Polymer-based micellar-hydrogels and applications thereof to treat oral squamous cell carcinoma

Abstract

The present invention relates to the field of medicine, relating to hydrogel compositions containing therapeutic agents and their application in cancer treatment. The object of this invention is an hydrogel composition comprising a poloxamer-188 block copolymer and a polyethylenimine cationic polymer, wherein the molar ratio of said poloxamer-188 block copolymer to polyethylenimine cationic polymer is 1:2 and water is present at a concentration of 25% to 50% of the total weight of the hydrogel composition. Delivery systems can be derived from the hydrogel composition, in which a negatively charged active compound, and / or genetic material, preferably RNA, may be complexed with the hydrogel composition, to be internalized by cancer cells. The proposed invention aims to improve the delivery of therapeutic agents, such as miRNA, to oral squamous cell carcinoma (OSCC) cells for use in the treatment of OSCC.

Description

[0001] DESCRIPTION

[0002] POLYMER-BASED MICELLAR-HYDROGELS AND APPLICATIONS THEREOF TO TREAT ORAL SQUAMOUS CELL CARCINOMA

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the field of medicine, and mainly relates to hydrogel compositions containing therapeutic agents and their application in cancer treatment.

[0005] PRIOR ART

[0006] Oral squamous cell carcinoma (OSCC) develops on the mucosal epithelium of the oral cavity, with a substantial impact on the patient's quality of life, as it can contribute to alterations in physiognomy and functional activity, namely in pronunciation, swallowing, and flavor perception.

[0007] OSCC is associated with various risk factors, including tobacco, alcohol, and betel quid consumption; oral dysbiosis, related to the human papillomavirus (HPV) infection or poor oral hygiene; malnutrition; immune system imbalance; hereditary conditions; and aging. In 2020, the estimated number of OSCC cases exceeded 377,000, with a projected rise of approximately 40 % by 2040, accompanied by the increase in mortality. OSCC accounts for approximately 90 % of oral malignancies, with more than 60 % of the cases diagnosed at an advanced stage, linked to elevated metastatic potential, treatment failure, and subsequent recurrence, contributing to the meager 30 % five-year survival rate. Indeed, a network between epithelial-mesenchymal transition (EMT) regulators and key conductors of cancer cell proliferation, metabolism, motility, invasion, metastasis, and blockers of cell death have been intricated in the web of OSCC tumor microenvironment orchestrating its malignant behavior.Polymer-based materials have been studied extensively as drug delivery systems (DDSs) for a variety of biomedical applications, including cancer therapy, antimicrobial, and tissue engineering purposes. Currently, the design of DDSs is mostly set toward fulfilling only one programed task, for example, targeting one specific type of cancer or exclusively combatting bacterial infections. Indeed, common sense in the biomedical and materials science field is to develop compounds with specific functions that target specific sites for predetermined applications. In reality, the design, synthesis, and evaluation of a DDS requires tremendous efforts. Emphasis should rather be placed on a modular approach, that is, starting with relatively basic and readily available materials that can be adapted to meet a multitude of biomedical applications by a simple switch of the configuration, for example, the biopolymer chitosan. In this way, human, energy, and capital resources can be conserved.

[0008] To achieve this, the intended configurations should be compatible to allow for simple switching. Among the numerous polymer-based self-assembled structures, spherical micelles and nanogels are closely related because in most cases, nanogels can be regarded as crosslinked micelles.

[0009] The present invention aims to provide a safe polymer-based solution for the local and parenteral treatment of OSCC.

[0010] SUMMARY OF THE INVENTION

[0011] The object of this invention is a hydrogel composition comprising a poloxamer-188 block copolymer, which is a difunctional block copolymer surfactant terminating in primary hydroxyl groups, and a polyethylenimine (PEI) cationic polymer, wherein the molar ratio of said poloxamer-188 block copolymer to polyethylenimine cationic polymer is 1:2 and water is present at a concentration of 25% to 50% of the total weight of the hydrogel composition.In an advantageous aspect of the present invention, the hydrogel composition is able to form supramolecular structures, resulting in cloudy emulsions when said hydrogel composition is above 14 °C, and being transparent at 4 °C, which indicates that the formation of supramolecular particles requires higher temperatures.

