Porous nanofiber microspheres and methods of use thereof

Abstract

Nanofibrous microspheres and composites comprising the same are provided as well as methods of use thereof and methods of making.

Description

[0001] POROUS NANOFIBER MICROSPHERES AND METHODS OF USE THEREOF

[0002] Jingwei Xie Johnson Vitharikunnil John

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 609,918, filed December 14, 2023. The foregoing application is incorporated by reference herein.

[0004] This invention was made with government support under Grant Nos. R01 DE031272 and R01 DK134903 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] FIELD OF THE INVENTION

[0006] This application relates to the field of nanofiber structures. More specifically, this invention provides nanofibrous microspheres (NMs), composites comprising the nanofibrous microspheres, methods of synthesizing, and methods of use thereof.

[0007] BACKGROUND OF THE INVENTION

[0008] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

[0009] The International Diabetes Federation estimates that 783 million adults will have diabetes mellitus by 2045 (Sun, et al. (2022) Diabetes Res. Clin. Pract., 183: 109119). Approximately 20-25% of these patients suffer from diabetic foot ulcers (DFUs), which often lead to prolonged inflammation, severe infection, and, ultimately, lower extremity amputation (Lin, et al. (2020) PLoS One 15(9):e0239236; Huang, et al. (2023) Molecules 28(3): 1110). The current gold standard treatments for DFUs, which include debridement, antibiotic management, wound dressing, and ulcer off-loading, primarily offer pain relief but fail to significantly improve long-term outcomes (He, et al. (2022) Bioactive Mate., 10:460-473; Yang, et al. (2020) Int. J. Nanomed., 2020:5911-5926; Niculescu, et al. (2022) Polymers 14(3):421). Clinical surgical interventions such as graft transplantation and revascularization surgery do not only cause substantial pain due to secondary procedures but also increase the fiscal burden on patients and healthcare systems (Yang, et al. (2020) Int. J. Nanomed., 2020:5911-5926; Sloan, et al. (2021) Nature Rev. Endocrin., 17(7):400-420). Effective management and innovative strategies are imperative to treat diabetic wounds, prevent infections, and promote optimal healing. Tissue engineering, utilizing biomaterials and advanced technologies, offers promising solutions for DFU patients (Deng, et al. (2022) J. Biomed. Mater. Res., 110(11):2542- 2573). An ideal wound dressing should restore damaged tissue rapidly. The Centers for Medicare & Medicaid Services (CMS) currently recognizes 76 commercial skin replacement products for chronic wounds. Among these, products like Derma Gide®, Dermagraft®, Apligraf®, Graftjacket™, LeucoPatch®, and Regranex® are employed as grafts for DFUs (Armstrong, et al. (2021) Plast. Reconstr. Surg. Glob. Open 9(5):e3596; Hart, et al. (2012) Adv. Wound Care 1(3): 138-141; Zaulyanov, et al. (2007) Clin. Interven. Aging 2(l):93-98; Martin, et al. (2005) Inti. Wound J., 2(2): 161-165; Game, et al. (2017) Trials 18: 1-8). However, their overall success rates are limited due to issues such as ineffectiveness, the necessity for secondary surgeries, fibrosis, and delayed healing processes (Wang, et al. (2018) Acta Biomaterialia 69: 156-169). Therefore, there is a need for more effective and less invasive treatment options.

[0010] Biomaterials have been instrumental in diabetic wound tissue repair, serving as scaffolds for over two decades to enhance therapeutic effects. Various materials, including 2D electrospun nanofiber mats, films, sponges, microfibers, and hydrogels, have been extensively studied for wound healing (John, et al. (2023) Adv. Funct. Mater., 33(l):2206936; Chen, et al. (2020) J. Mater. Chem., 8(17):3733-3746). However, 2D nanofiber membranes often suffer from limited thickness and poor cellular penetration, making them less effective in mimicking 3D structures and limiting their application in tissue repair (Jiang, et al. (2018) Acta biomaterialia 68:237-248; Jiang, et al. (2016) Adv. Healthc. Mater., 5(23):2962-2962). Although hydrogels are favored for their 3D structure, their high-water absorption capacity, low porosity, and minimal extracellular matrix (ECM) microstructure restrict their widespread clinical use (Ahmadian, et al. (2021) J. Biomater. App., 36(1): 179-190; Tang, et al. (2023) ACS omega 8(11): 10030- 10039). Furthermore, their low mechanical strength and limited nutrient and waste diffusion can negatively affect cell growth and morphology (Cao, et al. (2021) Signal Transduc. Targeted Ther., 6(1 ):426). Despite these advances, bioengineered scaffolds typically require surgical procedures for implantation and targeted medication to prevent infection (Deng, et al. (2022) J. Biomed. Mater. Res., 110(11):2542-2573). Therefore, developing effective, safe, and less invasive treatment methods to enhance healing processes is crucial. SUMMARY OF THE INVENTION

[0011] In accordance with the instant invention, nanofibrous microspheres and nanofibrous microsphere hydrogel composites are provided. In certain embodiments, the nanofibrous microspheres and nanofibrous microsphere hydrogel composites comprise electrospun nanofibers (e.g., uniaxially-aligned, random, entangled, and / or electrospun fibers). The nanofibrous microspheres and nanofibrous microsphere hydrogel composites may comprise a material that enhances water absorption, such as a hydrogel. In certain embodiments, the nanofibrous microsphere hydrogel composites comprise nanofibrous microspheres in a hydrogel. In certain embodiments, the nanofibrous microspheres are crosslinked. In a particular embodiment, the nanofibrous microspheres are mineralized. The nanofibrous microspheres and nanofibrous microsphere hydrogel composites may also comprise one or more agents or compounds such as therapeutic agents. In certain embodiments, the nanofibrous microspheres and nanofibrous microsphere hydrogel composites comprise cells and / or tissue. Compositions comprising the nanofibrous microspheres and / or nanofibrous microsphere hydrogel composites are also provided. Methods of synthesizing the nanofibrous microspheres and nanofibrous microsphere hydrogel composites of the instant invention are also provided.

[0012] In accordance with another aspect of the instant invention, methods of using the nanofibrous microspheres and nanofibrous microsphere hydrogel composites are provided. For example, the nanofibrous microspheres and nanofibrous microsphere hydrogel composites may be used to enhance wound healing, build tissue constructs, promote tissue regeneration (e.g., bone regeneration, cardiac tissue regeneration, etc.), reduce, inhibit, prevent, and / or eliminate infection, local delivery of drugs, and / or inhibit bleeding orother like uses.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figures 1A-1C provide schematics of the preparation and in vitro characterization of nanofibrous microspheres (NMs) and their application in diabetic wound healing. Fig. 1 A: Porous nanofiber microspheres (P-NMs) were fabricated as follows: i) produce PLGA:gelatin (1 : 1) nanofiber mat by electrospinning, ii) create short nanofibers by cryocutting, and iii) achieve P-NMs using co-axial electrospraying of short fibers in the shell and air in the core, followed by gelatin coating / crosslinking. Fig. IB: Schematic representation of nonporous nanofibrous microspheres (NP-NMs) and P-NMs including 2D structures, 3D structures, functionalization, and cell behavior (e.g., adhesion, proliferation and migration). Nutrients, media, gas, and cellular waste are shown to freely interchange in P-NMs but not NP-NMs. Fig. 1C: Schematics illustrate that P-NMs serve as either injectable carrier for cell delivery or scaffold for host cell migrate into. Figure ID provides a schematic illustration of the fabrication of P-NMs using co-axial electrospraying. A homogenized short nanofiber solution was used for co-axial electrospraying. Electrosprayed NMs were freeze dried and crosslinked to be used in in vitro cell culture and in vivo implantation using minimally invasive therapy. Figures IE and IF provide photographic images of porous nanofibrous microsphere generation using the co-axial electrospraying technique with the spray at various airflow rates and voltages. Fig. IE: The airflow rate varied from 0 - 10 ml / hour while other parameters, such as solution flowrate 2 ml / hour and voltage, were kept constant. Fig. IF: Microsphere size and pore volume architecture were varied at different voltages while setting a constant solution (2 ml / hour) and air (10 ml / hour) flow rate.

[0015] Figures 2A-2D show the morphological analysis and water absorption capacity of prepared NP and P-NMs. Fig. 2A: SEM images showing the nanofibrous architecture of PLGA:gelatin (1 : 1) NMs. The operating parameters during co-axial electrospraying were as follows: short fiber solution flow rate = 2 ml / hour; airflow rate = 10 ml / hour; and voltage = 6 kV. Fig. 2B: Bar graph with scatter dots shows the diameters range of NMs and P-NMs quantified from SEM images using ImageJ software. Fig. 2C: Pore diameters of P-NMs quantified from SEM images. Fig. 2D: Water absorption ratio of NP-NMs and P-NMs. Figure 2E: Histogram showing the diameter range of NP-NMs (left bars) and P-NMs (right bars) measured from SEM images by using ImageJ software. Figure 2F: Scanning electron microscopy images displaying the cross- sectional view of NP-NMs (top row) and P-NMs (bottom rows).

[0016] Figures 3A-3C show the proliferation of HDFs on NP-NMs and P-NMs at different time points. Fig. 3A: Live / dead staining of HDFs on NP-NMs and P-NMs using calcein AM and ethidium homodimer at day 1, 3 and 7. Fig. 3B: Cell proliferation quantified by Prestoblue™ dye. P-NMs (left) and NP-NMs (right) for each day. Fig. 3C: Confocal microscopy images of HDFs on P-NMs after seeding for 1, 3, and 7 days. The actin filaments of HDFs were stained using Alexa Flour™ phalloidin and cell nuclei were stained with DAPI, indicating cells proliferated on the surface as well as migrated inside due to open pore structures of P-NMs. *p<0.05, **p<0.01 and ns - not significant. Figure 3D provides confocal microscopy images of HDF -loaded on NP-NMs. Cytoskeleton formation (actin filaments) shown using Alexa Flour™ phalloidin and nuclei DAPI staining of NP-NMs at different time points such as day 1, 3 and 7. The cells adhered on the surface of the fibrous microspheres.

[0017] Figures 4A-4C show the mechanical properties of the NMs measured using nanoindentation. Fig. 4A: Schematic illustrates the indentation process on NMs. Fig. 4B: Effective Young’s modulus calculated from load vs. nanoindentation depth curve, depicts cell-laden day 7 NMs (Day 7 with cells; right bar) demonstrated higher effective Young’s modulus than the samples without loaded cells at day 0 (Day 0 w / o cells; left bar). ***p<0.001 and ****p<0.0001 denotes statistically significant. Fig. 4C: Cell- loaded NP-NMs and P-NMs after 7 days of incubation exhibiting higher mechanical strength than the counterparts without loaded cells were attributed to the attached cells and their secreted extracellular matrix.

[0018] Figures 5A-5H provide injectability studies on NMs to examine the pressure protection on cells loaded to NMs in terms of their viability and morphology. Fig. 5A: Illustration of two different approaches (Inj lx and Inj2x) for pressure generation at day 1, 2, 3 and 7. Fig. 5B: Actin filament staining of cells on Inj lx samples at day 1, 3, and 7. Fig. 5C: Actin filament staining of cells on Inj2x samples at day 1, 3, and 7. Fig. 5D: Diagram illustrating the impact of pressure and friction on the survival of the cells during the injection of cell-loaded NMs. Figs. 5E-5H: Proliferation of HDFs on NP-NMs and P-NMs were quantified using Prestoblue™ after injection with two different strategies (Inj lx and Inj2x). *p<0.05 denotes statistically significant. Figures 51 and 5J show in vitro injection test was performed on NMs to check their stability pre-, during, and postinjection. Fig. 51: The various injection flow rates and injectability behavior of the microspheres are visualized in table format. Fig. 5J: Light microscopy images of NMs at different conditions such as pre-, during, and post-injection showing that NMs retained their structure after injection at a flowrate of 10 ml / hour. Figures 5K and 5L provide live / dead staining pictures of pressure protection study samples on day 1 (DI) and 3 (D3) after injection of HDF-loaded NP and P-NMs under two different injection conditions. Fig. 5K: Injection once (Inj Ix-DX) on DI and D3. Fig. 5L: Injection twice (Inj2x-DX) on DI and D3.

[0019] Figures 6A-6C show NMs recruit cells in an in vitro wound healing model. Fig. 6 A: Schematic illustrating an in vitro wound healing model: i) GFP -tagged HUVECs were encapsulated in fibrin gel and casted in 96-well plate and allowed to crosslink for 30 minutes at 37°C; ii) NMs were introduced; and iii) over time cells started sprouting and migrated towards NMs and wrapped the NMs completely. In the case of P-NMs, HUVECs aligned on pores and migrated into the pores. Figs. 6B-6C: NP and P-NMs samples images captured on different time points to show GFP -tagged HUVEC migration, (i) single plane image of NMs on day 1, (ii) Z-stack image captured on day 5 with lOx magnification image depicts HUVECs migration towards NP-NMs additionally through pores of P-NMs, and (iii) fluorescence image demonstrates that migrated cells towards NP-NMs and through pores of P-NMs on day 5 shown using depth coding imaging. Figures 6D-6F show in vitro wound closure experiment using a scratch assay on HDFs supplemented with NP and P-NMs spent media to determine their migration in the presence of NM released subproducts. Fig. 6D: Microscopic pictures of the wound closure at different time points such as 0, 5, 10 and 24 hours. ImageJ quantification of wound closure percentage (Fig. 6E) and rate of cell migration (pm / hour) (Fig. 6F) calculated from the captured optical images are provided, ns - not significant. Figure 6G shows the application of cell migration towards NMs. GFP-tagged HUVECs migration towards NP-NMs on day 3 and 10 depicting HUVECs coverage on the surface of the NP-NMs (left). P-NMs facilitating HUVECs penetration through micropores on day 3 and forming a vascular network after 10 days (right). Figure 6H: 3D volume rendered confocal microscope images of NP-NMs (top) and P-NMs (bottom) shows cell penetration in Z-axis.

