Plasma-Treated Hydrogel Compositions and Uses Thereof

Abstract

The present invention relates to a composition including a polymer aqueous solution, a bioceramic material and reactive oxygen and nitrogen species (RONS) and its use for the treatment of bone cancer and/or bone tissue regeneration.

Claims

1. A composition comprising a polymer aqueous solution, a bioceramic material comprising calcium, and reactive oxygen and nitrogen species (RONS), wherein said RONS comprise between 0.68 and 200.00 mg/L H.sub.2O.sub.2 and/or between 0.46 and 36.80 mg/L NO.sub.2.sup.−.

2. The composition according to claim 1, wherein said RONS comprise between 12.00 and 150.00 mg/L H.sub.2O.sub.2, preferably between 13.60 and 150.00 mg/L H.sub.2O.sub.2.

3. The composition according to claim 1, wherein said RONS comprise 13.80 and 36.80 mg/L NO.sub.2.sup.−, preferably between 18.40 and 36.80 mg/L NO.sub.2.sup.−.

4. The composition according to claim 1, wherein the polymer is selected from gelatin and its derivatives, such as methacrylated gelatin, fibrin, fibronectin, collagen, and collagen derivatives, alginate, agarose, cellulose, modified cellulose, such as hydroxypropyl cellulose, methylcellulose, carboxymethylcellulose or hydroxyethyl cellulose, xantan gum, polyethylene glycol, hyaluronic acid, chitosan, polylactide-co-glycolide, polyhydroxyalcanoates and mixtures thereof, preferably is selected from gelatin, alginate, collagen and mixtures thereof.

5. The composition according to claim 1, wherein the composition comprises between 0.15 and 50.00 weight % of polymer in respect of the total weight of the composition, preferably between 0.50 and 20.00 weight % of polymer in respect of the total weight of the composition, more preferably between 1.00 and 1.50% of polymer in respect of the total weight of the composition.

6. The composition according to claim 1, wherein the bioceramic material comprising calcium preferably comprises calcium phosphate, and is selected from tetra-calcium phosphate, dicalcium phosphate anhydrous, dicalcium phosphate dihydrate, alpha-tricalcium phosphate, beta-tricalcium phosphate, monocalcium phosphate monohydrate, hydroxyapatite, calcium deficient hydroxyapatite, fluorapatite, amorphous calcium phosphate, calcium-sodium and potassium-phosphate, calcium- and sodium-phosphate, calcium- and potassium-phosphate, calcium pyrophosphate, calcium carbonate, calcium sulphate, calcium sulphate hemihydrate, calcium oxide and calcium hydroxide, and mixtures thereof.

7. The composition according to claim 1, wherein the bioceramic material is hydroxyapatite, brushite, tricalcium phosphate or mixtures thereof.

8. The composition according to claim 1, wherein the bioceramic material is in form of nanoparticles, microspheres, microparticles, foams or scaffolds, or mixtures thereof.

9. The composition according to claim 1, wherein the composition comprises between 0.5 and 99.5 weight % of bioceramic materials in respect of the total weight of the composition.

10. The composition according to claim 1, wherein the pH of the composition is between 5.0 and 8.0, preferably between 6.0 and 7.5, measured according to ASTM E70.

11. The composition according to claim 1, further comprising an active pharmaceutical ingredient.

12. The composition according to the claim 11, wherein the active pharmaceutical ingredient is a chemotherapeutic drug or a coadjuvant in the cancer therapy.

13. A method for the treatment of bone cancer, wherein the method comprises administering a composition according to claim 1 to a patient in need thereof.

14. A method for bone tissue regeneration, wherein the method comprises administering a composition according to claim 1 to a patient in need thereof.

15. The method according to claim 13, wherein the bone cancer is osteosarcoma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1: SEM image of the composition of Example 6 after freeze-drying.

[0030] FIG. 2: SEM image of the freeze-dried composition sample of example 8 without RONS (A) and with RONS (B).

[0031] FIG. 3: Example of reconstruction of the 3D scaffold (A), and SEM image of the freeze-dried composition of Example 9 (B).

[0032] FIG. 4: SEM image showing bone ingrowth within the composition of Example 9.

[0033] FIG. 5: SEM picture of freeze-dried composition of Example 10.

[0034] FIG. 6: Release of doxorubicin (DOX) from the composition of Example 11 (where calcium phosphate cement (CPC) microspheres have been previously loaded with doxorubicin), and untreated-hydrogel with DOX-loaded CPC microspheres to MilliQ water release media. 200 μL of composition were put in contact to 1 mL of MilliQ water.

EXAMPLES

[0035] The following examples are provided to further illustrate, but not to limit this invention.

[0036] Materials

[0037] Gelatin type B (Rousselot 250 LB8, Rousselot, France), sodium alginate (MW: 10000-600000 g/mol) (Panreac, USA), both in powder form, and MilliQ water (MilliPore, Merck) were used for preparation of polymer solutions. Argon (Ar 5.0, PRAXAIR, Spain) was employed as precursor gas for APP generation in polymer solutions.

[0038] Sulphanilamide (Sigma Aldrich, USA), N-(1-naphthyl)ethylenediamine dihydrochloride (Sigma Aldrich, USA) and Ortho-Phosphoric Acid 85%, pure, pharma grade (USP-NF, BP, Ph. Eur.) (H.sub.3PO.sub.4) (85%) (Panreac, USA) have been used for the preparation of Griess reagent, used for NO.sub.2.sup.− detection. NaNO.sub.2 (Sodium nitrite, Sigma Aldrich, USA) was used for calibration curves of nitrites. Amplex™Red reagent (Invitrogen™, Thermo Fisher Scientific) and Peroxidase from Horseradish (Type VI) (HRP) (Sigma Aldrich) were used for determination of H.sub.2O.sub.2 in liquid solutions. 30% (w/w) H.sub.2O.sub.2 solution (Sigma Aldrich) was used for calibration curve for H.sub.2O.sub.2 detection in MilliQ water.

