Injectable shear-reducing hydrogel containing polypeptide therapeutic agents for enhanced tumor treatment

Abstract

The present invention provides a composition comprising a novel shear-reducing biomaterial useful for the delivery of polypeptide therapeutic agents. [Solution] A shear-thinning hydrogel composition comprising gelatin, silicate nanoparticles, and a polypeptide therapeutic agent, wherein the gelatin, silicate nanoparticles, and therapeutic agent form a shear-thinning hydrogel. Preferably, a shear-thinning hydrogel composition is provided comprising about 1% (w / w) to about 5% (w / w) of gelatin and about 1% (w / w) to about 5% (w / w) of silicate nanoparticles.

Description

Technical Field

[0001] The present invention relates to shear-thinning biomaterials containing bioactive agents, as well as methods for making and using them.

Background Art

[0002] Shear-thinning hydrogels are non-Newtonian materials that behave as viscous fluids under shear stress and then recover solid-like properties when the stress is removed [1]. Due to these properties, injectable shear-thinning biomaterials (STBs) have attracted attention as a group of self-healing materials that enable fluid injection and local equilibrium after approaching the final application site. In clinical applications, STBs can be delivered into the body by hand pressure using a needle or a general / microcatheter [2-4]. To optimize clinical applications, it is necessary to adjust the physical properties of STBs according to specific clinical situations. Some physiological and clinical parameters, including but not limited to (1) the pH around the tumor (environmental pH of the tumor), (2) tumor cell density, (3) extracellular matrix synthesis of the tumor, (4) treatment period, (5) aggressiveness of the tumor, (6) tumor physiological functions including central necrotic lesion formation, (7) accessibility of the tumor, (8) drug solubility, and (9) hydrophilicity of the drug / agent, affect formulation design. By changing the physical properties, hydrogels suitable for specific clinical applications (e.g., occluding blood vessels of a specific size, controlled drug release, and adjusting the stiffness of tissue engineering scaffolds) can be synthesized.

[0003] The physical properties of conventional STBs can be modulated by combinations of several carbon-based, polymer, and inorganic nanomaterials [5–8]. Several biomaterials, such as gelatin, hyaluronic acid, chitosan, collagen, and alginates, have been previously used in conjunction with inorganic components to form STBs [14–17]. Gelatin, in particular, demonstrates substantially improved hemocompatibility of STBs in vitro by limiting the adsorption of nonspecific proteins, enhancing hemolysis, and ultimately prolonging coagulation time

[18] . Furthermore, the application of gelatin in tissue engineering and regenerative medicine has been approved by the Food and Drug Administration (FDA) [19–21]. Conventional STBs are prepared by mixing gelatin with LAPONITE®, a synthetic clay nanoparticle, for hemostasis and intravascular embolization [18, 22]. These STBs exhibit strong shear-thickening behavior, as well as biocompatibility ranging from blood coagulation to minimal inflammatory response. Other researchers have expanded this study to implement STBs as embolizers

[23] , functionalized scaffolds [24, 25], 3D bioinks

[26] , and drug delivery systems

[27] . Unfortunately, however, synthetic clay nanoparticles such as LAPONITE are crystalline nanoparticles, and their size and surface chemistry are not easily controlled. In addition, the biocompatibility of LAPONITE in various in vivo applications needs further investigation, and this fact makes its use in clinical applications difficult.

[0004] For the reasons stated above, there is a need in the art for new shear-thinning materials and methods for their fabrication and use. [Overview of the project]