[0012] In another advantageous aspect of the present invention, the hydrogel composition is able maintain the formed supramolecular structures at concentrations between 25 µg / mL and 20 mg / mL, in which reticular structure of said hydrogel composition is visible. The developed hydrogel composition is stable upon dilution down to a concentration below 25 µg / mL, with morphologic features of spheric nanoparticles with sizes lower than 100 nm, which makes the hydrogel composition suitable to deliver active compounds by systemic administration, as it avoids the burst release of the active compound before reaching target cells.

[0013] It is also an object of the present a process for obtaining the hydrogel composition, comprising the following steps:

[0014] a. Dissolving the poloxamer-188 block copolymer in a first organic solvent; b. Adding triethylamine and acryloyl chloride to the dissolved poloxamer- 188 block copolymer to form a first reaction mixture;

[0015] c. Stirring the first reaction mixture in reflux at 20 °C for at least 3 hours to form a first product mixture;

[0016] d. Filtering the first product mixture to obtain a filtered product mixture; e. Performing a liquid-liquid extraction and removing remaining organic solvents from the filtered product mixture to obtain an activated poloxamer-188 block copolymer;

[0017] f. Mixing the activated poloxamer-188 block copolymer with a polyethylenimine cationic polymer and a second organic solvent to form a second reaction mixture, in which the activated poloxamer-188 block copolymer and the polyethylenimine cationic polymer are present in a 1:2 molar ratio;

[0018] g. Stirring the second reaction mixture in reflux at 45 °C for at least 48 hours to form a second product mixture;h. Removing remaining organic solvents from the second product mixture to obtain a poloxamer-188-polyethylenimine polymer;

[0019] i. Diluting the poloxamer-188-polyethylenimine polymer in an aqueous solution to obtain an hydrogel composition, wherein water is present at a concentration of 25% to 50% of the total weight of said hydrogel composition.

[0020] The chemical synthesis process to obtain the hydrogel composition of the present invention is in a modified two-step reaction, in which the first reaction step consists in the activation of the poloxamer-188 block copolymer, preferably consisting in the block copolymer Pluronic® L121, by introducing two acrylate groups to its end caps; followed by the second reaction step in which the PEI cationic polymer is conjugated with the block copolymer Pluronic® L121, resulting in a cross-linked Pluronic® L121-PEI polymer having hydrogel properties upon dilution in aqueous solutions.

[0021] The first reaction to activate the block copolymer Pluronic® L121 produces triethylamine hydrochloride as a by-product, which is removed in the filtration step. The first organic solvent is removed from the filtered product mixture by evaporation, so to obtain a dried activated block copolymer Pluronic® L121 diacrylate to be subsequently dissolved in a second organic solvent for the conjugation reaction with the cationic polymer.

[0022] The second reaction corresponds to the conjugation of the activated block copolymer Pluronic® L121 diacrylate with the PEI cationic polymer through a Michael addition reaction in a 1:2 molar ratio. After total evaporation of the second organic solvent, a cross-linked Pluronic® L121-PEI polymer is obtained as a sticky yellow solid, which is then diluted in an aqueous solution to form the hydrogel composition of the present invention.

[0023] The hydrogel composition of the present invention is positive charged, which allows negatively charged molecules to be solubilized, internalized and complexed with the polymer. Therefore, it is also an object of the present invention a delivery systemcomprising the hydrogel composition and therapeutic agent, wherein the therapeutic agent comprises a negatively charged molecule.

[0024] In another advantageous aspect of the present invention, the positive charge of the hydrogel composition allows genetic material, comprising a highly negatively charged phosphate backbone, to be complexed with the hydrogel composition and thereby allowing the delivery of genetic material to target cells, such as cancer cells.

[0025] Therefore, in another embodiment of the delivery system, the therapeutic agent comprises genetic material in complex with the hydrogel composition. In a preferred embodiment of the present invention, the genetic material is RNA, preferably miRNA.

[0026] The ability of the hydrogel composition to interact with negatively charged molecules allows the simultaneous binding to genetic material and to active compound molecules, thereby constituting a delivery system comprising the hydrogel composition with a negatively charged molecule and genetic material, such as RNA, preferably miRNA.