[0039] Sarcoma osteogenic cells (SaOs-2, ATCC, USA) were expanded in McCoy's 5A culture medium (Sigma Aldrich). Foetal Bovine Serum (FBS) and Penicillin/Streptomycin (P/S) (50 U/ml and 50 μg/ml, respectively) were purchased from Invitrogen. Bone marrow-derived Mesenchymal Stem Cells (hMSC, PCS-500-012, #70014245, ATCC, USA) were cultured in Advanced Dulbecco's Eagle Medium (1×) (AdvDMEM) (Gibco, ThermoFisher Scientific). Cells from passage between 24 and 32 were used in all experiments. Cell Proliferation Reagent WST-1 (Roche Diagnostics GmbH, ref. 05015944001) and PrestoBlue™ Cell Viability Reagent (Invitrogen™, Thermo Fisher Scientific, ref. A13261) were used for cell viability determination.

[0040] Methods

[0041] Preparation of Polymeric Solutions.

[0042] Different polymeric solutions were prepared by dissolving in water or aqueous saline solutions suitable concentrations polymers such as fibrin, fibronectin, collagen, alginate, gelatin, etc, and mixtures thereof. As an example, a detailed description of the procedure followed for the preparation of alginate and gelatin solutions is included below:

[0043] Alginate solutions were obtained by mixing the dry sodium-alginate powder with DI water in a SpeedMixer (DAC 150.1 FVZ-k, 3500 rpm) for 15 min at 0.5% w/w. The solutions can also be obtained by stirring with a conventional stirrer for longer times.

[0044] For the preparation of gelatin solutions, gelatin in powder was mixed with MilliQ water at 37° C. using magnetic stirring for 2 hours to obtain a 2% wt gelatin solution. Gelatin solutions were stored at 4° C. and used within a lifespan of 2 weeks. Both gelatin and alginate powder solutions were filtered at 37° C. using a 0.22 μm syringe filter before cell experiments (Millipore, Merck). For cell experiments, all the processes leading to the preparation of the formed polymer solution or hydrogel were carried out under sterile conditions.

[0045] Plasma Treatments.

[0046] In the examples presented here, two kinds of atmospheric plasma jet were used: a commercially available cold atmospheric plasma jet kINPen IND (NEOPLAS Tools, Germany), operating with argon and an atmospheric pressure plasma jet (APPJ) using He as plasma gas in a jet design based on a single electrode. Gas flow was regulated between 1 and 2.5 L/min for kINPen and between 1 and 5 L/min for APPJ by using Ar and He Bronkhorst Mass View flow controllers (BRONKHORST, Netherlands), respectively. All plasma treatments of polymeric solutions for RONS quantification were performed on 200 μL of the polymeric solution in 96-well plates, with a distance between the nozzle and the sample surface between 10 and 20 mm. These plasma treatments were done with ungrounded samples. Both grounded and ungrounded samples can be used to obtain the compositions of the present invention, since the skilled person can set the plasma treatment conditions to obtain the desired RONS concentrations.

[0047] Preparation of the Compositions

[0048] To prepare the compositions of the present invention, the plasma-treated polymeric solutions were blended with the calcium containing biomaterial. The method for blending and preparing the compositions may vary depending on the actual morphology/shape of the calcium comprising material. In the case of calcium phosphate nanoparticles, mixing with the polymer solution can be done manually, in a double-syringe system, using a SpeedMixer or any other method ensuring homogeneous dispersion. In this case, the mixture can be done with polymer solution containing RONS or alternatively treat the composition with plasma to transfer the RONS to the composition after mixing of the polymer solution and the calcium phosphate phase. If the plasma treatment is applied to the composition comprising the polymer solution and the calcium comprising material, then the treatment has to be applied before polymerization or crosslinking (gelation) of the polymer.

[0049] In the case of bioceramics in the shape of scaffolds, mixing should be done with the RONS— containing polymer solution, and different methods may be considered, namely by immersion, employing two syringes, dropwise addition, etc.

[0050] Detection of RONS in the Polymer Solutions.

[0051] Determination of NO.sub.2.sup.− concentration in plasma-treated polymer solutions was performed using Griess reagent. The Griess reagent used was obtained by dissolving 1% wt/v of sulphanilamide, 0.1% wt/v of N-(1-naphthyl)ethylenediamine dihydrochloride (NEED) and 5% w/v of phosphoric acid in de-ionized water. 200 μL of Griess reagent were added on 200 μL of sample in 96 well-plates. The plates were incubated for 10 min at room temperature protected from the light. The absorbance was measured at λ.sub.abs=540 nm using a Synergy HTX Hybrid Multi Mode Microplate Reader (BioTek Instruments, Inc., USA). The [NO.sub.2.sup.−] in each sample was determined from the absorbance values by using a calibration curve made from NaNO.sub.2 dilutions in the corresponding polymer solutions.

[0052] The concentration of hydrogen peroxide was determined by reaction of H.sub.2O.sub.2 with Amplex Red in presence of HRP enzyme that leads to the creation of resorufin, a fluorescent product. Amplex Red/HRP reagent consists in 100 μM of Amplex Red and 0.25 U/mL HRP in DI water. Since the higher concentration of H.sub.2O.sub.2 able to be processed properly by this reagent is around 10 μM of H.sub.2O.sub.2, plasma-treated polymer solutions were diluted 200 times previously to the addition of the reagent. In this case, for hydrogen peroxide detection, 50 μL of the Amplex Red/HRP reagent was added to 200 μL of the 200×-diluted polymer solution sample in a 96-well plate and incubated for 30 min at 37° C. Subsequent fluorescence measurements were performed by means of a Synergy HTX Hybrid Multi Mode Microplate Reader (BioTek Instruments, Inc., USA), with fluorescence filters centred at λ.sub.ex=560/20 nm and λ.sub.em=590/20 nm as excitation and emission wavelengths, respectively. Concentrations of H.sub.2O.sub.2 in polymer solution generated by plasma treatment were obtained from the fluorescence values using a calibration curve made from 30% hydrogen peroxide solution in the corresponding polymer solutions.