[0020] Figures 7A-7F show NMs for diabetic wound healing in vivo. Fig. 7A: H & E staining images of NP-NMs treated diabetic wounds after day 7 and 14 (circle refers to the structure of implanted NP-NMs on wound site on day 7). Fig. 7B: Magnified images of cells infiltrated around the NP-NMs (arrow mark). Fig. 7C: H & E staining images of P-NMs treated diabetic wounds after day 7 and 14 (circle refers to the structure of implanted P-NMs on wound site on day 7). Fig. 7D: Magnified images of cells infiltrated around and inside the P-NMs (arrow mark), granulation tissue formation (arrow mark), and blood vessel formation (arrow mark). Figs. 7E-7F: Percentage of cell infiltration (Fig. 7E) and neovascularization (Fig. 7F) in wound area after the treatment of NP (left bar) and P-NMs (right bar) for 7 and 14 days. ** p<0.01, ***p<0.001 and ****p<0.0001 denotes statistically significant. Figures 7G-7I show the application of NMs for wound healing by cell recruitment toward the center. Fig. 7G: Schematic illustrating the NMs with bFGF that enhances wound healing by guiding and accelerating cell migration. The in vitro wound model showed cell migration towards NMs at different time points observed under confocal microscopy. Fig. 7H: HDF migration towards NP-NMs. Fig. 71: HDF migration towards P-NMs. White dotted lines depict the NMs boundary, circle depicts pores in the P-NMs and the arrow denotes cell migration towards NMs in the wound model. Fig. 7J: Detailed wound modeling and treatment plan for in vivo studies on NMs. In vivo experiment of injected NMs on diabetic mouse wounds. Fig. 7K: Photographs of wounds in diabetic mice filled with NP or P-NMs. Fig. 7L: Photographs of treated wounds after 7 and 14 days of implantation of NP-NMs (top row) and P-NMs (bottom row). Figures 7M and 7N show injected NMs on diabetic mice wounds at 7 and 14 days. Trichrome staining of the wounds and surrounding tissues in diabetic mice after NMs treatment for 7 (left panels) and 14 days (right panels). Fig. 7M: NP-NMs after 7 days and 14 days. Fig. 7N: P-NMs after 7 days and 14 days of implantation.

[0021] Figure 8 provides a schematic of a mechanism of enhanced healing by injectable P-NMs at the diabetic mouse wound site. NP-NMs demonstrated host cell infiltration with lower levels of vascularization and re-epithelization due to insufficient exchange of substances which ultimately impact the cell survival and maturation. Conversely, P- NMs exhibited enhanced cell penetration, neovascularization and re-epithelialization due to the advantage of porous architecture, in the same way, production of extracellular matrix contributed to the fast wound closure.

[0022] Figure 9A provides a schematic overview of the fabrication of porous or nonporous nanofibrous microspheres composed injectable hybrid composite. Figure 9B provides photographs and SEM images of various concentration of GelMA (0.1% to 0.5%) composed porous nanofiber microspheres hybrid composites.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] Current research methods are focused on improving treatment efficacy for patients with DFUs, aiming to reduce amputations and complications associated with chronic wounds (Zhang, et al. (2020) Front. Bioeng. Biotech., 8:516; Chen, et al. (2019) NPG Asia Mater., 11 (1 ): 3). Recently, attention has been given to minimally invasive approaches in both soft and hard tissue repair (Zawani, et al. (2021) Biomed., 9(5): 527). Notably, these therapies utilize biomaterials to deliver cells and drugs through injectable carriers that can adapt to the shape of the defect site and can carry and deliver multiple therapeutic agents (Chen, et al. (2019) NPG Asia Mater., 11 (1 ): 3 ; Sarviya, et al. (2023) Macromol. Biosci., 23(l):2200347; John, et al. (2019) Nanomed., 22: 102081; Wei, et al. (2018) Adv. Mater., 30(31): 1802273; Kankala, et al. (2019) Small 15(25): 1901397). Thus, granular scaffolds, such as microgels and microspheres, are emerging as promising options for therapeutic agent delivery, offering capabilities akin to 3D bioscaffolds (John, et al. (2020) Small 16(19): 1907393; Chen, et al. (2023) Acta Biomaterialia 157:593-608).

[0025] Granular microspheres may be suitable carriers to deliver cells or pharmaceuticals, given their injectability and carrying capacity. Several methods for microsphere fabrication have been explored, including self-assembly, microfluidic assembly, and electrospraying (John, et al. (2019) Nanomed., 22: 102081; Liu, et al. (2011) Nat. Mater., 10(5):398-406; Liu, et al. (2023) Adv. Healthc. Mater., 2300823). For instance, hollow, star-shaped PLLA microspheres designed for knee repair were successfully produced using self-assembly. However, the complexity of polymer synthesis in self-assembly makes this approach more challenging and limits its application to functional groups such as -OH, -NH2, and -COOH to self-assemble into microspheres (Liu, et al. (2011) Nat. Mater., 10(5):398-406).

[0026] Recently, attention has been shifted towards microgel annealed particles (MAPs) as candidates for wound dressing materials. Their appeal lies in their injectability and porous structures (Liu, et al. (2023) Adv. Healthc. Mater., 2300823; Griffin, et al. (2015) Nat. Mater., 14(7): 737-744). Although MAPs have shown promise in facilitating cell migration and collagen matrix formation, their porosity remains suboptimal for enhancing cell adaptability and regeneration.

[0027] To address these limitations in MAPs, nanofibrous microspheres (NMs) with precisely controlled porosity and predetermined structures have been developed. The NMs are designed to emulate the microarchitecture of the ECM and have shown potential in promoting cell migration during tissue healing (John, et al. (2020) Small 16(19): 1907393). The fabrication method to produce open porous NMs through co-axial electrospraying presents a scalable method for producing large quantities of NMs. This technique allows for the customization of the NMs’ architecture, which is crucial for influencing cellular behaviors (John, et al. (2020) Small 16(19): 1907393). Furthermore, the effectiveness of NMs as a vehicle for growth factor delivery has been demonstrated. This approach has been shown to modulate cellular responses, indicating that NMs are a viable strategy for enhancing tissue healing (John, et al. (2019) Nanomed., 22: 102081). Compared to self-assembly methods, producing porous NMs via a combined approach of electrospinning and electrospraying demonstrates significant potential for large-scale manufacturing. This advantage stems from the fact that self-assembly methods are heavily dependent on specific functional groups. In vivo studies have particularly highlighted the superior performance of porous NMs in facilitating cell migration and integration with host tissues, especially when comparing subcutaneous implantations of porous NMs with their nonporous counterparts (John, et al. (2020) Small 16(19): 1907393).

[0028] Herein, the applicability of porous NMs for diabetic wound healing is demonstrated, both in vitro and in vivo, where they function as granular scaffolds. The mechanical stability of these NMs is demonstrated herein along with the ability to use the NMs in minimally invasive therapies due to their injectable properties. The interconnected pores in these injectable NMs enhance cell loading capacity and tissue integration. Furthermore, the mechanical robustness of the NMs protects loaded cells from shear stress during injection in the cell delivery in various tissue repair studies.

[0029] Diabetic foot ulcers (DFUs) are a significant challenge in the clinical care of diabetic patients, often necessitating limb amputation and compromising the quality of life and life expectancy of this cohort. Minimally invasive therapies, such as modular scaffolds, are at the forefront of current DFU treatment, offering an efficient approach for administering therapeutics that accelerate tissue repair and regeneration. The herein study reports a facile method for fabricating granular nanofibrous microspheres (NMs) with predesigned structures and porosities. The proposed technology combines electrospinning and electrospraying to develop a therapeutic option for DFUs. Specifically, porous NMs were constructed using electrospun poly (lactic-co-glycolic acid) (PLGA): gelatin short nanofibers, followed by gelatin crosslinking. These NMs demonstrated enhanced cell adhesion to human dermal fibroblasts (HDF) during an in vitro cytocompatibility assessment. Notably, porous NMs displayed superior performance owing to their interconnected pores compared to nonporous NMs. Cellladen NMs demonstrated higher Young's modulus values than NMs without loaded cells, indicating improved material resiliency attributed to the reinforcement of cells and their secreted extracellular matrix. Dynamic injection studies on cell-laden NMs further elucidated their capacity to safeguard loaded cells under pressure. In addition, porous NMs promoted host cell infiltration, neovascularization, and re-epithelialization in a diabetic mouse wound model, signifying their effectiveness in healing diabetic wounds. Taken together, porous NMs are a minimally invasive, injectable treatment that effectively promote tissue integration and regeneration. In accordance with the instant invention, porous nanofibrous microspheres are provided. The porous nanofibrous microspheres comprise electrospun nanofiber segments. In certain embodiments, the porous nanofibrous microspheres further comprise a hydrogel (e.g., gelatin). In certain embodiments, the electrospun nanofiber segments are crosslinked. The porous nanofibrous microspheres of the instant invention may have one or more (or all) of the following features: i) can be administered in a minimally invasive manner, therefore decreasing the risk of infection and improving comfort; ii) capable of filling any irregularly shaped defects; iii) provide a platform technology capable of delivering active agents such as signaling molecules and / or cells for any disease or disorder (e.g., bone healing, myocardial infarction repair, etc.); iv) provide a biomimetic structure (e.g., nanofibrous architecture); and v) have no limitations to compositions unlike self-assembled porous NMs.

[0030] As stated herein, the electrospun nanofiber segments of the instant invention may be crosslinked (e.g., to enhance their stability). As used herein, crosslinking may be done using a variety of techniques including thermal crosslinking, chemical crosslinking, and photo-crosslinking or other like crosslinking methods. For example, the compounds may be crosslinked with a crosslinker such as, without limitation: formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, a photocrosslinker, genipin, and natural phenolic compounds (Mazaki, et al., Sci. Rep. (2014) 4:4457; Bigi, et al., Biomaterials (2002) 23:4827-4832; Zhang, et al., Biomacromolecules (2010) 11 : 1125-1132; incorporated herein by reference). The crosslinker may be a bifunctional, trifunctional, or multifunctional crosslinking reagent. In certain embodiments, the crosslinker is glutaraldehyde.

[0031] Composites comprising the porous nanofibrous microspheres are also encompassed by the instant invention. In certain embodiments, the composite further comprises a hydrogel (referred to herein as nanofibrous microsphere hydrogel composites). In certain embodiments, the nanofibrous microspheres are encompassed by or contained within the hydrogel. Any hydrogel can be mixed with the porous nanofibrous microspheres to create the compositions or composites. In certain embodiments, the hydrogel is a fibrillar hydrogel or a fibrillar hybrid hydrogel (FHG). In certain embodiments, the hydrogel is matrix metalloproteinase (MMP) responsive (degradable) fibrillar network. Examples of hydrogels include, without limitation: gelatin, gelatin methacryloyl (GelMA), hyaluronic acid, hyaluronic acid methacrylate (HAMA), alginate, chitosan, collagen, starch, pectin, cellulose, methylcellulose, sodium polyacrylate, starch-acrylonitrile co-polymers, other natural or synthetic hydrogels, and derivatives thereof or combinations thereof. In certain embodiments, the hydrogel is selected from the group consisting of gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA), collagen, or combinations thereof. Varying concentrations of hydrogel may be used in the compositions or composites. In certain embodiments, the percentage of hydrogel is between about 0.01% and about 5%, about 0.05% and about 1%, about 0.1% and about 1%, or about 0.2% and about 0.5% (e.g., by w / v). In certain embodiments, the hydrogel is crosslinked. In certain embodiments, the hydrogel is crosslinked in situ (e.g., in vivo or in the host). In certain embodiments, the hydrogel is crosslinked after administration to the subject. The liquid composition can be injected into a desired site (e.g., cavity) and then crosslinked to solidify or harden in place. In certain embodiments, the crosslinker is added or administered after the nanofibrous microsphere and hydrogel composition. In certain embodiments, the crosslinking is photocrosslinking (e.g., UV crosslinking). In certain embodiments, the method comprises applying UV radiation to the added or administered nanofibrous microsphere and hydrogel composition (e.g., in situ). In certain embodiments, the hydrogel comprises a photoactive group. Photoactive groups are known in the art. Examples of photoactive groups include, without limitation: acrylate, methacrylate, 2-chloroacrylate, 2- phenyl acrylate, acrylamide, methacrylamide, 2-chloroacrylamide, 2 -phenyl acrylamide, N-lower alkyl substituted acrylamide, N-lower alkyl substituted methacrylamide, N- lower alkyl substituted 2- chloroacrylamide, N-lower alkyl substituted 2- phenyl acrylamide, vinyl ether, vinyl ester, styrene, diamine, amide, imide, siloxane, amic ester, amic acid, and derivatives thereof. In certain embodiments, the photoactive group is acrylate, methacrylate, 2-chloroacrylate, 2 -phenyl acrylate, vinyl ether, vinyl ester, styrene, siloxane, or derivatives thereof. In certain embodiments, the photoactive group is acrylate or methacrylate. In certain embodiments, the photoactive group is acrylate (CH2=CH(CO)O-; wherein the terminal oxygen may be part of the polymer).

[0032] In certain embodiments, to facilitate photocrosslinking, a photoinitiator may be present. For example, the nanofibrous microspheres and hydrogel composition may further comprise a photoinitiator. Photoinitiators are known in the art. Generally, a photoinitiator absorbs energy such as visible light or UV light and starts a chemical reaction that solidifies a liquid. In certain embodiments, the photoinitiator is a crosslinker or photocrosslinker. Examples of photoinitiators include, without limitation: lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), benzophenone, diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (DPPO), phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide (BAPO, Irgacure 819), 2-hydroxy-2-methyl-l-phenyl-propan-I-one (Irgacure 1173), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), 2,2'-azobis[2-methyl-n-(2-hydroxyethyl) propionamide) (VA-086), 2,2- dimethoxy-2-phenylacetophenone (Irgacure 651 or DMPA), diphenyl(2,4,6- trimethylbenzoyl)phosphine oxide (Darocure TPO; Lucirin TPO), and ethyl (2,4,6- trimethylbenzoyl) phenylphosphinate (Lucirin TPO-L). In certain embodiments, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). In certain embodiments, the photoinitiator is Irgacure 2959. In certain embodiments, the concentration of the photoinitiator is at least 0.05%. In certain embodiments, the photoinitiator is present at about 0.01% to about 0.5%, about 0.05% to about 0.3%, or about 0.1% (e.g., w / v).

[0033] The nanofibrous microsphere hydrogel composites of the instant invention can be used as an injectable wound dressing. In certain embodiments, the nanofibrous microsphere hydrogel composites mimics extracellular matrix (ECM). In certain embodiments, the nanofibrous microsphere hydrogel composites comprises antibacterial (e.g., W379) and / or angiogenic agents (e.g., QK peptides) or other like molecules to accelerate wound healing and avoid or reduce infections. Notably, to reduce or avoid unwanted swelling, a low concentration of hydrogel composition (e.g., <1-5%) can create a fibrillar hydrogel network at the sub-micron level. In the hybrid gel systems, the porous nanofibrous microspheres can maintain wound structure and provide space for sufficient cell migration to accelerate wound healing. Therefore, the nanofibrous microsphere hydrogel composites of the instant invention comprising porous nanofibrous microspheres incorporated into stimuli -responsive shear-thinning hybrid hydrogels will allow for the avoidance of wound contraction and allow for rapid cell migration, thereby being an ideal wound dressing.