[0027] In an advantageous aspect of the present invention, the delivery mechanism of the genetic material or active compounds bound to the hydrogel composition is sensitive to pH, being triggered by acidic pH, as the acrylate linker is broken thereby releasing PEI from the Pluronic® L121-PEI polymer upon cellular internalization and lysosomal degradation. The products of lysosomal degradation are then released in the cytoplasm, in which genetic material such as miRNA or active compounds can exert a therapeutic function. In addition, the released polymers, Pluronic® L121 and PEI, are excreted by renal clearance mechanisms without bioaccumulating.

[0028] DESCRIPTION OF THE FIGURES

[0029] Figure 1 – Chemical synthesis of cross-linked Pluronic® L121-PEIpolymer, comprised in the hydrogel composition of the present invention.Figure 2 – Container of the synthesized hydrogel composition, showing a yellow opaque appearance.

[0030] Figure 3 –1H-NMR and FTIR spectra of the synthesized hydrogel composition – L121-PEI.

[0031] Figure 4 – Thermal characterization of the synthesized hydrogel composition (PP) in comparison with Pluronic® L121 and PEI polymers.

[0032] Figure 5 – Turbidimetric assay to assess the mucoadhesive properties of the synthesized hydrogel composition (PP)in mucin dispersions. In the presence of saliva (B), the synthesized hydrogel composition outperforms the reference mucoadhesive polymer chitosan (Ch) and hyaluronic acid (HA).

[0033] Figure 6 – Critical micellar concentration (CMC) of the synthesized hydrogel composition (PP)at 25 °C and 37° C (A); and reticular structure of the synthesized hydrogel composition at different concentrations (B). Advantageously, the micellar hydrogel composition can be diluted at values below the CMC, which was determined to be 68 µg / mL at 25 °C and 38 µg / mL at 37 °C.

[0034] Figure 7 – Buffering capacity and complexation of the synthesized hydrogel composition (PP) with genetic material. (A) Titration of the synthesized hydrogel composition, in which the hydrogel composition presents similar buffer capacity as the PEI used for its chemical synthesis. (B, C) Capacity of the synthesized hydrogel composition polymer to complex genetic material (D) in the presence of 10% fetal bovine serum (FBS).

[0035] Figure 8 – (A, B) In vitro reduction of metabolic activity of the synthesized hydrogel composition (PP), present at a concentration between 25 µg / mL and 150 µg / mL in highly invasive oral cancer cells, HSC-3 cells; (C) Hemolysis assay in the presence of 5 mg / mL of the synthesized hydrogel composition.Figure 9 – Cellular uptake assay of model drug coumarin-6 (C6) in the presence or absence of the synthesized hydrogel composition (PP). To test the capacity of the synthesized hydrogel composition as a vehicle for a hydrophobic cargo, C6 was used, wherein it was observed that the synthesized hydrogel composition increases significantly the uptake of C6 more than 6 times comparing to the free C6.

[0036] Figure 10 – In situ delivery of genetic material in complex with the synthesized hydrogel composition (PP) to OSCC cells (SCC-9) and to HSC-3 cells with high metastatic potential.

[0037] Figure 11 – (A) Internalization profile of miRNA in complex with the synthesized hydrogel composition (PP)by HSC-3 cells. (B) Representation of the cellular internalization of miRNA in complex with the synthesized hydrogel composition, followed by the release of miRNA cargo inside the cytoplasm and excretion of the PEI and Pluronic® L121 polymers. In acidic (pH 5.0) and neutral pH (pH 7.4), the ester pH-sensitive bound of the crosslinked Pluronic® L121-PEI polymer is broken down and it may be an explanation for the advantage release of the miRNA cargo complexed with the synthesized hydrogel composition.

[0038] Figure 12 – (A) Therapeutic effect of the administration of miRNA in complex with the synthesized hydrogel composition (PP) in a 3D OSCC homotypic spheroid model, during 18 days. (B) OSCC homotypic spheroid size reduction after treatment with the miRNA in complex with the synthesized hydrogel composition. (C) Metabolic activity reduction of the 3D OSCC homotypic spheroids after treatment with miRNA in complex with the synthesized hydrogel composition. (D) Reduction of the viability of 3D OSCC homotypic spheroids after treatment with miRNA in complex with the synthesized hydrogel composition, showing signs of blebbing which is an indication of cell death by apoptosis.