[0005] As discussed below, the inventors have developed an immune checkpoint inhibitor (ICI)-loaded injectable shear-thinning biomaterial (STB) using silicate nanoplatelets (LAPONITE XLG) and a gelatin-based polymer. Injectable STBs are a class of hydrogels that can be injected by applying shear stress during injection and rapidly self-heal after the stress is removed. STBs have attracted considerable attention in the biomedical field, particularly for drug and cell delivery, as well as for minimally invasive intravascular embolization, due to their ability to be easily applied through syringes and undergo rapid sol-gel transition at the target site. LAPONITE (LAPONITE XLG) is a synthetic disc-shaped silicate nanoparticle with a diameter of 25 nm and a thickness of approximately 1 nm. The surface of LAPONITE is negatively charged, while its flakes are positively charged. These unique characteristics not only allow this clay to form a hydrogel with shear-thinning properties, but also give it promising potential for drug delivery by adsorbing various drugs via electrostatic interactions. In this specification, the inventors describe a strategy for enhancing tumor treatment by combining checkpoint blockade and intravascular embolization using an injectable STB containing laponite and gelatin. As shown in Scheme 1 (Figure 4), a PD-1 blocking antibody (anti-PD-1) molecule is first adsorbed onto the surface of laponite via electrostatic interaction and then delivered to vascular structures near the tumor via a syringe in the form of an STB. The anti-PD-1 is released from the STB and then enters the interior of the tumor.

[0006] The invention disclosed herein has many embodiments. Embodiments of the invention include, for example, compositions comprising gelatin, silicate nanoparticles, and polypeptides, which are typically therapeutic agents such as immune checkpoint inhibitor antibodies (e.g., anti-PD-1). In such compositions, the amounts of gelatin, silicate nanoparticles, and polypeptides are selected to form a shear-thinning hydrogel. In a particular embodiment of the present invention, the composition comprises about 1% (w / w) to about 5% (w / w) of gelatin (e.g., gelatin in any of the ranges of about 1% (w / w) to about 2% (w / w) to about 3% (w / w) to about 4% (w / w) to about 5% (w / w)) (in other words, a range between any two of the above values), and about 1% (w / w) to about 5% (w / w) of silicate nanoparticles (e.g., silicate nanoparticles in any of the ranges of about 1% (w / w) to about 2% (w / w) to about 3% (w / w) to about 4% (w / w) to about 5% (w / w)). In certain embodiments of the present invention, the composition comprises about 0.5% (w / w) to about 85% (w / w) of gelatin and silicate nanoparticles (e.g., gelatin and silicate nanoparticles in any of the following ranges: about 0.5% (w / w) to about 1% (w / w) to about 2% (w / w) to about 5% (w / w) to about 10% (w / w) to about 25% (w / w) to about 50% (w / w) to about 75% (w / w) to about 85% (w / w)). In certain embodiments of the present invention, the ratio of silicate nanoparticles to gelatin is about 1.0 to about 0.1 (e.g., in any of the following ranges: about 1.0 to about 0.9 to about 0.8 to about 0.7 to about 0.6 to about 0.5 to about 0.4 to about 0.3 to about 0.2 to about 0.1). In certain embodiments, the gelatin is methacrylated gelatin (GelMA), acrylic gelatin, or thiolated gelatin. In certain embodiments of the present invention, the composition contains about 0.5% (w / w) to about 99% (w / w) of water (for example, water in any of the following ranges: about 0.5% (w / w) to about 1% (w / w) to about 2% (w / w) to about 5% (w / w) to about 10% (w / w) to about 25% (w / w) to about 50% (w / w) to about 75% (w / w) to about 90% (w / w) to about 95% (w / w) to about 97.5% (w / w) to about 99% (w / w)).In certain embodiments, the composition comprises about 0.01% (w / w) to about 20% (w / w) of polypeptide therapeutic agent (for example, polypeptide therapeutic agent in any of the following ranges: about 0.01% (w / w) to about 0.02% (w / w) to about 0.5% (w / w) to about 1% (w / w) to about 2% (w / w) to about 5% (w / w) to about 10% (w / w) to about 15% (w / w) to about 20% (w / w)). In certain embodiments, the polypeptide therapeutic agent is selected from the group consisting of cytokines, antibodies, anti-cancer enzymes, tumor antigens, and pro-apoptotic proteins or peptides. In certain embodiments, the polypeptide therapeutic agent is selected from the group consisting of IL-2, IL-12, IFN-γ, TNF-α, trastuzumab, partuzumab, bevacizumab, rituximab, atezolizumab, durvalumab, nivolumab, caspase-3, recombinase (Cre), L-asparaginase, RNase A, DNase I, cGAMP synthase, granzyme B, catalase, OVA, TRP2, Hpg10025-33, p-AH1-A5, MAGE-A3, p53, cytochrome c, KLAKLAKKLAKLAKGG, PTEN, and saporins. Typically, these compositions further include pharmaceutically acceptable excipients selected from the group consisting of preservatives, tonicity modifiers, surfactants, viscosity modifiers, sugars, and pH modifiers. In some embodiments of the present invention, the composition further includes human cancer cells. The compositions of the present invention comprise one or more polypeptide therapeutic agents or other agents. In an exemplary embodiment of the present invention, the therapeutic agent is an anti-PD-1 antibody.

