Temperature responsive in SITU gelling composition

Abstract

The present invention relates to a temperature-responsive in-situ gelling composition comprising an amphiphilic triblockcopolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO–PPO–PEO), a water soluble hydrophilic polymer whereby the composition further comprises at least a lipidic agent. The lipidic agent is selected from the group consisting of phospholipids, sterols, glycerides, fatty acids or esters and derivatives, fatty alcohols, cholesterol and derivatives, or a combination of more than one lipidic agent. The invention also relates to a vascular embolizing agent comprising the temperature- responsive in-situ gelling composition.

Description

[0001] TEMPERATURE RESPONSIVE IN SITU GELLING COMPOSITION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a temperature responsive in situ gelling composition. The invention also relates to a temperature responsive formulation comprising the composition. The invention further relates to medicinal delivery systems locally applied through the formulation which forms gel in-situ and enables drugs to be released in a controlled fashion over a period of time and at a designated concentration. Furthermore the invention relates to an embolizing agent comprising the in situ gelling composition or formulation.

[0004] BACKGROUND OF THE INVENTION

[0005] Hydrogels are three-dimensional networks formed by water-soluble polymers that are interconnected through physical or chemical interactions. Due to their porous structure, they have the ability to absorb a significant amount of water when placed in aqueous environments. The porosity of hydrogels allows for the loading of drugs into the gel matrix and subsequent drug release, which is dependent on the diffusion rate of small or large molecules through the gel network.

[0006] Hydrogels can be classified into two categories based on the mechanism of their polymer network formation. The first category is based on physical networks, gel networks formed by temporary interactions such as ionic forces, hydrogen bonds, or non- covalent hydrophobic linking. The second category is based on chemical networks, gel networks composed of more permanent formations through chemical reactions such as covalent cross-linked bonding. Physical hydrogels tend to be more easily reversible while chemical hydrogels can even be irreversible.

[0007] Hydrogel drug delivery has a number of documented benefits such as slow drug release, sustained high concentration at the target tissue for extended durations, bioadhesive properties, biodegradability, shape adaptability, biocompatibility with the biologic environments, similarity to extracellular fluids, and utility in systemic drug delivery regimens.

[0008] However, hydrogels suffer from certain limitations that impede certain applications. Common limitations include loading drugs uniformly through the network, rapid loss of the drug from networks with large amounts of water and / or larger pore structures, and low mechanical strength.

[0009] Hydrogels, within the main polymer networks, comprise functional groups that typically respond to stimuli which are commonly referred to as “stimuli-responsive polymers” or “smart polymers.” Some hydrogels are also referred to as “smart hydrogels,” due to their capability of responding to environmental stimuli. This includes physical stimuli such as temperature, electric or magnetic fields, light, pressure, or sound, and chemical stimuli such as acid-alkaline reactions (pH), solvent composition, ionic strength, and molecular species. Smart hydrogels find application in various fields, including wound dressings, cell immobilization, drug delivery systems, tissue engineering, and embolization.

[0010] For smart hydrogels, the polymer structure in solution is formed through polymerpolymer and polymer-solvent interactions. In suitable solvents, polymer-solvent interactions dominate, and intrachain entanglements are minimized, resulting in polymer chains adopting a free state. However, if a polymer is placed in an unsuitable solvent, the polymers aggregate due to increased polymer-polymer interactions, leading to reduced polymer chain mobility. Chemical and physical stimuli, by altering polymer-solvent interactions, cause hydrogel swelling.

[0011] The aim of the present invention is to overcome the above mentioned problems. In the present invention, temperature-responsive hydrogels are in particular of interest. More in particular negative temperature-sensitive hydrogels that have the lower critical solution temperature (LCST) are of interest. Polymers of the LCST type display favorable interactions with solvents at lower temperatures in which they undergo a sudden transition from a coil-like structure to a globule-like structure once the temperature surpasses their LCST. This transition results in their precipitation from the solution due to an increase in entropy. In essence, the hydrogel is in a gel state above the LCST and a soluble state while below the LCST. Conversely, UCST-type polymers are non-soluble at low temperatures and become soluble above the UCST by an enthalpic-driven process. Thermally reversible LCST-type hydrogels transition from a soluble phase to a gel phase as the temperature increases. These amphiphilic polymers are soluble in aqueous environments at lower temperatures. As the temperature increases, they attain hydrophobic properties in certain regions which aggregate and reduce the water present in those areas. Further, the gelation temperature of these classes of hydrogels depends on several factors including the length of the hydrophobic polymer chain, the chemical structure of the polymer, and the concentration of polymers present.