[0039] DETAILED DESCRIPTION

[0040] The more general and advantageous configurations of the present invention are described in the Summary of the Invention. Such configurations are detailed below inaccordance with other advantageous and / or preferred embodiments of implementation of the present invention.

[0041] Preferably, the first organic solvent to dissolve the poloxamer-188 block copolymer, Pluronic® L121, comprises anhydrous benzene.

[0042] In a preferred embodiment of the present invention, the second organic solvent used to dissolve both activated poloxamer-188 block copolymer and the PEI cationic polymer, present in a 1:2 molar ratio, comprises dichloromethane.

[0043] In a preferred embodiment of the present invention, the PEI cationic polymer to be conjugated with the poloxamer-188 block copolymer consists in low molecular weight-polyethyleneimine (LMW-PEI).

[0044] In an advantageous aspect of the present invention, the hydrogel composition revealed to be mucoadhesive, translated in the increase of work of adhesion and the detachment force, which is relevant in the context of using therapeutic approaches based on the synthesized hydrogel composition to treat OSCC, considering that the primary site of development of OSCC is in the buccal mucosa. Based on turbidimetric assays, the mucoadhesive properties of the synthesized hydrogel composition were evaluated using mucin dispersions, in which the synthesized hydrogel composition performs similarly to the mucoadhesive polymer used as reference, Chitosan - Fig. 5A-and outperforms said reference mucoadhesive polymer in the presence of saliva, the most abundant fluid in buccal mucosa - Fig. 5B.

[0045] Regarding the ability to deliver active compounds or genetic material, the synthesized hydrogel composition interacts with negatively charged molecules, that could be either active compounds, such as drugs, or genetic material, such as RNA. The supramolecular structures formed in aqueous environment allow negative charged molecules to be solubilized in the core of the micelles in the hydrogel composition, or to be electrostatic interacting with the polyethyleneimine shell of the synthesized the hydrogel composition. Using a model drug, rosmarinic acid, that is negatively charged atphysiological pH, it was found that he solubility of rosmarinic acid in the synthesized hydrogel composition increases without statistical significance, having a mass polymer:active compound ratio of 3:1. Interestingly, the release and permeability profile of free rosmarinic acid, or in the absence of synthesized hydrogel composition, shows a burst release was observed in the first hours of the experiment. However, when rosmarinic acid is conjugated with the synthesized hydrogel composition, a modified release was observed at least during 48 h, in the presence of saliva. Interestingly, as rosmarinic acid molecules could interact with the positive charges of the synthesized hydrogel composition, the hydrodynamic diameter of the micelles decreases approximately 2.5-fold, which facilitates cellular uptake and internalization of the micelles. However, a considerable decrease on the charge was also observed, being approximately + 10 mV in the presence of rosmarinic acid. Nevertheless, the remaining positive charge is of benefit to electrostatically promote the electrostatic interaction with genetic material, which allows the delivery system based on the hydrogel composition to transport and deliver active compounds and genetic material simultaneously.

[0046] In an advantageous aspect of the present invention, the synthesized hydrogel composition is found to induce the reduction of metabolic activity in a representative model of highly invasive oral cancer, HSC-3 cells - Fig. 8A, the hydrogel composition being present at a concentration between 25 µg / mL and 150 µg / mL. The half maximal inhibitory concentration (IC50) of the synthesized hydrogel composition is of approximately 55 µg / mL. This effect is likely due to the highly positive surface charge, in which the interaction with the negatively charged components present in the cell membrane of cancer cells may result in its destabilization and may compromise cellular membrane integrity.

[0047] Moreover, the reduction of metabolic activity by the hydrogel composition is selective for tumor cells and time dependent, in which the impact in normal mucosa is only achieved from 10-fold the concentration necessary to reduce the metabolic activity in cancer cells, as observed in the ex vivo study for a possible buccal administration, Fig.

[0048] 8B. In addition, no hemolysis was observed after the exposure to 5 mg / mL of thesynthesized hydrogel composition, Fig. 8C, which is relevant when considering a parenteral administration of the synthesized hydrogel composition.

