HYDROGELS BASED ON BLOOD PLASMA COMPONENTS, PROCESS AND USES THEREOF

Abstract

The present disclosure relates to bioactive hydrogels derived from human blood plasma. More particularly, the disclosure relates to multifunctional materials for cell encapsulation, cell culture platforms, medical treatment apparatus and methods, more particularly, hydrogels derived from human blood components and technologies for use of such materials in research, biomedical treatment, biotech and pharmaceutical industry. The disclosure further relates to 3D printable scaffolds, sponges, foams, fibers, particles, capsules, membranes and injectable systems comprising said hydrogel. Additionally, this disclosure allows for the controlled placement of biologically active components that may be delivered by the hydrogel compositions.

Claims

1. A hydrogel, comprising: a human plasma-derived element selected from the group consisting of: human platelet rich plasma, human platelet lysate, human plasma protein, and combinations thereof, wherein the human plasma-derived element is directly linked to at least one polymerizable moiety selected from the group consisting of: a methacrylate, acrylate, ethacrylate, thiol, acrylamide, aldehyde, azide, amine reactive group, cyclic oligosaccharides, and combinations thereof.

2. The hydrogel according to claim 1, further comprising a biocompatible polymer, wherein the biocompatible polymer comprises at least one polymerizable moiety selected from the group consisting of: a methacrylate, acrylate, ethacrylate, thiol, acrylamide, aldehyde, azide, amine reactive group, cyclic oligosaccharides, and combinations thereof.

3. The hydrogel according to claim 2, wherein the biocompatible polymer is selected from the group consisting of: chitosan, alginate, gelatin, collagen, laminarin, hyaluronic acid, polyethylene glycol, and combinations thereof.

4. The hydrogel according to claim 3, wherein the biocompatible polymer is linked to the human plasma-derived element or is mixed with the human plasma-derived element.

5. The hydrogel according to claim 3, wherein the biocompatible polymer is cross-linked with the plasma-derived element.

6. The hydrogel according to claim 1, wherein the human plasma-derived element has a concentration of one of: 5-90% w.sub.human plasma-derived element/V.sub.hydrogel; 5-90% w.sub.human platelet rich plasma/V.sub.hydrogel; 5-90% w.sub.human platelet lysate/V.sub.hydrogel; and 5-90% w.sub.human plasma protein/V.sub.hydrogel.

7. (canceled)

8. (canceled)

9. (canceled)

10. The hydrogel according to claim 1, wherein the human plasma-derived element has a degree of substitution between 10%-90%.

11. The hydrogel according to claim 1, wherein the human plasma protein is selected from the group consisting of: serum albumin, fibrinogen, angiotensinogen, vitronectin, apolipoprotein A, complement factors, immunoglobulins, serotransferrin, keratin, and combinations thereof.

12. The hydrogel according to claim 1, wherein the hydrogel further comprises a growth factor.

13. The hydrogel according to claim 1, wherein the growth factor is selected from the group consisting of: platelet-derived growth factor (PDGF), transforming growth factor (TGF), platelet factor interleukin (IL), platelet-derived angiogenesis factor (PDAF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor IGF, and fibroblast growth factor (FGF).

14. The hydrogel according to claim 1, wherein the hydrogel further comprises inorganic materials selected from the group consisting of: calcium phosphate, magnetic particles, bioglass particles, fibers, and combinations thereof.

15. The hydrogel according to claim 1, further comprising a biological agent selected from the group consisting of: a cell, a stem cell, a protein, a therapeutic agent, a biomolecule, a diagnostic marker, a probe, and combinations thereof.

16. The hydrogel according to claim 1, wherein the hydrogel is cross-linked via chemical crosslinking, guest-host complexes, or crosslinked enzymatically via transglutaminase, or by combinations thereof.

17. The hydrogel according to claim 1, further comprising: 10-15% w.sub.human platelet lysate/V.sub.hydrogel wherein the human platelet lysate is directly linked to at least one polymerizable moiety selected from a methacrylate; and wherein the degree of substitution is between 14-25.

