Hydrogel microparticles, compositions and uses thereof

Abstract

The invention relates to hydrogel microparticles, compositions and uses thereof. The present invention provides spherical or elliptical non-alginate hydrogel microparticles for local delivery and sustained release of therapeutic cells and / or tissues at an implantation site, the microparticles comprising a 3-dimensional substrate of a covalently crosslinked non-alginate polymer compound and a therapeutically effective amount of cells and / or tissues embedded therein, wherein the cells have a viability of at least 50%, the microparticles have a size of greater than 30 [mu] m, the non-alginate hydrogel microparticles are formed without using an oil emulsion, the polymer compound is polyethylene glycol vinyl sulfone or acrylated hyaluronic acid, the matrix further includes a cross-linking agent selected from the group consisting of dithiothreitol, polyethylene glycol dithiol, and ethylene glycol bis-mercaptoacetate, cross-linked with the non-alginate polymer compound. Compositions comprising such hydrogel microparticles and their use for the preparation of medicaments are also described.

Description

[0001] This application is a divisional application of the invention patent application filed on June 5, 2020, entitled "Tuned Degradation in Hydrogel Microparticles" with application number 2020800418980 (international application number PCT / US2020 / 036361).

[0002] Cross-reference to related applications

[0003] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 62 / 858,578, filed June 7, 2019, entitled “Tunable Degradation in Hydrogel Microparticles,” which is incorporated herein by reference in its entirety. Invention Field

[0004] This invention relates to hydrogel-based microparticles for encapsulating biological products, and methods for modulating the physical and chemical characteristics of the hydrogel matrix, for example, to increase or decrease the degradation profile of the microparticles. Background Technology

[0005] Cellular microencapsulation is a rapidly expanding field with broad potential applications in treating diseases such as diabetes, cancer, heart disease, and immune disorders, as well as in tissue engineering research and regenerative medicine. Traditional alginate microspheres still suffer from poor biocompatibility, and microencapsulation using more advanced hydrogels is challenging due to their slow gelation rates. What is needed are non-cytotoxic, non-emulsion-based hydrogel microparticles that can be fabricated from a variety of hydrogel materials. Summary of the Invention

[0006] This document describes non-alginate hydrogel microparticles for the local delivery and sustained release of therapeutic cells and / or tissues (including homogeneous or heterogeneous cell clusters) at the implantation site. The microparticles comprise a 3D matrix of a covalently cross-linked non-alginate polymer compound and a therapeutically effective amount of cells and / or tissue embedded therein, wherein the cells have at least 50% viability, and wherein the microparticles have a size greater than about 30 μm. Exemplary polymer compounds are selected from slow-gelling polymer precursors selected from the group consisting of: branched or unbranched hyaluronic acid, branched or unbranched functionalized hyaluronic acid, branched or unbranched functionalized polyethylene glycol, hyaluronan, fibrin, chitosan, collagen, polylactic acid, poly(L-lactic acid), polylactic-co-glycolic acid, polycaprolactone, polyvinyl alcohol, and combinations thereof. The matrix may further comprise a crosslinking agent crosslinked with the non-alginate polymer compound, the crosslinking agent being selected from the group consisting of: dithiothreitol, branched or unbranched functionalized polyethylene glycol, dithiol, ethylene glycol bis-mercaptoacetate, and combinations thereof. Exemplary functionalized polyethylene glycols include, but are not limited to, polyethylene glycol dithiol, polyethylene glycol diacrylate, polyethylene glycol divinyl sulfone, polyethylene glycol dimaleimide, and combinations thereof. The matrix may be homogeneous, comprising a single type of covalently crosslinked polymer backbone (with or without additional crosslinking agents of different polymer types). The matrix may also be heterogeneous, comprising a mixture of two or more polymer precursors, such as high ( > 500kDA, preferred > 100kDA) and low (<100kDA, preferred) < A combination of polymer precursors with a molecular weight of 50 kDa, and / or a mixture of two or more crosslinking agents.

[0007] This document also describes compositions comprising multiple such microparticles. Such compositions may also include multiple “empty” microparticles dispersed together with microparticles loaded with cells or tissue. The compositions may include mixtures of different microparticles, such as mixtures of degradable and durable hydrogel microparticles, and / or mixtures of microparticles of different sizes (e.g., multiple larger microparticles and multiple smaller microparticles), and / or mixtures of microparticles each containing different types of cells and / or tissues (e.g., insulin-producing cells in one group of microparticles and supporting stem cells in another group of microparticles). It is also possible to combine or co-administer two different compositions, rather than mixing two different types of particles into a single composition.

[0008] This document also considers various methods for the local delivery and sustained release of therapeutic cells and / or tissues at the implantation site by administering the microparticles and / or compositions containing such microparticles (alone or in combination with other types of therapeutic compounds if desired). Typically, the microparticles or compositions are injected directly into or implanted at or near the treatment site (adjacent to the treatment site).

[0009] This article also describes various methods and techniques for regulating the degradation rate of microparticles by altering various parameters of the microparticles, including formulation and / or processing parameters such as polymer precursor mass fraction or molecular weight, crosslinker molecular weight, crosslinker to polymer precursor ratio, crosslinker hydrolysis, crosslinking kinetics, crosslinking time (e.g., UV exposure time), and combinations thereof.

[0010] Furthermore, the embodiments described herein contemplate the use of multiple empty microparticles implanted in the joint for joint lubrication and / or viscoelastic supplementation. Attached Figure Description

[0011] Figure 1 This is an illustration of an example of covalent crosslinking of a hydrogel precursor compound.

[0012] Figure 2 The diagram illustrates a method for forming microparticles, including (A) droplet formation, (B) incubation in an alginate bath, (C) formation and thickening of the alginate shell, and (D) crosslinking of the hydrogel core followed by removal of the alginate shell to produce hydrogel microparticles.

[0013] Figure 3 Images are shown for A: the alginate shell surrounding the microparticles; and B: the hydrogel microparticles after the alginate shell has been removed.

[0014] Figure 4 Images are shown for: A: PEG-based spherical durable microparticles loaded with pancreatic islets; B: PEG-based elliptical microparticles loaded with pancreatic islets; C: MeHA-based microparticles with flat edges loaded with stem cells; and D: MeHA-based elliptical microparticles loaded with stem cells.

[0015] Figure 5 Results are shown from the following: cytotoxicity studies and A: the effects of calcium concentration and exposure time on canine islet viability, and B: the effects of photoinitiator concentration and UV light exposure time on cell viability.

[0016] Figure 6 It is a graphical representation of the diameters and good monodispersity of 100 representative particles composed of ThHA, MeHA, PEGDA, or AHA.

[0017] Figure 7 Images show fluorescently labeled dextran diffusing into PEGDA, ThHA, and MeHA hydrogel microparticles.

[0018] Figure 8A It is a degradation map of different particles based on the remaining particulate polymer mass over time.

[0019] Figure 8B This is a graph showing how the Q value (swelling ratio) of different particles changes over time.

[0020] Figure 8C This is a degradation diagram of different particles based on particle mass loss over time.

[0021] Figure 9 This is a graph showing the average daily blood glucose levels in each group of mice during the initial study period after transplantation.

[0022] Figure 10 Images of microparticles recovered from mouse IP space are shown: A: PEDGA, B: MeHA, C: PEDGA, where viability is indicated as by staining with deep red dithizone, and D: PEDGA, where viability is indicated as by co-staining with deep red dithizone and green calcein.

[0023] Figure 11 Images of healthy fat cells are shown, indicating the absence of fibrotic tissue after implantation of PEGDA microparticles.

[0024] Figure 12 This is a graph of average non-fasting blood glucose levels obtained from measurements taken in dogs each morning and afternoon.

[0025] Figure 13 The following images are shown: A: HEK293 cells aggregated in durable PEGDA microparticles; B: Cell morphology or number from A in PEGDA microparticles after 34 days with little change; C: HEK293 cells dispersed in degradable MeHA microparticles; and D: Proliferation of cells from C in MeHA microparticles after 34 days.

[0026] Figure 14 Images from microplate wells are shown, in which collected used culture medium shows A: very few HEK293 cells diffusing from PEGDA microparticles, and B: a large number of live cells diffusing from MeHA microparticles.

[0027] Figure 15 This is a graph summarizing average data from studies on cumulative cell release.

[0028] Figure 16The following images are shown: A: MSCs encapsulated in PEGDA microparticles, B: MSCs encapsulated in MeHA microparticles, C: islets encapsulated in PEGDA microparticles, D: islets encapsulated in MeHA microparticles, and E: MSCs encapsulated in AHA microparticles.

[0029] Figure 17 This is a cell viability map of a rat insulinoma cell line encapsulated in PEGDA and AHA microparticles.

[0030] Figure 18 Images of islets encapsulated in PEGDA or MeHA are shown, and stained with dithizone to demonstrate that they are functional islets that produce insulin.

[0031] Figure 19 The following images are shown: A: PEDGA microparticles infused into and around the omentum of the abdomen; B: microparticles attached to the tissue of the first dog; and C: microparticles attached to the tissue of the second dog, demonstrating the "stickiness" of the particles.

[0032] Figure 20 The following images are shown: A: Blue-stained PEGDA microparticles in the rat knee joint, visible through the muscle; B: Dissected knee joint, showing blue-stained PEGDA microparticles in the knee bursa area; C: Further dissected knee joint, showing blue-stained PEGDA microparticles remaining at the site of the initial implantation.

[0033] Figure 21 Images of explant tissues stained with hematoxylin and eosin from three microgroups are shown.

[0034] Figure 22 This is a graph showing the response of particles in the retina to (no) foreign matter.

[0035] Figure 23 This is a graph showing the effect of the mass fraction of the core (main chain) polymer material used in the hydrogel precursor solution on the Q value.

[0036] Figure 24A This is a graph showing the formulation in which increasing the concentration of the crosslinking agent results in a lower Q value.

[0037] Figure 24B This is a graph showing that increasing the concentration of the crosslinking agent results in a higher Q value for the formulation.

[0038] Figure 25A This is a diagram showing the formulation in which increasing pH leads to a higher Q value.

[0039] Figure 25B This is a diagram showing a formulation in which increasing pH results in a lower Q value.

[0040] Figure 26A This is a diagram showing formulations in which increasing the molecular weight of the crosslinking agent results in a higher Q value.

[0041] Figure 26B This is a graph showing formulations in which increasing the molecular weight of the crosslinking agent does not cause a change in the Q value.

[0042] Figure 27A This is a graph showing the effect of changing the molecular weight of a nuclear polymeric substance with a constant number of reaction sites per gram on the Q value.

[0043] Figure 27B This is a graph showing the effect of changing the molecular weight of nuclear polymeric substances with different reaction sites / grams on the Q value.

[0044] Figure 28A This is a graph showing the effect of UV crosslinking time on the Q value (swelling ratio) and diameter of different microparticle formulations.

[0045] Figure 28B This is a graph showing the effect of UV crosslinking time on the Q value of MeHA particles. Detailed Implementation

[0046] This invention broadly relates to hydrogel microparticles for the local delivery and sustained release of therapeutic cells and tissues. The hydrogel microparticles protect implanted cells from initial degradation and flushing (biodegradation) under physiological conditions and control the kinetics of cell (and its therapeutic proteins and molecules) release to local or systemic areas over time periods ranging from hours to weeks or months. Hydrogel microparticles designed for degradation ultimately degrade in vivo through normal physiological processes over time periods of days or weeks, and typically within less than 3 months. This means that the entire hydrogel has been degraded and flushed away under normal physiological conditions within this timeframe, leaving virtually no hydrogel residue in the implantation area (it should be understood that trace amounts of gel or polymer may still remain in the area, but are generally no longer sufficient to act as an encapsulating agent for the implanted cells). It should also be understood that in some indications, particularly in cases of implantation at joints, the microparticles also undergo mechanical degradation through movement and impact at the joint site.

[0047] The hydrogel microparticles comprise a hydrogel matrix in which a therapeutically effective quantity of cells is encapsulated. The cells can be eukaryotic or prokaryotic. Examples of prokaryotic cells include bacteria and archaea. Eukaryotic cells include animal cells, plant cells, and fungi. The cells can be non-proliferative or proliferative. Before implantation and / or during the implantation period, or after the proliferative cells are released from the degrading hydrogel, the proliferative cells will further proliferate (increase) from the initial number of cells encapsulated within the hydrogel. Non-proliferative cells have limited in vitro expansion capacity and generally do not increase from the initial cell number. However, due to the favorable conditions within the hydrogel microparticles, very little cell death occurs in the encapsulated cells, and the initial number of non-proliferative cells encapsulated by the hydrogel is not significantly reduced. In one or more embodiments, the initial viability of the cells encapsulated in the microparticles is maintained at at least 50%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, and even more preferably at least about 95% viability after encapsulation, compared to the viability of the initial cell population included in the precursor solution. In other words, less than 50% of the cell population is lost during encapsulation, preferably less than 30%, more preferably less than 20%, even more preferably less than 90%, and even more preferably less than 5% of the cell population is lost during the encapsulation process. This is because the encapsulation process is mild and the cell culture medium and nutrients can diffuse into the microparticles during storage. In other words, in one or more embodiments, the microparticles have a low diffusion barrier.

[0048] In one or more embodiments, the cell population included in the hydrogel precursor solution will comprise approximately 1 million to approximately 500 million cells / mL of the hydrogel precursor solution, preferably approximately 4 million to approximately 300 million cells / mL, and in some embodiments, approximately 5 million to approximately 150 million cells / mL. In one or more embodiments, the hydrogel precursor solution will comprise approximately 15% by volume of the cells, cell clusters, or tissue to be encapsulated / mL of the hydrogel precursor solution.