[0034] While the nanofibrous microsphere hydrogel composite is generally described herein as comprising porous nanofibrous microspheres, the nanofibrous microsphere hydrogel composite may comprise nonporous nanofibrous microspheres. In certain embodiments, the nanofibrous microsphere hydrogel composite comprises porous nanofibrous microspheres and nonporous nanofibrous microspheres. Nonporous nanofibrous microspheres of the instant invention are generally the same as porous nanofibrous microspheres, but lack the pores. Nonporous nanofibrous microspheres may be synthesized in the same way except that air or gas bubbles are not added during electrospraying (e.g., coaxial electrospraying). Nonporous nanofibrous microspheres tend to be slightly smaller than porous nanofibrous microspheres and may have a diameter (e.g., average longest distances across the microspheres) of about 200 pm to about 700 pm or about 400 pm to about 500 pm.

[0035] The instant invention also encompasses methods of synthesizing porous nanofibrous microspheres. In certain embodiments, the method comprises i) cutting (e.g., cryocutting) electrospun nanofibers into electrospun nanofiber segments, ii) dispersing the electrospun nanofiber segments in a solution (e.g., an aqueous solution), and iii) electrospraying the solution comprising the electrospun nanofiber segments with air or gas bubbles to generate porous nanofibrous microspheres. In certain embodiments, the method further comprises lyophilizing and / or freeze drying the synthesized porous nanofibrous microspheres or other like methods. In certain embodiments, the method further comprises crosslinking (e.g., by exposing to glutaraldehyde vapors) the synthesized porous nanofibrous microspheres (e.g., after lyophilization and / or freeze drying or other like methods). In certain embodiments, the method further comprises synthesizing (e.g., electrospinning) the electrospun nanofibers (e.g., an electrospun nanofiber mat) prior to step i). In certain embodiments, the method further comprises modifying the nanofibers, segments, and / or porous nanofibrous microspheres, as described herein.

[0036] Step i) comprises cutting and / or breaking the nanofibers into shorter nanofiber segments. In certain embodiments, the electrospun nanofibers of step i) are in an electrospun nanofiber mat. Cutting or breaking of the nanofibers to produce short nanofiber segments can be done using a variety of methods including but not limited to cryocutting, homogenization, wet milling, and cryomilling or other like methods. In certain embodiments, the nanofibers are cryocut. In certain embodiments, the nanofibers are cut in liquid nitrogen. In certain embodiments, the nanofibers are frozen (e.g., in a liquid such as water (e.g., at about -20°C or lower or at about -80°C or lower)) and then the frozen block containing the nanofibers is cut (e.g., with a cryotome (e.g., at about - 20°C or lower)). In certain embodiments, the nanofibers can be broken or homogenized into nanofiber segments by sonication or other like methods. For example, the nanofibers can be placed into water or an aqueous solution and homogenized using an ultrasonic probe sonicator (e.g., equipped with a microtip probe (e.g., 1 / 8 mm)). In certain embodiments, the nanofibers or nanofiber mat are cut into nanofiber segments using cryomilling (e.g., in liquid nitrogen). In certain embodiments, the electrospun nanofiber segments are crosslinked (e.g., by exposing to glutaraldehyde vapors) prior to step ii). In certain embodiments, the electrospun nanofiber segments are lyophilized and / or freeze dried prior to step ii). In certain embodiments, step ii) comprises homogenizing the solution. In certain embodiments, step ii) comprises sonicating the solution. In certain embodiments, the solution of step ii) further comprises a hydrogel (e.g., gelatin). In certain embodiments, step iii) comprises air or gas-induced core-shell electrospray microdripping of solution droplets, particularly into a freezing media (e.g., liquid nitrogen). In certain embodiments, step iii) comprises co-axial (e.g., core-shell) electrospraying. In certain embodiments, step iii) comprises electrospraying into a freezing media (e.g., liquid nitrogen).

[0037] Methods of synthesizing the nanofibrous microsphere hydrogel composites are also encompassed by the instant invention. In certain embodiments, the method comprises mixing or combining porous nanofibrous microspheres with a hydrogel. In certain embodiments, the method further comprises synthesizing the porous nanofibrous microspheres. In certain embodiments, the method further comprises crosslinking the mixture of the porous nanofibrous microspheres and hydrogel.

[0038] Compositions comprising a compound of the instant invention and at least one carrier (e.g., a pharmaceutically acceptable carrier) are also encompassed by the instant invention. In certain embodiments, the composition comprises i) a nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite and ii) at least one carrier (e.g., a pharmaceutically acceptable carrier).

[0039] In certain embodiments, the porous nanofibrous microspheres of the instant invention have a diameter (e.g., average longest distances across the microspheres) of about 100 pm to about 2000 pm, about 250 pm to about 1500 pm, about 400 pm to about 1000 pm, about 400 pm to about 800 pm, about 500 to about 900 pm, about 600 pm to about 800 pm, or about 500 to about 700 pm. In certain embodiments, the pores of the porous nanofibrous microspheres of the instant invention have a diameter (e.g., average longest distances across the pores) of about 10 pm to about 500 pm, about 20 pm to about 400 pm, about 25 pm to about 300 pm, about 50 pm to about 250 pm, about 60 pm to about 200 pm, or about 70 pm to about 150 pm. In certain embodiments, the electrospun nanofiber segments have a length (e.g., average length) of about 1 pm to about 200 pm, about 5 pm to about 100 pm, about 10 pm to about 75 pm, about 15 pm to about 60 pm, about 20 to about 40 pm, or about 50 to about 100 pm. The nanofibers of the instant invention can be fabricated by any method. For example, the nanofibers may be manufactured using a variety of methods including but not limited to electrospinning, phase separation, centrifugal force spinning, hypersonic spinning, and freeze-casting of short fiber solutions or other like methods. However, the nanofibers will be generally synthesized by electrospinning and are referred to as such. The nanofibers may be aligned fibers (e.g., uniaxially aligned), random fibers, and / or entangled fibers. In certain embodiments, the nanofibers comprise aligned fibers (e.g., uniaxially, radially, vertically, or horizontally). While the application generally describes nanofibers (fibers having a diameter less than about 1 pm (e.g., average diameter)) and the synthesis of porous nanofibrous microspheres, the instant invention also encompasses microfibers (fibers having a diameter greater than about 1 pm (e.g., average diameter)) and the synthesis of porous microfibrous microspheres.

[0040] The nanofibers of the instant invention may comprise any polymer. In certain embodiments, the polymer is biocompatible. In certain embodiments, the polymer is biodegradable. The polymer may by hydrophobic, hydrophilic, or amphiphilic. In certain embodiments, the polymer is hydrophobic. In certain embodiments, the polymer is hydrophilic. The polymer may be, for example, a homopolymer, random copolymer, blended polymer, copolymer, or a block copolymer. Block copolymers are most simply defined as conjugates of at least two different polymer segments or blocks. The polymer may be, for example, linear, star-like, graft, branched, dendrimer based, or hyperbranched (e.g., at least two points of branching). The polymer of the invention may have from about 2 to about 10,000, about 2 to about 1000, about 2 to about 500, about 2 to about 250, or about 2 to about 100 repeating units or monomers. The polymers of the instant invention may comprise capping termini.

[0041] Examples of hydrophobic polymers include, without limitation: poly(hydroxyethyl methacrylate), poly(N-isopropyl acrylamide), poly(lactic acid) (PLA (or PDLA)), poly(lactide-co-glycolide) (PLG), poly(lactic-co-glycolic acid) (PLGA), polyglycolide or polyglycolic acid (PGA), polycaprolactone (PCL), poly(aspartic acid), polyoxazolines (e.g., butyl, propyl, pentyl, nonyl, or phenyl poly(2-oxazolines)), polyoxypropylene, poly(glutamic acid), polypropylene fumarate) (PPF), poly(trimethylene carbonate), polycyanoacrylate, polyurethane, polyorthoesters (POE), polyanhydride, polyester, polypropylene oxide), poly(caprolactonefumarate), poly(l,2- butylene oxide), poly(n-butylene oxide), poly(ethyleneimine), poly(tetrahydrofurane), ethyl cellulose, polydipyrolle / dicabazole, starch, polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polydioxanone (PDO), polyether poly(urethane urea) (PEUU), cellulose acetate, polypropylene (PP), polyethylene terephthalate (PET), nylon (e.g., nylon 6), polycaprolactam, PLA / PCL (PLCL), poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV), PCL / calcium carbonate, and / or poly(styrene) or combinations thereof.

[0042] Examples of hydrophilic polymers include, without limitation: polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene glycol) and poly(ethylene oxide) (PEO), chitosan, collagen, chondroitin sulfate, sodium alginate, gelatin, elastin, hyaluronic acid, silk fibroin, sodium alginate / PEO, silk / PEO, silk fibroin / chitosan, hyaluronic acid / gelatin, collagen / chitosan, chondroitin sulfate / collagen, and chitosan / PEO or combinations thereof.

[0043] Amphiphilic copolymers or polymer composites may comprise a hydrophilic polymer (e.g., segment) and a hydrophobic polymer (e.g., segment) from those listed above (e.g., gelatin / polyvinyl alcohol (PVA), PCL / collagen, chitosan / PVA, gelatin / elastin / PLGA, PDO / elastin, PHBV / collagen, PLA / hyaluronic acid, PLGA / hyaluronic acid, PCL / hyaluronic acid, PCL / collagen / hyaluronic acid, gelatin / siloxane, PLLA / MWNTs / hyaluronic acid) or combinations thereof.

[0044] Examples of polymers particularly useful for electrospinning are provided in Xie et al. (Macromol. Rapid Commun. (2008) 29:1775-1792; incorporated by reference herein; see e.g., Table 1). Examples of compounds or polymers for use in the fibers of the instant invention, particularly for electrospun nanofibers include, without limitation: natural polymers (e.g., chitosan, gelatin, collagen type I, II, and / or III, elastin, hyaluronic acid, cellulose, silk fibroin, phospholipids (Lecithin), fibrinogen, hemoglobin, fibrous calf thymus Na-DNA, virus M13 viruses), synthetic polymers (e.g., PLGA, PLA, PCL, PHBV, PDO, PGA, poly(L-lactide-co-s-caprolactone) (PLCL), PLLA-DLA, PEUU, cellulose acetate, PEG-b-PLA, EVOH, PVA, PEO, PVP), blended (e.g, PLA / PCL, gelatin / PVA, PCL / gelatin, PCL / collagen, sodium alginate / PEO, chitosan / PEO, Chitosan / PVA, gelatin / elastin / PLGA, silk / PEO, silk fibroin / chitosan, PDO / elastin, PHBV / collagen, hyaluronic acid / gelatin, collagen / chondroitin sulfate, collagen / chitosan), and composites (e.g, PDLA / HA, PCL / CaCCL, PCL / HA, PLLA / HA, gelatin / HA, PCL / collagen / HA, collagen / HA, gelatin / siloxane, PLLA / MWNTs / HA, PLGA / HA). In certain embodiments, the nanofiber comprises polymethacrylate, poly vinyl phenol, polyvinylchloride, cellulose, polyvinyl alcohol, polyacrylamide, PLGA, collagen, polycaprolactone, polyurethanes, polyvinyl fluoride, polyamide, silk, nylon, polybennzimidazole, polycarbonate, polyacrylonitrile, polyvinyl alcohol, polylactic acid, polyethylene-co-vinyl acetate, polyethylene oxide, polyaniline, polystyrene, polyvinylcarbazole, polyethylene terephthalate, polyacrylic acid-polypyrene methanol, poly(2-hydroxyethyl methacrylate), polyether imide, polyethylene glycol, poly(ethylene- co-vinyl alcohol), polyacrylnitrile, polyvinyl pyrrolidone, polymetha-phenylene isophthal ami de, gelatin, chitosan, starch, pectin, cellulose, methylcellulose, sodium polyacrylate, starch-acrylonitrile co-polymers, and / or combinations of two or more polymers.

[0045] In certain embodiments, the nanofibers are made from a variety of polymers including but not limited to polymethacrylate, poly vinyl phenol, polyvinylchloride, cellulose, polyvinyl alcohol, polyacrylamide, poly(lactic-co-glycolic) acid (PLGA), poly(glycolide-co-lactide) (PGLA), collagen, polycaprolactone (PCL), poly(lactic acid) (PLA), polydioxanone (PDO), polyurethanes, polyvinyl fluoride, polyamide, silk, nylon, polybennzimidazole, polycarbonate, polyacrylonitrile, polyvinyl alcohol, polylactic acid, polyethylene-co-vinyl acetate, polyethylene oxide, polyaniline, polystyrene, polyvinylcarbazole, polyethylene terephthalate, polyacrylic acid-polypyrene methanol, poly(2-hydroxyethyl methacrylate), polyether imide, polyethylene gricol, polyethylene glycol, poly(ethylene-co-vinyl alcohol), polyacrylnitrile, polyvinyl pyrrolidone, polymetha-phenylene isophthalamide, gelatin, alginate, chitosan, hyaluronic acid, heparin, starch, pectin, cellulose, methylcellulose, sodium polyacrylate, starchacrylonitrile co-polymers, bioactive glass, and combinations of two or more polymers. Multiple polymers may be mixed to form the nanofibers. In certain embodiments, the nanofibers comprises PCL, PLGA, PGLA, PLA, chitosan, hyaluronic acid, and / or heparin or combinations thereof. The polymers may be mixed evenly or in various ratios depending on the desired properties of the nanofibers.

[0046] In certain embodiments, the nanofibers are made from a variety of inorganic materials including but not limited to bioactive glass, metal oxide, and hydroxyapatite. In certain embodiments, the nanofibers are made from a variety of composite materials including polymers, inorganic materials, carbon, and / or metals.

[0047] The nanofibers of the instant may further comprise a hydrogel (e.g., a material which enhances water absorption properties). A hydrogel may be a polymer matrix which is able to retain water, particularly large amounts of water, in a swollen state. The hydrogel may be with the polymer during electrospinning. In certain embodiments, the hydrogel is present at a 1:1 ratio (e.g., w / w) with the polymer. In certain embodiments, the nanofibers are coated or covered with the hydrogel (e.g., the hydrogel is on the surface of the polymer). While a coating may cover 100% of the nanofibers, a coating may also cover less than 100% of the surface of the nanofibers (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more the surface may be coated). Hydrogels include, without limitation: gelatin, gelatin methacryloyl (GelMA), hyaluronic acid, hyaluronic acid methacrylate (HAMA), alginate, chitosan, collagen, starch, pectin, cellulose, methylcellulose, sodium polyacrylate, starch-acrylonitrile co-polymers, other natural or synthetic hydrogels, and derivatives thereof (e.g., del Valle et al., Gels (2017) 3:27). In certain embodiments, the hydrogel is selected from the group consisting of gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA), collagen, or combinations thereof. In certain embodiments, the hydrogel is gelatin, gelatin methacryloyl (GelMA), and / or chitosan. In certain embodiments, the hydrogel is gelatin. In certain embodiments, the hydrogel is gelatin methacryloyl (GelMA).