[0053] Also, the chemical probe coumarin (Sigma Aldrich, USA) was employed to detect hydroxyl radicals (OH). Different polymer solutions were prepared in 1 mM coumarin, and different plasma-treatment times were evaluated. In solution, OH radicals react with coumarin giving a fluorescent product: 7-hydroxcoumarin (7-hC). The fluorescence intensity of 500 μL of plasma-treated solutions were measured with a Synergy™ HTX Multi-Mode Microplate Reader (λex/em=360/460). In order to calculate the production rate of this fluorescent product, calibration curves using 7-hC (Sigma Aldrich, USA) were prepared.

[0054] For certain polymer solutions, interferences may be found between the solution and the reactants, invalidating the measure. In such cases, another method was used to determine the concentration of H.sub.2O.sub.2, NO.sub.2.sup.− and NO.sub.2.sup.− in the polymer solutions after plasma treatment: QUANTOFIX® test strips, which were analyzed by means of a reflexion photometer (QUANTOFIX® Relax, of Macherey Nagel). The strips consist of plastic strips to which test paper has been sealed. Nitrite strips are also based in Griess reagent. Peroxide strips also use a redox reaction. The range of detection of the test strips used for H.sub.2O.sub.2, NO.sub.3.sup.− and NO.sub.2.sup.− were 1-100 mg/L, 10-500 mg/L and 1-80 mg/L, respectively. The plasma-treated polymer solutions were diluted, if necessary, to be within the measuring range.

[0055] To test if the values for RONS concentrations obtained with the two methods disclosed above were equivalent, different solutions were tested with both methods.

[0056] Solutions of known concentrations were prepared (100, 50, 25 y 12.5 mg/L for hydrogen peroxide and 8.28, 4.14, 2.07, 1.035 mg/L for nitrites), and the concentration of hydrogen peroxide was measured with both the AR/HRP method and the strips method, while the concentration of NO.sub.2.sup.− was measures with both the Griess reagent method and the strips method.

[0057] The four different dilutions of 30% H.sub.2O.sub.2 were prepared either in water or in a 0.5% wt alginate water solution, and H.sub.2O.sub.2 concentration was tested with both methods. Three replicates were tested for each point. As the following table shows, both methods give equivalent results:

TABLE-US-00001 H.sub.2O.sub.2 detection from 30% H.sub.2O.sub.2 solution DI water 0.5% alginate AR/HRP AR/HRP reagent reagent method Strips method method Strips method 100.0 ± 2.26  98.0 ± 19.6 100.0 ± 5.38 101.3 ± 20.3  50.0 ± 1.99 47.3 ± 9.5  50.0 ± 1.18  52.0 ± 10.4  25.0 ± 0.52 23.3 ± 4.7  25.0 ± 0.68 25.7 ± 5.1  12.5 ± 0.16 12.7 ± 2.5  12.5 ± 4.42 12.3 ± 3.5 0 Below minimum 0 Below minimum detection detection

[0058] The four different dilutions of NaNO.sub.3 were prepared either in water or in a 0.5% wt alginate water solution, and NO.sub.2.sup.− concentration was tested with both methods. Three replicates were tested for each point. As the following table shows, both methods give equivalent results:

TABLE-US-00002 NO.sub.2.sup.− detection from dilutions of NaNO.sub.3 powder (mg/L) DI water 0.5% alginate Griess reagent Griess reagent method Strips method method Strips method 8.28 ± 0.17 8.07 ± 1.61 8.28 ± 0.80 7.83 ± 1.57 4.14 ± 0.02 3.97 ± 0.79 4.14 ± 0.75 4.13 ± 0.83 2.07 ± 0.08 2.13 ± 0.43 2.07 ± 0.20 2.00 ± 0.40 1.04 ± 0.02 1.07 ± 0.21 1.04 ± 0.08 0.97 ± 0.19 0 Below minimum 0 Below minimum detection detection

[0059] Therefore, for the present invention, the RONS concentration is determined either using the AR/HRP reagent method and the Griess reagent method, or the strips method.

[0060] pH Monitoring.

[0061] The polymeric solution was placed in 24 well-plates and treated using kINPen or APPJ (10 mm, 1 L/min). pH was measured by using a PC80 Multiparameter instrument (XS Instruments, Italy) with a Crison 50 14 electrode (Crison, Spain).

[0062] SEM.

[0063] The compositions were freeze-dried and were C-coated using an EMITECH K950X Turbo Evaporator (Quorum Technologies Ltd., UK). All samples were imaged in a Phenom XL SEM (Phenom-World B.V., The Netherlands) under high vacuum at 5 kV and a 5 mm working distance.

[0064] Release of RONS.

[0065] 200 μL of the polymeric solution in 96-well plate were treated by kINPen for 90 s, 10 mm and 1 L/min and APPJ for 15 min, 10 mm and 1 L/min.

[0066] After plasma-treatment, the polymeric solution was transferred to CORNING Transwell polyester membrane cell culture insert (Sigma-Aldrich), with a 6.5 mm diameter and a 0.4 μm pore size and placed in suspension in 1 mL volume of cell culture media in 24-well plates. For the monitoring of the release kinetics of RONS from the hydrogels 100 μL of the cell culture medium used as release media were withdrawn at determined time points for subsequent quantification of NO.sub.2.sup.− and H.sub.2O.sub.2. 100 μL of fresh medium was replaced after each sample collection. Final volumes of release media have been measured at the end of release experiment to take into account the volume correction in the concentration calculations of NO.sub.2.sup.− and H.sub.2O.sub.2. NO.sub.2.sup.− and H.sub.2O.sub.2 were quantified as described in the previous section.