[0007] In some embodiments of the present invention, the amount of the component is such that less than 10% or less than 5% of the drug is released from the shear-thinned hydrogel over a period of 15 days when placed in an environment having a pH of 7.4, and more than 5% or more than 10% is released from the shear-thinned hydrogel over a period of 15 days when placed in an environment having a pH of 5.0. In certain embodiments of the present invention, the shear-thinned hydrogel requires an injection force of at least 5 Newtons but less than 30 Newtons to extrude the shear-thinned hydrogel from a 0.8 mm (2.4 Fr) catheter / 1 mL (1 cc) syringe.

[0008] Another embodiment of the present invention is a method for preparing a shear-thinning biocompatible composition disclosed herein, comprising the step of combining spherical silica nanoparticles, gelatin, and small therapeutic molecules such as an anti-PD-1 antibody, and optionally a pharmaceutically acceptable excipient, to form the shear-thinning biocompatible composition. In specific embodiments of these methods, the surface properties of the spherical silica nanoparticles, the median diameter of the spherical silica nanoparticles, the relative amount of the spherical silica nanoparticles, and / or the relative amount of the gelatin, etc., are selected to adjust or modulate one or more rheological properties or polypeptide release profile of the shear-thinning biocompatible composition. By adjusting the mechanical properties of the composition of the present invention in this manner, embodiments of the present invention can be adapted for use in a variety of different clinical applications.

[0009] Another embodiment of the present invention is a method for delivering a shear-thinning biocompatible composition disclosed herein to a pre-selected site (e.g., an in vivo site including cancer cells). Typically, such a method includes the steps of: placing the composition in a container (e.g., a catheter) having a first end and a second end including an opening; applying force to the second end of the container, such force is sufficient to liquefy the composition; and then delivering the composition from the container through the opening to the pre-selected site. In some embodiments, the container includes a syringe loaded with the composition, and the syringe is configured to be in fluid communication with a needle and / or catheter tube. Embodiments of such a method include therapeutic regimens using the shear-thinning biocompatible composition disclosed herein to deliver therapeutic polypeptides such as antibodies. In certain embodiments, the disclosure relates to a method for treating a solid tumor in a subject requiring it, comprising the step of administering to the subject a therapeutically effective amount of the shear-thinning biocompatible composition disclosed herein. In certain embodiments, the solid tumor is selected from vascular tumors, brain tumors (e.g., meningioma or glioblastoma), spinal cord tumors, carotid body tumors, liver cancer, lung cancer, neuroendocrine tumors, kidney tumors, pancreatic tumors, or prostate tumors. In certain embodiments, the solid tumor is selected from skin cancers (e.g., melanoma, mast cell tumors, squamous cell carcinoma, or basal cell tumors), breast cancer, sarcomatous tumors (e.g., fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, osteosarcoma, or chondrosarcoma), and lymphomatous tumors. In certain embodiments, the composition is administered to a topical area requiring treatment. In certain embodiments, the composition provides sustained release of a therapeutically effective amount of polypeptide therapeutic agent to the topical area. In certain embodiments, the topical area includes a solid tumor. In certain embodiments, the composition provides tumor embolization and sustained release of a therapeutically effective amount of polypeptide therapeutic agent to the tumor.