[0049] Examples of proliferating cells include stem cells and activated T cells, while examples of non-proliferating cells include pancreatic islets and differentiated neurons. In either case, cells (and their associated cell products) are slowly released from the hydrogel into the implanted local area. Exemplary cell products that may be secreted with the cells include signaling molecules, therapeutic proteins, vesicles, antibodies, viruses, extracellular bodies, etc. These protein-based components may be in their natural form or may be genetically modified. Similarly, cell product components may be natural or genetically modified. Compared to other carriers or unencapsulated cells, hydrogel microparticles allow cells to remain in the implanted local area for a longer period of time and provide that area with a temporary but sustained release of cells and / or their signaling molecules, proteins, etc., to enhance the therapeutic efficacy of the implanted cells. It should be understood that the cell load will depend on the size of the cells used and the size of each microparticle. In one or more embodiments, up to 50% of the volume of the microparticle is composed of encapsulated cells, cell clusters, and / or tissue, preferably, the cell, cluster, and / or tissue load ranges from about 10% to about 20% of the microbody volume. Typically, about 50 to about 1x10 5 Each cell can be encapsulated in each 1-mm particle, preferably about 100 to about 1x10. 5 Each particle will be encapsulated within a 1-mm particle.

[0050] In one or more embodiments, the microparticles are formed from relatively weak hydrogels that eventually decompose in vivo within approximately 3 months (90 days) from the date of implantation and are referred to herein as “degradable” hydrogels (it should be understood that most hydrogels eventually degrade). As used herein, “degradable” hydrogel microparticles are defined as “continuously releasing” but “rapidly degrading” or “short-term” hydrogels that will maintain their self-sustaining microsomes in PBS for at least 24 hours and up to 3 months upon implantation in vivo. Such degradable hydrogel microparticles will also decompose and degrade within approximately 6 months under in vitro storage conditions (PBS and 37°C). In other words, the hydrogel matrix has “in vitro storage stability” of less than 6 months, meaning that it begins to hydrolyze and detach from its self-sustaining body within 6 months or less when stored in PBS at 37°C. In one or more embodiments, the degradable hydrogel microparticles are characterized by having a Q value greater than 150, preferably greater than 200. However, as illustrated below, further modification of the crosslinking chemicals (e.g., using crosslinking agents that are hydrolytically unstable) can produce microparticles with low Q values ​​(strong initial matrix), but which degrade rather rapidly and are still characterized as degradable hydrogel microparticles.

[0051] These biodegradable microparticles contrast with alternative embodiments involving durable hydrogel microparticles. Such durable hydrogel microparticles are also continuously released, but for longer-term applications, they remain stable for at least 6 months under storage conditions in PBS at 37°C. In other words, the durable microparticles have “in vitro storage stability” of more than 6 months. In one or more embodiments, the durable hydrogel particles are stable in PBS at room temperature (27°C) for one year or longer. Preferably, when such durable hydrogel microparticles are implanted, they remain stable for at least 3 months, and more preferably at least 6 months, under normal physiological conditions (also known as normal in vivo foreign body clearance through phagocytosis, degradation, adsorption, etc.).

[0052] In any case, hydrogel microparticles may also be characterized as microspheres, microbeads, or hydrogel microparticles. They exist as individual self-supporting entities comprising a 3D hydrogel matrix and cells suspended, embedded, or encapsulated within the hydrogel matrix. This article also describes sustained-release compositions for implantation in subjects. Sustained-release compositions comprise multiple 3D hydrogel microparticles suspended in a pharmaceutically acceptable delivery carrier.

[0053] Suitable hydrogel precursor compounds for use in forming microparticles include polymers, oligomers, and / or monomers that form hydrogels and are thus capable of forming cross-linked or network structures or matrices (i.e., “hydrogels”) through cross-linking, wherein liquids and cells can be retained, suspended, embedded, and / or encapsulated in the interstitial spaces or pores of the resulting gelled structure or matrix. The hydrogel precursor compounds used in this invention are preferably non-alginate hydrogel precursor compounds. That is, the hydrogel precursor solution is preferably substantially free of alginate compounds (i.e., compounds based on alginate, alginic acid, or their salts or derivatives). As used herein, the term “substantially free” means that the component was not intentionally added to the composition, although incidental impurities may be present, or residues / trace amounts may be left from the manufacturing process. In such embodiments, the hydrogel precursor solution composition comprises less than about 0.05% by weight, preferably less than about 0.01%, and more preferably about 0% by weight of such component, based on a total weight of the solution being 100% by weight.

[0054] Hydrogel microparticles can advantageously be manufactured from a variety of slow-gelling polymer precursors, such as hyaluronic acid (HA) and polyethylene glycol diacrylate (PEGDA), polyethylene glycol maleimide (PEGMAL), multi-arm PEG, hyaluronic acid polysaccharides, fibrin, chitosan, collagen, heparin, polylactic acid (PLA), poly(L-lactic acid) (PLLA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), methacrylic acid (MAA), 2-hydroxyethyl methacrylate (HEMA), polyacrylamide (PAM), extracellular matrix and their functionalized forms (e.g., acrylated, methacrylated, thiolized, etc.) or their multi-arm forms (e.g., 4-arm PEG, 8-arm PEG). However, any crosslinkable hydrogel precursor compound will be suitable for use with the present invention, wherein preferred compounds are biocompatible non-alginate homopolymers or copolymers, and particularly non-alginate block copolymers, as well as other types of crosslinkable monomers and / or oligomers. Typically, the amount of polymer precursor included in the precursor solution will be less than 50% w / w. For high molecular weight ( > 500kDa, preferred > For polymers with molecular weights of 100 kDa and multiple substitutions, such as hyaluronic acid, the amount will range from about 1% to about 50% w / w, and for lower molecular weight polymers (<100 kDa, preferably...), the amount will be ... < 50kDa), such as PEGDA or PEGMAL, is about 5% to about 50% w / w.

[0055] The crosslinking profile of the microparticles can be tuned by adjusting the molecular weight of the precursor compound, as well as the chosen crosslinking agent, crosslinking conditions, and crosslinking process. For example, in some embodiments, “tighter” or faster crosslinking may be desired to achieve a smoother bead surface and / or a stronger gel by limiting the ability of molecules to leak from the droplet / core into the surrounding environment during crosslinking. This can be achieved by both increasing the molecular weight of the precursor material (e.g., >40 kDa) and / or by reducing the crosslinking time (by adjusting the crosslinking chemicals or initiation method). However, increasing the molecular weight of a bifunctional precursor material (i.e., PEGDA) generally results in more diffuse gel due to the reduced crosslinking density. Furthermore, hydrolytically unstable crosslinking agents can be used to generate initially strong microparticles with low initial Q values ​​to facilitate cell implantation at the site. However, such crosslinking agents will rapidly degrade under normal physiological conditions, thereby rapidly releasing their payload to the implantation site. Crosslinking can occur through various mechanisms, depending on the specific hydrogel precursor compound. In one or more embodiments, the core / shell microparticles are combined with a hydrogel matrix crosslinking agent, preferably in solution. A crosslinking agent is leached into the core / shell microparticles through the alginate shell, causing the hydrogel precursor compound to gel (crosslink) to form a 3D hydrogel matrix. The crosslinking agent will correspond to the hydrogel precursor compound but can be varied to control the rate and level of crosslinking achieved within the resulting crosslinked matrix. Typically, depending on the selected crosslinking agent and polymer system, the crosslinking dosage used to form the microparticles will range from about 0.5 mM to about 30 mM, preferably from about 1 mM to about 20 mM, more preferably from about 2 mM to about 15 mM, even more preferably from about 2.5 mM to about 10 mM, and even more preferably from about 2.5 mM to about 5 mM.

[0056] Suitable crosslinking agents include photo- or thermally initiated crosslinking agents and chemical crosslinking agents such as acrylates, methacrylates, acrylamide, vinyl sulfone, dithiols, etc., which will be included as part of the alginate bath. Self-crosslinking hydrogel precursors can also be used. In these embodiments, photoinitiators such as 2959 (2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone) or phenyl-2,4,6-trimethylbenzoyl-lithium phosphine (LAP) can be included as a catalyst in the hydrogel precursor solution. Photoinitiators can also be included in the alginate bath. For chemically crosslinked systems, the crosslinking agent is typically provided in the alginate bath. For UV-initiated crosslinked systems, the crosslinking agent can be included together with the main polymer (backbone) in the alginate bath or hydrogel precursor solution.

[0057] Table 1 provides examples of reactive groups from different main-chain chemicals that can be used to form hydrogels with appropriate crosslinking agents. Some of the reactions are initiated by UV light, while others are chemical reactions.

[0058] Table 1. Reactive Groups and Crosslinking Chemistry

[0059]

[0060]

[0061] Additional exemplary precursor compounds include, but are not limited to, non-alginate polysaccharides, collagen / gelatin, chitosan, agarose, etc. These precursor compounds can be branched polymers with multiple arms or unbranched / linear polymer chains with a single backbone. They can be further functionalized by attaching proteins, drugs, or enzymes to the hydrogel to achieve control over cell attachment, function, proliferation, or viability. Particularly preferred hydrogel precursor compounds are hyaluronic acid, or hyaluronic acid / PEG mixtures. One or more precursor compounds can also be functionalized with various chemical entities added to the precursor compounds during hydrogel fabrication. These chemicals will bind to the hydrogel matrix, rather than being encapsulated in interstitial spaces, to allow for simple diffusion of the microparticles. Examples would include the addition of cell support agents that can be functionalized, such that they react with crosslinking agents or the hydrogel backbone. In rapidly degrading microparticles, these agents will be locally released along with the cells and cell products encapsulated therein.

[0062] Similarly, preferred hydrogels are slow-gelling hydrogels, which, as used herein, mean hydrogels having a gelation rate insufficient to form a generally smooth, generally spherical construct upon contact of the gel precursor droplet with a solution containing a crosslinking agent. Slow-gelling hydrogels are generally considered to be those that do not form gel droplets immediately (or nearly immediately, e.g., within about 5-10 seconds) upon exposure to a crosslinking agent. Biocompatible hydrogels are also particularly preferred, depending on the intended end use of the hydrogel. As used herein, “biocompatible” means that it is harmless to living tissue or cells encapsulated within a matrix, and more particularly, that it is not biologically or otherwise undesirable in that it can be administered to a subject without excessive toxicity, irritation, or allergic or immunogenic reactions, and without causing any undesirable biological effects or interacting in a harmful manner with any other component of the composition comprising it. As will be well known to those skilled in the art, biocompatible hydrogels will be selected to minimize any adverse side effects in the subject. Additional optional components that may be included with the hydrogel precursor include fibronectin, laminin, collagen, other components of the extracellular matrix, including their synthetic forms.

[0063] Typically, techniques for preparing microparticles are described in detail in U.S. Patent No. 9,642,814, filed June 3, 2015, which is incorporated herein by reference. The method includes preparing a hydrogel precursor solution comprising a hydrogel precursor compound dispersed or dissolved in a solvent system, cells to be encapsulated, and divalent cations (e.g., calcium, barium, strontium, and combinations thereof). The divalent cations are dispersed or dissolved together with the hydrogel precursor compound in the solvent system. The divalent cations should be included in the solution at a level of about 0.025 mol / L to about 0.25 mol / L, based on a total volume of 100% of the solution.

[0064] The hydrogel precursor solution may also include optional hydrogel crosslinking agents, catalysts, additives, culture media, nutrients, pH buffers, density adjusters, viscosity adjusters, etc. The hydrogel precursor solution is then combined with alginate to initiate the gelation of alginate around the hydrogel precursor solution (through an "inside-out" gelation process) to produce core / shell microparticles. Each core / shell microparticle comprises an alginate shell surrounding a liquid core, which includes the hydrogel precursor solution. Figure 2 As illustrated, this typically involves (A) adding a precursor solution dropwise to (B) an alginate bath, such as by generating / extruding droplets of the precursor solution into or spraying it into the alginate bath. The amount of alginate in the solution can vary, but can range from about 0.1% to about 2.0% by weight / volume, based on taking the total volume of the solution as 100%. Generally, the viscosity of the alginate solution should be less than the viscosity of the hydrogel precursor solution. The viscosity of the alginate solution depends on the alginate concentration and the average molecular weight of the alginate polymer (i.e., the length of the alginate molecule or the number of monomer units in the chain); at similar concentrations, longer chains result in higher viscosity. In one or more embodiments, the viscosity of the alginate solution will range from about 1 to about 20 cP at room temperature (~20 to 25°C), and preferably from about 1 to about 4 cP. More particularly, at room temperature, the ratio of the viscosity of the hydrogel precursor solution to the viscosity of the alginate solution should be greater than 1. In another embodiment, the ratio of the viscosity of the hydrogel precursor solution to the viscosity of the alginate solution is from about 1:1 to about 1000:1. In one or more embodiments, the viscosity ratio of the hydrogel precursor is about 20:1. In one or more embodiments, the viscosity of the hydrogel precursor solution is about 1 to about 500 cP, of which about 40 to about 100 cP at room temperature is particularly preferred. The pH range of the alginate bath should be about 6.2 to about 7.8, and preferably about 6.6 to about 7.4.