18. The hydrogel according to claim 1, wherein the hydrogel is in the form of a foam, sponge, particle, capsule, fiber, membrane, or disc.

19. The hydrogel according to claim 18, wherein the foam, sponge, particle, capsule, fiber, membrane, or disc is lyophilized.

20. The hydrogel according to claim 19, wherein the foam, sponge, particle, capsule, fiber, membrane, or disc has one of: a 5-100% w.sub.human plasma-derived element/V.sub.hydrogel; a 5-100% w.sub.human platelet rich plasma/V.sub.hydrogel; a 5-100% w.sub.human platelet lysate/V.sub.hydrogel; and a 5-100% w.sub.human plasma protein/V.sub.hydrogel.

21. (canceled)

22. (canceled)

23. (canceled)

24. The hydrogel according to claim 1, wherein said hydrogel is further combined with a second hydrogel, forming a double-network or an inter-penetrating network.

25. The hydrogel according to claim 24, wherein the second hydrogel is selected from the group consisting of: chitosan, gelatin, alginate, laminarin, hyaluronic acid, poly(vinyl alcohol), polyacrylamide, carboxymethylcellulose, sodium starch glycolate, sodium carboxymethyl starch, dextran, dextran sulfate, xanthan, gellan, pectinic acid, deoxyribonucleic acids, ribonucleic acid, albumin, polyacrolein potassium, sodium glycine carbonate, poly(acrylic acid) and its salts, polyacrylonitrile, poly(styrene sulfonate), poly(aspartic acid), polylysine, polyvinylpyrrolidone, polyvinyl alcohol, CARBOPOL, ultramylopectin, poly(ethylene glycol), neutral cellulose derivatives, microcrystalline cellulose, powdered cellulose, cellulose fibers, and starch.

26. (canceled)

27. (canceled)

28. A method for preparing a hydrogel of the type comprising a human plasma-derived element selected from the group consisting of: human platelet rich plasma, human platelet lysate, human plasma protein, and combinations thereof, wherein the human plasma-derived element is directly linkable to at least one polymerizable moiety, the method comprising the step of: directly linking the human plasma-derived element to at least one polymerizable moiety selected from the group consisting of: a methacrylate, acrylate, ethacrylate, thiol, acrylamide, aldehyde, azide, amine reactive group, cyclic oligosaccharides, and combinations thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of disclosure.

[0065] FIG. 1: A) Possible route for the reaction of a protein/peptide with methacrylic anhydride. B) .sup.1H-NMR spectra for PL, PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) and PL with a degree of substitution, in particular a degree of methacrylation, of 25% (high modification) with distinctive peaks characteristics of methacrylate groups: double bound methacrylate (1) and CH.sub.3 of methacrylate group (2).

[0066] FIG. 2: Plasma derived hydrogels formed from PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) and PL with a degree of substitution, in particular a degree of methacrylation, of 25% (high modification), at 10% (w/v) and 15% (w/v).

[0067] FIG. 3: A) Representative compressive stress-strain curves for PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) and PL with a degree of substitution, in particular a degree of methacrylation, of 25% (high modification) hydrogels at 10% (w/v) and 15% (w/v), B) Young's modulus, C) ultimate strain and D) ultimate stress. Statistical analysis through unpaired t test showed significant differences (*p<0.05) between the analyzed groups.

[0068] FIG. 4: A) Representative cross-section SEM images of PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) and PL with a degree of substitution, in particular a degree of methacrylation, of 25% (high modification) hydrogels at 10% (w/v) and 15% (w/v). B) Pore size values obtained for PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) and PL with a degree of substitution, in particular a degree of methacrylation, of 25% (high modification) at 10% (w/v) and 15% (w/v) hydrogels. C) Swelling ratio for PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) and PL with a degree of substitution, in particular a degree of methacrylation, of 25% (high modification) hydrogels at 10% (w/v) and 15% (w/v). Statistical analysis through unpaired t test showed significant differences (*p<0.05) between the analyzed groups.

