INJECTABLE SHEAR-THINNING HYDROGEL CONTAINING THERAPEUTIC AGENT FOR ENHANCED TUMOR THERAPY

Abstract

We have developed novel shear-thinning biomaterials using silica nanoparticles, gelatin-based polymers and small molecules such as doxorubicin. Shear-thinning biomaterial technology offers enables polymers and drugs loaded inside such polymers to be easily delivered directly through catheters into target area for use, for example, in cancer therapy and immunotherapy. When a force above a certain threshold is applied to inject such materials, they “thin” and behaves as a semi-solid, allowing the material to readily flow through a catheter. When the force is removed, the material instantly becomes a soft solid with significant cohesive properties that prevent it from dislodging or breaking up.

Claims

1. A composition of matter comprising: a polymeric gelatin; silicate nanoparticles; and a therapeutic agent having a molecular weight less than 1000 Daltons; wherein: the gelatin, the silicate nanoparticles and the therapeutic agent form a shear thinning hydrogel.

2. The composition of claim 1, wherein the composition comprises: from about 1% to about 5% polymeric gelatin; and from about 1% to about 5% silicate nanoparticles.

3. The composition of claim 2, wherein when disposed in an environment having a pH of 7.4, less than 15% or 20% of agent is released from the shear thinning hydrogel over a period of 15 days; and when disposed in in an environment having a pH of 5.0, more than 15% or 20% is released from shear thinning hydrogel over a period of 15 days.

4. The composition of claim 3, wherein the therapeutic agent is doxorubicin.

5. The composition of claim 1, 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 2.4Fr Catheter/1 cc syringe.

6. The composition of claim 1, further comprising a human cancer cell.

7. The composition of claim 1, further comprising a pharmaceutical excipient selected from the group consisting of: a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar and a pH adjusting agent.

8. The composition of claim 1, wherein the composition is disposed within a catheter.

9. The composition of claim 1, wherein the silicate nanoparticles comprise a LAPONITE.

10. A method of delivering a composition to a preselected site, the composition comprising a polymeric gelatin, silicate nanoparticles, and a therapeutic agent having a molecular weight less than 1000 Daltons, wherein the gelatin, the silicate nanoparticles and the therapeutic agent form a shear thinning hydrogel, the method comprising: disposing the composition in a vessel having a first end comprising an opening and a second end; applying a force to the second end of the vessel, wherein the force is sufficient to liquify the composition; delivering the composition out of the vessel through the opening and to the preselected site.

11. The method of claim 10, wherein the site is an in vivo site.

12. The method of claim 11, wherein the site is at an in vivo location comprising cancerous cells.

13. The method of claim 10, wherein the vessel is a catheter.

14. The method of claim 10, wherein the composition comprises: from about 1% to about 5% polymeric gelatin; and from about 1% to about 5% silicate nanoparticles.

15. The method of claim 14, wherein when disposed in an environment having a pH of 7.4, less than 15% or 20% of agent is released from the shear thinning hydrogel over a period of 15 days; and when disposed in in an environment having a pH of 5.0, more than 15% or 20% is released from shear thinning hydrogel over a period of 15 days.

16. The method of claim 15, wherein the therapeutic agent is doxorubicin.

17. The method of claim 10, 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 2.4Fr Catheter/1 cc syringe.

18. The method of claim 10, wherein the composition further comprises a human cancer cell.

19. A method of making a composition in accordance with claim 1, comprising combining together silica nanoparticles, gelatin, a therapeutic agent having a molecular weight less than 1000 Daltons and optionally a pharmaceutical excipient so as to form a shear-thinning biocompatible composition.

20. The method of claim 19, wherein a relative amount of silica nanoparticles; and/or a relative amount of gelatin is selected to tune or modulate one or more rheological properties of the composition and/or a release profile of the therapeutic agent from the shear thinning hydrogel.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1A, FIG. 1B and FIG. 1C show release profile data from various compositions of doxorubicin-loaded STBs under different pH conditions.

[0016] FIG. 2A, FIG. 2B and FIG. 2C show injection force data from various compositions of STBs.

[0017] FIG. 3A, FIG. 3B, and FIG. 3C show the rheological properties of various compositions of STBs.