[0067] In Vitro Cell Experiments.

[0068] Cell Culture.

[0069] Sarcoma Osteogenic (SaOS-2) were used to study the cytotoxicity of the hydrogels and the compositions. The cell culture medium consisted of McCoy's 5A with 10% FBS and 1% P/S. Cells were grown in 75 cm.sup.2 cell culture flasks at 37° C. in a 5% CO.sub.2 incubator and upon reaching 80% confluence. SaOS-2 were detached from the flask using trypsin (Invitrogen, Thermofisher) and 10000 cells/well were seeded into 24-well plates with 1 mL volume of culture medium. After 6 h-adhesion, plasma-treated sterile polymer solutions, previously prepared in sterile conditions, were introduced into a CORNING Transwell polyester membrane cell culture insert and placed in suspension in the well, to evaluate the effect of kINPen and APPJ plasma treatment of the polymeric solution on the SaOs-2 cell viability. As positive control, the same number of cells was placed without adding polymeric solution or composition. The cells were grown at 37° C. in a 5 CO.sub.2 incubator for another 72 h.

[0070] Bone marrow-derived Mesenchymal Stem Cells (hMSC) were used to evaluate the selectivity of the cytotoxicity of plasma-treated hydrogels between cancer and healthy cell lines. Cell culture medium of hMSC consisted of AdvDMEM supplemented with 10% FBS and 1% P/S. Seeding, cell density and experimental design of hMSC were reproduced in the exact same conditions such as presented above with SaOS-2. hMSC cell viability was evaluated at 72 hours for cells in presence of untreated polymer solution (UT), and plasma-treated polymer solution at different treatment times.

[0071] Cell viability at 24 and 72 hours was evaluated with WST-1 reagent following supplier's protocol. Absorbance was measured at λ.sub.abs=440 nm using a Synergy HTX Hybrid Multi Mode Microplate Reader (BioTek Instruments, Inc., USA). Normalization of the absorbance values was made with respect to cells only to determine the effects of untreated and plasma-treated Gel/Alg polymer solution on SaOS-2 cell viability.

[0072] Cell Viability.

[0073] Influence of plasma-treated polymeric solutions on SaOs-2 or hMSCs cell viability was evaluated for kINPen and APPJ (10 mm, 1 L/min) for 90 and 180 s of plasma treatments. Plasma-treated polymeric solutions were also studied for 180 s APPJ and kINPen plasma treatment. Cell viability was evaluated at 0, 24 and 72 hours. Cell culture media was replaced by preparation consisting of 250 μL of Cell Proliferation Reagent WST-1 in Mc Coy's 5A culture medium (1:10) and incubated for 1 hour at 37° C. Afterward, 100 μL of the supernatant were transferred to another well for absorbance measurement at 440 nm. To evaluate the effects untreated and plasma-treated polymer solutions on SaOs-2 cell viability, normalization of the values was made with respect to the well containing cells only.

[0074] Range of Concentrations of Reactive Species Generated in the Polymer Solution

[0075] The concentrations of reactive species generated by atmospheric pressure plasma treatment in 200 μL of polymer solution (gelatin/alginate as in Example 1) and in 1 mL of polymer solution, at different treatment times are shown below.

TABLE-US-00003 kINPen Detection method: Strips 200 μL Treat. time (s) H.sub.2O.sub.2 (mg/L) NO.sub.2.sup.− (mg/L) NO.sub.3.sup.− (mg/L) 0 0 0 0 15 1.9 ± 0.4 1.3 ± 0.3 <10 30 2.4 ± 0.5 2.6 ± 0.5 19 ± 3.8 45 4.5 ± 0.9 4.0 ± 0.8 25 ± 5.0 60 5.3 ± 1.1 4.3 ± 0.9 27 ± 5.4 90 7.7 ± 1.5 6.7 ± 1.3 44 ± 8.8 180 16.7 ± 3.3  17.0 ± 3.4  124 ± 24.8 EXAMPLE 1) 300 31.5 ± 6.3  22.0 ± 4.4  190 ± 38.0 (EXAMPLE 2) 1 mL t (s) H.sub.2O.sub.2 (mg/L) NO.sub.2.sup.− (mg/L) NO.sub.3.sup.− (mg/L) 0 0 0 0 180 15.8 ± 3.2  6.0 ± 1.2 48.7 ± 9.7 300 24.3 ± 4.9  9.1 ± 1.8  59.3 ± 11.9 600  62.0 ± 12.4 13.7 ± 2.7 103.7 ± 20.7 900  97.0 ± 19.4 21.3 ± 4.3 214.0 ± 42.8

TABLE-US-00004 APPJ Detection method: Strips 200 μL Treat. time (s) H.sub.2O.sub.2 (mg/L) NO.sub.2.sup.− (mg/L) NO.sub.3.sup.− (mg/L) 0 0 0 0 15  2.7 ± 0.5 1.3 ± 0.3 <10 30  4.8 ± 1.0 2.1 ± 0.4 24 ± 4.8 45  6.0 ± 1.2 3.2 ± 0.6 40 ± 8.0 60  6.6 ± 1.3 3.8 ± 0.8  55 ± 11.0 90  8.2 ± 1.6 5.0 ± 1.0  55 ± 11.0 180 13.6 ± 2.7 8.7 ± 1.7 49 ± 9.8 300 22.4 ± 4.5  11 ± 2.2  53 ± 10.6 1 mL t (s) H.sub.2O.sub.2 (mg/L) NO.sub.2.sup.− (mg/L) NO.sub.3.sup.− (mg/L) 0 0 0 0 180  7.1 ± 1.4  6.0 ± 1.2  53.0 ± 10.6 300 13.0 ± 2.6 11.0 ± 2.2  92.3 ± 18.5 600 21.7 ± 4.3 15.2 ± 3.0 134.0 ± 26.8 900 37.8 ± 7.6 18.5 ± 3.7 220.3 ± 44.0