[0010] Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. However, it should be understood that the detailed description and specific examples illustrate, but are not limiting, some embodiments of the present invention. Many changes and modifications can be made within the scope of the present invention without departing from the spirit of the invention, and the present invention includes all such modifications. [Brief explanation of the drawing]

[0011] [Figure 1A] This shows anti-PD-1 released from STH with different laponite / gelatin ratios in PBS (pH=7.4). [Figure 1B] This shows the release of anti-PD-1 from various compositions loaded with different antibody concentrations in PBS (pH=7.4). [Figure 1C] Same as above. [Figure 1D] This shows release profile data from various compositions containing STBs loaded with anti-PD-1 antibodies under different pH conditions. [Figure 1E] Same as above. [Figure 2A] Shear stress data from various compositions of STB with different antibody concentrations are shown. [Figure 2B] Same as above. [Figure 2C] This shows the mechanical properties of various compositions of STB with different antibody concentrations. [Figure 2D] Same as above. [Figure 3A] A photograph of a tumor treated according to an embodiment of the present invention is shown. [Figure 3B] This report provides data from an in vivo study evaluating the direct contact anticancer effect of STBs loaded with anti-PD-1 antibodies. [Figure 3C] The data presented here comes from a study of tumor weight on the day of slaughter. [Figure 4] Scheme 1 illustrates the dual function of injectable STH for enhancing tumor treatment by combining anti-PD-1 delivery and intravascular embolization. [Modes for carrying out the invention]

[0012] The description of embodiments may refer to accompanying drawings illustrating specific embodiments that form part of this specification and enable the implementation of the invention. It should be understood that other embodiments may be utilized and structural modifications may be made without departing from the scope of the invention. Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have meanings generally understood by those skilled in the art to which the invention pertains. In some cases, terms having generally understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from those generally understood in the art. Many of the aspects of techniques and procedures described or referenced herein are well understood and commonly used by those skilled in the art. The following text describes various embodiments of the invention.

[0013] Shear-thinning biomaterial (STB) technology offers unique properties that allow internally loaded solid polymers and drugs to be easily delivered directly to target regions by means such as catheters. Considering this, the inventors have developed a novel class of shear-thinning biomaterials using silica nanoparticles and gelatin-based polymers. Embodiments of the present invention include, for example, shear-thinning biocompatible compositions comprising silica nanoparticles, gelatin, and polypeptides such as antibodies. Among inorganic composite compositions, silica is classified as "Generally Recognized As Safe (GRAS)" by the FDA and is considered one of the most biocompatible materials. Considering this, silicate nanoparticles have been used in the pharmaceutical, cosmetic, and food industries as active ingredients or rheological modifiers due to their uniform particle size and surface charge and bioactive properties [9, 10]. In addition, the size, structure, and surface properties of silica nanoparticles can be easily controlled, and these properties allow silica nanoparticles to provide a class of useful materials for a wide variety of biomedical applications.

[0014] The compositions of the present invention may comprise further components (e.g., further polymers, excipients, therapeutic agents, etc.). For example, the compositions of the present invention may comprise one or more FDA-approved polymers or cell-compatible polymers. Examples of such polymers include alginates, chitosan, collagen, hyaluronic acid (HA), chondroitin sulfate (ChS), dextrin, gelatin, fibrin, peptides, and silk. Synthetic polymers (e.g., poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poloxamer (Pluronic®) (PEO-PPO-PEO), polyoxamine (Tetronic®) (PEO-PPO), poly(vinyl alcohol) (PVA), poly(lactic acid-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), polycaprolactone (PCL), poly(L-glutamic acid) (PLga), polyanhydride, poly(N-isopropylacrylamide) (PNIPAAm), polyaniline, etc.) may also be included in the compositions of the present invention. As is known in the art, hydrogel preparations may be made to include materials that are crosslinked either chemically or physically.