[0065] like Figure 2As illustrated in (B) and (C), the alginate shell forms from the inside out and thickens around the droplet as cations leach from the precursor solution droplet. In other words, the presence of cations in the droplet causes the alginate in the bath to aggregate to the surface and crosslink around the droplet. Next, gelation of the hydrogel precursor compound in the liquid core is initiated, such as by crosslinking and / or polymerization to produce core / shell crosslinked microparticles. Each core / shell crosslinked microparticle comprises an alginate shell and a gel core, the gel core comprising a crosslinked 3D hydrogel matrix and embedded cells. Crosslinking can be chemically induced, thermally induced, or photoinitiated, depending on the specific precursor solution prepared. For example, a hydrogel crosslinking agent can be included in the alginate bath and diffused through the alginate shell to crosslink the hydrogel microparticles in the core. Alternatively, the core / shell microparticles can be subjected to a UV radiation source to initiate crosslinking in the hydrogel microparticle core. The UV exposure time ranges from about 1 min to about 10 min, preferably from about 2 min to about 8 min, and even more preferably from about 2.5 min to about 5 min. The wavelength of UV exposure will depend on the photoinitiator and / or polymer system selected, but will typically range from about 100 nm to about 400 nm, preferably from about 315 nm to about 400 nm, and more preferably from about 365 nm.

[0066] Unlike the ionic bonds of alginate-type microparticles, the microparticles of this invention involve covalent cross-linking of hydrogel precursor compounds, such as... Figure 1 The illustration shows an example of PEGDA with diacrylate chemicals at the ends of the PEG backbone (black lines), where reactive sites are indicated by double red lines. PEGDA molecules crosslink with each other to produce the final hydrogel formulation. In contrast, the MeHA backbone (blue lines) with reactive groups shown as dispersed on the chain (red double lines) is covalently crosslinked with PEGDA. Figure 1 Therefore, it should be understood that the precursor compounds in the final product are chemically altered through a crosslinking reaction to produce a crosslinked hydrogel matrix. For example, in one or more embodiments, the hydrogel matrix consists essentially (or primarily (event) of) a crosslinked network of PEG and HA polymers containing thioether and ester crosslinks (e.g., for thiolized HA). However, it should be understood that the matrix may include trace amounts of unreacted precursor compounds, functional groups, etc., remaining in the network. Table 1 provides other examples of possible hydrogel matrices and crosslinks. This list provides examples but is not exhaustive. Examples of main-chain molecules that can be used with the chemicals listed in Table 1 include chitosan, agarose, chondroitin sulfate, or combinations thereof. Bonds formed between the main chains include chemical bonds with thioether esterification, thioether esterification, and methyl, thioether sulfone, or thioether succinimide on the β-carbon. However, other chemical bonds may be used. Photo (radical) bonds may include ester-etherification or amide-etherification. It should be understood that increasingly complex combinations exist that can be utilized, making the possible combinations virtually infinite.

[0067] Once the nucleus has been gelled, the alginate shell is then removed (e.g., with chelating agents and / or mechanical agitation, such as sonication) to produce self-sustaining hydrogel microparticles or beads and the cells embedded within them, such as... Figure 2 As illustrated in (D) of the diagram. In other words, the alginate shell is not part of the final product and is always removed before the microparticles are used. The resulting hydrogel microparticles can be collected from the solution using a mesh screen or other device and can be rinsed or suspended in a culture medium or suitable nutrients as needed. Similarly, the resulting biodegradable hydrogel microparticles are preferably substantially free of alginate.

[0068] This technology can be used to embed / encapsulate cells, cell clusters, tissues, combinations thereof, and fragments thereof in hydrogel microparticles. Cells can be primary cells of natural origin or freshly isolated, cultured or expanded cells, established cell lines, differentiated or undifferentiated, engineered or genetically modified, etc. Non-limiting examples of cells and tissues include pancreatic islets, islet clusters, hepatocytes, stem cells and related cells and tissues, as well as endocrine cells, stem cell clusters, thyroid clusters, adrenal clusters, pituitary clusters, and other 3D cell clusters for tissue engineering or cell-based therapies. Combinations of cell types and / or tissues (e.g., primary cell types combined with stem cells) can also be used in the microparticles. Depending on the cell type, the microparticles may also include a hydrogel matrix component that supports cell maintenance and growth. Exemplary culture medium components include one or more of the following: serum, nicotinamide, antibiotics (penicillin, amphotericin B, streptomycin, gentamicin sulfate), amino acids (alanine, arginine, aspartic acid, glutamine, etc.), pH buffer (sodium bicarbonate), inorganic salts (sodium, potassium, magnesium and calcium ion sources), carbon sources (glucose, galactose, fructose, maltose, sodium pyruvate), proteins and peptides (albumin, transferrin, fibronectin, activin, insulin), fatty acids and lipids, vitamins (A, D, E, K, B, nicotinamide), minerals and trace elements (zinc, copper, selenium), hormones, growth factors, etc. Complete culture media, basal culture media, and culture medium components are commercially available, including but not limited to Eagle's Minimum Essential Medium (EMEM), Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640, Ham's Nutrient Mixtures, Iscove's Modified Dulbecco's Medium (IMDM), CMRL-1066, etc.

[0069] The resulting hydrogel matrix is ​​characterized as a semi-rigid network permeable to liquids and gases, but it is non-flowing and maintains its integrity in a steady state. Hydrogels can be considered solids or described as having near-infinite viscosity. The hydrogel matrix is ​​a 3D self-sustaining body. The term "self-sustaining body" means that once formed, the hydrogel matrix retains its shape without external support structures and is not easily deformed solely by its own internal forces or weight. Unlike jelly, putty, or paste, which are flexible, permanently deformable, or flowable, self-sustaining bodies are elastic, allowing the matrix to temporarily yield or deform under force. In other words, a self-sustaining body will retract or spring back to its original shape after slight compression and / or bending—it should be understood that a hydrogel matrix will crack, break, or shear under sufficient external pressure or force (and will not recover afterwards).

[0070] Hydrogel microparticles are matrix-type capsules that retain the filling material throughout the bead, rather than having a distinct shell like core-shell capsules. As mentioned above, hydrogel microparticles are also self-sustaining. In one or more embodiments, the resulting microparticles are substantially spherical in shape. Advantageously, the particle size is highly customizable depending on the capability of the selected droplet generator. In one or more embodiments, the resulting hydrogel microspheres or microparticles have an average maximum cross-sectional surface area (i.e., in the case of spherical or elliptical microspheres) greater than 30 μm, and in some cases greater than 300 μm. In one or more embodiments, the resulting hydrogel microspheres or microparticles have an average maximum surface area (mean) of less than about 5 mm. Preferably, the resulting hydrogel microspheres or microparticles have an average maximum surface area (mean) of less than about 2 mm, more preferably about 30 μm to about 2 mm, even more preferably ranging from about 50 μm to about 1.5 mm, more preferably about 150 μm to about 1.5 mm, and even more preferably about 300 μm to about 1.4 mm. In some cases, smaller particles with sizes ranging from about 30 μm to about 750 μm or 500 μm can be formed. For ease of reference, this cross-sectional dimension is simply referred to herein as the “size” of the particle. In any case, the hydrogel particles of the present invention are not nanoscale and are not considered to be nanoparticles or any other kind of nanocrystal shape.

[0071] Hydrogel microparticles are particularly suitable for localized therapeutic treatment of acute or chronic conditions, where short-term treatment regimens can be used to alleviate such conditions. Examples include local delivery (e.g., direct injection) of cells, cell clusters, and / or tissues for bone or cartilage repair (e.g., in joints) and transplanted therapeutic cells, cell clusters, and / or tissues to protect them from the host immune system in general. The microparticles protect the encapsulated cells, cell clusters, and / or tissues from the immune system for a short period (e.g., days, weeks, up to three months), releasing their therapeutic benefits until the hydrogel breaks down and is washed away through normal physiological processes. Systemic therapy is also envisioned. In one or more embodiments, the microparticles are not used to deliver small molecule therapeutic agents and preferably do not contain such drugs, unless such drugs are included in the microparticles as cell-supporting activators (e.g., to ensure cell and implant success, such as immunosuppressants), but not to the extent that they are activators or therapeutic agents themselves.

[0072] For treatment methods, microparticles are suspended or dispersed in a suitable delivery carrier for administration to a subject. Exemplary delivery carriers may include a biocompatible liquid suspension, viscous solution, putty, paste, or gel in which microparticles are distributed, and preferably include nutrients and components to support the maintenance and growth of the cells, cell clusters, and / or tissues to be administered. Cell culture media as described above, which may be further supplemented depending on the cell or tissue type, are preferred carriers. Saline solutions or other buffered solutions may also be used as delivery carriers. The method typically includes (or comprises) administering a therapeutically effective amount of the obtained microparticle composition to a patient, such as administering a therapeutically effective amount of the obtained microparticle composition to a site of inflammation, injury, arthritis, degeneration, etc., in the patient. Administration typically involves direct injection of the microparticle composition at or near the site of inflammation, injury, arthritis, degeneration, etc. Advantageously, sustained-release microparticle compositions confine the administered cells, cell clusters, and / or tissues to a local area for a sustained therapeutically effective period, such that slowly released cells, cell clusters, and / or tissues and / or their cellular components or products are released locally to alleviate or reduce the severity of the patient's condition. As used herein, the term "therapeuticly effective" refers to the amount and / or duration of time that will elicit a biological or medical response in an tissue, system, animal, or human that an investigator or clinician is seeking, and particularly to the amount and / or duration of time that will elicit some desired therapeutic effect. For example, in one or more embodiments, a therapeutically effective amount and duration is one that reduces inflammation and induces or promotes healing of sites of inflammation, injury, arthritis, degeneration, etc. Those skilled in the art will recognize that an amount or duration of time can be considered therapeutically effective even if the condition is not completely eradicated but partially improved.

[0073] If necessary, treatment can be repeated via additional injections or infusions. Those skilled in the art will understand that treatment regimens can vary depending on the specific inflammation, injury, arthritis, degeneration, condition, or healing status, as well as the preferences of a medical or veterinary practitioner or researcher. For example, neuronal repair, such as the repair of spinal cord injuries or degenerative diseases, can potentially be treated by injecting microencapsulated stem cells into the epidural space or epidural lesion area. Degradable hydrogel microparticles are designed to immediately release stem cells into the area, allowing the stem cells to interact directly with the environment, or are designed to persist for a longer period (e.g., ~1-3 months) to microencapsulate stem cells to release growth-promoting factors into the area while slowly degrading, releasing cells for destruction and degradation until nothing remains. Similarly, cardiac repair after a heart attack can be improved by locally releasing cells or growth factors from microencapsulated cells, cell clusters, and / or tissue placed in the injured area. Another example includes treating nonfunctional glands with hormone therapy, such as restoring thyroid function by placing microencapsulated thyroid cells, cell clusters, and / or tissue in or near / adjacent areas (if scar tissue exists after removal or reduction of the thyroid gland). Additional examples of durable hydrogel microparticles include local delivery of neurotransmitters associated with neuroendocrine disorders, release of bone-promoting factors near unhealed fractures, systemic release of molecules such as atrial natriuretic peptide to treat congestive heart failure, release of clotting factors in the treatment of hemophilia, or encapsulation of engineered or transgenic cells that produce a variety of bioactive substances.

[0074] In one or more embodiments, “empty” hydrogel microparticles can also be administered as part of joint lubrication therapy. In other words, the presence of the hydrogel itself provides therapeutic relief to the patient without the need for cells or any other active agents within the particles themselves.

[0075] Compared to alginate or other types of microparticles, the compositions and microparticles of the present invention offer a particular advantage for implantation due to their improved "adhesion." That is, the microparticles tend to adhere to or attach to tissues within the body at the implantation site, as illustrated in the photographs of the following examples. This further enhances therapeutic efficacy by maintaining the local release of cells, cell clusters, and / or tissues and their cellular products at the implantation site. Furthermore, the microparticles have demonstrated no induction of any inflammatory response and / or any foreign body reaction at the implantation site, including lymphocyte or collagen ring formation.

[0076] Upon reviewing the disclosure herein and the working examples below, additional advantages of various embodiments of the invention will be apparent to those skilled in the art. It should be understood that, unless otherwise stated herein, the various embodiments described herein are not necessarily mutually exclusive. For example, a feature described or depicted in one embodiment may be included in, but not necessarily included in, other embodiments. Therefore, the invention encompasses various combinations and / or arrangements of the specific embodiments described herein.

[0077] As used herein, the phrase “and / or” when used in a list of two or more items means that any one of the listed items may be used alone or any combination of two or more of the listed items may be used. For example, if a composition is described as containing or excluding components A, B and / or C, the composition may contain or exclude: A only; B only; C only; a combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B and C.

[0078] This specification also uses numerical ranges to quantify certain parameters relevant to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be interpreted as providing literal support for claims limiting only the lower limit of the range and claims limiting only the upper limit of the range. For example, the disclosed numerical range of about 10 to about 100 provides literal support for claims listing "greater than about 10" (no upper limit) and claims listing "less than about 100" (no lower limit).

[0079] Example

[0080] The following embodiments illustrate the method according to the present invention. However, it should be understood that these embodiments are provided by way of illustration and should not be construed as limiting the overall scope of the invention.

[0081] We demonstrate that one or more HA formulations used in the examples degrade within a timeframe of less than 3 months. In contrast, the PEGDA formulation exhibits greater durability. We compare the characteristics of products manufactured in this manner by comparing degradable microparticles to durable microparticles. These comparisons include physical and chemical differences, in vitro and in vivo stability, cell morphology, function, and migration within the final product microparticles, and the ability of the final product microparticles to attach to tissues in vivo, thereby localizing their application within the body.