TABLE-US-00005 kINPen Detection method: Strips 1 mL 3 L/min 1 L/min Treat. time H.sub.2O.sub.2 NO.sub.2.sup.− NO.sub.3.sup.− H.sub.2O.sub.2 NO.sub.2.sup.− NO.sub.3.sup.− (min) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) 0 0 0 0 0 0 0 10 25.2 ± 4.4 2.8 ± 1.4 27.9 ± 1.2 21.4 ± 6.5 15.2 ± 1.8 158.7 ± 18.0 15 54.1 ± 8.2 6.0 ± 0.5 71.9 ± 5.0 47.6 ± 6.1 23.5 ± 1.8 249.3 ± 22.3 20 83.3 ± 2.0 23.5 ± 3.7  288.3 ± 45.3  65.0 ± 11.2 62.1 ± 6.4 449.5 ± 23.6

TABLE-US-00006 APPJ 1 mL Detection method: Strips Gas flow Treat. 1 L/min time H.sub.2O.sub.2 NO.sub.2.sup.− NO.sub.3.sup.− (min) (mg/L) (mg/L) (mg/L)  0 0 0 0  5 16.3 ± 2.5 12.1 ± 3.5 115.6 ± 28.3 10  38.7 ± 10.1 28.7 ± 6.5 385.7 ± 32.3 15 58.3 ± 7.1 38.6 ± 2.9 409.7 ± 43.7 20 67.7 ± 7.1 41.3 ± 7.4 442.0 ± 32.2 30 138.7 ± 20.5 68.0 ± 8.7  624.0 ± 110.5

Example 0

[0076] Gelatin in powder was mixed with MilliQ water at 37° C. using magnetic stirring for 2 hours to obtain a 2% wt gelatin gel. 200 μL of this gelatine solution was treated with two types of atmospheric pressure plasma jet: i) kINPen IND® (Neoplas, Germany) operating with Argon, 1 L/min gas flow and 10 mm distance and ii) APPJ (a home-made atmospheric pressure plasma jet) operating with helium, 1 L/min gas flow and 10 mm distance. The reactive species generated after different plasma treatment times were quantified. Said plasma-treated gelatin solution was used in cell viability assays in both an osteosarcoma cell line (SaOS-2).

TABLE-US-00007 kINPen APPJ Treat. (200 μL, 1 L/min) (200 μL, 1 L/min) time H.sub.2O.sub.2 NO.sub.2.sup.− H.sub.2O.sub.2 NO.sub.2.sup.− (s) (mg/L) (mg/L) (mg/L) (mg/L)   0 0 0 0 0  15 12.0 ± 6.3  3.8 ± 0.7  5.1 ± 1.2 1.0 ± 0.1  30 12.7 ± 2.7  7.2 ± 1.1  6.3 ± 0.9 1.6 ± 0.3  45 16.0 ± 4.9  9.6 ± 0.6  7.7 ± 1.3 2.7 ± 0.1  60 23.4 ± 3.6 11.7 ± 1.6  9.9 ± 1.1 3.3 ± 0.2  90 25.2 ± 5.2 10.4 ± 2.0 13.2 ± 1.2 5.1 ± 0.2 120 33.5 ± 1.9 12.3 ± 1.3 18.2 ± 3.2 6.0 ± 0.6 180 47.7 ± 3.9 19.1 ± 0.8 26.4 ± 3.9 7.2 ± 0.1 300 79.1 ± 5.2 27.0 ± 1.5 37.5 ± 1.1 7.6 ± 0.5

[0077] The concentrations of reactive species generated in the gelatin solutions upon plasma treatment are higher than those disclosed until now, and the gelatin hydrogels comprising said RONS concentrations display an enhanced cytotoxicity on osteosarcoma cell line SaOS-2.

TABLE-US-00008 Plasma treatment Cell viability at 24 h (%) Cell viability at 72 h (%) conditions (200 μL, 1 L/min) (200 μL, 1 L/min) Cells only 100 100 Untreated 101.74 ± 1.87  88.76 ± 10.98 APPJ 30 s  70.93 ± 7.72 71.35 ± 2.33 APPJ 90 s  48.84 ± 0.65 23.88 ± 0.32 APPJ 180 s  46.51 ± 2.61 22.75 ± 0.33 kINPen 30 s  57.56 ± 0.31 44.94 ± 3.30 kINPen 90 s  36.63 ± 3.73 15.73 ± 2.07 kINPen 180 s  26.74 ± 4.24 12.36 ± 0.71

Example 1

[0078] A 50/50 blend of 0.5 weight % alginate and 2 weight % gelatin solutions were prepared (final concentration of 0.25% wt alginate and 1 wt gelatin).

[0079] The mixture of alginate/gelatin was prepared is by vortexing in a ratio 1:1, 2% wt gelatin with 0.5% wt alginate for 2 minutes. Gelatin in powder is mixed with MilliQ water at 37° C. using magnetic stirring for 2 hours to obtain a 2% wt gelatin gel. 0.5% alginate was prepared by mixing alginate powder with MilliQ water using a SpeedMixer™ DAC 150.1 FVZ-K (SpeedMixer™, Germany) at 3500 r.p.m. for 15 min.