[0015] Certain embodiments of the compositions of the present invention include, for example, pharmaceutically active ingredients (selected from the group consisting of, for example, preservatives, tonicity modifiers, surfactants, viscosity modifiers, sugars, and pH modifiers). For compositions suitable for administration to humans, the term “excipients” means, but is not limited to, the components described in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2006) (the contents of which are incorporated herein by reference).

[0016] If necessary, the compositions of the present invention may comprise one or more polypeptide therapeutic agents (e.g., immune checkpoint inhibitors). In embodiments of the present invention disclosed herein, the polypeptide therapeutic agent is an anti-PD-1 antibody. The compositions of the present invention may be formulated for use as carriers or scaffolds for therapeutic agents such as drugs, cells, proteins, and bioactive molecules (e.g., enzymes). As carriers, such compositions can incorporate drugs and deliver them to desired sites in the body for the treatment of various pathological conditions. Pathological conditions include, for example, infectious and inflammatory diseases (e.g., Parkinson's disease, bacterial and antimicrobial infections, diabetes, etc.), as well as cancers (e.g., colon cancer, lung cancer, breast cancer, ovarian cancer, lymphoma, etc.). Furthermore, as scaffolds, the compositions of the present invention can provide flexible retention spaces for cells and other agents for use in tissue repair and regeneration of desired tissues (e.g., repair of cartilage, bone, retina, brain, and nerve tissue, vascular regeneration, wound healing, etc.). Furthermore, embodiments of the present invention may comprise immunomodulators useful in immunotherapy, for example, to modulate components of the immune system. Specific exemplary materials and methods that may be suitable for use in such embodiments of the present invention are, for example, *Hydrogels: Design, Synthesis and Application in Drug Delivery and Regenerative Medicine 1st Edition*, Singh, Laverty and Donnelly Eds; and *Hydrogels in Biology and Medicine (Polymer Science and Technology) UK*. This can be found in the ed.Edition by J. Michalek et al.

[0017] Embodiments of the present invention include, for example, compositions comprising gelatin, silicate nanoparticles, and polypeptides. In such compositions, the amounts of gelatin, silicate nanoparticles, and polypeptides are selected to form a shear-thinning hydrogel. In certain embodiments of the present invention, the composition comprises from about 1% to about 5% gelatin and from about 1% to about 5% silicate nanoparticles. Typically, these compositions further comprise a pharmaceutical excipient selected from the group consisting of preservatives, tonicity modifiers, surfactants, viscosity modifiers, sugars, and pH adjusters. In some embodiments of the present invention, the composition further comprises human cancer cells. In an exemplary embodiment of the present invention, the polypeptide is an anti-PD-1 antibody.

[0018] In some embodiments of the present invention, the composition is disposed within a container (e.g., a catheter) selected for its ability to facilitate the user's adjustment of one or more rheological properties of the composition. Specific exemplary materials and methods that may be adapted for use in embodiments of the present invention are found, for example, in Biomedical Hydrogels: Biochemistry, Manufacture and Medical Applications (Woodhead Publishing Series in Biomaterials) 1st Edition; Steve Rimmer (Editor). As shown in FIGS. 2A - 2D, which are exemplary embodiments of the present invention, the components of the composition are selected to provide the desired rheological properties.

[0019] Another embodiment of the present invention is a method for delivering a shear-thinning biocompatible composition disclosed herein to a pre-selected site (e.g., an in vivo site where an individual has experienced trauma or injury, or exhibits a lesion such as cancer). Typically, such a method includes the steps of: placing the composition in a container (e.g., a catheter) having a first end and a second end including an opening; applying a force to the second end of the container, such that the force is sufficient to liquefy the composition; and then delivering the composition from the container through the opening to the pre-selected site. Embodiments of such a method include, for example, therapeutic regimens using the shear-thinning biocompatible composition disclosed herein to deliver a therapeutic agent, as shown in Figures 3A-3C.