[0018] FIG. 4A shows photographs of various doxorubicin-loaded STBs. FIG. 4B provides a schematic illustration of in vitro study for assessing direct-contact anti-cancer effect of doxorubicin-loaded STBs. FIG. 4C provides a schematic illustration of in vitro study for assessing anti-cancer effect of doxorubicin-loaded STBs through transwell release.

[0019] FIG. 5A, FIG. 5B and FIG. 5C show data from an in vitro anti-cancer efficacy evaluation through direct-contact of doxorubicin-loaded STBs.

[0020] FIG. 6A, FIG. 6B and FIG. 6C shows data from an in vitro anti-cancer efficacy evaluation through transwell release of doxorubicin-loaded STBs.

[0021] FIG. 7A shows data from an in vivo anti-cancer efficacy study of DOX-STB for melanoma. FIG. 7B shows data from a tumor growth curve for the melanoma-bearing mouse with doxorubicin-loaded STBs. FIG. 7C shows data from a tumor weight study on the day of sacrifice

DETAILED DESCRIPTION OF THE INVENTION

[0022] In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the aspects of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. The following text discusses various embodiments of the invention.

[0023] Shear-thinning biomaterial (STB) technology offers unique properties enabling a solid polymer and drugs loaded inside to be easily delivered directly through means such as catheters into a target area. In view of this, we have developed a class of novel shear-thinning biomaterials using silica nanoparticles and gelatin-based polymers. Embodiments of the invention include, for example, a shear-thinning biocompatible composition of matter comprising silica nanoparticles and gelatin. Among inorganic composite compositions, silica is classified as “Generally Recognized As Safe (GRAS)” FDA, and is considered one of the most biocompatible materials. In view of this, silicate nanoparticles have been employed in pharmaceutical, cosmetic and food industries as active ingredients or rheological modifiers due to their uniform particle size with electrical surface charges and bioactive properties [9, 10]. In addition, the size, structure, surface properties of silica nanoparticles can be easily tuned, properties which allow silica nanoparticles to provide a class of materials that are useful in a wide variety of biomedical applications.

[0024] The compositions of the invention can include further constituents such as additional polymers, excipients, therapeutic agents and the like. For example, compositions of the invention can include one or more Food and Drug Administration (FDA) approved or cytocompatible polymers. Such polymers include alginate, chitosan, collagen, hyaluronic acid (HA), chondroitin sulfate (ChS), dextrin, gelatin, fibrin, peptide, and silk. Synthetic polymers such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poloxamer (Pluronic®) (PEO-PPO-PEO), polyoxamine (Tetronic®) (PEO-PPO), poly(vinyl alcohol) (PVA), poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), polycaprolactone (PCL), poly(L-glutamic acid) (PLga), polyanhydrides, poly(N-isopropylacrylamide) (PNIPAAm), polyaniline and the like can also be included in compositions of the invention. As is known in the art, preparations of hydrogels can be made to include either chemically or physically crosslinked materials.

[0025] Certain embodiments of the compositions of the invention include, for example a pharmaceutical excipient such as one selected from the group consisting of a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar and a pH adjusting agent. For compositions suitable for administration to humans, the term “excipient” is meant to include, but is not limited to, those ingredients described in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2006) the contents of which are incorporated by reference herein.

[0026] Optionally, the compositions of the invention include one or more small molecule therapeutic agents (i.e. ones having a molecular weight less 1000 Daltons) such as an anti-inflammatory agent, an agent that modulates coagulation, an antibiotic agent, a chemotherapeutic agent or the like. In a working embodiment of the invention disclosed herein, the small molecule therapeutic agent is doxorubicin. Compositions of the invention can be formulated for use as carriers or scaffolds of therapeutic agents such as drugs, cells, proteins, and bioactive molecules (e.g., enzyme). As carriers, such compositions can incorporate the agents and deliver them to a desired site in the body for the treatments of a variety of pathological conditions. These include, for example, infectious and inflammatory diseases (e.g. Parkinson's disease, bacterial and antimicrobial infection, diabetes and the like) as well as cancers (e.g. colon, lung, breast, ovarian, lymphoma cancers and the like). In addition, as scaffolds, compositions of the invention can provide a flexible dwelling space for cells and other agents for use in tissue repair and the regeneration of desired tissues (e.g. for cartilage, bone, retina, brain, and, neural tissue repair, vascular regeneration, wound healing and the like). Moreover, embodiments of the invention can include immunomodulatory agents useful for immunotherapy in order to, for example, enhance components of the immune system. Certain illustrative materials and methods that can be adapted for use in such embodiments of the invention are found, for example in 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 ed. Edition by J. Michalek et al.