[0048] In certain embodiments, the nanofibers comprise PLGA. Notably, the degradation rate of PLGA can be tailored by varying the ratio of lactide / glycolide. In certain embodiments, the PLGA is PLGA (50:50). In certain embodiments, the nanofibers comprise PLGA and gelatin. In certain embodiments, the nanofibers comprise PCL. In certain embodiments, the nanofibers comprise PCL and gelatin. In certain embodiments, the nanofibers comprise PDO. In certain embodiments, the nanofibers comprise PDO and gelatin. In certain embodiments, the nanofibers comprise PLCL. In certain embodiments, the nanofibers comprise PLCL and gelatin. In certain embodiments, the nanofibers further comprise GelMA. In certain embodiments, the nanofibers comprise GelMA instead of gelatin. In certain embodiments, the nanofibers comprise collagen. In certain embodiments, the nanofibers comprise bioactive glass.

[0049] The nanofibers, porous nanofibrous microspheres, and / or nanofibrous microsphere hydrogel composites of the instant invention may be sterilized. For example, the nanofibers, porous nanofibrous microspheres, and / or nanofibrous microsphere hydrogel composites can be sterilized using various methods (e.g., by treating with ethylene oxide gas, gamma irradiation, or 70% ethanol). In certain embodiments, the nanofibers, porous nanofibrous microspheres, and / or nanofibrous microsphere hydrogel composites are sterilized by treating with ethylene oxide.

[0050] The nanofibers, porous nanofibrous microspheres, and / or nanofibrous microsphere hydrogel composites of the instant invention may be modified. The nanofibers, porous nanofibrous microspheres, and / or nanofibrous microsphere hydrogel composites of the instant invention may have one or more or the following modifications.

[0051] In certain embodiments, the nanofibers, porous nanofibrous microspheres, and / or nanofibrous microsphere hydrogel composites comprise (e.g., encapsulate, loaded, and / or coated) an active agent such as a drug (e.g., small molecule) and / or biologic molecule. Active agents (e.g., biologies) include, but are not limited to: proteins, peptides, antibodies, antibody fragments, DNA, RNA, and other known biologic substances, particularly those that have therapeutic use. In certain embodiments, the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite is conjugated to the active agent (e.g., directly or via a linker).

[0052] The active agent may be conjugated to nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite by any means. As stated above, the active agent may be directly conjugated or conjugated via a linker. In certain embodiments, active agent is crosslinked to the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite. In certain embodiments, the greater the crosslinking and / or conjugation of the active agent results in longer retention of the active agent to the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite (e.g., in vivo). In certain embodiments, the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite comprises a methacrylic group such as gelatin methacryloyl (GelMA) which provides binding sites for covalent conjugation of active agents (e.g., peptides) via photocrosslinking. For example, active agents such as peptides (e.g., BMP-2 peptides and / or VEGF peptides) can be modified with octenyl alanine (OCTAL) (e.g., at a terminus of the peptide (e.g., N-terminus), optionally via a pendant amino acid), which allows for conjugation to GelMA. In certain embodiments, the OCTAL-peptide (e.g., BMP-2-0CTAL and / or VEGF-OCTAL) and a photo initiator (e.g., Irgacure 2959) is photocrosslinked (e.g., UV crosslinked (e.g., at 365 or 405 nm)) to the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite (e.g., John et al., Nanomedicine (2019) 22: 102081).

[0053] In certain embodiments, the active agent is a therapeutic agent, growth factor, signaling molecule, vitamin, cytokine, hemostatic agent, angiogenic agent, analgesic, pain medication, anti-inflammatory, antimicrobial, and / or antibiotic. Specific examples of active agents include but are not limited to growth factors (including but not limited to VEGFs, FGFs, EGFs, PDGFs, bone morphogenic proteins (ex. BMP -2, BMP -2 peptides, BMP -2 fragments and / or analogs thereof), GM-CSF, G-CSF, EPO, GDNFs, HGFs, and IGFs), growth factor mimicking peptides (such as VEGF mimicking peptides), chemokines (including but not limited to CCL21, CCL22, CCL2, CCL3, CCL5, CCL7, CCL8, CCL13, CCL17, CXCL9, CXCL10, and CXCL11), cytokines (including but not limited to IL-2 subfamily cytokines, interferon subfamily cytokines, IL- 10 subfamily cytokines, IL-1, 1-18, IL-17, tumor necrosis factor, and transforming-growth factor beta superfamily cytokines), blood clotting factors (including but not limited to thrombin and fibrinogen) or combinations thereof. In certain embodiments, the active agent is a vitamin (e.g., vitamin D3). In certain embodiments, the active agent is a hemostatic agent or a blood clotting factor (e.g., for treatment of hemorrhages or bleeding). In certain embodiments, the growth factors stimulate wound healing.

[0054] In certain embodiments, the active agent is an antibiotic. In certain embodiments, the active agent is an antimicrobial peptide. The antibiotic and / or antimicrobial peptide may prevent and / or inhibit biofilm formation and / or promote wound healing (e.g., in chronic wounds). In certain embodiments, the antimicrobial peptide is selected from the antimicrobial peptide database (aps.unmc.edu). In certain embodiments, the antimicrobial peptide is a fragment of LL37 or an LL37 analog. In certain embodiments, the antimicrobial peptide comprises W379 (RRRWWWWV; SEQ ID NO: 3). In certain embodiments, the antimicrobial peptide has activity against Gram-positive and / or Gramnegative bacteria. In certain embodiments, the antimicrobial peptide has activity against biofilms. In certain embodiments, the antimicrobial is selected from the group consisting of W379, antibiotics, Aurein 1-2, Mellitin, Brevinin, Maculatins, Citropin, Buforin, Cathelicidins, LL37, W379, BMAP-27, 28, 34, Magainins, Cecropin, Protegrins, Bactenecin, Defensins, Tachyplesins, Polyphemusin, PR-39, TRitrpticin, Indolicidn, Crotalcidin, Histatins, Pexiganan, OP145, Omiganan, PAC 113, Iseganan, IMX942, Dalbavancin, Dalvance, PAC-113, P-113, Fuzeon, Baciim, Vancocin, Daptomycin, Telavancin, Colistin, Gramicidin, D2A21, PXL01, Omiganan, NISin, or combinations thereof.

[0055] In certain embodiments, the active agent is a drug. Drugs include but are not limited to anti-inflammatory drugs, antimicrobials (including but not limited to antibacterials, antivirals, and antifungals), pain medications, and other drugs useful for treating specific diseases or disorders. In certain embodiments, the active agent is a bone morphogenic proteins (BMP) or segment, fragment, derivative, or analogs thereof. In certain embodiments, the BMP stimulates bone regeneration (e.g., periodontal bone regeneration for the treatment of periodontal diseases). In certain embodiments, the bone morphogenetic protein is BMP- 2, BMP-7, BMP-12, or BMP-9. In certain embodiment, the bone morphogenetic protein is BMP -2. In certain embodiments, the BMP is human. In certain embodiments, the BMP is a BMP -2 fragment, peptide, and / or analog thereof. In certain embodiments, the BMP is a BMP-2 peptide such as KIPKASSVPTELSAISTLYL (SEQ ID NO: 1). In certain embodiments, the BMP is a BMP -2 fragment (e.g., up to about 25, about 30, about 35, about 40, about 45, about 50 amino acids, or more of BMP -2), particularly comprising the knuckle epitope (e.g., amino acids 73-92 of BMP-2 or SEQ ID NO: 1). In certain embodiments, the BMP-2 peptide is linked to a peptide of acidic amino acids (e.g., Asp and / or Glu; particularly about 3-10 or 5-10 amino acids such as E7, E8, D7, D8) and / or bisphosphonate (e.g., at the N-terminus).

[0056] In certain embodiments, the active agent is an angiogenic agent. In certain embodiments, the active agent is a vascular endothelial growth factor (VEGF) or segment, fragment, derivative, or analogs thereof. In certain embodiments, the VEGF induces endothelial cell proliferation and / or stimulates vascularization. In certain embodiments, the vascular endothelial growth factor binds the VEGF1 receptor and / or VEGF2 receptor. In certain embodiment, the VEGF is human. In certain embodiments, the VEGF is a VEGF fragment, peptide, and / or analog thereof. In certain embodiments, the VEGF is a QK peptide such as KLTWQELYQLKYKGI (SEQ ID NO: 2).

[0057] In certain embodiments, the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite comprises bone morphogenic proteins (BMP) or segment, fragment, derivative, or analogs thereof and a vascular endothelial growth factor (VEGF) or segment, fragment, derivative, or analogs thereof. In certain embodiments, the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite comprises a QK peptide and a BMP-2 peptide. In certain embodiments, the presence of the QK and BMP-2 peptides mimic bone healing and / or allow incorporation of bone marrow mesenchymal stem cells (BMSCs) to promote bone regeneration (e.g., alveolar bone regeneration). In certain embodiments, the BMP-2 peptide promotes the osteogenic differentiation of BMSCs and the QK peptide promotes the tubular network formation of endothelial cells (e.g., human umbilical vein endothelial cells (HUVECs)). In certain embodiments, the QK peptide and the BMP-2 peptide are released sequentially from the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite. As explained herein, by varying the crosslinking time or amount of conjugation, the release kinetics of conjugated active agents (e.g., peptides) can be varied. For example, angiogenic growth factors are predominantly expressed during the early phases for re-establishing the vascularity, whereas osteogenic growth factors are continuously expressed during bone regeneration and remodeling. In certain embodiments, the QK peptide can be conjugated with a shorter crosslinking time, while the BMP-2 peptide will be conjugated with a longer crosslinking time. This will result in sequential release of QK peptide and BMP -2 peptide to recapitulate bone healing. Notably, the QK peptide and BMP-2 peptide can be conjugated to the same nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite or can be conjugated to separate or different nanofibers, porous nanofibrous microspheres, and / or nanofibrous microsphere hydrogel composites. For example, a nanofibrous microsphere hydrogel composite may comprise first and second porous nanofibrous microspheres, wherein the first porous nanofibrous microspheres are conjugated to QK peptide and the second porous nanofibrous microspheres are conjugated to BMP -2 peptides. In certain embodiments, the QK peptide is incorporated or conjugated to the shell of the nanofiber and the BMP -2 peptide is incorporated or conjugated to the core of nanofiber, thereby leading to sequential delivery.

[0058] In certain embodiments, the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite comprises an antimicrobial agent and an angiogenic agent (e.g., for the acceleration of wound healing).

[0059] In certain embodiments, the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite are mineralized (e.g., comprise minerals and / or coated with minerals). Mineralization, for example, with hydroxyapatite, can enhance the adhesion of osteogenic precursor cells in vitro and in vivo (Duan, et al., Biomacromolecules (2017) 18:2080-2089). In certain embodiments, the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite are coated with Ca, P, and O. In certain embodiments, the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite are coated with hydroxyapatite, fluorapatite, or chlorapatite, particularly hydroxyapatite. In certain embodiments, the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite are immersed in simulated body fluid (SBF) for the mineralization (e.g., a solution comprising NaCl, CaCE, NaFhPC , and NaHCCh).

[0060] In certain embodiments, the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite comprise and / or encapsulate cells or tissue. In certain embodiments, the cells are autologous to the subject to be treated with the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite. The nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite may comprise and / or encapsulate any cell type. Cell types include, without limitation: embryonic stem cells, adult stem cells, bone marrow stem cells, induced pluripotent stem cells, progenitor cells (e.g., neural progenitor cells), embryonic like stem cells, mesenchymal stem cells, CAR-T cells, immune cells (including but not limited to T cells, B cells, NK cells, macrophages, neutrophils, dendritic cells and modified forms of these cells and various combinations thereof), cell based vaccines, and cell lines expressing desired therapeutic proteins and / or genes. In certain embodiments, the cells comprise stem cells. In certain embodiments, the cells comprise fibroblasts (e.g., dermal fibroblasts). In certain embodiments, the cells comprise bone marrow mesenchymal stem cells (BMSCs). In certain embodiments, the cells comprise endothelial cells (e.g., human umbilical vein endothelial cells (HUVECs)). In certain embodiments, the cells comprise BMSCs and endothelial cells (e.g., human umbilical vein endothelial cells (HUVECs)). In certain embodiments, the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite comprises and / or encapsulates cell spheroids. In certain embodiments, the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite comprises and / or encapsulates tissue samples (e.g., minced tissue), such as skin tissue samples or bone samples. The cells or tissue may be cultured within the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite (e.g., the cells or tissue may be cultured for sufficient time to allow for growth within and / or infiltration into the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite). For example, the cells or tissue may be cultured with the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite for 1 day, 2 days, 3 days, 4 days, 5 days, or more.

[0061] The nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite of the present invention may also be modified with targeting moieties (e.g., to enhance delivery to specific sites within the body (e.g., tissue types, disease areas, etc.)). Examples of targeting moieties include but are not limited to peptides, proteins, antibodies, antibody fragments, and small molecules. In a particular embodiment, a nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite is linked to a targeting ligand. A targeting ligand is a compound that specifically or preferentially binds to a specific type of tissue or cell type. For example, a targeting ligand may be used for engagement or binding of a target cell (e.g., a surface marker or receptor). In a particular embodiment, the targeting ligand is a ligand for a cell surface marker / receptor. The targeting ligand may be an antibody or fragment thereof immunologically specific for a cell surface marker (e.g., protein or carbohydrate) preferentially or exclusively expressed on the targeted tissue or cell type. The targeting ligand may be linked directly to the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite or via a linker.

[0062] The nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite of the present invention may also be modified or coated with additional materials to enhance their properties. Examples of coating include but are not limited to, collagen, proteoglycans, elastin, glycosaminoglycans (hyaluronic acid, heparin, chondroitin sulfate, and keratan sulfate), etc.