[0082] Example 1

[0083] Durability comparison of the physical and chemical differences between biodegradable hydrogel microparticle end products

[0084] introduce

[0085] The concept of cell encapsulation was first popularized by Lim and Sun in 1980, who demonstrated that islets embedded in alginate hydrogel microparticles could reverse diabetes in rats without immunosuppression, albeit for only a few weeks. This initial success was followed by research aimed at better understanding and refining the process. Over time, the field has made significant progress; however, alginate microspheres have long suffered from known biocompatibility issues. Despite its limitations, alginate continues to be a prominent material choice for cell encapsulation due to its unique, near-instantaneous cross-linking kinetics, enabling the direct fabrication of convenient injectable microparticles.

[0086] Conversely, other hydrogels are typically only prepared into bulk macroscopic structures due to their slower cross-linking rates. Several schemes have been developed to produce microspheres using alternative hydrogels such as polyethylene glycol, agarose, chitosan, or hyaluronic acid. However, these methods are based on oil emulsion techniques, which are generally unsuitable for cell encapsulation and transplantation due to their reliance on non-aqueous solvents and poor process control.

[0087] As the number and complexity of cell encapsulation applications continue to grow, the ability to manufacture convenient, injectable, and biocompatible microparticles using advanced biomaterials will be decisively useful. Here, a novel method for producing hydrogel microparticles, called core-shell spheroidization (CSS), as described in U.S. Patent No. 9,642,814, filed June 3, 2015 and incorporated herein by reference, is tested. This method is strategically designed to utilize hydrogels with a much slower gelation rate compared to alginate, enabling the production of hydrogel microparticles with a wide range of chemical, physical, and biologically active properties. Furthermore, this method does not employ oil emulsion techniques but is developed for standard GMP-ready equipment and materials to better facilitate accessibility, scale, and regulatory compliance. We illustrate the microparticle production via CSS using two popular hydrogels: hyaluronic acid (HA) and polyethylene glycol diacrylate (PEGDA).

[0088] method

[0089] Cytotoxicity of CSS crosslinking procedure

[0090] The cytotoxicity of calcium (a component of the CSS method) was tested by exposing canine islets to a solution containing 100 mM or 200 mM calcium chloride, 10 mM HEPES buffer, and 20% PEGDA, MW 3,400 Da (Laysan Bio, Inc.), for 5, 10, or 15 minutes. Groups were also exposed to 100 mM calcium chloride without PEGDA to assess the effect of calcium alone. Islets were suspended in the test solution at approximately 5,000 IEQ / mL to simulate the high cell load density associated with the encapsulation process. Following calcium exposure, the islets were washed twice with supplemented CMRL 1066 islet medium and then incubated at 37°C and 5% CO2 for 3 hours before evaluation.

[0091] Dead cells were identified using propidium iodide staining and fluorescence microscopy. Fluorescence micrographs were captured using a Cytation 5 Imaging Multi-Mode Reader (Biotek Instruments, Inc., 531 / 647nm ex / em). Cell death was quantified by calculating the ratio of red (dead) pixels to total islet pixels using Adobe Photoshop. Twenty-five individual islets were analyzed in each group and distinguished from non-islet tissue by dithizone staining. Results were reported as mean viable cell fraction and normalized to islet viability in matched untreated controls (i.e., islets from the same batch in culture medium).

[0092] For PEGDA and methacrylated hyaluronic acid (MeHA) microparticles, photocrosslinking was used. Therefore, the toxicity of ultraviolet (UV) light and the photoinitiator was first investigated. Canine islets were suspended in a photoinitiator containing 0, 0.025, or 0.05% (w / v) of photoinitiator. 2959 μL of Dulbecco's phosphate-buffered saline (DPBS) was used. Approximately 50 IEQs were loaded into the wells of a 24-well plate and irradiated with long-wave ultraviolet (UV) light for 3, 5, or 10 minutes. According to the manufacturer's data (Uvitron International), the plate was irradiated at a distance of 6” using a PortaRay 400 UV lamp in low power mode, corresponding to approximately 40 mW / cm². 2 Following exposure, the islets were washed twice with supplemented CMRL 1066 islet medium and incubated at 37°C and 5% CO2 for 3 hours before assessment. Cell death was assessed by propidium iodide staining and quantified as described in the previous section.

[0093] Manufacturing MeHA hydrogel microparticles

[0094] Once the non-toxicity level of the crosslinking agent is determined, particle production begins. Figure 2 A general schematic diagram of the CSS method for the fabrication of hydrogel microparticles is provided. MeHA is first synthesized by reacting HA (MW 1MDa, Lifecore Biomedical) with a 50-fold molar excess of glycidyl methacrylate (Sigma) for 5 days in a 50:50 water:DMSO mixture in the presence of triethylamine and tert-butylammonium bromide (Sigma). The MeHA is then dialyzed with deionized (DI) water for 2 days and subsequently lyophilized. 1 The degree of methacrylate esterification was determined to be 54-72% by calculating the ratio of the relative peak areas of methacrylate protons to methyl protons using ¹H NMR (Avance AV-III 500, Bruker).

[0095] The solution contained 100 mM calcium chloride, 15 mM HEPES, and 0.05% (w / v) of OptiPrep. A 2.5%:1% (w / w) methacrylated HA:PEGDA polymer blend (MW 3.4 kDa, Laysan Bio, Inc.) was prepared in a custom buffer solution of 2959 (CosmoBioUSA, Inc.). In this embodiment, a low concentration of PEGDA was added to the MeHA solution as a crosslinking agent to increase the crosslinking density of the hydrogel. The solution was passed through a 0.22-micron filter. The viscosity of the precursor solution was measured at room temperature using a Cannon-Manning semi-micro calibrated glass capillary viscometer.

[0096] The precursor was extruded via an automated droplet generator into a stirred bath containing 300 mM mannitol, 0.1% Tween 20, and 0.1% sodium alginate (Protanal LF 10 / 60, FMC Corp.), and adjusted to pH 7.6 using a custom-made 15 mM HEPES buffer. For the MeHA precursor, 0.05% (w / v) sodium alginate was also added. 2959 initiator was added to the alginate bath to maintain Equilibrium between droplets and bath, and minimize Leaching was performed after the droplets were dropped into the bath (this would reduce crosslinking). A Buchi 395-Pro ​​encapsulator (Buchi Corporation, Newcastle, Delaware (DE)) equipped with an air jet nozzle system and a 400-micron diameter internal fluid nozzle within a 1.5 mm concentric air nozzle was used for droplet generation. The droplets were extruded using compressed nitrogen. MeHA droplets formed core-shell constructs with their respective alginate shells encapsulating the hydrogel precursor solution / droplet core. The hydrogel precursor solution / droplet core was then irradiated with long-wave UV light to initiate free radical photocrosslinking of MeHA and PEGDA. The irradiance at the center of the bath was approximately 40 mW / cm². 2 (PortaRay 400, Uvitron International). Irradiation was applied continuously during the extrusion of the precursor solution and for 1 minute after extrusion to ensure complete cross-linking of the hydrogel core in the core-shell construct. The core-shell construct was then washed for 5 minutes in 25 mM citrate buffer in DPBS with gentle agitation to dissolve the alginate shell. The resulting microparticles were collected using a steel mesh sieve and suspended in DPBS. A second wash was performed in 50 mM citrate for 5 minutes to ensure complete dissolution and removal of the alginate shell.

[0097] Manufacturing ThHA hydrogel microparticles

[0098] The ThHA precursor was prepared as follows: 1.2% (w / w) of thiolized HA (HyStem, Biotime Inc) was dissolved in a custom buffer (CosmoBioUSA, Inc.) containing 100 mM calcium chloride and OptiPrep to adjust the solution density. The pH of the precursor solution was adjusted to approximately 7.0 using a custom 15 mM HEPES buffer to reduce disulfide bridging of the ThHA macromonomer. The solution viscosity was measured as described above. The core-shell construct was produced as described above. However, crosslinking was achieved chemically (in contrast to free radical photocrosslinking using MeHA microparticles). The terminal acrylate groups of the PEGDA molecule were bonded to the thiol groups on the ThHA via Michael addition. In short, the precursor solution, upon contact with an alginate bath, produced a core-shell construct with a ThHA precursor core and an alginate shell. The alginate bath also contained 0.4% PEGDA as a crosslinking agent (MW 3.4 kDa, LaysanBio, Inc.) and was gently stirred for 5 minutes. Both the bath and the hydrogel precursor can contain a crosslinking agent (e.g., PEGDA), but in this scheme, it is preferably only included in the bath. Crosslinking is achieved by the diffusion of smaller 3.4 kDa PEGDA molecules through the alginate shell into the core. The bath solution is then diluted by half with DPBS, which lowers the solution pH to 7.4. Stirring continues for an additional 30 minutes to crosslink the HA precursor within the core. The core-shell construct is collected, and the alginate shell is removed as described above to produce microparticles.

[0099] Manufacturing PEGDA hydrogel microparticles

[0100] The PEGDA hydrogel precursor solution was prepared as follows: PEGDA 3.4kDa and 20kDa (Laysan Bio, Inc.) were dissolved at 18% and 12% (w / w) respectively in a solution containing 100mM calcium chloride, 10mM HEPES, and 0.025% (w / v) HEPES. In a buffer solution of 2959. Filter the solution using a 0.22-micron syringe and measure the viscosity as described above. For MeHA microparticles, the precursor was extruded into a stirred alginate bath, but with a concentration of 0.025% (w / v). 2959. All solutions used for the fabrication of MeHA and PEGDA microparticles were prepared in water degassed by ultrasonic treatment to remove excess oxygen (a known inhibitor of photocrosslinking). The core-shell construct was irradiated as described above to achieve core crosslinking. The construct was further processed as described above to dissolve the alginate shell and produce hydrogel microparticles.

[0101] Manufacturing AHA hydrogel microparticles

[0102] First, AHA was synthesized in the presence of triethylamine and N-dimethylformamide (Sigma) by reacting HA (MW 1MDa or 200kDa, Lifecore Biomedical) with acryloyl chloride and glycidyl chloride (TCI) for 5 days. The AHA was then dialyzed against deionized (DI) water for 5 days and subsequently lyophilized. 1 ¹H NMR (Avance AV-III 500, Bruker) determined the degree of acrylation to be 53–72% by calculating the ratio of the relative peak areas of the acylated protons to the methyl protons. The AHA precursor solution was then prepared by dissolving 4% acrylated HA in a buffer containing 100 mM calcium chloride, 15 mM HEPES, and 0.05% (w / v) [amount missing]. In a custom buffer solution of 2959. The precursor solution was filtered using a 0.22-micron syringe and the viscosity was measured as described above. For the MeHA particles, the precursor was extruded into a stirred alginate bath. The core-shell construct was irradiated as described above to achieve core crosslinking. The alginate shell was removed using a citrate buffer as described above to collect the AHA hydrogel microparticles.

[0103] Physical properties and size distribution of hydrogel microparticles

[0104] Images of 100 individual microparticles were captured using a Biotek Cytation 5 multimodal imaging reader. The microparticles were imaged in multi-well plates of PBS, and the images were analyzed using Adobe Photoshop to determine the average microparticle diameter and size distribution for each microparticle type (N = 100 microparticles). The hydrogel microparticles were further characterized by determining the swelling ratio “Q” (i.e., the ratio of swollen hydrated mass to dry mass) for each microparticle type. The microparticles were immersed in excess DI water for 24 hours prior to measurement to remove dissolved salts and ensure equilibrium swelling was achieved. Excess surface moisture was removed from the microparticles, and they were then weighed on pre-weighed petri dishes. Subsequently, the microparticles were dried overnight at 60°C and reweighed to obtain the dry mass (N = 3).

[0105] Diffusion characteristics of particles

[0106] Following previously published procedures, hydrogel microparticles with molecular weights of 10, 40, 70, and 500 kDa (Invitrogen Molecular Probes) were incubated overnight in a DPBS solution containing 0.1 mg / mL FITC-labeled dextran. The microparticles were rinsed with DPBS and imaged using a laser scanning confocal microscope (Olympus Fluoview 300) to monitor probe efflux. Micrographs were captured at set time points between 3 and 30 minutes after the microparticles were removed from the FITC-dextran incubation solution.

[0107] Aperture Calculation

[0108] Empty PEGDA and MeHA particles were examined using environmental scanning electron microscopy (ESEM) on a FEIQuanta 600F ESEM instrument. Samples were immersed in water on a cooling stage, and the temperature in the SEM chamber was set to 6°C. The chamber was then evacuated to 1000 Pa, and the relative humidity was maintained at 100%. Images were captured at 10,000x magnification. Standard SEM images were also collected. For these images, samples were critically dried or lyophilized and coated with 10 nm platinum, and imaged at 5 kV in high vacuum mode on the same ESEM instrument. SEM images were captured at 1000x magnification.

[0109] result

[0110] Core-shell spheroidization procedure

[0111] Hydrogel microparticles are obtained through Figure 2 The CSS method summarized in the text is used to fabricate the hydrogel. This method begins with a slowly hardening hydrogel precursor solution (functionalized PEG or HA). When it impacts an alginate bath, it is embedded as a droplet within an alginate shell, providing time for the functionalized PEG or HA to harden. The alginate shell formed around the nucleus is characterized by a concentric ring connection morphology. Figure 3 (A in the original text). Subsequently, the alginate shell was removed, leaving only the core hydrogel (…). Figure 3 (B in the middle). Figure 3 B in the diagram illustrates a predominantly spherical shape for the particles. However, other shapes are acceptable for the final product. Figure 4 Additional instances of acceptable final product features are provided. Figure 4 The A in the example provides an instance of a more durable (aka long-lasting) PEGDA-based microparticle with a slower degradation profile, loaded with canine pancreatic islet cells, and having a fully spherical shape. Figure 4 B in the diagram shows PEGDA-based microparticles loaded with islets but with an elongated shape; similarly durable microparticles with a 3D ellipsoidal shape. Figure 4 The diagram in Figure C illustrates a rapidly degrading (aka short-term) MeHA-based microparticle with a rapid degradation profile, loaded with stem cells, and with a flattened edge on the left. Figure 4 The 'D' in the image represents the same type of particle, but with a 3D elliptical shape. All of these particles, along with other related shapes such as teardrops or eggs, are functional instances of the CSS particle final product.