[0027] Embodiments of the invention include, for example, a composition of matter comprising a polymeric gelatin, silicate nanoparticles and a therapeutic agent having a molecular weight less than 1000 Daltons. In such compositions, amounts of the gelatin, the silicate nanoparticles and the therapeutic agent are selected to form a shear thinning hydrogel. In certain embodiments of the invention, the composition comprises from about 1% to about 5% polymeric gelatin, and from about 1% to about 5% silicate nanoparticles. Typically, these compositions further comprise a pharmaceutical excipient selected from the group consisting of a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar and a pH adjusting agent. In some embodiments of the invention, the composition further comprises a human cancer cell. In illustrative embodiments of the invention, the therapeutic agent is doxorubicin.

[0028] In some embodiments of the invention, the composition is disposed within a vessel (e.g. a catheter) selected for its ability to facilitate a user modulating one or more rheological properties of the composition. Certain illustrative materials and methods that can be adapted for use in embodiments of the 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. 3A-3C, illustrative embodiments of the invention, the components of the composition are selected to provide desirable rheological properties.

[0029] Another embodiment of the invention is a method of delivering a shear-thinning biocompatible composition disclosed herein to a preselected site (e.g. an in vivo location where an individual has experienced trauma or injury or exhibits a pathology such as a cancer). Typically, such methods comprise disposing the composition in a vessel having a first end comprising an opening and a second end (e.g. a catheter); applying a force to the second end of the vessel, wherein the force is sufficient to liquify the composition; and then delivering the composition out of the vessel through the opening and to the preselected site. Embodiments of such methods include treatment regimens that use a shear-thinning biocompatible composition disclosed herein to deliver a therapeutic agent as shown, for example, in FIGS. 4A-4C.

[0030] Yet another embodiment of the invention is a method of making a shear-thinning biocompatible composition disclosed herein comprising combining together silica nanoparticles and gelatin, a small molecule therapeutic agent and optionally a pharmaceutical excipient so as to form a shear-thinning biocompatible composition. In certain embodiments of these methods, a surface property of the silica nanoparticles, a median diameter of the silica nanoparticles, a relative amount of silica nanoparticles; and/or a relative amount of gelatin or the like is selected to tune or modulate one or more rheological properties of the shear-thinning biocompatible composition. By modulating the mechanical properties of the compositions of the invention in this manner, embodiments of the invention can be tailored for use in a variety of different clinical applications. In this context, a wide variety of art accepted materials and methods can be adapted for use in embodiments of the invention, for example those disclosed in U.S. Patent Publication Nos.: 20050227910, 20100120149, 20120315265, 20140302051 and 20190290804; and 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.

Illustrave Materials and Methods of the Invention

[0031] As an illustrative working embodiment of the invention, we developed a doxorubicin-loaded injectable shear thinning biomaterials (STB) using silicate nanoplatelet (LAPONITE XLG) and gelatin-based polymers. Shear-thinning biomaterial technology offers unique properties enabling drugs loaded inside to be easily delivered directly through needles or catheters into target area. We focused on the fact that shear thinning biomaterial is 1) injectable and easy to apply, and 2) exhibits high localization due to the high mechanical stability after injection. Therefore, we loaded anti-cancer drug, doxorubicin to the shear thinning material to treat solid tumor.