[0069] FIG. 5: Representative fluorescence images for: A) L929 and hASCs live/dead at 1 and 7 days of culture. B and C) L929 and hASCs DAPI/Phalloidin staining at 3 and 7 days of cell culture. (D) DAPI/Phalloidin staining for hydrogel microstructures of encapsulated L929 at 7 days of culture. DNA (E) and MTS (F) results for L929 and hASCs at 1 day, 3 and 7 days of cell culture. Statistical analysis through unpaired t test showed significant differences (*p<0.05) between the analyzed groups. L929 cells were encapsulated in PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) (10% w/v). hASCs cells were encapsulated in PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) (15% w/v).

[0070] FIG. 6: MG-63 spheroids embedded into Matrigel (A) and embedded into PL gels (B). C) Live-dead imaging of a MG-63 spheroid on PL gel.

[0071] FIG. 7: Plasma derived hydrogels formed from PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) and PL with a degree of substitution, in particular a degree of methacrylation, of 25% (high modification), at 10% (w/v), 15% (w/v) and 20% (w/v).

[0072] FIG. 8: A) Representative curves for storage modulus (G) for PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) hydrogels at 10% (w/v), 15% (w/v) and 20% (w/v). (B) Representative curves for storage modulus (G) for PL with a degree of substitution, in particular a degree of methacrylation, of 25% (high modification) hydrogels at 10% (w/v), 15% (w/v) and 20% (w/v). (C) t1/2 and tan for PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) hydrogels at 10% (w/v), 15% (w/v) and 20% (w/v) and for PL with a degree of substitution, in particular a degree of methacrylation, of 25% (high modification) hydrogels at 10% (w/v), 15% (w/v) and 20% (w/v). Statistical analysis through two-tailed unpaired t test showed significant differences (*p<0.01) between the analyzed groups.

[0073] FIG. 9: A) Representative compressive stress-strain curves for PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) and PL with a degree of substitution, in particular a degree of methacrylation, of 25% (high modification) hydrogels at 10% (w/v), 15% (w/v) and 20% (w/v), B) Young's modulus, C) ultimate strain and D) ultimate stress. Statistical analysis through two-tailed unpaired t test showed significant differences (*p<0.01) between the analyzed groups.

[0074] FIG. 10: A) Representative cross-section SEM images of PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) and PL with a degree of substitution, in particular a degree of methacrylation, of 25% (high modification) hydrogels at 10% (w/v), 15% (w/v) and 20% (w/v). B) Pore size values obtained for PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) and PL with a degree of substitution, in particular a degree of methacrylation, of 25% (high modification) at 10% (w/v), 15% (w/v) and 20% (w/v) hydrogels. C) Swelling ratio for PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) and PL with a degree of substitution, in particular a degree of methacrylation, of 25% (high modification) hydrogels at 10% (w/v), 15% (w/v) and 20% (w/v). Statistical analysis through two-tailed unpaired t test showed significant differences (*p<0.01) between the analyzed groups.

[0075] FIG. 11: Representative fluorescence images for: A) L929 and hASCs live/dead at 1 and 7 days of culture. B and C) L929 and hASCs DAPI/Phalloidin staining at 3 and 7 days of cell culture. (D)Immunocytochemistry images of hASCs with CD90/DAPI and CD73/DAPI at 7 days of cell culture. DNA (E) and MTS (F) results for L929 and hASCs at 1 day, 3 and 7 days of cell culture. Statistical analysis through two-tailed unpaired t test showed significant differences (*p<0.01) between the analyzed groups. L929 cells were encapsulated in PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) (10% w/v). hASCs cells were encapsulated in PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) (15% w/v). Scale bar: 100 m.

[0076] FIG. 12: Representative fluorescence images of DAPI/Phalloidin staining of BM-MSC, MG-63, SaOS-2 and A549 spheroids embedded into PL with a degree of substitution, in particular a degree of methacrylation, of 14% (low modification) hydrogels at 10% (w/v), 15% (w/v) and 20% (w/v), embedded into PEGDA hydrogel (10% (w/v)) and embedded into Matrigel at 14 days of culture.