[0112] Cytotoxicity studies

[0113] During microparticle fabrication, although the alginate shell remains, the gelation of the core requires cross-linking to maintain a 3D, self-sustaining structure after shell removal. To optimize the CSS approach, the cytotoxic effects of the cross-linking components in the microparticle fabrication process on freshly dispersed cells (canine islets) were evaluated. Figure 5 Figure A describes the effect of calcium concentration and exposure time on canine islet viability. Three conditions were tested: 100 and 200 mM calcium with PEGDA or 100 mM calcium alone. At the shortest measured exposure time of 5 minutes, no significant differences in cytotoxicity were observed in any group. Viability in all groups exceeded 97.5% compared to a matched untreated control (islets from the same batch in culture medium). However, at each subsequent time point, a significant decrease in viability was observed in the 200 mM calcium group compared to the 5-minute measurement. At both 10 and 15 minutes, the viability of the 200 mM calcium group was significantly lower than that of the two 100 mM groups. Within 10 minutes, no differences were observed in either 100 mM group. However, at 15 minutes, a significant decrease in viability was shown in the 100 mM group containing PEGDA compared to the 5-minute and 10-minute time points, and compared to the 100 mM group without PEGDA at 15 minutes. Interestingly, no significant changes in viability were observed at any time point in the 100 mM group without PEGDA, with viability at 15 minutes being 97.6% compared to the control.

[0114] The effects of photoinitiator concentration and UV light exposure time on cell viability, such as Figure 5 As shown in B, canine islets were prepared at concentrations of 0, 0.025, and 0.05% (w / v) of [a solution / method / treatment]. 2959 with ~40mW / cm 2 Long-wave UV exposure was performed for 3, 5, and 10 minutes, and cytotoxicity was assessed. Interestingly, no photoinitiator was included. The group containing 2959 showed the highest degree of cytotoxicity. Compared to a 3-minute exposure time, the survival of the 0% group was significantly reduced at 5 and 10 minutes. Furthermore, compared to two groups containing [unclear text - possibly a specific ingredient or group name], [unclear text - possibly a specific ingredient or group name] exhibited the highest degree of cytotoxicity. Compared to the control group, the 0% group had significantly lower viability at 5 and 10 minutes. The only significant difference between the 0.025% and 0.05% groups was at 10 minutes, with viable cell fractions of 94.5% and 87.2%, respectively.

[0115] Physical properties and size distribution of hydrogel microparticles

[0116] To fully test the new CSS platform, the microparticles were generated from two different starting materials: HA and PEGDA. HA production was further divided into thiol-modified HA (ThHA) and methacrylate-modified HA (MeHA) to evaluate both chemically crosslinked and photocrosslinked hydrogels using the CSS platform. Table 2 provides the physical properties of the hydrogel formulations before and after crosslinking, as well as the average microparticle diameter and diameter range.

[0117] Table 2. Characteristics and physical properties of particle manufacturing

[0118]

[0119]

[0120] 1. The swelling ratio "Q" is the ratio of the hydrated equilibrium mass of the gel to its dry mass.

[0121] 2. The diameters of HA and PEGDA beads were significantly different (p<0.001).

[0122] For PEGDA, the precursor mass fraction of the gel precursor was 30%, while ThHA and MeHA were prepared from solutions of 1.2% and 2.5%, respectively. The high PEGDA precursor concentration was necessary to achieve a solution viscosity suitable for forming well-shaped microparticles. The gel formulations and corresponding precursor viscosities are shown in Table 2. ThHA was the only chemically crosslinked hydrogel, and it required significantly more time to crosslink (35 minutes) compared to the photocrosslinked gels (MeHA and PEGDA). MeHA microparticles had the largest average diameter, followed by PEGDA, and then ThHA microparticles, which were smaller than the other two groups. PEGDA microparticles exhibited the lowest equilibrium swelling ratio "Q", indicating a more compact overall hydrogel. This is in Figure 6 Further illustration shows the diameters of 100 representative microparticles composed of ThHA, MeHA, PEGDA, or AHA, illustrating the good monodispersity of the microparticles, especially for the HA materials. Despite being fabricated using the same droplet generation equipment and parameters prior to crosslinking, the ThHA microparticles are significantly smaller than those of PEGDA or MeHA and have a narrower diameter range compared to PEGDA. The average diameters of MeHA and AHA microparticles are the highest among the four groups. Interestingly, however, PEGDA particles containing islets exhibit a slightly larger average diameter but improved monodispersity compared to empty PEGDA microparticles (938 μm for PEGDA with islets [CV: 8.4%], and 904 μm for empty PEGDA microparticles [CV: 15.7%]).

[0123] Diffusion characteristics of particles

[0124] Figure 7The diffusion of fluorescently labeled dextran into PEGDA, ThHA, and MeHA hydrogel microparticles is illustrated. Microparticles were incubated overnight in fluorescent dextran of different molecular weights, rinsed, and immediately examined by confocal microscopy to assess the extent of dextran penetration and the efflux rate as a measure of diffusion characteristics. The 10 kDa probe was able to penetrate all microparticles. However, fluorescence was weaker in the PEGDA microparticles, particularly near the center of the construct, indicating a lower probe penetration rate compared to the HA microparticles (both exhibiting strong fluorescence). Furthermore, the fluorescence of the 10 kDa probe rapidly diminished in the HA group and was absent or almost absent 30 minutes after rinsing. In contrast, localized fluorescence was still observed in the PEGDA group at 30 minutes, again indicating a higher diffusion barrier for the 10 kDa probe compared to the two HA gels.

[0125] Low levels of fluorescence from the 40 kDa probe were detected around the periphery of the PEGDA particles, but almost no signal was observed in the center, indicating a diffusion barrier to the probe. Figure 7 Larger dextran (70 and 500 kDa) showed almost no fluorescence signal in the PEGDA microparticles, suggesting that the penetration of these probes into the gel matrix during the overnight incubation period was negligible.

[0126] Both ThHA and MeHA microparticles exhibited strong fluorescence from all evaluated probes, indicating minimal diffusion barriers even for macromolecules. Furthermore, compared to ThHA, the relative fluorescence signal in MeHA appeared significantly higher immediately after rinsing for all probes, suggesting a higher probe concentration and therefore an even lower diffusion barrier. This is best illustrated by a 70 kDa probe, which exhibits moderate infusion into ThHA microparticles but appears to be saturated in MeHA microparticles. All additional studies focused on hydrogel formulations with the largest and smallest diffusion barriers, PEGDA and MeHA, respectively.

[0127] discuss

[0128] We have developed and evaluated a novel method for fabricating hydrogel microparticles for cell encapsulation and delivery. Current methods for cell microencapsulation are primarily based on alginate spheres due to their rapid gelation rates. Other hydrogels used for microencapsulation involve harsh oil emulsion techniques. In contrast, the CSS method can be used with a wide range of hydrogel materials, is compatible with standard, commercially available, and GMP-ready equipment, and exhibits minimal cytotoxicity to pancreatic islets.

[0129] Low cytotoxicity is a key characteristic of cell encapsulation strategies. Therefore, we evaluated the effects of some key licensing components of the CSS method, particularly calcium content and UV exposure, on the cytotoxicity of islets. Unsurprisingly, 200 mM calcium was rapidly cytotoxic. However, we found that islets tolerated 100 mM calcium for at least 10 minutes even in highly concentrated and viscous PEGDA solutions. Exposure to 40 mW / cm²... 2 Long-wavelength (~365 nm) UV light, lasting up to 10 minutes, showed the least cytotoxicity to canine pancreatic islets. Surprisingly, the group without a photoinitiator exhibited the most cell death after UV exposure, contrasting with other similar studies. However, these other studies used much lower UV intensities, in the range of 4–10 mW / cm². 2 Between these concentrations, and the photoinitiator concentration used in this paper is relatively low. Therefore, in our experiments, the photoinitiator can provide protection by preferentially absorbing UV photons. Nevertheless, in the presence of... A concentration-dependent increase in cytotoxicity was still observed in group 2959, consistent with previously published findings.

[0130] Despite being fabricated using the same 400-micron nozzle system, the particle sizes varied significantly, with MeHA beads nearly twice the diameter of ThHA beads, corresponding to a difference of approximately eight times in total volume per particle. The size differences between groups are most likely a result of variations in post-fabrication volume swelling kinetics. Hydrogel swelling is governed by many variables, but is strongly correlated with the initial (i.e., pre-crosslinked) polymer concentration and the crosslink density within the gel post-fabrication. More specifically, solvent molecules (e.g., water) adsorbed onto the gel polymer matrix exert outward pressure, causing the gel to swell (swell), while crosslinks within the gel matrix resist this outward swelling. For example, hydrogels with high initial polymer concentrations and low crosslink densities will be expected to swell significantly in volume post-fabrication. Conversely, gels with high crosslink-to-polymer concentration ratios tend to resist swelling post-fabrication. In our study, the smaller final size of the ThHA particles is likely a result of a low initial polymer fraction (1.2%) coupled with a relatively high crosslink density. It is important to note that post-fabrication volume swelling should not be confused with the swelling ratio “Q” (hydrated mass / dry mass). Specifically, Q describes the final gel structure after volume swelling has reached equilibrium, where a lower Q value generally indicates a stronger and more compact gel. Therefore, the gel can undergo significant volume swelling but still have a relatively low Q value, which appears to be the case with the PEGDA microparticles in this study. In contrast, the ThHA microparticles experience relatively less volume swelling (if any) compared to the PEGDA microparticles but have a higher Q value. Furthermore, the MeHA microparticles, despite having a low initial polymer concentration (3.5%), swell significantly and have a high Q value, indicating a very low relative crosslinking density.

[0131] The diffusion characteristics differed significantly among the microparticle groups. ThHA and MeHA microparticles exhibited stronger permeability to dextran probes up to 500 kDa, with MeHA microparticles appearing to have the highest diffusivity among all three groups. Conversely, PEGDA particles appeared to strongly restrict the diffusion of dextran probes with a MW of 40 kDa and larger. Although various factors can influence the gel's diffusivity, the swelling ratio "Q" (the ratio of the gel's equilibrium hydrated mass to its dry mass) is strongly correlated with the hydrogel's permeability. The Q values ​​for PEGDA, ThHA, and MeHA microparticles were 17.3, 27.7, and 105.7, respectively. Therefore, the diffusion behavior of the dextran probes observed in the microparticles is consistent with their corresponding Q values ​​and measured surface pore sizes.

[0132] Example 2

[0133] Durability and stability of the final product of hydrogel microparticles that degrade rapidly

[0134] introduce

[0135] The description of the durability of rapidly degrading hydrogel microparticles depends on the stability of the microparticles' physical composition. Both in vitro and in vivo stability studies were conducted to determine if there were differences between the three formulations (PEGDA, MeHA, and AHA). While the CSS approach provides a detailed tuning for hydrogel degradation at different rates, examples of two durable hydrogels are presented below, which can last from months to years compared to hydrogels that degrade more rapidly.

[0136] method

[0137] In vitro durability testing

[0138] For MeHA, PEGDA, and AHA, 1,000 g of hydrated microparticles were collected and placed in a degradation buffer consisting of 10 mM HEPES-buffered phosphate-buffered saline and 1% gentamicin. The degradation solution was maintained at pH 7.3 ± 0.10. The microparticles in solution were stored at 37°C for 1, 3, and 6 weeks, then collected and dried overnight at 60°C to obtain the degraded dry mass. The degraded dry mass was compared with the dry mass of the microparticles on day 0 to determine the percentage of mass loss. Furthermore, the swelling ratio was measured at each time point to assess gel strength and degradation.

[0139] In vivo durability testing

[0140] Canine pancreatic islet separation

[0141] Canine islets were isolated from pancreas locally obtained from euthanized donors at a local veterinary clinic with the consent of the organ donor's owner. The procurement and digestion protocols have been previously detailed. After retrieval and cleaning of the organ, collagenase digestion was performed using the standard published protocol, followed by density gradient purification. The isolated islets were converted to islet equivalents, or "IEQ," for quantification purposes by dithizone staining, according to the standard published protocol. The canine islets were cultured in CMRL 1066 at 37°C and 5% CO2 in a solution supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 mM nicotinamide, and 1% antibiotic antifungal agent.

[0142] Encapsulated islet xenotransplantation

[0143] NOD / SCID mice (NOD.CB17-Prkdc) induced with diabetes were treated with streptozotocin (STZ, 220-250 mg / kg). scid The study, conducted at the Jackson Laboratory, evaluated the functional capacity of canine islets encapsulated via CSS. Six mice received islets encapsulated in PEGDA, two mice received islets encapsulated in PEGMAL (synthesis procedure below), and four mice received the same dose of islets encapsulated in MeHA. Two mice received an equal dose of unencapsulated islets as controls. At week 10 of the study, all mice receiving MeHA-encapsulated islets, along with the unencapsulated islet group, developed hyperglycemia and were discontinued. Four mice from the PEGDA group were also discontinued at week 10, serving as a comparison for subsequent histological studies. The remaining two PEGDA mice were discontinued after an additional six weeks. Monitoring of the two PEGMAL mice is ongoing (more than 16 weeks post-transplantation).