[0020] Yet another embodiment of the present invention is a method of making a shear-thinning biocompatible composition disclosed herein, the method comprising combining silica nanoparticles and gelatin, a polypeptide (e.g., an anti-PD-1 antibody), and optionally a pharmaceutical excipient together to form a shear-thinning biocompatible composition. In certain embodiments of these methods, the surface properties of the silica nanoparticles, the median diameter of the silica nanoparticles, the relative amount of the silica nanoparticles, and / or the relative amount of gelatin, etc., are selected to adjust or regulate one or more rheological properties or polypeptide release profile properties of the shear-thinning biocompatible composition. By adjusting the mechanical properties of the compositions of the present invention in this manner, embodiments of the present invention can be adapted for use in a variety of different clinical applications. In this regard, for example, U.S. Patent Application Publication Nos. 20050227910, 20100120149, 20120315265, 20140302051, and 20190290804; as well as Lee, Biomaterials Research volume 22, Article number: 27 (2018); Thambi et al., J Control Release. 2017 Dec 10;267:57-66.doi:10.1016 / j.jconrel.2017.08.006.Epub 2017 Aug 4; and Gianonni et al., Biomater.Sci., 2016 (the contents of which are incorporated by reference), various materials and methods recognized in the art, such as those disclosed therein, can be adapted for use in embodiments of the present invention.

[0021] Exemplary Materials and Methods of the Present Invention As an exemplary embodiment of the present invention, the inventors developed an injectable shear-thinning biomaterial (STB) loaded with an anti-PD-1 antibody using silicate nanoplatelets (LAPONITE XLG) and a gelatin-based polymer. Shear-thinning biomaterial technology offers unique properties that allow the internally loaded drug to be easily and directly delivered to a target region through a needle or catheter. The inventors focused on the fact that shear-thinning biomaterials are 1) injectable and easy to apply, and 2) exhibit high localization due to high mechanical stability after injection. Therefore, the inventors loaded the anticancer drug anti-PD-1 antibody onto the shear-thinning biomaterial to treat solid tumors.

[0022] The inventors tested three different STB compositions (gelatin 4.5% / LAPONITE 1.5% [6NC25]; gelatin 3.0% / LAPONITE 3.0% [6NC50]; gelatin 1.5% / LAPONITE 4.5% [6NC75]) on ICI-loaded biomaterials and analyzed the anti-PD-1 antibody release profiles for 30 days (Figure 1A). The release rate of anti-PD-1 from the STB decreased sharply with increasing LAPONITE content in the STB. As shown in Figures 1B and 1C, when the antibody concentration was adjusted to the range of 0.5 μg / mg STB to 5.0 μg / mg STB, the release rate of anti-PD-1 significantly increased from 1 μg / mg STB (2.58 ± 0.05%) to 5.0 μg / mg STB (8.92 ± 0.14%) in the case of 6NC50 (Figure 1C). On the other hand, the highest release of anti-PD-1 was observed at an antibody concentration of 1 μg / mg STB for 6NC25 (Figure 1B). The effect of PBS with different pH values ​​(7.4, 6.0, and 5.0) on anti-PD-1 release was evaluated (Figures 1D and 1E). The results show that anti-PD-1 release was significantly improved under acidic conditions.

[0023] As shown in Figures 2A and 2B, similar shear stress-shear rate curves were observed in 6NC25 loaded with different concentrations of anti-PD-1, indicating no significant effect on the shear viscosity reduction behavior of 6NC25 and 6NC50 after antibody loading at 37°C. The storage modulus reflects the recovery from liquid-like behavior to solid-like behavior after application of high strain (100%) and low strain (1%). Figures 2C and 2D show the storage modulus of STB loaded with different concentrations of anti-PD-1 after 5 cycles of high (100%) to low (1%) vibration strain amplitude. For 6NC25 and 6NC50, the storage modulus during 100% strain vibration was slightly lower than the initial value during high strain after 5 cycles, indicating a rapid recovery of the organic-inorganic hybrid network consisting of laponite and gelatin. These results indicate that both the shear reduction capacity and storage modulus of STB remain stable at physiological temperatures, regardless of the load of anti-PD-1 at different concentrations.