[0063] In accordance with the instant invention, the porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite may be used in inducing and / or improving / enhancing wound healing and inducing and / or improving / enhancing tissue regeneration. The porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite of the present invention can be used for the treatment, inhibition, and / or prevention of any injury or wound. In a particular embodiment, the method comprises administering a porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite comprising an agent and / or cell as described herein. Porous nanofibrous microspheres and / or nanofibrous microsphere hydrogel composites of the instant invention can be loaded with different cell types as necessary for regeneration of various tissues. In a particular embodiment, the porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite comprises blood clotting factors (e.g., for accelerating blood clot formation and / or preventing blood loss). For example, the porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite can be used to induce, improve, or enhance wound healing associated with surgery (including non-elective (e.g., emergency) surgical procedures or elective surgical procedures). Elective surgical procedures include, without limitation: liver resection, partial nephrectomy, cholecystectomy, vascular suture line reinforcement and neurosurgical procedures. Non-elective surgical procedures include, without limitation: severe epistaxis, splenic injury, liver fracture, cavitary wounds, minor cuts, punctures, gunshot wounds, and shrapnel wounds. The porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite of the present invention can also be incorporated into delivery devices (e.g., a syringe or spray) that allow for their injection / delivery directly into a desired location (e.g., a wound). The porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite also may be delivered directly into a cavity (such as the peritoneal cavity) using a needle, syringe, or pressurized cannula.

[0064] In accordance with the instant invention, the porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite of the present invention can be used to treat and / or prevent a variety of diseases and disorders. Examples of diseases and / or disorders include but are not limited to wounds, ulcers, infections, hemorrhage, tissue injury, tissue defects, tissue damage, bone fractures, bone degeneration, cancer (e.g., the use of docetaxel and curcumin for the treatment of colorectal cancer (Fan, et al., Sci. Rep. (2016) 6:28373)), neurologic diseases (e.g., Alzheimer’s and Parkinson’s), ischemic diseases, inflammatory diseases and disorders, heart disease, myocardial infarction, and stroke. Methods for inducing and / or improving / enhancing wound healing in a subject are also encompassed by the instant invention. Methods of treating, inhibiting, and / or preventing a diabetic wound or diabetic ulcer (e.g., diabetic foot ulcer) in a subject are also encompassed by the instant invention. Methods of inducing and / or improving / enhancing tissue healing and / or tissue regeneration (e.g., blood vessel growth, neural tissue regeneration, and bone regeneration) in a subject are also encompassed by the instant invention. Methods of inducing and / or improving / enhancing hemostasis in a subject are also encompassed by the instant invention. The methods of the instant invention comprise administering or applying a porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite of the instant invention to the subject (e.g., at or in a wound). In a particular embodiment, the method comprises administering a porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite comprising an agent and / or cell as described herein. Porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite of the instant invention can be loaded with different cell types as necessary for regeneration of various tissues. In a particular embodiment, the porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite comprises blood clotting factors (e.g., for accelerating blood clot formation and / or preventing blood loss). In a particular embodiment, the method comprises administering porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite to the subject and an agent as described herein (i.e., the agent is not contained within the nanofiber microsphere or composite). When administered separately, the porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite may be administered simultaneously and / or sequentially with the agent. The methods may comprise the administration of one or more porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite. When more than one porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite is administered, the porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite may be administered simultaneously and / or sequentially.

[0065] The porous nanofibrous microspheres and / or nanofibrous microsphere hydrogel composites can also be used to expand and increase cell numbers (e.g., stem cell numbers) in culture. In a particular embodiment, microtissues can be grown in situ by prolonged culture of cell laden porous nanofibrous microspheres and / or nanofibrous microsphere hydrogel composites (e.g., in confined microfluidic channel devices). These microtissues are injectable or transplantable into a tissue defect to promote wound healing in a subject (e.g., the porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite comprise autologous cells).

[0066] The porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite of the present invention may be administered by any method. The porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite described herein will generally be administered to a subject or a patient as a pharmaceutical composition. The compositions of the instant invention comprise porous nanofibrous microsphere and / or nanofibrous microsphere hydrogel composite and a pharmaceutically acceptable carrier. The term “patient” as used herein refers to human or animal subjects. These compositions may be employed therapeutically, under the guidance of a physician.

[0067] The compositions of the instant invention may be conveniently formulated for administration with any pharmaceutically acceptable carrier(s). For example, the agents may be formulated with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. The concentration of the porous nanofibrous microspheres and / or nanofibrous microsphere hydrogel composites in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical preparation. Except insofar as any conventional media or agent is incompatible with the agents to be administered, its use in the pharmaceutical preparation is contemplated.

[0068] Compositions of the instant invention may be administered by any method. For example, the compositions of the instant invention can be administered, without limitation, parenterally, subcutaneously, orally, topically (ex. using a cream or spray), pulmonarily, rectally, vaginally, intravenously, intraperitoneally, intrathecally, intracerbrally, epidurally, intramuscularly, intradermally, intratumoral, intracarotidly, or by direct injection (e.g., a localized injection into a specific tissue or organ). In certain embodiments, the composition is administered by injection (e.g., directly to the desired site). In certain embodiments, the porous nanofibrous microspheres and / or nanofibrous microsphere hydrogel composites are implanted or surgically implanted. Selection of a suitable pharmaceutical preparation will also depend upon the mode of administration chosen. For example, the compositions of the invention may be administered parenterally. In this instance, a pharmaceutical preparation comprises the porous nanofibrous microspheres and / or nanofibrous microsphere hydrogel composites dispersed in a medium that is compatible with the parenteral injection. The porous nanofibrous microspheres and / or nanofibrous microsphere hydrogel composites may be formulated in a variety of solutions and formats, such as, without limitation, a cream or ointment, a spray such as an aerosol, a powder, colloidal dispersion, emulsion, gels, and a liquid for injection or other form of administration.

[0069] Pharmaceutical compositions containing an agent of the present invention as the active ingredient in intimate admixture with a pharmaceutically acceptable carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., parenterally.

[0070] In a particular embodiment of the instant invention, methods for modulating (increasing) hemostasis; inhibiting blood loss; and / or treating hemorrhage are provided. In a particular embodiment, the method comprises administering the porous nanofibrous microspheres and / or nanofibrous microsphere hydrogel composites to the wound or site of bleeding. In a particular embodiment, the porous nanofibrous microspheres and / or

[0071] T1 nanofibrous microsphere hydrogel composites comprise a blood clotting factor such as thrombin and / or fibrinogen.

[0072] In a particular embodiment of the instant invention, methods for stimulating bone regeneration and / or treating bone loss are provided. In a particular embodiment, the method comprises administering the porous nanofibrous microspheres and / or nanofibrous microsphere hydrogel composites to the site of bone loss. In a particular embodiment, the site of bone loss is periodontal. In a particular embodiment, the bone loss is alveolar bone loss. In a particular embodiment, the porous nanofibrous microspheres and / or nanofibrous microsphere hydrogel composites are mineralized. In a particular embodiment, the porous nanofibrous microspheres and / or nanofibrous microsphere hydrogel composites comprise a bone growth stimulating growth factor such as a bone morphogenic protein or fragment or analog thereof.

[0073] The invention includes, but is not limited to, the embodiments of the following numbered paragraphs:

[0074] 1. A composite comprising nanofibrous microspheres and a hydrogel.

[0075] 2. The composite of paragraph 1, wherein said nanofibrous microspheres are porous nanofibrous microspheres.

[0076] 3. The composite of paragraph 1 or 2, wherein said nanofibrous microspheres comprises electrospun nanofiber segments.

[0077] 4. The composite of paragraph 3, wherein said electrospun nanofiber segments are crosslinked.

[0078] 5. The composite of paragraph 3 or 4, wherein said electrospun nanofiber segments comprise a polymer and a hydrogel.

[0079] 6. The composite of paragraph 5, wherein said polymer comprises poly(lactic-co- glycolic acid) (PLGA), polycaprolactone (PCL), polydioxanone (PDO), or poly(lactide- co-caprolactone) (PLCL). 7. The composite of paragraph 5, wherein said polymer comprises poly(lactic-co- glycolic acid) (PLGA).

[0080] 8. The composite of paragraph 5, wherein the hydrogel is gelatin or gelatin methacryloyl (GelMA).

[0081] 9. The composite of any one of paragraphs 1-8, wherein the hydrogel is selected from the group consisting of gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA), collagen, and combinations thereof.

[0082] 10. The composite of any one of paragraphs 1-9, wherein the hydrogel is present between 0.1% and 1%.

[0083] 11. The composite of any one of paragraphs 1-10, wherein said composite and / or nanofibrous microspheres are mineralized.

[0084] 12. The composite of any one of paragraphs 1-11, wherein said composite and / or nanofibrous microspheres comprises cells.

[0085] 13. The composite of any one of paragraphs 1-12, wherein said composite and / or nanofibrous microspheres comprise an agent is selected from the group consisting of a therapeutic agent, an analgesic a growth factor, a growth factor mimicking peptide, a signaling molecule, a cytokine, a hemostatic agent, an antimicrobial, and an antibiotic.

[0086] 14. A composition comprising the composite of any one of paragraphs 1-13 and a pharmaceutically acceptable carrier.

[0087] 15. A method for treating and / or preventing a disease or disorder in a subject in need thereof, said method comprising administering to said subject the composite of any one of paragraphs 1-13, optionally wherein the composite is crosslinked after administration.

[0088] 16. The method of paragraph 15, wherein the disease or disorder includes but is selected from the group consisting of wounds, ulcers, infections, hemorrhage, tissue injury, tissue defects, tissue damage, bone fractures, bone degeneration, cancer, neurologic diseases, ischemic diseases, inflammatory diseases and disorders, heart disease, myocardial infarction, and stroke.

[0089] 17. The method of paragraph 15, wherein the disorder is a diabetic foot ulcer.

[0090] 18. The method of any one of paragraphs 15-17, wherein the administration is injection into the area in need of treatment.

[0091] 19. The method of paragraph 15, wherein said disease or disorder is bone loss.

[0092] 20. A method for synthesizing the composite of paragraph 1, comprising i) cutting electrospun nanofibers into electrospun nanofiber segments, ii) dispersing the electrospun nanofiber segments in a solution, iii) electrospraying the solution comprising the electrospun nanofiber segments with air or gas bubbles to generate porous nanofibrous microspheres, and iv) mixing the porous nanofibrous microspheres with a hydrogel, thereby synthesizing said composite.

[0093] 21. The method of paragraph 20, further comprising crosslinking the composite generated after step iv).

[0094] Definitions

[0095] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0096] As used herein, the term “electrospinning” refers to the production of fibers (i.e., electrospun fibers), particularly micro- or nano-sized fibers, from a solution or melt using interactions between fluid dynamics and charged surfaces (e.g., by streaming a solution or melt through an orifice in response to an electric field). Forms of electrospun nanofibers include, without limitation, branched nanofibers, tubes, ribbons and split nanofibers, nanofiber yarns, surface-coated nanofibers (e.g., with carbon, metals, etc.), nanofibers produced in a vacuum, and the like. The production of electrospun fibers is described, for example, in Gibson et al. (1999) AlChE J., 45: 190-195. “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0097] A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., TrisHCl, acetate, phosphate), water, aqueous solutions, oils, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington.

[0098] As used herein, the term “polymer” denotes molecules formed from the chemical union of two or more repeating units or monomers. The term “block copolymer” most simply refers to conjugates of at least two different polymer segments, wherein each polymer segment comprises two or more adjacent units of the same kind.

[0099] “Hydrophobic” designates a preference for apolar environments (e.g., a hydrophobic substance or moiety is more readily dissolved in or wetted by non-polar solvents, such as hydrocarbons, than by water). In certain embodiments, hydrophobic polymers may have aqueous solubility less than about 1% wt. at 37°C. In certain embodiments, polymers that at 1% solution in bi-distilled water have a cloud point below about 37°C, particularly below about 34°C, may be considered hydrophobic.

[0100] As used herein, the term “hydrophilic” means the ability to dissolve in water. In a particular embodiment, polymers that at 1% solution in bi-distilled water have a cloud point above about 37°C, particularly above about 40°C, may be considered hydrophilic.

[0101] As used herein, the term “amphiphilic” means the ability to dissolve in both water and lipids / apolar environments. Typically, an amphiphilic compound comprises a hydrophilic portion and a hydrophobic portion.

[0102] The term “antimicrobials” as used herein indicates a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, viruses, or protozoans.

[0103] As used herein, the term “antiviral” refers to a substance that destroys a virus and / or suppresses replication (reproduction) of the virus. For example, an antiviral may inhibit and or prevent: production of viral particles, maturation of viral particles, viral attachment, viral uptake into cells, viral assembly, viral release / budding, viral integration, etc.

[0104] As used herein, the term “antibiotic” refers to antibacterial agents for use in mammalian, particularly human, therapy. Antibiotics include, without limitation, betalactams (e.g., penicillin, ampicillin, oxacillin, cioxacillin, methicillin, and cephalosporin), carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides (e.g., gentamycin, tobramycin), glycopeptides (e.g., vancomycin), quinolones (e.g., ciprofloxacin), moenomycin, tetracyclines, macrolides (e.g., erythromycin), fluoroquinolones, oxazolidinones (e.g., linezolid), lipopetides (e.g., daptomycin), aminocoumarin (e.g., novobiocin), co-trimoxazole (e.g., trimethoprim and sulfamethoxazole), lincosamides (e.g., clindamycin and lincomycin), polypeptides (e.g., colistin), and derivatives thereof.

[0105] As used herein, an “anti-inflammatory agent” refers to compounds for the treatment or inhibition of inflammation. Anti-inflammatory agents include, without limitation, non-steroidal anti-inflammatory drugs (NSAIDs; e.g., aspirin, ibuprofen, naproxen, methyl salicylate, diflunisal, indomethacin, sulindac, diclofenac, ketoprofen, ketorolac, carprofen, fenoprofen, mefenamic acid, piroxicam, meloxicam, methotrexate, celecoxib, valdecoxib, parecoxib, etoricoxib, and nimesulide), corticosteroids (e.g., prednisone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, tramcinolone, and fluticasone), rapamycin, acetaminophen, glucocorticoids, steroids, beta-agonists, anticholinergic agents, methyl xanthines, gold injections (e.g., sodium aurothiomalate), sulphasal azine, and dapsone.

[0106] As used herein, the term “analgesic” refers to an agent that lessens, alleviates, reduces, relieves, or extinguishes pain in an area of a subject's body (i.e., an analgesic has the ability to reduce or eliminate pain and / or the perception of pain).

[0107] As used herein, the term “small molecule” refers to a substance or compound that has a relatively low molecular weight (e.g., less than 2,000). Typically, small molecules are organic, but are not proteins, polypeptides, or nucleic acids.

[0108] As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human.

[0109] As used herein, the term “prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition resulting in a decrease in the probability that the subject will develop the condition. The term “treat” as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.

[0110] The term “hydrogel” refers to a water-swellable, insoluble polymeric matrix (e.g., hydrophilic polymers) comprising a network of macromolecules, optionally crosslinked, that can absorb water to form a gel.