[0049] In an advantageous aspect of the present invention, the hydrogel composition outperforms the reference PEI polymer in the delivery of genetic material, in particular therapeutic miRNA, to OSCC cells in situ (SCC-9) and with highly metastatic potential cancer cells (HSC-3), Fig. 10.

[0050] In addition, using a 3D OSCC homotypic spheroid model that could reliably mimic a tumor microenvironment, it was demonstrated that the synthesized hydrogel composition also outperforms the reference PEI polymer in the delivery of a therapeutic miRNA, in which it was observed a decrease in size of the OSCC spheroids over the course of 18 days, Figs. 12A and 12B. Moreover, the metabolic activity, and consequently cell viability, of the OSCC spheroids were also decreased using the synthesized hydrogel composition as delivery system, compared to the reference PEI polymer and control cells, Fig. 12C, in which it blebbing of treated cells was observed by SEM morphological, Fig. 12D.

[0051] Together, these results revealed that solutions based on the synthesized hydrogel composition can outperform PEI polymer-based solutions and, thereby, constitute a smart strategy to use in the treatment of OSCC, having versatile properties for local or intravenous administration without affecting normal tissue or induced hemolysis in therapeutic doses.

[0052] Of course, the preferred embodiments shown above are combinable, in different possible configurations, being the present invention not limited to the embodiments previously described.EXAMPLES

[0053] Synthesis of hydrogel composition

[0054] For the first reaction, the block copolymer Pluronic® L121 (15.1 g) was dissolved in anhydrous benzene (60 mL) into a three-neck round bottom flask. After, triethylamine (2.75 mL) and acryloyl chloride (1.60 mL) were added dropwise using a graduated dropping funnel, and the mixture was kept under stirring at 20 ± 1 °C in reflux for 3 h. The reaction mixture was then filtered to remove the produced triethylamine hydrochloride, and benzene was evaporated in a rotary evaporator in a fume hood. The obtained product was subjected to liquid-liquid extraction using a separatory funnel, the organic phase was collected and preserved, and the aqueous phase was subjected to the same protocol another three times using ultrapure water. The acquired organic phase was collected and evaporated using a rotary evaporator, and the obtained residue was dried at 40 °C in a vacuum oven overnight to remove any remaining organic solvent.

[0055] The resultant product was structurally characterized prior to the following step reaction. The successfully activated Pluronic ® L121 diacrylate was conjugated with LMW-PEI through a Michael addition reaction in a 1:2 molar ratio. In brief, 2.0 g of low molecular weight branched polyethylenimine (LMW-bPEI) was dissolved in 6.0 mL of dichloromethane in a round bottom flask and Pluronic® L121 diacrylate dissolved in 6.0 mL of dichloromethane was added dropwise using a graduated dropping funnel. The reaction was carried out in quintuplicate in a Radleys carrousel reaction station, under stirring at 45 ± 0.1 °C for 48 h. The remaining dichloromethane was evaporated in a rotavator. The resultant products, Pluronic® L121-PEI, were isolated as yellow residues and left in a vacuum-drying oven overnight at 25 ± 0.1 °C to remove any residual organic solvent.

[0056] A sticky yellow solid was then obtained, corresponding to the cross-linked hydrogel composition Pluronic® L121-PEL 6 mg of each final reaction product was structurally characterized by ATR-FTIR and1H-NMR using D2O. The lack of proton peaks between2.57 and 2.72 ppm, may indicate that all the unreacted PEI was removed. The reaction products were kept at 4 °C in a desiccator until further use.

[0057] Upon visual observation, the hydrogel composition appeared as cloudy emulsions above 14 °C, whereas yellow transparent at 4°C, indicating the formation of particles for temperatures higher than it.

[0058] Structural characterization of the synthesized hydrogel composition

[0059] 1H-NMR and FTIR

[0060] FTIR analysis showed that Pluronic® L121 diacrylate was successfully synthetized as a new band around 1730 cm−1(C=O bond) appears. The conjugation of Pluronic® L121 diacrylate with PEI was confirmed by the presence of a band between 3380 and 3390 cm−1(N–H bond).1H-NMR results showed characteristic proton peaks from Pluronic (-CH3at δ1.1 ppm) and from PEI (-CH2-CH2NH- between δ2.7–3.4 ppm) and the molar ratio Pluronic-PEI was 1:2 - Fig. 3. This estimation was corroborated by a TNBS assay that reveals, based on a standard calibration curve using the PEI present in the innovative nano system, that the synthesized compound was constituted by approximately 40% of PEI.