[0077] FIG. 13: A) Fluorescence microscopy images of DAPI/Phalloidin staining of the established 3D mono- and co-culture models at 14 days of culture and after a doxorubicin (DOX) treatment (24 days of culture). (B) Schematics representation of the established 3D co-culture OS model and fluorescence microscopy images of DAPI/Phalloidin staining demonstrating the cellular network formed by hBM-MSC and MG-63 tumor cells at (a, b) 14 days of culture and (c) 24 days of culture, after drug treatment.

DETAILED DESCRIPTION

[0078] The present disclosure provides tunable crosslinked blood plasma derived hydrogel, their processing method and use in tissue regeneration, drug delivery, organ development, cell culture and tissue growth.

[0079] Different functional groups in proteins from PL and PRP are sensitive to chemical modifications as shown in FIG. 1A. In particular, the reactive functional groups existing in proteins are located on the side groups of amino acid residues, including hydroxyl groups (from serine, threonine, hydroxyproline, and hydroxylysine residues), amino groups (from lysine and hydroxylysine residues), and carboxylic acid substitutes (from aspartic acid and glutamic acid residues). In particular, methacryloyl substitution occurs rapidly and with high yield with reactive functional groups present in all proteins. Addition of acryloyl groups to the amine/hydroxyl/carboxylic acid-containing side groups of proteins present in PL was used to make it light polymerizable. PL were converted to a photo-polymerizable material through the reaction with methacrylic anhydride at controlled pH, in particular at pH=8 and temperature, in particular at 18-25 C.

[0080] In an embodiment, the use of different ratios PL:methacrylic anhydride, which stands for methacrylation degree, allows for tailoring the physicochemical and biological properties of the hydrogels for specific applications. The properties of the material can also be controlled with irradiation time and concentration of photo reactive PL hydrogel precursor. These will allow obtaining hydrogels possessing a wide range of physical properties, e.g., strength, stiffness, toughness, durability, degradability, mass transport and water uptake, according to the desired use. A ratio PL:methacrylic anhydride between 10:110.sup.3 (v/v)-10:5 (v/v) is suitable for the preparation of precursors for hydrogels.

[0081] In an embodiment, different degrees of methacrylation were obtained by varying the molar ratio of methacrylic anhydride to PL concentration, in particular the following degrees of methacrylation were obtained 14% (low modification) and 25% (high modification).

[0082] In an embodiment, the insertion of acrylate groups in the PL was verified by proton nuclear magnetic resonance (.sup.1H-NMR) spectroscopy performed before and after modification. Methacrylation was confirmed by the peaks at 1.8-2.0, 5.7-5.9, 6.1-6.3 ppm from methacrylate group (FIG. 1B).

[0083] In an embodiment, the polymer is crosslinked by UV light in the presence of a photo-initiator (2-Hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone) at mild temperatures (FIGS. 2 and 7).

[0084] In an embodiment, to determine the effect of methacrylation degree, hydrogel precursor concentration and irradiation time on the mechanical properties of the PL hydrogels, compression assays were performed on samples with methacrylation degrees of 14% (low modification) and 25% (high modification) and concentrations of 10 w.sub.plaquet lysate/v.sub.hydrogel% and 15 w/v.sub.hydrogel% and irradiation time of 30 s and 60 s.

[0085] In general, increasing the degree of methacrylation increased the stiffness at all strain levels (FIGS. 3, 8 and 9). Similarly, maintaining a constant degree of methacrylation while increasing the PL concentration significantly increased the stiffness under all conditions tested. Apparently, the increase of irradiation time from 30 s to 60 s do not change the mechanical properties of the hydrogels.

[0086] The degree of methacrylation is defined as the degree of methacryloyl substitution on the proteins from platelet lysates or platelet rich plasma.

[0087] The degree of methacrylation may be determined using the following method or methods: .sup.1H NMR, mass spectroscopy, fluoraldehyde assay, Habeeb method.

[0088] In an embodiment, structural analysis by scanning electronic microscopy (SEM) was performed. Platelet hydrogels have a porous network influenced by precursor concentration as showed in FIGS. 4A, 4B, 10A and 10B. For lower (10% w/v) precursor concentration, hydrogels have larger porous than in higher (15% w/v or 20% w/v) PLMA concentrations.