[0012] Formulations that gel in response to temperature fluctuations have polymers that bind together and effectively trap water molecules. As the free water content decreases and leads the concentration of polymer to reach the critical micelle concentration (CMC), micellar structures form. This process creates a hydrogel system and uses the unique properties of these micelle structures to provide various benefits.

[0013] Among temperature-responsive hydrogels, a class of amphiphilic triblock copolymers with the base molecular structure polyoxyethylene-polyoxypropylene- polyoxyethylene (PEO-PPO-PEO) (known as Pluronics or poloxamers) is one of the most commonly used. These hydrogels are known in the art and for example described in EP2734187. In these types of hydrogels, additives such as cosolvents, salts, and surfactants can alter the gelation temperature by affecting polymer-solvent interactions. For example, surfactants, due to their amphiphilic nature, can adsorb polymer molecules upon addition to the solution, leading to a change in the balance between hydrophilic and hydrophobic interactions. This can result in either the prolongation or elimination of the gelation phenomenon. Hydrogels comprising for example poloxamer, and a hydrophilic polymer such as hyaluronic acid (HA)-chitosan is disclosed in CN112516075. Also LIS2014005306 discloses a HA-gelatin-pluronic based hydrogels and methods for making the HA-gelatin-pluronic hydrogels. Also US9364545 discloses a thermosensitive injectable hydrogel based on HA and a copolymer of polyethylene oxide (PEO) and polypropylene oxide (PPO), which has a gel formation temperature from 30 degrees centigrade to 37 degrees centigrade The thermosensitive injectable hydrogel of the invention provides a potential drug delivery system that can increase therapeutic efficacy of the drug.

[0014] In the present invention, responsive hydrogel systems are preferred, particularly those based on polyoxyethylene-polyoxypropylene- polyoxyethylene (PEO-PPO-PEO) copolymers, which we have found to exhibit unique properties benefiting in-situ gelling systems. The object of the present invention is to provide a thermo-responsive hydrogel composition that results in a hydrogel which means the hydrogel with enhanced stability and structural features which means the hydrogel provides long-term stability under physiological conditions and improved mechanical properties such as increased elasticity and strength. It is a further object of the present invention to provide a composition that forms a hydrogel in reduced gelation time. It is still a further object of the present invention to control gelation behavior in formulations designed for biomedical applications. .It is still a further object of the present invention to provide a composition that is capable of carrying both hydrophilic and hydrophobic bioactive agents, enabling versatile and sustained delivery within physiological environments

[0015] SUMMARY OF THE INVENTION

[0016] The present invention in its various embodiments describes the composition of an in-situ gel forming network which acts as a localized delivery vehicle for bioactive agents or medications. The invention utilizes several unique properties of polyoxyethylene-polyoxypropylene- polyoxyethylene (PEO-PPO-PEO) such as poloxamer copolymers to form hydrogels with micellar structures enabling improved performance as an in-situ gel drug delivery technique. Embodiments of the present invention can be used for a variety of pharmaceutical tasks, particularly those where the medication needs to be delivered in high-local concentrations or over an extended period of time. Embodiments of the present invention can also be used as a liquid embolic system for the embolization of lesions in the peripheral vasculature.

[0017] The term "hydrogel" as used herein refers to a three-dimensional, hydrophilic or amphiphilic polymeric network capable of taking up large quantities of water. The networks are composed of homopolymers or copolymers, and are insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interactions, entanglements) crosslinks. The crosslinks provide the network structure and physical integrity. Hydrogels exhibit a thermodynamic compatibility with water that allow them to swell in aqueous media.

[0018] The term "lipidic agent" as used herein refers to a component within the formulation that primarily consists of lipophilic molecules. These agents can include various types of lipids, such as phospholipids, sterols, fatty acids, and lipid-based surfactants. The lipidic agents may contribute to the stability, structural integrity, and functional properties of the hydrogel by interacting with the polymer network, either by embedding within or enhancing the formation of micellar or vesicular structures. Their inclusion can enhance the formulation's mechanical properties, control the gelation time, and potentially improve the system’s biocompatibility and efficacy in biomedical applications.