[0061] Thermal characterization of the synthesized hydrogel composition

[0062] The thermal characterization revealed that the synthesized hydrogel composition presents characteristics of amorphization indicating it is fully crosslinked, as no glass transition was observed in the ramp temperature used, after colling and heating and vice-versa - Fig. 4.

[0063] Colloidal Properties and morphology of the synthesized hydrogel composition

[0064] DLS / ELS

[0065] The colloidal properties of the synthesized hydrogel composition were evaluated in terms of hydrodynamic diameter (nm), polydispersity index (Pdl) and zeta potential, asa mean of stability after dilution, upon filtration, at different temperatures (25 °C and 37 °C) and using different aqueous dispersants (nuclease free water and HEPES buffer, pH 7.4). The nanoparticles' hydrodynamic diameter was approximately 125 nm with a Pdl below 0.250, and a charge nearby +40 mV, even upon dilution. Samples of the synthesized hydrogel composition were placed on glass slides and examined using light microscopy revealing the supramolecular spheric shape of the synthesized cross-linked hydrogel composition. Moreover, as denoted by the evaluation of the CMC and the transmission electron microscopy (TEM) images upon a dilution below 30 pg / mL the structural properties were maintained, which is of importance to delivery active compounds by systemic administration, indicating its stability instead upon dilution to low concentration avoiding the burst release of the active substance - Fig. 6. Upon visual observation, the synthesized hydrogel composition appeared as cloudy emulsions.

[0066] Interestingly it is notorious in the TEM microscopy images that a reticular structure is present in the hydrogel composition for concentrations of 20 mg / mL, which is also visible for concentrations of 50 pg / mL with less visible cross-linking for concentrations below 25 pg / mL.

[0067] Buffering capacity of the synthesized hydrogel composition and complexation with genetic material

[0068] The buffering capacity of the synthesized hydrogel composition was evaluated by acid–base titration, Fig. 7A, to assess the possible in vitro translation and efficacy to deliver cargos and promote endosomal escape. The pH of the hydrogel composition, equivalent to 0.4 mg / mL of branched polyethylenimine (bPEI1.8k) was adjusted to 12 by the addition of NaOH IM, and the solution was titrated with 1 M HCI until reaching pH = 2. The pH values were recorded using a pH meter.

[0069] The ability of hydrogel composition to complex miRNA (22 bp) was examined by electrophoresis. Freshly prepared polyplexes (N / P 5-100) were applied on agarose gel (1% in tris acetate EDTA buffer) stained with GreenSafe Premium (NZYTech, PT).Electrophoresis was performed at a constant voltage (80 V) for 40 min. The gels were visualized using a StepOne® detection system (Thermo Fisher Scientific, Winsford, UK), and also using the displacement assay with instructions, SYBR Gold (Invitrogen). The Yellow fluorescence resulting from the hybridization of SYBR Gold to free mRNA was measured in opaque black well-plates, using a microplate reader (Biotek) set with excitation wavelength = 495 nm and emission wavelength = 537 nm.

[0070] As observed in Fig. 7A, the synthesized hydrogel composition presents similar buffer capacity as the PEI used for its chemical synthesis, which protects the hydrogel composition from lysosomal degradation. Moreover, the capacity to complex genetic material was considerable higher for the synthesized hydrogel composition compared to PEI, which is of paramount importance to carrier and protect the genetic material to the target site (Fig. 7B, 7C). Moreover, as revealed in Fig. 7D, instead in the presence of 10 % of FBS, that mimics serum proteins it is notorious that in a ratio higher than 5 / 1 (Nitrogen / Phosphate) the synthesized hydrogel composition can complex microRNA.

[0071] Delivery of Genetic material

[0072] The cellular uptake of coumarin-6 (C6) significantly increases after the entrapment in the synthesized hydrogel composition, indicating its cellular internalization.