[0144] As described above, durable PEGDA and rapidly degrading MeHA formulations of islet-encapsulating microparticles were fabricated, except that canine islet cells were incorporated into the precursor. A chemically cross-linked (i.e., non-UV) durable PEGMAL formulation was prepared using a similar protocol to that used for the ThHA microparticles described above. Briefly, a hydrogel precursor consisting of 25% (w / w) 8-arm 40kDa PEGMAL (JenKemUSA) and canine islets was extruded into an alginate bath at pH 6.6 containing 0.25% (m / v) of a 1kDa SH-PEG-SH cross-linking agent (BiochemPEG), and stirred for 5 minutes. After 5 minutes, the cross-linked alginate bath solution was diluted by half with HEPES-buffered islet medium to a final pH of ~7.2 and stirred for another 30 minutes to allow complete cross-linking of the hydrogel core. The alginate shell was then removed as described.

[0145] Microparticles containing islets were administered into the intraperitoneal (IP) space of mice via an 18G catheter. The islet dosage was based on previous escalation studies, which indicated that approximately 4000 islet equivalents / mouse resulted in normal blood glucose levels and were consistent with previous publications on canine islet transplantation into mice. Blood glucose levels were measured daily in all mice for 14 days post-transplantation, and then every two weeks thereafter.

[0146] Following animal termination (10 and 16 weeks), islet-containing microparticles were recovered from the IP space and placed in culture medium for evaluation. Islet-containing microparticles were stained with dithizone to detect insulin presence, and calcein (for live cells) and propidium iodide (for apoptotic / necrotic cells) to assess cell viability of the explant tissues. Color, bright-field, and fluorescence images of the stained microparticles were captured using a Cytation 5 imaging multimodal reader (Biotek Instruments, Inc.).

[0147] Before transfer to PBS, pancreatic and tissue samples were removed from the peritoneum containing attached microparticles and fixed overnight in 10% neutral buffered formalin. The tissues were embedded in paraffin blocks and sectioned appropriately for staining. For hematoxylin / eosin (H&E) staining, sections were deparaffinized sequentially by placing sections in Clear Rite 3 solution three times for 3 minutes each time, followed by subsequent dehydration and rehydration steps before staining with hematoxylin and eosin. Sections were rinsed in water, followed by further rinsing with tap water using Bluing Reagent. Finally, sections were immersed in 100% anhydrous ethanol and Clear Rite 3 to improve color stability, and coverslips were applied accordingly. The slides were examined and images captured on a BioTekCytation 5 cell imaging multimodal reader.

[0148] Encapsulated islet allogeneic transplant

[0149] Two recently diagnosed adult male dogs received canine islet grafts encapsulated with either durable PEDGA or rapidly degrading MeHA (N=1 / group). Encapsulation of the canine islets followed the protocol described earlier. Each dog received a graft consisting of approximately 60 mL of microparticles, which were delivered directly to the peritoneum via catheter. The dogs' weight and blood glucose levels were monitored daily for the first 20 days post-transplantation.

[0150] result

[0151] In vitro degradation

[0152] MeHA, PEGDA, and AHA microparticles were placed in a HEPES buffer degradation solution at 37°C for 1, 3, or 6 weeks, and then dried to determine the dry polymer mass. After 6 weeks of degradation, it was found that MeHA microparticles retained only 24% of their initial mass, while PEGDA and AHA retained 37% and 53%, respectively. Figure 8A Although PEGDA loses a significant amount of mass ( Figure 8C However, it maintained its gel strength over 6 weeks, as indicated by no change in the swelling ratio. Figure 8B As shown in the figure. In contrast, MeHA and AHA microparticles exhibit a sharp increase in their swelling ratio, indicating a significant decrease in gel strength as the microparticles degrade. The variation in gel strength between PEGDA and HA-based microparticles can likely be attributed to both crosslinking density and polymer chemistry. For PEGDA microparticles, the polymer chains are tightly held together by several covalent bonds at a higher crosslinking density than HA microparticles. Furthermore, PEGDA polymer chains contain regions with some hydrophobic portions. Both the crosslinking density and the hydrophobic regions result in a slower rate of water diffusion into the microparticles than the rate of hydrolytic degradation occurring on the microparticle surface. Naturally, if degradation only occurs on the surface, the internal gel network will remain intact and maintain gel strength. However, in MeHA and AHA microparticles, the crosslinking density is much lower, and the polymer chemistry results in a more hydrophilic structure compared to PEGDA. The lower crosslinking density and hydrophilicity of HA microparticles both promote a higher rate of water diffusion into the microparticles, leading to a bulk degradation mechanism. In bulk degradation, water penetrates deep into the hydrogel network and causes hydrolytic degradation, which compromises the integrity of the gel network. This type of degradation could explain why the remaining AHA microparticles have a higher polymer mass than PEGDA after 6 weeks of degradation. The loss of gel strength in HA particles over time indicates that hydrolytic degradation is occurring, but the degradation products have not yet diffused out of the gel network. In this case, the remaining polymer mass cannot accurately assess the degree of degradation because, although the polymer is inside the gel network, it no longer contributes to the integrity of the hydrogel.

[0153] Function of canine pancreatic islet-loaded microparticles in diabetic mice

[0154] Canine islets encapsulated in PEGDA or MeHA were transplanted into diabetic NOD / SCID mice. The control group received unencapsulated islets. Figure 9The mean daily blood glucose levels for each group during the initial study period post-transplantation are shown. All mice in the MeHA, PEGMAL, and PEGDA groups had normal blood glucose levels at week 2 post-transplantation. However, despite reintroduction of exogenous insulin, animals receiving rapidly degrading MeHA microparticles gradually reverted to hyperglycemic levels (starting approximately 3 weeks post-transplantation). These mice were euthanized at approximately 10–11 weeks. Mice receiving durable PEGDA and PEGMAL-encapsulated islets maintained normal blood glucose levels throughout the study period (PEGMAL in progress, 16 weeks), exhibiting excellent glycemic control in the absence of exogenous insulin.

[0155] In contrast, all mice that received unencapsulated islets failed to achieve sustained normal blood glucose levels at any time point. These mice had an initial average blood glucose level of 386 ± 23 mg / dL before transplantation and 494 ± 61 mg / dL on the day of transplantation. After receiving islets of the same volume as the other two groups, one animal had only one normal blood glucose reading on day 2, while the other mouse showed fluctuations in blood glucose levels on days 19–30 but then reverted to sustained hyperglycemia for the remainder of the study.

[0156] Following necropsy, microparticles were recovered from the IP space of the mice. Results are shown in... Figure 10 In the AD (adrenal insufficiency) of the pancreas, durable PEGDA microparticles containing islets were readily recovered after approximately 10 weeks, but rapidly degrading MeHA microparticles were few in number and difficult to identify. Samples containing recovered PEGDA microparticles were found to be randomly dispersed throughout the IP cavity and remained transparent and structurally intact. The recovered PEGDA microparticles showed almost no signs of degradation (i.e., smooth edges, no significant size change, and mechanical stability), and the transplanted cells were still contained within the microparticles. Figure 10 (A) In contrast, rapidly degrading MeHA particles are deformed and smaller in diameter, indicating significant hydrogel degradation. MeHA particles do not appear to contain intact islets of Langerhans (A). Figure 10 (B in the text). At a later time point (16 weeks), the durable PEDGA microparticles continued to be readily recoverable upon necropsy. Islets within the PEGDA microparticles were stained with dithizone, calcein, and propidium iodide (PI) to confirm the presence of viable, functional islets.

[0157] PEGDA microparticles recovered approximately 16 weeks post-transplantation contained healthy, viable islets identified by deep red dithizone staining. Figure 10 (C in the text). Identical islets are co-labeled with green calcein, a commonly used indicator of living cells. Figure 10D). Some positive PI staining (red; apoptotic / necrotic cells) was observed, but these were generally located in non-islet (i.e., dithizone-negative) tissues that appeared to adhere to the surface of the recycled particles. Conversely, the islets (dithizone-positive cells) showed almost no PI (dead cell) staining. Figure 10 (D in the middle).

[0158] Durable PEGDA microparticles were found to adhere to tissues such as the liver and abdominal wall, but typically did not adhere to cavities and floated freely within them. Histology at the transplant site determined the location of the PEGDA microparticles. Although histological processing methods disrupted the integrity of the PEGDA microparticles, no significant fibrosis was observed at the attachment sites. Figure 11 The illustration shows healthy adipocytes at the intersection of two microparticles, with the microparticle locations indicated by black lines. The blue-gray membrane at the center of each microparticle location is the hydrogel remaining after treatment. In contrast, no gel material was identifiable in histological sections of the MeHA group. For the PEGDA group, fibrosis was generally absent at the microparticle-membrane interface. To ensure that the normalization of blood glucose levels in PEGDA-treated mice was due to graft transplantation rather than endogenous islets remaining in the pancreas after streptozotocin treatment, the presence of islets was examined in histological sections of the pancreas. Only one islet-like structure was identified in six pancreatic sections from each mouse (results not shown).

[0159] A similar study was conducted using canine islets transplanted into two diabetic dogs. Both animals had diabetes prior to transplantation and were not regularly receiving exogenous insulin. One dog received canine islets encapsulated in durable PEGDA microparticles, while the other received canine islets encapsulated in a biodegradable MeHA hydrogel. Figure 12 The mean non-fasting blood glucose levels from measurements obtained each morning and afternoon are shown. Dogs receiving islets in a durable hydrogel slowly recovered to lower blood glucose levels within the normal range (solid line) and maintained these levels throughout the experimental period (8 weeks). In contrast, animals receiving a rapidly degrading hydrogel slowly recovered to full diabetes within 20 days post-transplantation (dashed line).

[0160] discuss

[0161] Interestingly, while both mouse groups achieved normal glycemic levels after IP microparticle injection, unencapsulated islets at the same dose and transplant site did not. Since the animal models included immunocompromised mice, these results suggest that the hydrogel matrix provided a physically supportive environment promoting better cell function and survival, particularly in cases of transplantation via IP cavity injection. This conclusion was further confirmed by observing the degradation of MeHA microparticles over time and a simultaneous return to hyperglycemia. More notably, compared to MeHA transplantation, PEGDA and PEGMAL islet microparticles (which are structurally more robust) resulted in superior peri-transplantation glycemic control before hyperglycemic relapse in the MeHA group.

[0162] Although the canine study involved only two animals, the results are significant because they compared the differences in response between immunocompromised mice and immune-active dogs. In the case of an intact canine immune system, the weaker hydrogel (MeHA) degraded more quickly, resulting in a faster return to hyperglycemic blood glucose levels. The results do indeed suggest that certain formulations of this degradable hydrogel microparticle have an in vivo duration of approximately several days to several weeks, depending on the substance. However, durable hydrogel microparticles exhibit an in vivo duration of at least two months.

[0163] Example 3

[0164] Durability comparison: Rapidly degrading microparticles proliferate within beads in the final product.

[0165] introduce

[0166] Manipulating the physical and chemical properties of cell encapsulating agents has become a powerful tool in in vitro tissue engineering research. It is known that the mechanical and chemical properties of the microenvironment alter the behavior of various cell types, but most notably, stem cells are affected by the mechanical properties surrounding them. The ability of stem cells to continue proliferating within hydrogels is a crucial feature for therapeutic treatment of soft tissue defects caused by trauma or secondary to surgical interventions. For example, muscle or skin injuries would benefit from locally delivered microbeads containing proliferating stem cells. This would result in smaller doses of microbeads, as the cell number would continue to increase while within the microbeads. However, not all hydrogels allow for cell proliferation within microbeads. In this paper, we demonstrate that rapidly degrading hydrogel microbeads allow for robust cell proliferation within the microbeads.

[0167] method

[0168] Human embryonic kidney cells (HEK293) were cultured in DMEM medium with high glucose and fetal bovine serum and maintained in an incubator at 37°C and 5% CO2. When the cells reached an appropriate density, they were separated from the culture flasks by adding 0.5% trypsin-EDTA for 2 minutes. After separation, the cells were transferred to growth medium. To test the ability of cells to proliferate within the hydrogel, microparticles were fabricated using the CSS fabrication method described above, consisting of two types of hydrogel polymers, rapidly degrading MeHA, and durable PEGDA. An equal amount of polymer precursor (1 mL) was mixed with the same number of proliferating human embryonic kidney cells (HEK293; 4 million cells), followed by microparticle formation using the method described previously. The microparticles were maintained under culture conditions for 3 weeks. To assess cell proliferation within the microparticles, images were captured over 34 days using a BioTek Cytation 5 cell imaging multimodal reader.

[0169] result

[0170] When formulating the microparticles, it was noted that the cell morphology and its distribution within the microparticles are unique, depending on the hydrogel. Initially, HEK293 cells aggregated together in durable PEGDA microparticles. Figure 13 (A) In contrast, the same cells loaded at the same density remain as single cells in degradable (MeHA) microparticles. Figure 13 (C in the middle).

[0171] Over time, the morphology or number of cells in PEGDA microparticles remained almost unchanged. Figure 13 (B in the text). In contrast, cells in the degradable MeHA microparticles proliferate within the hydrogel, producing large clusters within the microparticles, as can be seen after 34 days of culture. Figure 13 (D in the text). The clusters do not appear to protrude from or attach to the surface of the MeHA beads, but are still encapsulated within the bead matrix. However, the same cells within durable PEGDA microparticles do not proliferate rapidly or form clusters of proliferating cells within the PEGDA beads.