[0111] As used herein, a linker is generally a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches two compounds. The linker can be linked to any synthetically feasible position of the two compounds. Exemplary linkers may comprise at least one optionally substituted; saturated or unsaturated; linear, branched or cyclic aliphatic group, an alkyl group, or an optionally substituted aryl group. The linker may be a lower alkyl or aliphatic. The linker may also be a polypeptide (e.g., from about 1 to about 10 amino acids, particularly about 1 to about 5). The linker may be non-degradable and may be a covalent bond or any other chemical structure which cannot be substantially cleaved or cleaved at all under physiological environments or conditions.

[0112] The term “crosslink” refers to a bond or chain of atoms attached between and linking two different molecules (e.g., polymer chains). The term “crosslinker” refers to a molecule capable of forming a covalent linkage between compounds. A “photocrosslinker” refers to a molecule capable of forming a covalent linkage between compounds after photoinduction (e.g., exposure to electromagnetic radiation in the visible and near-visible range). Crosslinkers are well known in the art (e.g., formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, etc.). The crosslinker may be a bifunctional, trifunctional, or multifunctional crosslinking reagent.

[0113] The following examples illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

[0114] EXAMPLE 1

[0115] Materials and Methods

[0116] Materials PLGA (50:50) was purchased from EVONIK (Vernon, CA). Type A Gelatin from porcine skin, agar, and 1,1,1,3,3,3-Hexafhioroisopropanol (HFIP) were purchased from Sigma Aldrich (St. Louis, MO). Glutaraldehyde (alcoholic solution) was purchased from LADD research (Williston, VT). Dulbecco’s Modified Eagle Medium with GlutaMAX™, fetal bovine serum, penicillium streptomycin, 1X DPBS, and TrypLE™ were procured from Gibco (USA). Calcein AM, ethidium homodimer, PrestoBlue™, Alexa Fluor™ 594 phalloidin, and DAPI were procured from Invitrogen™ (USA).

[0117] Preparation of PLGA / Gelatin electrospun nanofiber

[0118] 8% PLGA:gelatin (1 :1) was dissolved in 10 ml of HFIP to fabricate a nanofiber electrospun mat using the electrospinning method. A typical electrospinning setup was employed with a current voltage of 15 kV, rotating mandrel set at high speed to get an aligned nanofiber and solution flow rate at 600 pl / hour. The electrospun polymer fiber mat was crosslinked overnight with 25% glutaraldehyde (glutaraldehyde in ethanol) vapors.

[0119] Fabrication of nanofibrous microspheres

[0120] Nanofibrous microspheres (NMs) were fabricated by an electrospraying technique utilizing electrospun short fibers. Briefly, crosslinked PLGA:gelatin electrospun fiber mat was cut into 2 mm pieces and frozen at -80 °C, followed by cryosectioning. These segmented fibers were lyophilized overnight to obtain nanofiber short fibers and stored at 4°C until further use. NMs were fabricated from cryosectioned fibers by dispersing 20 mg / ml in distilled water followed by sonication to get a homogenous solution of nanofibrous short fiber with an amplitude of 20% and 10 seconds ON and 20 seconds OFF cycle for 20 minutes. Next, 500 pl of 5-8% gelatin solution was added during the homogenization process, and sonication was continued for another 20 minutes. Finally, the homogenized short fiber solution was utilized for NMs fabrication using the electrospraying technique. NMs were fabricated with the newly invented technology, co-axial electrospraying of gas bubbles in the core and short nanofiber solutions in the shell. An electrospraying setup was used comprising a solution ejector connected to a positive voltage of 6-6.5 kV and a liquid nitrogen collector container connected to a negative current. The needle tip-to-collector distance was set at 10 cm, and a 21G blunt needle was used with a solution flow rate of 2 ml / hour. NP-NMs were prepared with 0 airflow rate with optimized parameters. For P-NMs, a co-axial electrospraying setup was used with a 50 pm diameter microcapillary tube, and airflow rate and voltage were varied to obtain an optimal size NMs. First, the airflow rate was varied from 0 to 10 ml / hour to check bubble formation. Secondly, the solution and airflow rate were kept constant, and voltage was varied from lower to higher to examine the effect on NMs size and pore formation. Finally, the optimized parameters of 2 ml / hour solution flow rate, 10 ml / hour airflow rate, and 6-6.5 kV voltage were used to prepare P-NMs. These collected frozen NMs were immediately transferred to a lyophilizer and freeze-dried overnight. Freeze-dried NP-NMs and P- NMs were crosslinked with glutaraldehyde vapors (25% glutaraldehyde in ethanol solution) to reinforce the microsphere structure.

[0121] Morphological characterization

[0122] The morphological features of NMs were characterized using environmental scanning electron microscope (SEM) (FEI Quanta 600). The fabricated NMs fixed onto double sided conductive carbon tape then sputter coated (Magnetron Ion Sputtering Coater, MSE PRO™) using gold target at peak current of 15 A for 3 minutes. For analyzing the cross-sectional view, the electrosprayed NMs were dispersed in water, frozen at -80°C for 2-3 hours, and then cryo-sectioned into 50-pm thick slices. These cryo-cut samples were subsequently collected, freeze-dried, and mounted onto carbon tape before being coated with gold using the same sputter coating parameters. The gold coated samples were imaged at an accelerating voltage of 30 kV and spot size of 5 with a working distance of 10 mm.

[0123] Water uptake ratio measurement

[0124] The water absorptions of the NP-NMs and P-NMs were measured using the weight gain measurement. The dry samples were weighed and immersed in distilled water for 5 minutes, then retrieved, and the wet weight (Wwet) was measured. After that, samples were lyophilized, and dry (Wdry) measurements were taken. The water uptake ratio was measured according to the following equation,

[0125] Water uptake ratio = (Wwet- Wdry) / Wdryx100%

[0126] Human dermal fibroblast (HDF) cell culture

[0127] HDFs were cultured in Dulbecco’s Modified Eagle Medium + GlutaMAX™ (DMEM) with 10% fetal bovine serum and 1% penicillin streptomycin at 37°C with 5% C02humidified atmosphere. Once the cells reached the confluency, they were trypsinized using TrypLE™ and utilized for future experiments. NP-NMs and P-NMs scaffolds were UV sterilized for 1 hour, followed by IX Tris HCL buffer treatment for 45 minutes, then washed with IX DPBS. The processed scaffolds were homogenously distributed in an agar-coated 24-well plate. HDF cells were seeded at a seeding density of 1 x 105cells per well, and cell-laden scaffolds were cultured with DMEM complete media at 37°C with a 5% CO2 incubator. Cell-seeded NMs were used for further assay at the indicated time point, and half-media was replenished every 24 hours.

[0128] In vitro cell adhesion and proliferation on microspheres

[0129] Cell-laden NP-NMs and P-NMs were studied for cell adhesion and proliferation at different culture times (days 1, 3, and 7). Cell-laden NMs media was withdrawn at indicated time points and washed with IX DPBS followed by calcein AM (1 pM) and ethidium homodimer (1 pM) staining at room temperature for 20 minutes. Then, fluorescently stained samples were washed with IX DPBS and visualized under confocal microscopy (Zeiss LSM 700) at 488 nm for calcein AM (green color, viable cells) and 561 nm for ethidium homodimer (red color, dead cells).

[0130] The proliferation of cells seeded on NMs was quantified using PrestoBlue™ cell viability reagent at different time points. In short, PrestoBlue™ (Invitrogen™) working concentration solution (1: 10 dilution with complete media) was prepared according to the manufacturer’s instructions. Cell culture complete media was removed from respective samples, followed by the addition of PrestoBlue™ solution and incubated for 3 hours at 37°C with 5% CO2 incubator. After that, the solution was collected from each sample, and absorbance was measured at 570 nm and 600 nm using a Varioskan™ Lux plate reader (Thermo Fisher Scientific). The cell proliferation rate is directly proportional to the dye reduction percentage, which was calculated from the absorbance data and presented as a bar graph with standard deviation (triplicate samples were used for each time point).

[0131] Fluorescence staining of actin fdaments

[0132] The morphology of the adhered cells was visualized using actin staining. At indicated time points, HDFs seeded on NMs (P-NMs and NP-NMs) were obtained and fixed with 4% formaldehyde solution overnight at 4°C then washed with IX DPBS followed by 0.1 % Triton® X-100 treatment for 10 minutes. These permeabilized samples were washed with IX DPBS thrice and blocked with 1 % bovine serum albumin for 30 min, then washed with IX DPBS. Then, Alexa Fluor™ 594 phalloidin fluorescent dye was added and incubated for 30 minutes at room temperature. These fluorescently stained microspheres were washed with IX DPBS, and finally, nuclei were stained with DAPI for 5 minutes. Fluorescently stained microspheres were imaged under confocal microscopy (Zeiss LSM 700) for fluorescent channels 594 nm (cytoskeleton, red) and 405 nm (nuclei, blue).

[0133] Mechanical strength evaluation of microspheres using nanoindentation

[0134] The Pavone nanoindenter (Optics 11 Life) was used to measure the effective Young’s modulus of single NMs based on published protocols with minor modifications (Zhang, et al. (2022) Macromol. Biosci., 22(4):2100498; Carvalho, et al. (2021) Biofabrication 13(3):035008). Both NMs groups, such as NP-NMs and P-NMs without cells and day 7 cell-laden samples (n=3), were utilized for mechanical strength evaluation. Initially, the probe was calibrated in IX DPBS against a petri dish. In short, microspheres were immobilized on the petri dish containing IX DPBS solution and employed for single-point indentations with a tip radius of 10 pm and stiffness of 0.022 N / m. The obtained data was fit to a linear Hertzian model to calculate the effective Young’s modulus.

[0135] Injectability experiments on pressure protection

[0136] The injectability of the NMs was studied for their pressure protection on cell viability after injection based on literature with minor modifications to the protocol (Wei, et al. (2018) Adv. Mater., 30(31): 1802273). Previously, the injection flow rate was optimized to maintain a constant pressure / load, which can provide a relatable condition during injection on both sample groups. In short, NMs (non-cell-loaded) were dispersed in IX DPBS, loaded in a 3 ml syringe with a needle gauge of 16G, and injected at a 10 ml / hour flow rate. For observation of NMs movement and morphological change during injection under an optical microscope (BZ-X series, Keyence microscope), a transparent PTFE tube with an inside diameter of 1 mm was connected to the injector needle, and images were recorded during injection. Further, two different injection conditions were modeled for in vitro injectability studies, such as injection once (Inj Ix-DX) and injection twice with a 6 hour time interval on the same day (Inj2x-DX) for long-time observation of cell viability. Here, DX denotes time points (DI, D2, D3, and D7) after injection experiments to assess the cell viability and proliferation. The same passage of cells was used for two different sets of injection experiments such as Inj lx and Inj2x. At indicated time points, the samples were retrieved and stained for live / dead staining using calcein AM and ethidium homodimer. At longer run, samples were also employed for cell proliferation using the PrestoBlue™ protocol.

[0137] In vitro wound healing model to study cell migration

[0138] In vitro direct wound healing experiments were performed using a scratch assay. For this experiment, engineered NMs underwent UV sterilization followed by washing with IX DPBS. These sterilized NMs were incubated in a complete cell culture medium for approximately one week and the spent media such as NMs treated media was collected and utilized for cultivating HDF. Secondly, HDFs were seeded on a 24-well plate at a seeding density of 5 * 104cells per well and cultured till they attained confluency. Once cells are 70% confluent a direct line of scratch (wound) was made using a sterile 10 pl tip. The collected spent from NP-NMs and P-NMs was supplemented to HDFs for the culturing them. The spent was employed to cells to examine whether the subproducts released from NMs into the media impacted cell survival, proliferation and migration of cells to attain wound closure. DMEM complete media without introducing NMs treatment was used as a control group. Bright-field images were captured using the Keyence™ microscope (BZ-X series, Keyence) at different time points such as 0, 5, 10, and 24 hours to compare the cell migration between sample groups. Collected images were quantified for the rate of cell migration and wound closure percentage using a mentioned formula with ImageJ software,

[0139] Rate of cell migration = (Wi - Wt) / t Where Wi is the initial wound width average in pm, Wt is wound width at different time points, and t is the time point of the assay in hours.

[0140] Wound closure % = (At=o - At=At) / At=ox100% In this case, At=o is the initial wound area, and At= t is the wound area at an indicated time point after a time span of initial hours, both in pm2.

[0141] In order to examine the migration of encapsulated cells towards NMs, an in vitro model was created using fibrin gel (6 mg / ml, Sigma-Aldrich) and HUVECs. GFP- tagged HUVECs were encapsulated in fibrin gel at a concentration of 1 x 106cells per 100 pl of gel and cast in a 96-well plate followed by crosslinking at 37°C with 5% CO2 incubator. UV sterilized and collagen-coated NMs were introduced on top of HUVECs encapsulated fibrin gels, and images were captured periodically using ECHO Revolution (Discover ECHO) and confocal microscope (Zeiss LSM 700) to examine the HUVECs sprouting and migration toward NMs.

[0142] To investigate the cell migration from 2D surface into 3D NMs, an in vitro model was created using GelMA. 8% (w / v) of GelMA with 0.1 % (w / v) of Irgacure 2959 photoinitiator was prepared in sterile distilled water. In a 24-well plate, 400 pl of GelMA solution was added to each well and crosslinked with 405 nm wavelength UV light for 60 seconds. Fabricated GelMA constructs were washed with IX DPBS, then 1 x 105HDF cells were added to each well and continuously cultured at 37°C with 5% CO2. A 4 mm hole was created using a biopsy punch when cells reached the confluency stage, then sterilized, collagen-coated NMs were loaded inside the void with basic fibroblast growth factor (bFGF) and cultured continuously to examine the migration of fibroblast towards the wound site. Cell migration towards NMs were imaged under confocal microscope on different time points. 2.5D images were processed from z-stack scan images using ImageJ software.

