of total solution) of about 1 :3 to about 1 :200.

17. The method of claim 16, the ratio of raw Pi silk to salt solution ratio is about 1:3 to about 1:50; or about 1 to about 4.

18. The method of any one of claims 13-17. wherein dissolving the raw Pi silk comprises incubating the raw Pi silk in the salt solution at a temperature of about 25 °C to about 100 °C.

19. The method of claim 18, wherein the raw Pi silk is incubated in the salt solution at a temperature of about 60 °C to about 80 °C.

20. The method of any one of claims 13-19, wherein dissolving the raw Pi silk comprises incubating the raw Pi silk in the salt solution for about 2 hours to about 48 hours.

21. The method of claim 13, wherein dissolving the raw- Pi silk comprises: (a) incubating the raw Pi silk in an about 7 to about 9.3 M LiBr solution at a raw Pi silk:salt solution ratio of about 1 :30 to about 1:50 at about 25 °C to about 100 °C for about 2 to about 3 h, either with or without mixing;

(b) incubating the raw Pi silk in an about 8 to about 9.3 M LiBr solution at a raw Pi silk:salt solution ratio of about 1:30 to about 1:50 at about 25 °C to about 100 °C for about 2 to about 24 h, either with or without mixing;

(c) incubating the raw Pi silk in an about 9.3 M LiBr solution at a raw Pi silk:salt solution ratio of about 1 :4 at about 60 °C for about 2 to about 3 h, either with or without mixing;

(d) incubating the raw- Pi silk in an about 5 to about 8 M Ca(NOs)2 solution at a raw Pi silk:salt solution ratio of about 1:30 to about 1 :50 at about 25 °C to about 100 °C for about 48 h. either with or without mixing;

(e) incubating the raw Pi silk in an about 5 to about 8 M Ca(NCh)2 solution at a raw Pi silk: salt solution ratio of about 1:30 to about 1:50 at about 60 °C to about 80 °C for about 48 h, either with or without mixing;

(f) incubating the raw Pi silk in an about 6to about 8 M Ca(NOs)2 solution at a raw Pi silk:salt solution ratio of about 1:30 to about 1:50 at about 60 °C to about 80 °C for about 3 h, either with or without mixing;

(g) incubating the raw Pi silk in an about 6 an about 8 M Ca(NOs)2 solution at a raw Pi silk: salt solution ratio of about 1:30 to about 1:50 at about 20 °C to about 26 °C for about 24 h to about 48 h, either with or without mixing;

(h) incubating the raw Pi silk in an about 1 to about 2 M LiNCh solution at a raw Pi silk:salt solution ratio of about 1 :30 to about 1:50 at about 25 °C to about 100 °C;

(i) incubating the raw Pi silk in an about 3 to about 5 M Mg(NOs)2 solution at a raw Pi silk:salt solution ratio of 1:30 to 1:50 at about 25 °C to about 100 °C;

(j) incubating the raw Pi silk in an about 5 to about 7 M MgCh solution at a raw Pi silk: salt solution ratio of about 1:30 to about 1 :50 at about 25 °C to about 100 °C; or

(k) incubating the raw Pi silk in an about 5 to about 8 M CaCk solution at a raw Pi silk:salt solution ratio of about 1 :30 to about 1:50 at about 25 °C to about 100 °C.

22. The method of any one of claims 13-21, wherein filtering the dissolved /7 silk comprises passing the dissolved Pi silk through a 22-25 pm pore filter or a 1. 5, 10, 15, 20, and 30 pm pore Polyethylene terephthalate filter.

23. The method of claim 22, wherein the 22-25 m pore filter comprises a miracloth filter and the dissolved Pi silk is passed through filter using a gravity filtration method.

24. The method of any one of claims 13-23, wherein dialyzing the filtered Pi silk comprises dialyzing the filtered Pi silk using a 3.5 kDa MWCO dialysis membrane.

25. The method of any one of claims 13-24, wherein the dialyzing is performed at about

4 °C to about 25 °C for about 4 to about 48 h.

26. The method of any one of claims 13-25, wherein filtered Pi silk is the dialyzed against water, a salt solution, a buffered solution, a PEG-containing solution, or a combination thereof.

27. The method of any one of claims 13-26, wherein the dialyzing comprises dialyzing against about 2 to about 10 changes of a dialysis solution.

28. The method of claim 27, wherein the filtered Pi silk is dialyzing against 2 or more solutions of decreasing salt concentration.

29. The method of any one of claims 13-28, further comprising degumming the Pi silk to yield Pi silk fibroin.

30. The method of claim 29. wherein the degumming comprises incubating the Pi silk (a) in water; (b) in a solution containing an alkaline agent; (c) a solution containing a neutral soap solution; or (d) with an enzyme that degrades sericin; or a combination of two of more of (a)-(d).

31. The method of claim 30, wherein the alkaline agent is Na2CO. and/or the neutral soap is sodium laury l sulfate and/or the enzyme is papain.

32. The method or any one of claims 13-31, wherein the method further comprises: purifying the Pi silk or Pi silk fibroin, concentrating the Pi silk or PI silk fibroin, and/or size fractionating the Pi silk of Pi silk fibroin.

33. The method of any one of claims 13-31. wherein the raw Pi silk is produced using the method of claim 12.

34. An aqueous Pi silk or Pi silk fibroin solution made by the method of any one of claims 13-33.

35. Pi silk or Pi silk fibroin derived from the aqueous Pi silk or Pi silk fibroin solution of claim 34.

36. The aqueous Pi silk or Pi silk fibroin solution of claim 34, or the Pi silk or Pi silk fibroin of claim 35, wherein the Pi silk fibroin is genetically or chemically modified.

37. A biomaterial made using the aqueous Pi silk or Pi silk fibroin solution of claim 34 or 36 or the Pi silk or Pi silk fibroin of claim 35 or 36.

38. The biomaterial of claim 37, wherein the biomaterial comprises a sponge, a microparticle, a nanoparticle, a film, a hydrogel, an electrospun fiber, a porous silk fibroin material, an implant, or a scaffold.

39. The biomaterial of claim 38, wherein the biomaterial is linked to, is associated with, encapsulates, or provides a substrate for, one or more bioactive substances.

40. The biomaterial of claim 39, wherein the one or more bioactive substances comprises a biologically active molecule, a cell, or a combination thereof.

41. The biomaterial of claim 39, wherein the biologically active molecule comprises pharmaceutically active ingredient, a hormone, or a growth factor; and/or the cell comprises a stem cell, an immune cell, a somatic cell, a fibroblast, an epithelial cell, or an endothelial cell.

42. The biomaterial of any one of claims 37-41 for use in tissue regeneration, tissue engineering, wound repair, wound dressing, nerve regeneration, bone regeneration, coating material for protection or modulation of growth, encapsulating a payload, drug delivery⁷.

43. The biomaterial of any one of claims 37-42, wherein the biomaterial is made using a form or is 3D printed.

44. The biomaterial of any one of claims 34-43, wherein the biomaterial contains a second polymer.