[0172] discuss

[0173] In the final product form of microparticles, microparticles may inhibit or allow cell proliferation within the microparticles. Using the data presented in this paper, we show that durable hydrogels inhibit cell proliferation, while softer, rapidly degrading MeHA hydrogels allow significant proliferation of embryonic cells. Furthermore, cell morphology can be altered depending on the formulation used. This may be important when encapsulating proliferating cells in contrast to non-proliferating cells and can be tailored using CSS techniques.

[0174] Example 4

[0175] Durability comparison of cell migration in biodegradable microparticle end products

[0176] introduce

[0177] In some therapeutic cases, the migration of cells or their products from microparticles is an important aspect of treatment. For example, endogenous stem cells or progenitor cells have been shown to migrate to the brain during normal development and also under pathological conditions such as brain injury or disease. Some studies suggest that natural stem cells actually migrate or "hone" to the damaged area to aid in its repair. However, most studies have failed to confirm this claim when controlled in place to measure the number of stem cells in undamaged tissue.

[0178] Therefore, for cells to help repair any tissue, they must be positioned in the area for an extended period of time. Therapeutic practice involving the placement of unencapsulated stem cells in an area is challenging because the cells are small and they will rapidly spread out of the area or be attacked by the immune system. Therefore, localized methods that allow the controlled release of cells into specific areas would have significant therapeutic benefits. In this paper, we demonstrate these properties of degradable microparticles.

[0179] method

[0180] Three types of hydrogel polymers, MeHA, AHA, and PEGDA, were fabricated into microparticles using the CSS manufacturing method described above. Human embryonic kidney cells (HEK293) were cultured in DMEM medium with high glucose and fetal bovine serum and maintained in an incubator at 37°C and 5% CO2. Rat bone marrow stem cells (rBMSCs) were cultured in high glucose DMEM, fetal bovine serum, and Glutagro. When the cells reached an appropriate density, they were separated from the culture flasks by adding 0.5% trypsin-EDTA for 2 minutes. After separation, the cells were transferred to growth medium. An equal amount of polymer precursor (1 mL) was mixed with the same number of HEK293 cells or rBMSCs (4 million), and beads were then formed.

[0181] To assess the number of cells migrating from the microparticles, the microparticles were removed from the culture medium and washed on a sieve at defined time points. The washed culture medium and the culture medium surrounding the microparticles were collected. Cells were counted using a Cytation 5 imaging multi-mode reader (Biotek Instruments, Inc.) and an EVE cell counter.

[0182] result

[0183] Figure 14Typical images are provided of the used culture medium collected after 4 days of encapsulation, and the wash culture medium containing single cells. In the culture medium containing HEK293 cells encapsulated in PEGDA hydrogel, very few cells were counted, and many of them had died (in red). Figure 14 In contrast, biodegradable hydrogels allow for the release of high numbers of live cells, such as (A). Figure 14 The high number of live cells (green) remaining in well B is shown. However, these cell numbers are small compared to the accumulated cell release in week 2.

[0184] Figure 15 This summary summarizes average data from studies on cumulative cell release. During the first 6 days of culture, only a few thousand cells were released from the beads. By day 14, over 20 million cells were released from the rapidly degrading MeHA microbeads, compared to only 220,000 cells from the durable PEGDA microbeads. Cumulatively, by day 21, the MeHA beads released 30.8 million cells, while PEGDA released less than 5% of that.

[0185] discuss

[0186] The results elucidated a significant difference in the number of proliferating cells migrating from durable microparticles compared to degradable microparticles. The final microparticle product allows cells to migrate from the microparticles to surrounding tissues. Compared to durable hydrogels, rapidly degradable formulations achieve this effect in a faster and larger manner. These results demonstrate the utility of rapidly degradable hydrogels in allowing cells to migrate to localized injury sites, even before the microparticles undergo physical degradation.

[0187] Example 5

[0188] Durability comparison of the viability, morphology, and function of rapidly degrading particulate end products

[0189] introduce

[0190] Durable or rapidly degradable / short-term hydrogel formulations have numerous applications. Literature has demonstrated that microencapsulated mesenchymal stem cells (MSCs) significantly enhance paracrine-mediated healing in a mouse hindlimb injury model, exhibiting increased survival and pro-angiogenic activity compared to unencapsulated MSCs. Once encapsulated, cells must maintain viability and possess therapeutic function. It has been shown that increasing the stiffness of the encapsulating matrix guides MSC differentiation towards more osteogenic pathways compared to using softer materials for chondrogenic pathways. Furthermore, rat neurons cultured in 3D collagen hydrogels exhibit better survival and behave more like natural neural networks compared to cells grown in 2D on the same material.

[0191] method

[0192] Viability and Morphology

[0193] Two types of hydrogel polymers (rapidly degradable (MeHA and AHA) and durable (PEGDA)) were fabricated into microparticles using the CSS manufacturing method described above. Equal amounts of polymer precursors (1 mL) were mixed with equal numbers of mouse MSCs (16 million) or insulinoma cells (30 million cells), immediately forming beads. The beads were maintained under culture conditions for 2 weeks. For each formulation, cell viability and function were high when cultured within the microparticles. After encapsulation, the viability of MSCs and insulinoma cells within the microparticles was measured using a fluorescent viability staining agent. Calcein was added to the culture medium as a measure of viable cells, as calcein can only enter cells with intact cell membranes. Propidium iodide was added to the culture medium to identify dead cells. Images of the microparticles were acquired on a Cytation 5 imaging multimodal reader (Biotek Instruments, Inc.) after 30–60 minutes of incubation in fluorophores.

[0194] Similar experiments were also conducted using non-proliferating somatic cell sources (canine islets) encapsulated in two different hydrogels. Microparticles were fabricated as described above, in which cells (islets or cultured cells) were mixed into the precursor. For canine islets, the precursor at a concentration of 1.1 was mixed with canine islet serous fluid at a volume ratio of 10:1, just before droplet formation. The fabrication of the islet microparticles was performed aseptically in a closed, sterile bioreactor system.

[0195] The completed microparticles were stained with dithizone to detect the presence of insulin, and stained with calcein (for live cells) and propidium iodide (for apoptotic / necrotic cells) to assess cell viability of the explant tissue. Color, bright-field, and fluorescence images of the stained microparticles were captured using a Cytation 5 imaging multimodal reader (Biotek Instruments, Inc.).

[0196] result

[0197] MSCs encapsulated in PEGDA or MeHA exhibited high viability, such as Figure 16 As shown in A and B. A similar result can be seen in AHA (...). Figure 16 (E in the image). Green fluorophores represent live cells stained with calcein. Red / yellow cells represent dead cells stained with propidium iodide. Interestingly, these cells were loaded into the microparticles as individual cells, but they rapidly aggregated in the durable PEGDA gel, however, they did not in the degradable MeHA hydrogel, where they remained primarily as individual cells. Monitoring of cell morphology during the manufacturing process indicated that the cells aggregated during, rather than after, microparticle fabrication.

[0198] In studies using canine islets, cells exhibited high viability in both types of hydrogel microparticles. Figure 16 C-PEDGA and Figure 16 (D-MeHA in the sample). As demonstrated by staining for their insulin content, they continue to function in either hydrogel. Islets are loaded into microparticles as cell clusters. The aggregation frequency or cluster size appears to remain unchanged in either formulation.

[0199] Similar results were obtained using rat insulinoma cell lines. Figure 17 The study showed high cell viability within one day of encapsulation, particularly when encapsulated in AHA. Over the next 7 days, cell viability in AHA decreased to approximately 50%. In contrast, insulinoma cells in PEGDA started with lower viability but maintained near that average throughout the study.

[0200] Pancreatic islet cell function is assessed by staining intracellular insulin. Dithizone staining is a standard indicator of insulin production. It stains insulin-positive cells with a red / brown hue. Figure 18 The images illustrate positive insulin staining of islets in both durable PEGDA and degradable MeHA microparticles approximately one week after encapsulation, demonstrating their ability to continue producing insulin after encapsulation in either microparticle. Unlike mouse MSCs, the morphology of these somatic islet cells did not show significant changes in either hydrogel formulation.

[0201] Example 6

[0202] Durability comparison with rapidly degrading microparticle end products for topical cellular delivery

[0203] introduce

[0204] Durable or rapidly degradable hydrogel formulations have numerous applications. Literature has demonstrated that microencapsulated MSCs significantly enhance paracrine-mediated healing in a mouse hindlimb injury model, exhibiting increased survival and pro-angiogenic activity compared to unencapsulated MSCs. One problem with paracrine release using MSCs is that unencapsulated MSCs do not remain at the injury site and are rapidly removed from the body. Therefore, encapsulation with a viscous hydrogel captures cells at the implantation site, where they can secrete beneficial compounds. We conducted experiments to determine whether durable or rapidly degradable formulations of microparticles show differences in their ability to adhere to surrounding tissue and remain in the area.

[0205] method

[0206] Adhesion to tissues

[0207] Instantaneous tissue adhesion was tested during the transplantation procedure into the omentum of dogs. Four dogs were transplanted with microparticles containing canine islets. On the day of transplantation, the animals were anesthetized using standard veterinary procedures, including the use of a cephalic vein catheter for fluid infusion before, during, and after surgery. The surgeon injected the microparticles into the omental veil. Care was taken not to enter or damage any intestinal tissue, blood vessels, nodes, or lymphatic vessels. 1 mL of hydrogel microparticles contained a total of 10,000 islet equivalents. The fabrication of the PEGDA microparticles containing islets has been previously described. Photographs were taken immediately after the omentum was gently returned to the abdomen and the abdominal wall was closed, followed by intradermal sutures to close the skin.

[0208] Another example of tissue adhesion was demonstrated by injecting microbeads into the knee of a rat. In short, 100 μL of 300-micron PEGDA microparticles in PBS were extruded into the joint capsule of the knee joint of a Sprague-Dawley rat using a 25G needle. Prior to extrusion, the solution and the beads were stained blue to confirm the presence of the microparticles within the joint capsule.

[0209] Histology of Adhesive Tissue

[0210] Healthy Sprague-Dawley rats were used to assess the acute biocompatibility and location of the microparticles in the peritoneal cavity (N=2 for each microparticle formulation). Empty microparticles were implanted around the omentum following the previously described procedure. The microparticles were delivered via syringe as a suspension in DPBS. Approximately 1 mL of loosely packed microparticles deposited around and inside the omentum of the rats. Pain markers and activity levels in the rats were monitored and scored daily for 10 days following microparticle implantation.

[0211] Fourteen days post-implantation, rats were sacrificed to assess the implantation site, determine the location of intraperitoneal microparticles, and examine for possible tissue abnormalities. Tissue samples were collected and preserved in neutral buffered formalin, embedded in paraffin, and sectioned at a thickness of 7 micrometers. Sections were stained with H&E and evaluated under a microscope.

[0212] result

[0213] PEDGA microparticles were infused into and around the omentum, such as Figure 19 As shown in A and described in the Methods section, images were collected immediately after the microparticles in the culture medium were infused into and around the omentum, showing the microparticles adhering to the tissue (even when the omentum was manipulated manually). Figure 19 Figures B and C in the diagram show results from two different dogs, both of which demonstrate the viscous properties of the particles.

[0214] Stained PEGDA microparticles were injected into the knee of rats. The joint was dissected after manual manipulation. The blue color from the microparticles was visible even through the muscle. Figure 20(A in the text). Further dissection revealed that the particles were still visible and found within the knee joint. Figure 20 (B and C in the middle).

[0215] Durable PEGDA and degradable MeHA and AHA microparticles (cell-free) were implanted into healthy Sprague-Dawley rats to evaluate the safety and biocompatibility of the microparticles produced using the CSS method, as well as their location and attachment to surrounding tissues. Necropsy was performed at 2 and 10 weeks, and no signs of acute inflammation, excess fluid, or tissue abnormalities were observed throughout the peritoneum, although some tissue adhesion was identified at the omentum-gastric wall suture. Both microparticle formulations were found intact in the omentum and were visually translucent, appearing to be surrounded by a very thin membranous layer. A small number of microparticles were found adhering to the liver surface. However, the vast majority of microparticles were attached to the omentum, with some free-floating microparticles.

[0216] For the three microarrays, hematoxylin and eosin staining of explant tissues was shown in... Figure 21 No significant differences were noted between the groups. The microparticles were generally intact, but as a result of histological tissue processing, their size appeared to have shrunk and in some cases were crushed / deformed. A thin layer of cells, typically two to three cells thick, was usually found around the microparticles, and none were found inside, considering the injection of empty microparticles into the animals. Most individual microparticles were surrounded by normal omentum tissue, indicating no inflammatory response to the microparticles in response to various formulations.

[0217] Figure 22 The figure illustrates the quantification of foreign body responses in microparticles within the retinoid. The width of the fibrotic region, defined by low cellular structure and high collagen content, is smallest in AHA and even greater around PEGDA microparticles. Nevertheless, the thickness of the fibrotic ring around PEGDA microparticles remains smaller than that reported for other hydrogel formulations such as alginate. Similarly, there are fewer lymphocyte regions in AHA microparticles. Lymphocyte regions are defined as cellular immune cells migrating around the microparticles. They are also higher for PEGDA microparticles, but are still considered to be minimal. Figure 22 ).