[0080] The 0.25% wt alginate and 1 wt gelatin aqueous mixture was treated with an atmospheric pressure plasma jet kINPen IND® (Neoplas, Germany) operating with Argon to generate plasma. Treatment conditions: 1 L/min gas flow, 10 mm nozzle distance, and 180 seconds treatment. Treatment performed in 200 μL of mixture in a 96-well plate.

[0081] Said plasma-treated mixture produced the following concentrations of reactive species in the material:

TABLE-US-00009 H.sub.2O.sub.2 NO.sub.2.sup.− NO.sub.3.sup.− (mg/L) (mg/L) (mg/L) Water 10.3  2.6 — Example 1 16.7 17.0 124.0

[0082] All values have a ±20% variability due to the measuring method.

[0083] As shown in the table, the values of reactive species obtained in the composition of example 1 are several-fold higher than those generated in water.

[0084] Said plasma-treated mixture was used in cell viability assays in both an osteosarcoma cell line (SaOS-2) and in healthy cells (human bone marrow mesenchymal stem cells or hBM-MSC):

TABLE-US-00010 Example 1 Cell viability at 72 h (%) SaOS-2 40.94 ± 3.44 hBM-MSC 90.57 ± 8.19

[0085] The composition of example 1 shows selectivity of the plasma-treated polymer solution on cancer cell line, allowing the survival of healthy cells (hBM-MSC) after 72 hours.

Example 2

[0086] An aqueous mixture comprising 0.25% wt alginate and 1 wt gelatin was treated with an atmospheric pressure plasma jet kINPen IND® (Neoplas, Germany) operating with Argon to generate plasma. Treatment conditions: 1 L/min gas flow, 10 mm nozzle distance, and 300 seconds treatment. Treatment performed in 200 μL of mixture in a 96-well plate. Said plasma-treated mixture produced the following concentrations of reactive species in the material, which are much higher than in water:

TABLE-US-00011 H.sub.2O.sub.2 NO.sub.2.sup.− NO.sub.3.sup.− (mg/L) (mg/L) (mg/L) Water 29.3  2.7 — Example 2 31.5 22.0 190.0

[0087] All values have a ±20% variability due to the measuring method.

[0088] Said plasma-treated mixture was used in cell viability assays in both an osteosarcoma cell line (SaOS-2) and in control cells (human bone marrow mesenchymal stem cells or hBM-MSC):

TABLE-US-00012 Example 2 Cell viability at 72 h (%) SaOS-2  6.60 ± 0.27 hBM-MSC 94.38 ± 2.80

[0089] The composition of example 2 also shows selectivity of the plasma-treated polymer solution on cancer cell line, allowing the survival of healthy cells (hBM-MSC) after 72 hours.

Example 3

[0090] An aqueous mixture comprising 0.25% wt alginate and 1% wt gelatin was treated with an atmospheric pressure plasma jet operating with Helium to generate plasma. Treatment conditions: 1 L/min gas flow, 10 mm nozzle distance, and 180 seconds treatment. Treatment performed on 200 μL of mixture in a 96-well plate.

[0091] The said plasma-treated mixture produced the following concentrations of reactive species in the material, which are much higher than those produced in water:

TABLE-US-00013 H.sub.2O.sub.2 NO.sub.2.sup.− NO.sub.3.sup.− (mg/L) (mg/L) (mg/L) Water 9.2 1.7 — Example 3 13.6 8.7 49.0

[0092] All values have a ±20% variability due to the measuring method.

[0093] Said plasma-treated mixture was used in cell viability assays in both an osteosarcoma cell line (SaOS-2) and in control cells (human bone marrow mesenchymal stem cells or hBM-MSC):

TABLE-US-00014 Example 3 Cell viability at 72 h (%) SaOS-2 50.08 ± 1.99 hBM-MSC 95.03 ± 1.44

[0094] The composition of Example 3 also shows selectivity of the plasma-treated polymer solution on cancer cell line, allowing the survival of healthy cells (hBM-MSC) after 72 hours.

Example 4

[0095] An aqueous mixture comprising 0.25% wt alginate and 1% wt gelatin was treated with an atmospheric pressure plasma jet operating with Helium to generate plasma. Treatment conditions: 1 L/min gas flow, 10 mm nozzle distance, and 300 seconds treatment. Treatment performed on 200 μL of mixture in a 96-well plate.

[0096] Said plasma-treated mixture produced the following concentrations of reactive species in the material, which are much higher than those produced in water:

TABLE-US-00015 H.sub.2O.sub.2 NO.sub.2.sup.− NO.sub.3.sup.− (mg/L) (mg/L) (mg/L) Water 16.4 2.7 — Example 4 22.4 11.0 53.0

[0097] All values have a ±20% variability due to the measuring method.

[0098] Said plasma-treated mixture was used in cell viability assays in both an osteosarcoma cell line (SaOS-2) and in control cells (human bone marrow mesenchymal stem cells or hBM-MSC):

TABLE-US-00016 Example 4 Cell viability at 72 h (%) SaOS-2 11.24 ± 1.69 hBM-MSC 94.79 ± 2.01

Example 5

[0099] The compositions of Examples 1 to 4 were prepared comprising 5% wt of calcium deficient hydroxyapatite microspheres (MS), which were added and mixed in the vortex for 2 min. The diameter of the microspheres was 100 μm<Ø<150 μm. The compositions were freeze-dried to perform scanning electron microscopy. Example 5 corresponds to composition of Example 1 (5-min kINPen treatment of the alginate/gelatin blend)+5% wt of calcium deficient hydroxyapatite microspheres. The amount of reactive species in the composition is proportional to the percent of polymer solution of Examples 1 to 4. The amount of RONS was not affected by the addition of the bioceramic material.