[0032] We 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]) for the doxorubicin-loading biomaterials. The release profile was analyzed for 30 days under the three pH conditions (pH 5.0, 6.0, 7.4) (FIGS. 1A, 1B, 1C). Gelatin released doxorubicin within 3 days regardless of pH condition, while LAPONITE released less than 20% of doxorubicin only in acidic condition. Interestingly, all three STB compositions showed high release of doxorubicin under acidic conditions at pH 5, but little release at 7.4, which is normal physiological pH. It was also observed that the higher the LAPONITE ratio, the lower the release of doxorubicin. Injection force measurement showed that all three compositions we tested could be delivered via a 2.4Fr microcatheter and a 5Fr general catheter (FIGS. 2A, 2B, 2C). All STB compositions were within favorable injection force range (<30N), but the higher the LAPONITE content, the higher the injection force. Rheological measurements found that all STB compositions exhibit shear thinning properties (non-Newtonian behavior, decreasing viscosity upon shear rate increase) and the storage modulus is higher with higher LAPONITE content (FIGS. 3A, 3B, 3C).

[0033] We prepared two types of STB by mixing gelatin polymer and LAPONITE, and then added doxorubicin solution to make doxorubicin-loaded STB (DOX-STB) (FIG. 4A). To determine the in vitro anti-cancer efficacy of DOX-STB, we devised two types of in vitro tests. First, to analyze the anticancer efficacy of DOX-STB in contact with tumor, B16F10 melanoma cells were seeded on the culture plate, and then applied two STBs loaded with four ranges of doxorubicin (FIG. 4B). Other in vitro models were identified using a transwell release anticancer efficacy model to analyze anticancer efficacy due to doxorubicin released from DOX-STB rather than direct contact. (FIG. 4C). Both direct contact and transwell release in vitro models were analyzed for anticancer efficacy by performing live dead staining after 1, 3 and 7 days of DOX-STB application. Direct contact in vitro anti-cancer efficacy analysis showed that STB itself did not show anti-cancer effects, whereas most tumor cells were killed at a concentration of 10 ug/g or more of doxorubicin (FIGS. 5A, 5B, 5C) Similarly, indirect transwell release results showed that tumor cells were killed at 10 ug/g or more of DOX-STB (FIGS. 6A, 6B, 6C).

[0034] Finally, in vivo anti-cancer efficacy of DOX-STB was analyzed in a melanoma tumor model made by subcutaneous injection of B16F10 melanoma tumor cells into C57B1/6J mice (FIG. 7A). In gross, similar to in vitro experiments, STB itself did not show anti-cancer efficacy, while tumor size decreased in all groups loaded with doxorubicin (DOX, DOX-6NC25, DOX-6NC75). On the tumor growth curve measured by caliper every two days, both doxorubicin-loaded groups were found to inhibit melanoma growth (FIG. 7B). Interestingly, median tumor weights of DOX-6NC25 and DOX-6NC75 were less than DOX in tumor weight (FIG. 7C).

[0035] Taken together, our DOX-STB shows 1) pH-dependent doxorubicin release (doxorubicin release at acidic pH), 2) that DOX release is adjustable according to STB composition change, and 3) that direct and/or released doxorubicin from DOX-STB could kill the tumor cells, and 4) that DOX-STB could inhibit the tumor growth in animal models.

[0036] In summary, we developed a novel shear-thinning biomaterial with the composite of doxorubicin, gelatin and biocompatible silica nanoparticles with various properties. By tuning the properties of silica nanoparticles such as particles size and composition of gelation/silica nanoparticles, the mechanical and rheological properties can be carefully adjusted to meet different requirements. All the compositions used in these experiments are injectable through different sized catheters. The rheological tests showed rapid recovery and mechanical stability with shear-thinning characteristics. Gelatin-Silica nanoparticles-based STBs display superior biological stability, body temperature extrudability, shear-thinning behavior, and rapid network recoverability. These attractive physicochemical properties are favorable for easy administration in vivo, and a gelatin-Silica nanoparticle-based STB may hold great potential in drug delivery, endovascular embolization, tissue regeneration, bioprinting and for other biomedical applications.

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[0066] All publications mentioned herein (e.g. the references numerically listed above, and U.S. Patent Publication 20180104059) are incorporated herein by reference to disclose and describe aspects, methods and/or materials in connection with the cited publications.

[0067] The Appendix that is included with this provisional application provides invention disclosure that is formatted for publication in a journal. This disclosure shows various aspects and embodiments of the invention. This Appendix is incorporated by reference herein.