[0073] Moreover, as revealed by the kinetic profile of acridine orange the ratio between red to green fluorescence increase in the first 60 minutes indicating the internalization of the synthesized hydrogel composition complexed with a therapeutic miRNA. However, a decrease is observed after, indicating that the pH sensitive bound associated with the acrylate linker was broken and the polymers were released from the lysosome to the cytoplasm - Fig. 11A. Here, the therapeutic microRNA could exert its function by modulating different key mechanisms in OSCC, particularly epithelial-to mesenchymal transition and the polymers were release and excreted by renal clearance mechanisms, as reported before, without bioaccumulating - Fig. 11B.Sterility assay - parenteral administration of the synthesized hydrogel composition

[0074] Considering the possible parenteral administration of the hydrogel composition, a sterility assay was conducted. For this, hydrogel composition dispersions were sterilized by terminal filtration, recurring to 0.22 pm cellulose acetate membrane filters (VWR, Manati, Puerto Rico). Filtered hydrogel composition dispersions were directly inoculated Tryptic (Trypticase) Soy Agar (TSA) digest media in 150 mm plates and incubated at 37 °C, meeting the requirements of United States Pharmacopeia (USP), European Pharmacopoeia (EP) and Japanese Pharmacopoeia (JP)l-3 for performance specifications. Digest medium was used as negative control in parallel. Microbial growth was evaluated visually in terms of colony forming units (CFUs), in which no colonies were observed for 7 days at 37°C, confirming that the hydrogel composition dispersion is sterile.

Claims

CLAIMS1. A hydrogel composition comprising a poloxamer-188 block copolymer and a polyethylenimine cationic polymer, wherein the molar ratio of said poloxamer- 188 block copolymer to polyethylenimine cationic polymer is 1:2 and water is present at a concentration of 25% to 50% of the total weight of the hydrogel composition.

2. Hydrogel composition according to claim 1 wherein the polyethylenimine cationic polymer comprises low molecular weight-polyethylenimine.

3. Delivery system comprising the hydrogel composition according to any of the previous claims and a therapeutic agent, wherein the hydrogel composition is present at a concentration between 25 µg / mL and 20 mg / mL.

4. Delivery system comprising the hydrogel composition according to claim 3 and a therapeutic agent, wherein the hydrogel composition is present at a concentration between 25 pg / mL and 150 pg / mL.

5. Delivery system according to any of claims 3 – 4, wherein the therapeutic agent comprises a negatively charged molecule and / or genetic material.

6. Delivery system according to claim 5, wherein the genetic material is in complex with the hydrogel composition, the genetic material being comprised of RNA, preferably miRNA.

7. Delivery system according to any of claims 3 – 6 for use in the treatment of oral squamous cell carcinoma.

8. Process for obtaining a hydrogel composition comprising the following steps: a. Dissolving the poloxamer-188 block copolymer in a first organic solvent;b. Adding triethylamine and acryloyl chloride to the dissolved poloxamer- 188 block copolymer to form a first reaction mixture;c. Stirring the first reaction mixture in reflux at 20 °C for at least 3 hours to form a first product mixture;d. Filtering the first product mixture to obtain a filtered product mixture; e. Performing a liquid-liquid extraction and removing remaining organic solvents from the filtered product mixture to obtain an activated poloxamer-188 block copolymer;f. Mixing the activated poloxamer-188 block copolymer with a polyethylenimine cationic polymer and a second organic solvent to form a second reaction mixture, in which the activated poloxamer-188 block copolymer and the polyethylenimine cationic polymer are present in a 1:2 molar ratio;g. Stirring the second reaction mixture in reflux at 45 °C for at least 48 hours to form a second product mixture;h. Removing remaining organic solvents from the second product mixture to obtain a poloxamer-188-polyethylenimine polymer;i. Diluting the poloxamer-188-polyethylenimine polymer in an aqueous solution to obtain an hydrogel composition, wherein water is present at a concentration of 25% to 50% of the total weight of said hydrogel composition.

9. Process for obtaining a hydrogel composition according to claim 8 wherein the first organic solvent is selected from anhydrous benzene.

10. Process for obtaining a hydrogel composition according to any of claims 8 – 9, wherein the second organic solvent is selected from dichloromethane.

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