[0100] The concentration of reactive species generated by plasma in the polymer solution and in the composition after adding the bioceramic material is equivalent, as can be seen below:

TABLE-US-00017 [H.sub.2O.sub.2] [NO.sub.2.sup.−] [NO.sub.3.sup.−] Example (mg/L) (mg/L) (mg/L) 1 78.0 ± 15.6 20.0 ± 4.0 297.0 ± 59.4 5 (Example 1 + 84.7 ± 16.9 21.5 ± 4.3 270.0 ± 54.0 5% microspheres)

[0101] The species generated in the composition of Example 5 can be released to a surrounding media and preserved at least for 24 hours:

TABLE-US-00018 H.sub.2O.sub.2 concentration in 1 mL release media (mg/L) Time Example 5 with (h) Example 1 Example 5 DOX-loaded MS 0 0 0 0 0.5 2.37 ± 0.15 3.11 ± 0.19 3.18 ± 0.11 1 2.55 ± 0.37 4.18 ± 0.39 2.57 ± 0.09 2 1.99 ± 0.34 3.50 ± 0.25 2.64 ± 0.09 4 2.08 ± 0.33 3.76 ± 0.39 3.06 ± 0.10 24 1.95 ± 0.23 3.06 ± 0.64 2.21 ± 0.08

TABLE-US-00019 NO.sub.2.sup.− concentration in 1 mL release media (mg/L) Time Example 5 with (h) Example 1 Example 5 DOX-loaded MS 0 0 0 0 0.5 0.25 ± 0.02 0.25 ± 0.03 0.25 ± 0.05 1 0.31 ± 0.02 0.36 ± 0.04 0.29 ± 0.06 2 0.38 ± 0.01 0.46 ± 0.01 0.35 ± 0.07 4 0.43 ± 0.02 0.51 ± 0.03 0.29 ± 0.06 24 0.54 ± 0.06 0.60 ± 0.04 0.32 ± 0.06

[0102] Said Example 5 was used in cell viability assays in osteosarcoma cell line (SaOS-2):

TABLE-US-00020 SaOS-2 cell viability SaOS-2 cell viability at 24 h (%) at 72 h (%) Untreated composition 93.6 ± 6.8 96.7 ± 2.1 Example 5 13.8 ± 1.3  7.5 ± 5.5

Example 6

[0103] Example 6 corresponds to the composition of Example 2 (5-min kINPen treatment of the alginate/gelatin blend)+5% wt of calcium deficient hydroxyapatite microspheres. The composition was freeze-dried and photographed by SEM (FIG. 1). The amount of reactive species in the composition was proportional to the percent of polymer solution of examples 1 to 4. The amount of RONS was not affected by the addition of the bioceramic material.

[0104] The concentration of reactive species generated by plasma in the polymer solution and in the composition after adding the bioceramic material is equivalent, as can be seen below:

TABLE-US-00021 Example [H.sub.2O.sub.2] (mg/L) [NO.sub.2.sup.−] (mg/L) [NO.sub.3.sup.−] (mg/L) 2 118.3 ± 23.7 28.5 ± 5.7 346.0 ± 69.2 6 (Example 2 +  96.7 ± 19.3 30.0 ± 6.0 364.0 ± 72.8 5% microspheres)

[0105] Said Example 6 was used in cell viability assays in osteosarcoma cell line (SaOS-2):

TABLE-US-00022 SaOS-2 cell viability SaOS-2 cell viability at 24 h (%) at 72 h (%) Untreated material 93.6 ± 6.8 96.7 ± 2.1 Example 6  7.4 ± 0.1  2.6 ± 0.1

Example 7

[0106] The compositions of Examples 1 to 4 were prepared comprising 5% wt of hydroxyapatite nanoparticles. These compositions were tested for injectability and it was found that all were fully injectable.

Example 8

[0107] A composition comprising a polymeric aqueous solution containing gelatin 6.5% wt, fibrinogen 10 mg/mL and aprotinin 1 μg/mL and 0.5% wt hydroxyapatite nanoparticles was prepared. Plasma treatment conditions: 1 L/min gas flow, 10 mm nozzle distance, and 5 min treatment, performed on 1000 μL of the composition (FIG. 2). Injectability was always good, with slightly higher values for RONS comprising compositions, but still keeping proper injectability for use.

Example 9

[0108] A composition comprising a polymeric aqueous solution containing 0.25% wt alginate and 1% wt gelatin was treated with an atmospheric pressure plasma jet kINPen IND operating with Argon to generate plasma. Treatment conditions: 1 L/min gas flow, 10 mm nozzle distance, and 180 seconds treatment. Treatment performed in 200 μL of mixture in a 96-well plate, and then loaded to a calcium phosphate scaffold, to obtain a composition with a final 55% wt of calcium-deficient hydroxyapatite, in respect of the total weight of the composition. In this example, the polymer solution is embedded within the 3D-printed scaffold (FIG. 3). This composition was implanted into a 5 mm condyle defect of healthy New Zealand rabbits. The animals were euthanized two months after the surgical procedure and bone regeneration was assessed by micro-computed tomography and SEM.

[0109] The composition of Example 9 (Scaffold+hydrogel containing plasma-generated RONS) demonstrated in vivo safety, allowing equivalent bone regeneration than the same composition without RONS (Scaffold+hydrogel without plasmas treatment) (FIG. 4).

[0110] The amount of regenerated bone in the scaffolds was quantified from micro-computed tomography images. Considering that the degradation of the scaffold can be negligible, macropore volume corresponds to the sum of newly formed bone and void pixels. Therefore, the average bone regeneration was calculated as BV/MV, being BV the volume of the newly formed bone and MV the macropore volume. The average bone regeneration was calculated and reported as mean±standard deviation (SD).