[0218] discuss

[0219] A unique and advantageous feature of the final product is its viscous nature, regardless of whether it is composed of a durable or biodegradable hydrogel matrix. This viscous characteristic allows it to rapidly attach to surrounding tissue and remain in the area until degradation. In addition to attaching islets to the peritoneal space, the use of viscous hydrogels for administering MSCs or other cells that release anti-inflammatory or pro-healing cytokines can extend the delivery of cytokines to a localized area without concern for redistribution to other parts of the body.

[0220] Example 7

[0221] Research on parameters affecting hydrogel properties

[0222] In this embodiment, various parameters were investigated to better understand their relationships with each other and their impact on the properties and characteristics of the resulting hydrogel microparticles. As discussed in more detail below, various microparticle characteristics can be fine-tuned and adjusted according to specific desired treatment regimens and outcomes. It is worth noting that each of these parameters is a dynamic factor in hydrogel microparticle manufacturing, meaning that its influence will be related to the amount or ratio of other components used in the formulation and / or other processing parameters. Furthermore, modifications to certain parameters do not necessarily produce proportional changes in the resulting hydrogel. Similarly, however, subtle differences in different microparticle formulations and processing parameters allow for many different customizable characteristics, depending on the cell type to be encapsulated, the implantation site, the condition to be treated or prevented, and other desired outcomes.

[0223] To explore these different parameters, the swelling ratio (Q) was used as a representative of the relative degradation rate of the particles.

[0224] A. Mass fraction of nuclear polymeric substances

[0225] Mass fraction refers to the percentage by mass of the reactive polymeric material in the hydrogel precursor solution used for the polymer backbone (e.g., 2.5 g PEGMAL in 10 g of precursor solution = 25% mass fraction). Adjusting the mass fraction of the reactive polymer in the precursor solution has a consistent effect on the resulting Q value. The lower the mass fraction, the higher the Q value and the softer the gel. This is illustrated by crosslinking various multi-arm PEGMAL materials with 500 Da PEG dithiol. In this example, the crosslinking agent concentration was kept constant at 0.25% mass fraction in the alginate bath. Non-reactive PEG material (PEG 8000) was included in the lower mass fraction array to maintain a constant solution viscosity. The results are shown in... Figure 23 middle.

[0226] B. Concentration of cross-linking substances in the alginate bath

[0227] The concentration of crosslinking agents (e.g., PEG dithiol, DTT) in the alginate bath affects the resulting hydrogel microparticles. However, the relationship between crosslinking agents and the hydrogel matrix is ​​not straightforward, as too little or too much crosslinking agent can be present in the bath. Insufficient crosslinking agent results in a weak gel with insufficient crosslinking; however, too much crosslinking agent will saturate the reactive sites on the polymer backbone, similarly preventing crosslinking and producing a weak gel. Therefore, this parameter must also be balanced with the amount and type of polymer precursor used for the backbone.

[0228] 1. Increasing the crosslinking agent concentration leads to a lower Q value.

[0229] In this example (core polymer = 4-arm 10kDa PEGMAL), increasing the crosslinking agent concentration (DTT) resulted in a lower Q value. This indicates that the crosslinking agent concentration never reached a level where core polymer saturation would occur. The results are shown in... Figure 24A middle.

[0230] 2. Increasing the crosslinking agent concentration leads to a higher Q value.

[0231] In this example (core polymer = 8-arm 40kDa PEG-vinyl sulfone), increasing the concentration of the crosslinking agent (BMA, CAS123-81-9) initially resulted in a lower Q value, but further increases led to a higher Q value, compared to the formation observed in Example 1. Notably, the relative molar concentrations of the crosslinking agent (thiol) and the core reactive group (vinyl sulfone) differed from those in the previous examples. Therefore, the conditions in Example 2 may have resulted in core polymer saturation, and thus inefficient crosslinking, leading to a weaker gel network and a higher Q. The results are shown in... Figure 24B middle.

[0232] C. Alginate bath pH

[0233] Thiol-ene reactions are strongly influenced by ambient pH. Higher pH levels significantly increase the kinetics of thiol-ene reactions. Therefore, adjusting the pH of the alginate bath can greatly affect the kinetics of gel crosslinking and the resulting Q value.

[0234] 1. Increasing pH leads to a higher Q value.

[0235] In this embodiment (core polymer = 8-arm 40kDa PEG-vinyl sulfone; crosslinking agent = BMA), increasing the bath pH resulted in a higher Q value. The results are shown in... Figure 25A The increased reaction rate leads to faster reaction with the nucleopolymer and eventual partial saturation of the nucleopolymer, resulting in a weaker gel network and a higher Q value.

[0236] 2. Increasing pH results in a lower Q value.

[0237] In this example (core polymer = 8-arm 40kDa PEGMAL; crosslinking agent = 500Da PEG dithiol), increasing the pH resulted in a lower Q value. The polymer and crosslinking agent were balanced to avoid saturation. Therefore, in this case, increasing the pH led to faster reaction kinetics and more efficient crosslinking. The results are shown in... Figure 25B Note that the increase in Q value relative to pH in Example 2 is quite small compared to the very sharp increase in Example 1. Therefore, both the direction and magnitude of the effect of pH on Q value appear to be highly influenced by the relative amounts of crosslinking agent and core polymer reaction sites.

[0238] D. Molecular weight of cross-linked substances

[0239] The molecular weight of the crosslinking material affects both the rate of diffusion into the core-shell construct and the distance between crosslinks within the gel network. Therefore, differences in the molecular weight (MW) of the crosslinking material can influence the final Q value of the resulting gel microparticles, depending on other parameters used in the method.

[0240] 1. Increasing MW results in a higher Q value.

[0241] In this example (core polymer = 8-arm 40kDa PEG-vinyl sulfone), increasing the MW of the crosslinking agent from 154 Da to 500 Da resulted in a significant increase in the Q value. The molar concentration of the crosslinking agent was 10 mM in both formulations. The results are shown in... Figure 26A middle.

[0242] 2. Increasing MW does not change the Q value.

[0243] In this example (core polymer = 8-arm 40kDa PEGMAL), the increase in the crosslinking agent's molecular weight (MW) did not result in a significant difference in the Q value of the resulting gel. The effect of changes in the crosslinking agent's MW may offset the adjustment of the relative amount of crosslinking agent used, compared to the polymer reaction sites. The results are shown in... Figure 26B middle.

[0244] E. Molecular weight of nuclear polymeric substances

[0245] Assuming that the number of reactive sites per gram of polymer is maintained, the molecular weight of the core polymer material appears to influence the Q value more predictably than the molecular weight of the crosslinking agent. For example, an 8-arm 40kDa PEGMAL and a 4-arm 20kDa PEGMAL have the same number of reactive sites (maleimide) / gram. However, as shown below, when prepared under otherwise identical conditions, the 40kDa material produces a stronger gel (lower Q value).

[0246] 1. Effect of MW on nuclear polymers at constant reaction sites / gram.

[0247] This example uses three side-by-side comparisons to demonstrate the effect of the core polymer molecular weight on the Q value of gel microparticles prepared via CSS. In all formulations, despite having the same mass fraction and reactive sites / g, the Q value decreased as the core MW increased. Note that the effect of the core MW became more pronounced as the crosslinking agent concentration decreased (and the size increased). The results are shown in... Figure 27A middle.

[0248] 2. Effect of MW on nuclear polymers under unequal reaction sites / grams.

[0249] In this embodiment, microparticles made from 8-arm 40kDa or 4-arm 10kDa PEGMAL core materials were compared. In all cases, the mass fraction of the core polymer solution was 10%. The 4-arm 10kDa material had twice the number of reactive sites / gram compared to the 8-arm 40kDa; however, despite the lower potential crosslinking density, the 8-arm 40kDa microparticles had a lower Q value (i.e., stronger gel) compared to the 4-arm 10kDa microparticles. The results are shown in… Figure 27B middle.

[0250] F. Effect of UV crosslinking time

[0251] In this embodiment, AHA and MeHA microspheres of different molecular weights were produced under different UV exposure times. Briefly, 2.5% 1MDa or 200kDa AHA or 1MDa MeHA solutions (each containing 1% 3.4kDa PEGDA) were dropwise extruded into a stirred alginate bath as described above and exposed to UV light for 2.5, 5, or 7.5 minutes. The resulting diameter and hydrogel swelling ratio for each polymer type were quantified. The results are shown in… Figure 28A and Figure 28B The microsphere diameter and swelling ratio varied with UV exposure time. These results indicate that both the diameter and swelling ratio decreased with increasing UV exposure time. Notably, the swelling ratio of the 1MDa MeHA hydrogel microspheres decreased significantly after 7.5 minutes of UV exposure compared to 5 minutes, with swelling ratios of 509.1 and 113.4, respectively. This trend was constant for each polymer type and molecular weight, demonstrating the control of hydrogel durability through variations in UV exposure time.

[0252] G. Potential control over hydrogel degradation through the hydrolytic stability of cross-linked materials, regardless of the Q value.

[0253] 1. BMA is a crosslinking agent that is more unstable in hydrolysis.

[0254] Certain crosslinking agents can be used to influence the degradation profile of microparticles. For example, the dithiol ester crosslinking agent molecule BMA may potentially be well utilized as a means to accelerate the hydrolytic degradation of thiol-olefin hydrogels.

[0255] A series of hydrogel microparticles composed of PEG-vinyl sulfone crosslinked via DTT or BMA were prepared and stored in 50 mL polypropylene tubes in DPBS at room temperature (~27 °C) for at least 6 months. Table 3 below lists the composition and corresponding swelling ratio of each formulation for reference. After 6 months of storage, signs of microparticle degradation were qualitatively re-examined. Notably, all BMA-crosslinked microparticles were completely degraded within 6 months. Those crosslinked via DTT appeared relatively unchanged after 6 months, although formal characterization had not yet been performed at this time. Furthermore, two of the completely degraded BMA-crosslinked microparticles previously exhibited the lowest Q values ​​of all microparticles included in the table, but were still completely degraded compared to DTT-crosslinked microparticles with higher Q values. This result suggests that BMA crosslinking agents may be a useful tool for producing microparticles that are initially very strong (low Q) but degrade faster than other microparticles with similar initial strength crosslinked via DTT or PEG dithiol.

[0256] Table 3. Long-term durability of microparticles crosslinked with DTT or BMA.

[0257]

[0258] Table 4 below provides a list of additional dithiol substances and their corresponding pKa values ​​for one or more thiol groups. The hydrolytic instability of bonds formed by thiol-ene reactions is related to the pKa of the reactive thiol. Therefore, these substances can be used as chemical crosslinking agents in thiol-ene reaction schemes to control hydrolytic instability and thus the degradation rate of the particles.

[0259] Table 4. Examples of dithiol crosslinking agents with varying degrees of hydrolytic instability.

[0260] Chemical name pKa Dithioglycolic acid glyceride 7.9-8.1 Diol dithiocarboxylate 7.9-8.1 PEG-diester-dithiol-1 7.9-8.1 (S)-2-aminobutane-1,4-dithiol 8.5 Dithiothreitol 9.2 Diol di(3-mercaptopropionate) 9.4-9.5 2,2'-Thiodiethylthiol 9.4-9.5 PEG-diester-dithiol-2 9.4-9.5 2-Mercaptoethyl ether 10.5-10.7 2,2'-(Ethylenedioxy)diethylene glycol 10.5-10.7 Tetraethylene glycol dithiol 10.5-10.7 Hexaethylene glycol dithiol 10.5-10.7 PEG-dithiol 10.5-10.7 .

Claims

1. A spherical or elliptical non-alginate hydrogel microparticle for local delivery and sustained release of therapeutic cells and / or tissue at an implantation site, the microparticle comprising a 3D matrix of a covalently cross-linked non-alginate polymer compound and a therapeutically effective amount of cells and / or tissue embedded therein, wherein the cells have at least 50% viability and wherein the microparticles have a size greater than 30 μm, wherein the non-alginate hydrogel microparticles are not formed using an oil emulsion, wherein the polymer compound is polyethylene glycol vinyl sulfone or acrylated hyaluronic acid, the matrix further comprising a cross-linking agent cross-linked with the non-alginate polymer compound, the cross-linking agent being selected from the group consisting of: dithiothreitol, polyethylene glycol dithiol, and ethylene glycol bis-mercaptoacetate.

2. The particles according to claim 1, wherein, The particles have a size greater than 300 μm.

3. The particles according to claim 1, wherein, The matrix is ​​essentially composed of polyethylene glycol vinyl sulfone crosslinked with the dithiothreitol, polyethylene glycol dithiol, or ethylene glycol bis-mercaptoacetate.

4. A composition for local delivery and sustained release of therapeutic cells and / or tissues and / or cell products at an implantation site, comprising a plurality of hydrogel microparticles according to any one of claims 1-3 dispersed or suspended in a biocompatible delivery carrier.

5. The composition of claim 4, further comprising a plurality of hydrogel microparticles, said hydrogel microparticles comprising a 3D matrix of a covalently cross-linked non-alginate polymer compound having a size greater than 30 μm and substantially free of any cells or tissues.

6. The composition according to claim 4, wherein, The plurality of hydrogel microparticles comprise a mixture of degradable and durable hydrogel microparticles dispersed or suspended in the biocompatible delivery carrier.

7. Use of a plurality of particles according to any one of claims 1-3 or compositions according to any one of claims 4-6 in the preparation of a medicament for the local delivery and sustained release of therapeutic cells and / or tissues at an implantation site.

8. The use according to claim 7, wherein, The drug is formulated to be injected into or near the implantation site.

9. The use according to claim 8, wherein, The injection is an intra-articular injection.

10. The use according to claim 7, wherein, The implantation site is an area of ​​inflammation, injury, arthritis, or degeneration in the subject.

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