[0024] Finally, the in vivo anticancer efficacy of ICI-STB was analyzed in a melanoma tumor model created by subcutaneous injection of B16F10 melanoma tumor cells into C57Bl / 6J mice (Figure 3A). Generally, STB (6NC25) itself did not show anticancer efficacy, but tumor size was reduced in 6NC25 loaded with anti-PD-1. Tumor growth curves measured every two days by calypso were found to inhibit melanoma growth in the anti-PD-1 loaded STB group (Figure 3B). Interestingly, the median tumor weight of ICI-STB (anti-PD-1 loaded 6NC25) was smaller than that of other tumors (Figure 3C).

[0025] In summary, the inventors have developed a novel shear-thinning biomaterial having a composite of an anti-PD-1 antibody, gelatin, and biocompatible silica nanoparticles with selected desired properties. By adjusting the properties of the silica nanoparticles, such as the particle size and composition of the gelling / silica nanoparticles, the mechanical and rheological properties can be carefully tuned to meet different requirements. All compositions used in these experiments are injectable through catheters of different sizes. Rheological tests showed rapid recovery and mechanical stability due to the shear-thinning properties. Gelatin-silica nanoparticle-based STBs exhibit excellent biological stability, thermogenic extrusion, shear-thinning behavior, and rapid network recovery. These attractive physicochemical properties are advantageous for easy in vivo administration, and gelatin-silica nanoparticle-based STBs may hold great potential for drug delivery, intravascular embolization, tissue regeneration, bioprinting, and other biomedical applications.

[0026] References

[0027] [Table 1-1]

[0028] [Table 1-2]

[0029] [Table 1-3]

[0030] [Table 1-4]

[0031] All publications referenced herein (for example, the references listed numerically above, and U.S. Patent Application Publication No. 20180104059) are incorporated herein by reference to disclose and describe aspects, methods, and / or materials in relation to the cited publications.

[0032] The appendices included in this provisional application provide a disclosure of the invention formatted for publication in a journal. This disclosure illustrates various aspects and embodiments of the invention. These appendices are incorporated herein by reference.

Claims

1. Gelatin and, Silicate nanoparticles and A polypeptide therapeutic agent, A shear-thinning hydrogel composition comprising the gelatin, the silicate nanoparticles, and the therapeutic agent, wherein the gelatin, silicate nanoparticles, and the therapeutic agent form a shear-thinning hydrogel.

2. Approximately 1% (w / w) to approximately 5% (w / w) gelatin, Approximately 1% (w / w) to approximately 5% (w / w) silicate nanoparticles, The composition according to claim 1, comprising:

3. 7. When placed in an environment with a pH of 4, less than 10% or 5% of the drug is released from the shear-thinning hydrogel over a period of 15 days. The composition according to claim 1 or 2, wherein when placed in an environment having a pH of 5.0, 5% or more than 10% is released from the shear-thickened hydrogel over a period of 15 days.

4. The composition according to any one of claims 1 to 3, comprising approximately 0.5% (w / w) to approximately 85% (w / w) of gelatin and silicate nanoparticles.

5. The composition according to any one of claims 1 to 4, wherein the ratio of silicate nanoparticles to gelatin is about 1.0 to about 0.

1.

6. The composition according to any one of claims 1 to 5, wherein the gelatin is methacrylated gelatin (GelMA), acrylic gelatin, or thiolated gelatin.

7. The composition according to any one of claims 1 to 6, further comprising about 0.5% (w / w) to about 99% (w / w) of water.

8. A composition according to any one of claims 1 to 7, comprising approximately 0.01% (w / w) to approximately 20% (w / w) of a polypeptide therapeutic agent.

9. The composition according to any one of claims 1 to 8, wherein the polypeptide therapeutic agent is selected from the group consisting of cytokines, antibodies, anti-cancer enzymes, tumor antigens, and apoptosis-promoting proteins or peptides.