TABLE-US-00023 Kind of sample Bone regeneration (%) Example 9 without 39.97 ± 5.98 plasma treatment Example 9 40.10 ± 3.36

[0111] The percentage of bone regeneration being equivalent in both samples confirms the safety of the plasma-treated composition. The composition of the Example 9 does not hinder the proliferation of healthy bone cells of the rabbit and allows similar bone ingrowth to bioceramic-based bone grafts. Therefore, the composition of the Example 9 can be used to treat bone cancer since it does not damage healthy cells and allows bone regeneration.

Example 10

[0112] A composition comprising a 4 mg/mL collagen type I aqueous solution and 58% wt dry of hydroxyapatite nanoparticles was prepared. The composition was freeze-dried for SEM analysis (FIG. 5).

Example 11

[0113] The composition such as that of Example 5 was prepared where the calcium phosphate (CPC) microspheres had been previously loaded with doxorubicin. As control, untreated-hydrogel with DOX-loaded CPC microspheres were assayed for the release of RONS. 200 μL of composition were put in contact to 1 mL of MilliQ water. FIG. 6 shows that the loading of reactive species inside the hydrogel, does not affect the release of the active principle (doxorubicin) from the biomaterial.

Example 12

[0114] A composition comprising a 2% wt methacrylated-gelatin (GelMA) solution was treated with plasma. It was observed that higher amounts of RONS were obtained in said composition than a phosphate buffer saline (PBS) using the same treatment.

TABLE-US-00024 PBS t H.sub.2O.sub.2 NO.sub.2.sup.− (min) (mg/L) (mg/L)  0 0.00 0.00  2  3.96 ± 0.33 2.04 ± 0.46  4  7.33 ± 0.60 3.85 ± 1.12  6 10.76 ± 0.49 6.12 ± 1.54  8 13.99 ± 3.15 8.36 ± 1.93 10 16.94 ± 0.54 9.08 ± 1.82

TABLE-US-00025 2% GelMA t H.sub.2O.sub.2 NO.sub.2.sup.− (min) (mg/L) (mg/L)  0 0.00 0.00  2  3.63 ± 1.85  7.83 ± 3.82  4  5.71 ± 2.54 12.34 ± 1.51  6  9.72 ± 4.13 18.52 ± 1.22  8 15.62 ± 2.20 20.82 ± 3.50 10 23.68 ± 4.88 24.60 ± 3.39

Example 13

[0115] A composition comprising a polymeric solution containing methylcellulose 1% wt solubilized in a phosphate solution containing 200 mM of Na.sub.2HPO.sub.4 was treated with an atmospheric pressure plasma jet kINPen IND® (Neoplas, Germany) operating with Argon to generate plasma. Treatment conditions: 1 L/min gas flow, 10 mm nozzle distance. The treatment was performed in 1000 μL of solution in a 24-well plate. Said plasma-treated solution produced the following concentrations of RONS in the material:

TABLE-US-00026 treatment time (s) H.sub.2O.sub.2 (mg/L) NO.sub.2.sup.− (mg/L)   0 0 0  30  1.31 ± 0.23 0.50 ± 0.06  60  1.76 ± 0.39 0.87 ± 0.14 120  4.09 ± 0.55 1.53 ± 0.29 180  5.66 ± 0.66 2.09 ± 0.31 300 11.06 ± 0.40 2.66 ± 0.22

[0116] Moreover, the production rate of hydroxyl radical (OH*) during plasma treatment was estimated using the chemical probe coumarin. Said plasma-treated solution produced the following concentrations of 7-hydroxcoumarin (7-hC) in the material, which leads to a formation rate of 0.0002 μM/s:

TABLE-US-00027 treatment time (s) 7-hC (μM)   0 0  60 0.0146 180 0.0261 300 0.0563

Example 14

[0117] A composition comprising an alginate/gelatin blend such as the one described in Example 2, treated during 5 minutes with kINPen treatment was mixed with 1% wt of calcium deficient hydroxyapatite microspheres, which had been loaded with doxorubicin (1%). The amount of reactive species in the composition is that of examples 2 and 6, as the amount of RONS was not affected by the addition of the bioceramic material. The microspheres had a size of 100 to 150 microns diameter (from 0% to 5% drug load). The plasma treatment was performed in 1 ml in 24-well plates with kINPen; Argon; 10 mm; 1 L/min; 5 min.

[0118] A synergic effect can be observed in the cancer cell cytotoxicity with the combination of doxorubicin contained in the microspheres and RONS from the alginate/gelatin hydrogel. In this sense, the amount of doxorubicin can be reduced 4 times when RONS are delivered simultaneously by the hydrogel. The following table shows the MG63 cells viability in the presence of the hydroxyapatite microspheres (MS) loaded with 1, 2, 3, 4 or 5% doxorubicin and in the presence of the hydroxyapatite microspheres loaded with 1% doxorubicin in combination with untreated (UT) or with plasma treated (PT) alginate/gelatin hydrogels (HG):

TABLE-US-00028 Microspheres alone Composite Cells only 1% MS 2% MS 3% MS 4% MS 5% MS UT HG 1% MS PT HG 1% MS 24 h 100 ± 0 83.5 ± 0.8 72.4 ± 2.4  65.5 ± 3.8 55.4 ± 0.1 55.1 ± 1.3 88.6 ± 1.4 72.4 ± 1.6 72 h 100 ± 0 76.0 ± 9.1 40.2 ± 10.5 29.0 ± 2.1 26.1 ± 1.5 25.4 ± 0.8 80.0 ± 1.5 26.9 ± 0.5

[0119] 20.000 MG63 cells were plated per well in DMEM cell culture medium in 24-well plates and left for 24-hour adhesion. Prior to the material addition, the cell culture medium was changed (DMEM-Pyr). 2004 of material was added 2 hours after. The cells were kept in an incubator at 37° C.; 95% hum.; 5% CO.sub.2.