[0089] The water content of hydrogels was also evaluated. Results shown that this parameter is not significantly different between all the studied conditions. In general, plasma-based hydrogels have 90% of water content (FIGS. 4C and 10C).

[0090] In an embodiment, in vitro cytotoxicity/viability and proliferation screening was performed. The ability of the hydrogels of the present disclosure to sustain cells viability was assessed using L929 cells and human adipose derived stem cells (hASCs). The gels with encapsulated cells were then exposed to UV light to allow photo-polymerization. Afterwards, the discs were incubated for different time periods in cell culture medium. Cell viability after specific times of culturing (24 h and 7 days) was assessed using Calcein AM staining. Cell viability assay showed a uniform distribution of viable cells in the photo-crosslinked gels (FIGS. 5A and 11A). Cell proliferation and morphology on the gels was also evaluated. After specific time points the gels were fixed and the cells stained with dapi/phalloidin. An increasing density of live cells was observed which demonstrate that these hydrogels supports cells proliferation (FIGS. 5B, 5C, 5D, 11B, 11C, 11E and 11F).

[0091] Following cell encapsulation, cells on a solution of the material of the invention may be injected into the patient at the site of injury or defect and gellified in situ. In another application, the material of this invention may be used as a bioink to incorporate in bioprinters or similar apparatus to obtain hydrogels with controlled structure, or prepare hydrogels that find applications as supporting platforms for ex-vivo and in vitro biological studies, hydrogels and microgels for cell encapsulation and cell expansion for pharmaceutical studies (e.g. drug screening) or biotechnological (e.g. production of proteins) applications.

[0092] With several advantages, the autologous material now disclosed provides the basis for the development of a new autologous minimally-invasive system that could be used alone or seeded with cells suitable for restoring, maintaining or enhancing tissue/organ function. The hydrogels now disclosed are non-immunogenic, biodegradable under physiological conditions due to hydrolysable bonds in the polymer backbone, resulting in non-toxic fragments that are easily removed, from the body. Heterogeneous approaches can also be envisaged for the use of the proposed materials for ex-vivo and in vitro applications.

[0093] This novel hydrogel opens up new possibilities for drug discovery and development as it can be used for the generation of disease specific models for different tissue disorders. PRP/PL/plasma derived protein hydrogel precursor could be generated from patient-specific blood plasma. In addition, this could be combined with patient-specific cells.

[0094] The hydrogel now disclosed may be used as a 3D platform for spheroid invasion assessment.

[0095] In an embodiment, BM-MSCs, MG-63, SaOS-2 and A549 spheroids were embedded into Matrigel, embedded into PEGDA gels and embedded into PL gels. Tumor spheroid growth and invasion was improved when using PL derived gels (FIG. 12).

[0096] This novel hydrogel may be used for 3D in vitro model for disease development that: is physiologically relevant and/or patient-derived and meets the requirements of the pharmaceutical industry (HTS format, easy to manipulate, cost effective, reproducible and robust).

[0097] In an embodiment, MG-63 spheroids, BM-MSCs and osteoblasts were co-cultured to recapitulate tumor cell-microenvironment interaction of an invading tumor. PL hydrogels were able to support an invasive tumor morphology and produce an in vivo-like drug response (FIG. 12).

[0098] The synthesized PL derived material may be processed in the form of hydrogels, microfibers, particles, capsules, foams, sponges films or membranes with dimensions ranging from nanoscale to microscale. It can be also used as a coating substrate for cell culture and tissuegrowth.

[0099] The term comprising whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0100] It will be appreciated by those of ordinary skill in the art that unless otherwise indicated herein, the particular sequence of steps described is illustrative only and can be varied without departing from the disclosure. Thus, unless otherwise stated the steps described are so unordered meaning that, when possible, the steps can be performed in any convenient or desirable order.

[0101] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0102] The above described embodiments are combinable.

[0103] The following claims further set out particular embodiments of the disclosure.

REFERENCES

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