10. The polypeptide therapeutic agents include IL-2, IL-12, IFN-γ, TNF-α, trastuzumab, patuzumab, bevacizumab, rituximab, atezolizumab, durvalumab, nivolumab, caspase-3, recombinase (Cre), L-asparaginase, RNase A, DNase I, cGAMP synthase, granzyme B, catalase, OVA, TRP2, and Hpg100. ２５－３３ The composition according to claim 9, selected from the group consisting of p-AH1-A5, MAGE-A3, p53, cytochrome c, KLAKLAKKLAKLAKGG, PTEN, and saporin.

11. The composition according to any one of claims 1 to 8, wherein the polypeptide therapeutic agent comprises an anti-PD-1 antibody.

12. The composition according to any one of claims 1 to 11, wherein the shear-thinning hydrogel requires an injection force of at least 5 Newtons but less than 30 Newtons to extrude the shear-thinning hydrogel from a 0.8 mm (2.4 Fr) catheter / 1 mL (1 cc) syringe.

13. The composition according to any one of claims 1 to 12, further comprising human cancer cells.

14. The composition according to any one of claims 1 to 3, further comprising a pharmaceutically acceptable excipient selected from a preservative, a tonicity modifier, a surfactant, a viscosity modifier, a sugar, and a pH adjuster.

15. The composition according to any one of claims 1 to 14, wherein the composition is placed inside a catheter.

16. The composition according to any one of claims 1 to 15, wherein the silicate nanoparticles include LAPONITE.

17. A method for delivering a composition according to any one of claims 1 to 16 to a pre-selected site, The steps include placing the composition in a container having a first end and a second end, which include an opening, A step of applying force to the second end of the container, wherein the force is sufficient to liquefy the composition, The steps include delivering the composition from the container to the pre-selected portion through the opening, Methods that include...

18. The method according to claim 17, wherein the said site is an in vivo site.

19. The method according to claim 18, wherein the aforementioned site is located in vivo and contains cancer cells.

20. The method according to claim 17, wherein the container is a catheter.

21. A method for preparing a composition according to any one of claims 1 to 16, comprising combining silica nanoparticles, gelatin, polypeptide, and optionally a pharmaceutical excipient to form a shear-thinning biocompatible composition.

22. The method according to claim 21, wherein the relative amounts of silica nanoparticles and / or gelatin are selected to adjust or modulate one or more rheological properties of the composition and / or the release profile of the polypeptide from the shear-thinning hydrogel.

23. A method for treating a solid tumor in a subject requiring treatment, comprising administering a therapeutically effective amount of the composition described in any one of claims 1 to 16 to the subject.

24. The method according to claim 23, wherein the solid tumor is selected from the group consisting of vascular tumors, brain tumors, spinal cord tumors, carotid body tumors, liver cancer, lung cancer, neuroendocrine tumors, kidney tumors, pancreatic tumors, and prostate tumors.

25. The method according to claim 25, wherein the brain tumor is a meningioma or glioblastoma.

26. The method according to claim 23 or 24, wherein the solid tumor is selected from the group consisting of skin cancer, breast cancer, sarcomatous tumors, and lymphomatous tumors.

27. The aforementioned skin cancer is selected from the group consisting of melanoma, mast cell tumor, squamous cell carcinoma, or basal cell tumor. The method according to claim 27, wherein the sarcoma tumor is selected from the group consisting of fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, osteosarcoma, and chondrosarcoma.

28. The method according to any one of claims 23 to 28, wherein the composition is administered to a local area requiring treatment.

29. The method according to claim 29, wherein the composition provides a sustained release of a therapeutically effective amount of the polypeptide therapeutic agent to the local area.

30. The method according to claim 29, wherein the local region includes a solid tumor.

31. The method according to any one of claims 23 to 30, wherein the composition provides tumor embolus formation and sustained release of a therapeutically effective amount of the polypeptide therapeutic agent to the tumor.

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