[0143] In vivo wound tissue repair

[0144] Injections of NMs on TALLYHO type 2 diabetic mice (Male, 10-12 weeks, The Jackson Laboratory) wound site were carried out with ethical approval IACUC (Protocol #19-069-07-FC) at the University of Nebraska Medical Center (UNMC). Mice were anesthetized with 4% isoflurane in oxygen for approximately 2 min and placed on a heating pad to keep their body temperature stable. A 44 cm2 portion of each mouse's back was shaved, and the exposed skin was then treated three times with the povidone- iodine solution. Two circular wounds, each 8 mm in diameter, were created on the skin of each animal using a biopsy punch. NMs were suspended in saline with a concentration of 10 mg / ml and injected on the wound site. The circular wound splints and Tegaderm dressings were used to cover the wound to refrain NMs from falling off wound site. NP-NMs were used to treat one set of wounds (n = 8) and additional set of wounds (n = 8) received P-NM treatment. Each group consisted of four mice (two wounds per animal), and each group had a total of eight wounds at each time point. After the NMs were implanted, a digital camera was utilized to capture each mouse's wounds on days 7 and 14. After 14 days of implantation the wound and surrounding tissues were excised and fixed with 4 % paraformaldehyde. The fixed samples were dehydrated in a graded ethanol series starting from 70% to 100% then embedded in paraffin, and sectioned (50 pm thick). The samples were stained using Masson's trichrome or H&E according to standard protocols. The paraffin- embedded samples were deparaffinized and rehydrated with xylene, followed by a series of ethanol washes. The tissues were dehydrated in a series of ethanol washes to xylene before being mounted with Permount™ and covered with glass coverslips. The entire set of slides was then digitally scanned at UNMC’s Tissue Science Facility to obtain images for visualization. From the stained sections, cell infiltration area and neovascularization was counted per cm2was then quantified using image J software.

[0145] The H&E-stained images (n=3) from the different mice in each group were employed for calculating the area of cell infiltration. The percentage area of cell infiltration, or wound closure, was calculated using the below-mentioned formula,

[0146] Cell migration during the wound closure (%) = Aceii infiltration / ADefectx100 % Where, ADefect denotes the area of the implanted defect site (8 mm), then Aceii infiltration is the area of cells infiltrated inside the NMs based on migrated cells around the NP-NMs and inside and around the P-NMs.

[0147] Statistical analysis

[0148] All the cell culture experiments were performed in triplicate samples (n=3), and the data were expressed as mean ± standard deviation. The GraphPad Prism 9 software was used for performing statistical analysis. For determining statistical differences among the groups, a one-way ANOVA with Tukey test was performed. Statistical significance was set at * p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001 and ns for nonsignificant where p>0.05.

[0149] Results

[0150] Nanofibrous microspheres with tunable size and pore size

[0151] A co-axial electrospraying with bubble technology was used to generate NMs with microporous structures (John, et al. (2019) Nanomed., 22: 102081; John, et al. (2020) Small 16(19): 1907393). These NMs not only replicate the nanofibrous microstructural cues of the native ECM but also possess injectability, making them suitable for minimally invasive therapies. The fabrication process of PLGA:gelatin (1 : 1) NMs is represented schematically in Figure 1 A. Figure IB shows that these NMs feature a fibrous architecture that significantly enhances cell-biomaterial interactions, resulting in improved cell adhesion, proliferation, and maturation. Moreover, the porous nature of these NMs facilitates cell migration into the microspheres and allows for efficient transport of nutrients and waste in and out of the NM scaffolds, thereby improving cell viability and matrix maturation. Figure 1C demonstrates several applications of these NMs as dermal fillers and cell carriers, which can also be injected into a defect site in a minimally invasive way. The injectability of NMs allows for direct delivery to the defect site, where the porous NMs offer an increased area for host cell transmigration and ECM.

[0152] Initially, a PLGA:gelatin (1 : 1) electrospun nanofiber mat was synthesized with fiber diameters ranging from 500 to 600 nm (John, et al. (2019) Nanomed., 22: 102081; John, et al. (2020) Small 16(19): 1907393). The nanofiber mat was then segmented into 20 - 40 pm short nanofibers using cryosectioning (John, et al. (2019) Nanomed., 22: 102081). These fibers were homogenized in water using a probe tip ultrasonicator at a concentration of 20 mg / mL (%w / v). An 8% w / w gelatin solution related to fiber weight was added to the segmented nanofiber solution to impart stability (John, et al. (2019) Nanomed., 22: 102081). The electrospraying process was adjusted to match optimized conditions to electrospray non-porous microspheres (NP-NMs) and air bubbles were injected into a co-axial system, as shown in Figure ID to produce porous microspheres (P-NMs). This dynamic system allowed control over air and fluid flow rate and voltage, allowing for precise tuning of the NMs size and pore volume.

[0153] Varying airflow rates (0 - 10 ml / hour) influenced the architecture and pore density of the NMs (Figure IE). At an airflow rate of 0 ml / hour, hollow microspheres formed as no air passed through the capillary tubing. The airflow rate gradually increased from 2 ml / hour to 8 ml / hour, and a greater number of bubbles were observed in the droplet of the short nanofiber solution, resulting in a partially porous structure (Figure IE). At an airflow rate of 10 ml / hour, a fully open porous microsphere was obtained.

[0154] A range of voltages was tested, revealing that higher voltages produced fully open porous microspheres (Figure IF). At lower voltages, Janus microspheres formed, with air bubbles occupying half of the microsphere's volume, resulting in two distinct phases of top porous and bottom nonporous regions. Upon further increasing the voltage to 6 kV, fully open porous microspheres were produced, as illustrated in Figure IF.

[0155] The NP-NMs were engineered without the co-axial setup (John, et al. (2019) Nanomed., 22: 102081). SEM images in Figure 2A display the nanofibrous structure of the NMs. NP-NMs had a diameter of 588 ± 92, while P-NMs measured 698 pm ± 91 (Figure 2B) and their size range histogram shown in Figure 2E. P-NMs micropore sizes ranging from 70 to 150 pm as depicted in Figures 2C. Cross-sectional view of SEM images further confirms the internal fibrous structure in both NP- and P-NMs samples (Figure 2F). Additionally, internal porous architecture was observed in P-NMs samples (Figure 2F). The NMs demonstrated efficient water absorption due to their fibrous architecture (Figure 2D), making them ideal for wound site implantation.

[0156] Porous microstructure induced high cell proliferation and mechanical strength

[0157] Human dermal fibroblasts (HDFs) were seeded on P-NMs and NP-NMs to evaluate their cell adhesion and proliferation capabilities, an essential feature for efficient cell delivery. Cell viability and proliferation on each NMs type were assessed using live / dead staining and Prestoblue™ assay at 1, 3, and 7 days. Figure 3A shows a high number of viable cells attached to NP-NMs and P-NMs after 7 days. The metabolic activity of HDFs significantly increased from day 1 to day 7 in both the sample groups (Figure 3B). The morphology of HDFs on NMs, visualized through actin cytoskeleton staining, demonstrated cell attachment and cytoskeleton formation over time (Figures 3C, 3D). By day 7, cells covered the entire surface of the NMs in both sample groups. P- NMs displayed higher cell density, inferred from more intense actin staining, suggesting complete cell coverage (Figure 3C). Cells penetrated the micropores in P-NMs at later time points (Figure 3C).

[0158] Given the extensive cell coverage on day 7 HDF-loaded NMs (Figures 3C, 3D), it was hypothesized that cell-loaded NMs would exhibit enhanced mechanical properties via ECM secretion support (McCarthy, et al. (2023) Adv. Mater., 35(5):2207335). Nanoindentation was performed using the Pavone instrument to assess the mechanical strength of the cell-loaded NMs. Figure 4A illustrates the nanoindentation process on NMs. The indenter probe, immersed in liquid, applied minimal force upon contact with the NMs surface (Fmin). The NMs exhibited resistance to compression, from which effective Young’s modulus (Eeff) was calculated based on the load vs displacement curve (Figure 4A). Nanoindentation was conducted on day 0 and day 7 on HDF-loaded samples, yielding comparable values across multiple locations on the NMs samples. The Eeff of NMs alone (day 0 without cells) was 845 ± 49 Pa for NP-NMs and 870 ± 84 Pa for P-NMs. Day 7 HDF-loaded NMs showed a statistically significant increase in Eeff compared to day 0 samples for both NMs types (Figure 4B). NP-NMs on day 7 exhibited an Eeff of 2321 ± 131 Pa, while P-NMs recorded 3232 ± 228 Pa, marking a significant 1-fold increase between the two types. Cell-loaded NMs were expected to show higher resistance to the applied load, as explained in Figure 4C, due to a compact cell sheath and ECM secretion over time. This depicts that cell coverage and ECM secreted by adhered cells enhance the mechanical strength of the NMs.

[0159] NMs as cell carriers

[0160] Injectable systems, known for their minimally invasive approach, have the potential to significantly enhance the healing process (John, et al. (2019) Nanomed., 22: 102081; Liu, et al. (2017) Bone Res., 5(1): 1-20; Dimatteo, et al. (2018) Adv. Drug Deliv. Rev., 127: 167-184). The study explored HDF-loaded NMs as cell carriers for potential in vivo cell therapies and effective tissue regeneration. However, the injection process itself, involving pressure and friction, poses a risk of cell detachment or escape from the microspheres. This is particularly true for cells on the surface of the NMs, while those residing within the microspheres are more likely to remain intact (Wei, et al. (2018) Adv. Mater., 30(31): 1802273). This phenomenon suggests that partially cell- loaded carriers would improve the regeneration capability of the tissue by protecting the cells from shearing forces exerted during injection.

[0161] To assess the impact of injection -induced pressure on cell-loaded NMs, two pressure tests were conducted: a single injection (Inj Ix-DX) and a double injection (Inj2x-DX) with a 6-hour interval on the same day (Figure 5 A). For these tests, HDF- loaded NMs cultured under static condition for 7 days subsequently they were utilized for injection, and cell activity was monitored at various time points post-injection (1, 2, 3, and 7 days). Different injection rates were initially tested to identify the optimal flow rate for NMs injection without causing clumping or agglomeration. Figure 51 shows the impact of flow rate on the injection properties of NMs, indicating a preferred rate of 10 ml / hour. Bright-field images captured before, during, and after injection (Figure 5 J) indicated no noticeable structural changes in the microspheres, even under the applied pressure of injection.

[0162] Post-injection, cell activity was assessed using Live / dead staining, as displayed in Figure 5L, showed that the Inj2x-DX samples exhibited a reduced number of attached cells, and a significant presence of dead cells compared to the Inj Ix-DX group (Figure 5 J). Phalloidin actin staining was performed to further understand the cell morphology post-injection. Figure 5B reveals proper cell attachment and cytoskeleton formation in the Inj Ix-Dl, 3, and 7 samples, with fewer cells detached. In contrast, the Inj2x-Dl actin staining images showed cells detaching from the microspheres with disrupted cytoskeletons (Figure 5C). Additionally, both the Inj lx and Inj2x cases showed a decrease in the intensity of adherent cell cytoskeleton staining from day 1 to day 7, indicating that injection pressure adversely affects the cells (Figures 5B and 5C) and may render injectable cellular therapies less efficacious without proper cell carriers. Further injections in the Inj2x scenario caused more significant cell deterioration on microsphere samples (Figure 5C).

[0163] During injection, both NP-NMs and P-NMs undergo pressure and friction from the syringe wall and between microspheres, leading to cell abrasion. Notably, more cell damage was observed in NP-NMs (Figure 5D), where multidirectional pressure caused cells to detach from the microspheres. In contrast, P-NMs preserved cells that had migrated into their micropores during the period of cell growth, while surface-residing cells either detached or underwent apoptosis. The pressure during injection damages surface cells in both cases, resulting in impaired metabolism and proliferation, and in some cases, cells detach from the NMs during subsequent culture. To quantify cell proliferation in the injected samples, a Prestoblue™ assay was conducted at various time points. Cell viability gradually decreased over time after a single injection (Inj Ix-DX) on days 1, 2, 3, and 7 in both NP-NMs and P-NMs groups. Cell metabolic activity diminished by 79.4% in NP-NMs (Figure 5E) and 76.9% in P-NMs samples in the Inj lx scenario (Figure 5F), showing a statistically significant difference between NP and P- NMs groups. Greater cell losses were seen with the two-injection system (Inj2x): 84.6% for NP-NMs (Figure 5G) and 80.3% for P-NMs (Figure 5H), which showed a pattern like that of Inj lx. Meanwhile, the NP-NMs Inj lx samples showed statistically significant differences compared to the NP-NMs Inj2x samples, and similarly, the P- NMs samples demonstrated a similar trend. The greater loss of cell activity in the Inj2x scenario indicates that this mode of injection is more detrimental to cells; however, this is not applicable in clinical practice. It is evident under both conditions (Inj IX-DX and Inj2x-DX) that NP-NMs as cell carriers exhibit a higher loss of cell activity than P-NMs.

[0164] Studies on in vitro cell migration show that cells migrate toward microspheres

[0165] The primary objective of the scratch test was to evaluate the influence of NMs and the byproducts they release on cell behavior. As observed in Figure 6D, the wound closure area progressively decreased over time across all conditions, with complete closure occurring after 24 hours of culture, indicating active cell migration. The wound closure percentage and cell migration rate were quantified using Image! As shown in Figure 6E, the wound closure percentages were comparable across all groups, with no statistically significant differences under any of the conditions. Figure 6F inferred that while the overall cell migration rate was similar across groups, notable variations were observed at different time points. After ascertaining that the NMs byproducts did not negatively affect cell migration, any possible effects on cell chemotaxis was evaluated using a 3D in vitro cell migration study. Figure 6A depicts the cell recruitment model, consisting of a fibrin gel encapsulated with GFP -tagged human umbilical vein endothelial cells (HUVECs). NMs were deposited on the HUVEC-laden fibrin gel, and the directional migration of these cells and the formation of vascular networks in the presence of NP- and P-NMs were observed. Fluorescence images in Figures 6B and 6C show the dispersion of HUVECs throughout the hydrogel on day 1. By day 3, Figure 6G illustrates HUVECs migrating towards and enveloping the NP-NMs. Furthermore, day 5 3D volume rendered images indicates that cells migrate approximately 1 mm into P- NMs, indicating robust penetration into the pores (Figure 6H). In contrast, NP-NMs show a penetration distance of around 300 pm (Figure 6H). This makes it clear that cells penetrate deeper into P-NMs compared to NP-NMs, supported by the distance measurements from confocal microscopy images. Confocal images from day 5 reveal the directionality of HUVECs towards NP-NMs and the formation of vascular networks around these microspheres (Figure 6B). Conversely, P-NMs displayed a different interaction, with HUVECs aligning along the periphery of the pores and migrating into the micropores of P-NMs by day 3 (Figure 6G). Day 5 confocal images of P-NMs (Figure 6C) showed cells advancing towards and inside the pores, thereby forming a cellular network of HUVECs, indicating the potential for vascularization. This capability would facilitate neovascularization under in vivo conditions using P-NMs as implants.

[0166] Additionally, an in vitro wound model was developed using a gelatin methacryolyl (GelMA) construct seeded with HDFs to analyze cell migration from 2D surface to 3D NMs as depicted in Figure 7G. The migration of HDFs towards the center of the defect site and the NMs was assessed using confocal images. Figures 7H and 71 indicate an enhanced migration of fibroblasts towards both NP and P-NMs, marked by arrows. Furthermore, a more pronounced spatial distribution of cells was observed between loaded P-NMs (Figure 71), indicating that the porous architecture of P-NMs facilitates cell spreading throughout the defect site. This evidence underscores the utility of NMs, particularly P-NMs, in supporting and directing cell migration in tissue engineering applications.

[0167] In vivo experiments display host cell transmigration

[0168] To evaluate NMs as granular scaffolds for wound healing, both NP-NMs and P- NMs were injected into type 2 diabetic mice B6.BKS(D)-Leprdb / J (Male, 10-12 weeks, The Jackson Laboratory) (Figure 7J) as acellular scaffolds. Stem cell-laden 3D nanofiber scaffolds are effective in enhancing granulation tissue formation, promoting angiogenesis, and facilitating collagen deposition (John, et al. (2020) Small 16(19): 1907393). The open porous structure of the NMs, particularly P-NMs, prompted comparison of their wound healing efficacy against NP-NMs with data collected on days 7 and 14 post-implantation (Figure 7K). Hematoxylin and Eosin (H&E) staining images (Figures 7A-7D) provided insights into the response of diabetic mice to the treatment with these NMs over these periods. Figures 7A and 7B showcase the H&E staining of NP-NMs treated diabetic mice. The structure of the NP-NMs and their peripheral areas are evident (circle in Figure 7A). NP-NMs facilitated granulation tissue formation around their structure (arrow in Figure 7B), a finding consistent with observations with MAP hydrogels (Liu, et al. (2023) Adv. Healthc. Mater., 2023:2300823; Griffin, et al. (2015) Nat. Mater., 14(7):737-744.). Figures 7C and 7D represent the H&E staining images of the P-NMs treated diabetic mice. On day 7, the open porous structure of the P-NMs is visible (circle in Figure 7C), and by day 14, there was a notable migration of cells throughout the NMs (arrow in Figure 7D). This cell migration contributed to the formation of tissue structures through the P-NMs. The P-NMs treated mice group exhibited significant cell penetration from the surrounding areas and the bottom of the wound bed, showing more cell infiltration. Additionally, an increased number of blood vessels were observed in the samples treated with P-NMs (arrows in Figure 7D). The degree of cell migration and neovascularization during wound closure was quantitatively assessed. Over time, significant increases in cell migration were observed in both NP- NMs and P-NMs treated groups (Figure 7E). Approximately 80% of the wounds treated with P-NMs were closed by day 14, compared to only 35% in those treated with NP- NMs. Moreover, a higher amount of neovascularized sprouts was observed in the P- NMs treated diabetic mice, indicating a significant statistical difference from the NP- NMs group (Figure 7F). Further analysis using Masson's trichrome staining revealed that P-NMs (Figure 7N) showed more cell infiltration after 7 days of treatment compared to NP-NMs (Figure 7M), corroborating the findings from the H&E staining (Figures 7A-7D). Figures 7M and 7N revealed more collagen staining in day 14 tissue sections treated with P-NMs, while NP-NMs showed less ECM secretion. Based on these findings, the open porous microstructure of P-NMs is not only beneficial for diabetic wound care but would also be advantageous for treating a variety of wound types.

[0169] In the realm of DFU treatments, a myriad of methods have been explored to expedite wound healing (Deng, et al. (2022) J. Biomed. Mater. Res., 110(11):2542-2573; Liu, et al. (2022) Biomater. Sci., 10(13):3480-3492). Recent advancements in modular scaffolds have shifted towards minimally invasive treatments, particularly the direct injection of cells or therapeutic agents at the wound site. This approach, avoiding the need for secondary surgical interventions, has been gaining prominence due to its potential to accelerate wound healing (Liu, et al. (2022) Biomater. Sci., 10(13):3480- 3492; Kamaraj, et al. (2023) Trends Biotech., 45:631-647). Among these, hydrogels and microspheres have been extensively employed as injectable systems for tissue regeneration (John, et al. (2019) Nanomed., 22: 102081; Griffin, et al. (2015) Nat. Mater., 14(7): 737-744; Zhang, et al. (2022) ACS Appl. Mater. Interf. , 14(23):26404-26417; Zhao, et al. (2017) Biomaterials 122:34-47). Despite their advantages, challenges like low cell survival and limited host cell recruitment persist (Ahmadian, et al. (2021) J. Biomater. Appl., 36(1): 179-190; Tang, et al. (2023) ACS omega 8(11): 10030-10039). Researchers have attempted to overcome these by incorporating biochemical cues (growth factors, peptides, and drugs) into scaffolds (He, et al. (2022) Bioactive Mater., 10:460-473; John, et al. (2023) Adv. Funct. Mater., 33(l):2206936; Qian, et al. (2020) ACS Appl. Mater. Interf., 12(50): 55659-55674). However, issues such as the short halflife of these agents and high manufacturing costs often outweigh the benefits.

[0170] Modular hydrogel scaffolds, particularly microgels, have recently been recognized for their potential advantage in wound repair (Griffin, et al. (2015) Nat. Mater., 14(7):737-744; Liu, et al. (2023) Adv. Mater., 2023:2304049). These scaffolds offer spaces between particles for cell infiltration (Liu, et al. (2023) Adv. Healthc. Mater., 2023:2300823; Griffin, et al. (2015) Nat. Mater., 14(7):737-744; Liu, et al. (2023) Adv. Mater., 2023 :2304049), yet they lack interconnected porosity, limiting cell migration at the microscopic level and hampering 3D tissue reconstruction. Furthermore, microgel fabrication typically relies on microfluidic systems, which limit mass production and necessitate surface functionalization, complicating the process (Griffin, et al. (2015) Nat. Mater., 14(7):737-744; Wang, et al. (2022) Supramol ecul ar Mater., 1 : 100006). To address these limitations, porous NMs were developed using versatile electrospinning and electrospraying techniques. The substantial advantage of this technique has considerable potential for scalability over other approaches. The fabricated NMs showcase numerous beneficial properties, including a nanofibrous architecture, injectability, cell protection from shear stress, and an interconnected porous network facilitating cell migration. The use of PLGA:gelatin material in the NMs ensures safe application and promotes relatively quick degradation, facilitating host tissue reintegration for effective regeneration. In vitro experiments with HDFs confirmed the cytocompatibility of the NMs, showing no adverse effects on HDF adhesion and proliferation (Figures 3 A and 3B). Actin staining revealed that cells migrated into the micropores of the open P-NMs, providing a larger surface area for cell adhesion and proliferation (Figure 3C). This contrasts with MAP hydrogels, which restrict cell adhesion to macropores between adjacent microgels (Griffin, et al. (2015) Nat. Mater., 14(7):737-744; Cui, et al. (2022) Adv. Sci., 9(22):2201254). Moreover, nanoindentation studies indicated that P-NMs loaded with cells on day 7 displayed enhanced mechanical strength compared to NP-NMs (Figure 4C), indicating that larger cell adhesion spaces lead to increased ECM accumulation, reinforcing the NMs structure.

[0171] The use of these NMs into clinical applications would be a significant advancement in diabetic wound care. One challenge in this process is addressing the discrepancy between in vitro static cell culture experiments and the dynamic conditions experienced during therapeutic application, especially under the stresses of an injectable system. To bridge this gap, an in vitro dynamic injection system was used, providing insights into how cells behave under shear stress. The findings revealed that the NMs, particularly P-NMs, maintained their structural integrity without deformation during injection (Figure 5 J). This demonstrates their stability under load, a crucial factor for their effective in vivo application. P-NMs played a critical role in facilitating cell migration (Figure 3C) and in protecting against injection -induced pressure (Figure 5). The pressure exerted from the syringe wall and between microspheres significantly influenced the viability of HDFs loaded onto the NMs. NP-NMs tended to lose cells that were superficially attached, leading to reduced subsequent cell growth (Figures 5E and 5G). Injection conditions can affect cell viability and differentiation in stem cells loaded onto carriers (Wei, et al. (2018) Adv. Mater., 30(31): 1802273). The open P-NMs, in contrast, offered protection to the cells residing within, with dual or multiple injection modes resulting in higher cell loss (Figures 5G and 5H). Recently FDA approved cell therapy for type 1 diabetes called Lantidra™, which is the first allogeneic pancreatic islet cellular therapy by infusion method, made from deceased donor pancreatic cells (Harrison, C. (2023) Nat. Biotech., 41 : 1035-1046; Parums, et al. (2023) Med. Sci. Monitor 29:e941918-941911). Inspired by this advancement, NMs can serve as cellular carriers for such a treatment.

[0172] Wound healing is a complex process involving growth factors, cytokines, and cells through various stages: hemostasis, inflammation, proliferation, and remodeling (Qian, et al. (2020) ACS Appl. Mater. Interf. , 12(50): 55659-55674). The success of bioengineered scaffolds in this context depends on their integration at the defect site. The initial in vitro scratch experiment indicated that the byproducts of NMs did not adversely affect cell behavior (Figure 6D). Further in vitro studies showed that cells migrated towards implanted NMs, with P-NMs significantly enhancing cell migration into the microspores and aiding in forming vascular networks (Figure 6C). This ability to improve HDF migration into the defect location in the in vitro wound model is likely to expedite the healing process in vivo, as presented in Figure 8. The in vivo experiments corroborated these findings. NP-NMs exhibited low cell migration due to limited growth space and insufficient metabolic exchange (Figures 7A and 7B), leading to lower collagen accumulation around the implanted NP-NMs. Similarly, MAP exhibited cellular infiltration and neovascularization around the microgel space, inability to allow cellular infiltration inside microgels (Griffin, et al. (2015) Nat. Mater., 14(7):737-744; Cui, et al. (2022) Adv. Sci., 9(22):2201254; Peng, et al. (2023) ACS Appl. Mater. Interf., 15(15): 19560-19573). Conversely, P-NMs, with their interconnected micropores, provided ample growth space, enhanced nutrient and oxygen supply, and improved cell survival, thereby boosting collagen production. These characteristics of P-NMs facilitated cell adhesion and significantly influenced neovascularization, ECM formation, and re-epithelialization, culminating in the complete closure of diabetic wounds (Figure 8).

[0173] In conclusion, the work has successfully showcased the dual functionality of P- NMs as injectable granular scaffolds and 3D interconnected porous on the scaffolds for efficient wound tissue regeneration. P-NMs have been proven to promote high cell adhesion and proliferation in vitro. Unlike conventional hydrogels or microparticle systems, P-NMs also safeguard adhered cells from injection -induced damage and serve as an effective vehicle for therapeutic delivery. Their in vivo application led to increased cell penetration, migration, and survival within the interconnected micropores of P-NMs, facilitating collagen production and neovascularization, thereby accelerating the healing of diabetic wounds. This innovative approach can be used in chronic wound care and treating a broad spectrum of diseases, with the added capability of loading peptides onto NMs for directed cell maturation. For example, the NMs can be conjugated to biomimetic peptides for vascularization, anti-microbial, and MMP cleavable to enhance wound tissue repair in diabetic wound microenvironment by mimicking diabetic wound model in vivo.

[0174] EXAMPLE 2

[0175] Different gelation components like natural and synthetic polymers can be incorporated into nonporous and porous hybrid composites with the porous and non- porous microspheres for tissue repair and regeneration. Fig. 9A shows methods of making a hybrid composite via different gelation techniques such as UV-crosslinking and in situ gelation via different macromolecular chemistry (e.g., Michel addition, click chemistry, guest host chemistry, and thiol chemistry). For example, Fig. 9B shows methacrylated gelatin (GelMA) composed porous microsphere composite with various concentration of GelMA ranging from 0.1% to 0.5%. To increase the mechanical properties of the composite, the GelMA content and crosslinking density can be increased.

[0176] While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. A composite comprising nanofibrous microspheres and a hydrogel.

2. The composite of claim 1, wherein said nanofibrous microspheres are porous nanofibrous microspheres.

3. The composite of claim 1, wherein said nanofibrous microspheres comprises electrospun nanofiber segments.

4. The composite of claim 3, wherein said electrospun nanofiber segments are crosslinked.

5. The composite of claim 3, wherein said electrospun nanofiber segments comprise a polymer and a hydrogel.

6. The composite of claim 5, wherein said polymer comprises poly(lactic-co- glycolic acid) (PLGA), polycaprolactone (PCL), polydioxanone (PDO), or poly(lactide- co-caprolactone) (PLCL).

7. The composite of claim 5, wherein said polymer comprises poly(lactic-co- glycolic acid) (PLGA).

8. The composite of claim 5, wherein the hydrogel is gelatin or gelatin methacryloyl (GelMA).

9. The composite of claim 1, wherein the hydrogel is selected from the group consisting of gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA), collagen, and combinations thereof.

10. The composite of claim 1, wherein the hydrogel is present between 0.1% and 1%.

11. The composite of claim 1, wherein said composite and / or nanofibrous microspheres are mineralized.

12. The composite of claim 1, wherein said composite and / or nanofibrous microspheres comprises cells.

13. The composite of claim 1, wherein said composite and / or nanofibrous microspheres comprise an agent is selected from the group consisting of a therapeutic agent, an analgesic a growth factor, a growth factor mimicking peptide, a signaling molecule, a cytokine, a hemostatic agent, an antimicrobial, and an antibiotic.

14. A composition comprising the composite of any one of claims 1-13 and a pharmaceutically acceptable carrier.

15. A method for treating and / or preventing a disease or disorder in a subject in need thereof, said method comprising administering to said subject the composite of any one of claims 1-13.

16. The method of claim 15, wherein the disease or disorder includes but is selected from the group consisting of wounds, ulcers, infections, hemorrhage, tissue injury, tissue defects, tissue damage, bone fractures, bone degeneration, cancer, neurologic diseases, ischemic diseases, inflammatory diseases and disorders, heart disease, myocardial infarction, and stroke.

17. The method of claim 15, wherein the disorder is a diabetic foot ulcer.

18. The method of claim 15, wherein the administration is injection into the area in need of treatment.

19. The method of claim 15, wherein said disease or disorder is bone loss.

20. The method of claim 15, where the composite is crosslinked after administration to the subject.

21. A method for synthesizing the composite of claim 1, comprising i) cutting electrospun nanofibers into electrospun nanofiber segments, ii) dispersing the electrospun nanofiber segments in a solution,iii) electrospraying the solution comprising the electrospun nanofiber segments with air or gas bubbles to generate porous nanofibrous microspheres, and iv) mixing the porous nanofibrous microspheres with a hydrogel, thereby synthesizing said composite.

22. The method of claim 21, further comprising crosslinking the composite generated after step iv).

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