[0019] The term "crosslinked" as used herein refers to a composition containing intramolecular and / or intermolecular crosslinks, whether arising through covalent or noncovalent bonding. "Noncovalent" bonding includes hydrogen bonding, electrostatic (ionic) bonding, and hydrophobic interactions. These hydrophobic interactions occur when nonpolar segments within the polymer network cluster together in aqueous environments, enhancing network stability and physical integrity without the formation of covalent bonds.

[0020] The term "temperature sensitive" hydrogel as used herein refers to a block copolymer of the present disclosure and forms, to various degrees, a jelly-like or gelled product when heated to a particular temperature, for example body temperature (37 degrees centigrade), or a temperature higher than 34 degrees centigrade. The block copolymer soluble in water and the resulted solution is preferably a liquid at room temperature, but upon reaching a particular temperature, forms a hydrogel when mixed with water such that water is a dispersion medium forming the hydrogel.

[0021] The term "in situ" as used herein is defined as restricted to a specific site within a body without substantial invasion of surrounding tissues.

[0022] The term weight per volume (w / v%) herein defined means weight per volume, also known as m / v. For example: if 1g X is used for making 100ml solution, it is represented as 1%w / v solution of X.

[0023] The term volume per volume (v / v%) herein defined means volume per volume. For example: if 1 ml X is used for making 100ml solution, it is represented as 1%v / v solution of X.

[0024] The term "bioactive agent" is used herein to refer to a chemical material or compound suitable for administration to a human patient and that induces a desired beneficial effect, e.g., exhibits a desired pharmacological activity. The term includes, for example, agents that are therapeutically effective, prophylactic ally effective, and cosmetically (and cosmeceutical) effective. Also included are derivatives and analogs of those compounds or classes of compounds specifically mentioned which also induce the desired beneficial effect.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG 1: is a TEM image which reveals distinct lipidic agents within the in situ gelling composition.

[0027] FIG 2: is a (TEM) image showing a lipid bilayer Vesicles.

[0028] FIG 3: Atomic Force Microscopy (AFM) of the Gel Surface.

[0029] FIG.4 is a graph demonstrating the effects of Fluorouracil (5-Fll) on the cell viability of HEPG2 cells of embodiment 3.

[0030] FIG.5a is a graph depicting data comparing the drug concentration at the brain between free drug application as well as a methotrexate-gel, niosomal methotrexate, and a niosomal methotrexate gel embodiments of the invention at t = 60 min for embodiment 7.

[0031] FIG.5b is a graph depicting data comparing the drug concentration in plasma between free drug application as well as a methotrexate-gel, niosomal methotrexate, and a niosomal methotrexate gel embodiments of the invention at t = 60 min for embodiment 7 of the invention.

[0032] FIG.5c is a graph depicting data comparing the drug concentration for brain-to-plasma concentration ratio (Kp) between free drug application as well as a methotrexate-gel, niosomal methotrexate, and a niosomal methotrexate gel embodiments of the invention at t = 60 min for embodiment 7 of the invention.

[0033] FIG.6 is a CT scan which image demonstrates the radiopacity and in situ gel formation of an injected formulation in a comparative study involving the tails of two mice and the jugular vein of one rat.

[0034] FIG 7 shows a Flow Curve Analysis (Tau vs. Shear Rate) of 3 different compositions.

[0035] FIG 8 shows a Viscosity Curve Analysis (Viscosity vs. Shear Rate) of 3 different compositions.

[0036] FIG 9 shows a Creep Compliance Curve (J(t) vs. Time).

[0037] FIG 10 shows a measurement on gelation time of different compositions at different temperatures.

[0038] FIG 11 shows the results of an in vitro drug release test as disclosed in Example 8.

[0039] DETAIL DESCRIPTIONS OF THE INVENTION

[0040] All illustrations of the drawings are for the purpose of describing selected versions of the present invention and are not intended to limit the scope of the present invention. Unless otherwise stated, all percentages provided herein are weight percentages, based on the total weight of the medicinal delivery composition, except where noted otherwise.

[0041] The present invention thus provides a temperature-responsive in-situ gelling composition is provided comprising : a) an amphiphilic triblockcopolymer of polyoxyethylene-polyoxypropylene- polyoxyethylene (PEO-PPO-PEO) b) a water soluble hydrophilic polymer whereby the composition further comprises at least a lipidic agent.

[0042] Surprisingly it has been found that the thermo-responsive hydrogel composition comprising the lipidic agent(s) results in a stable hydrogel with notably enhanced structural integrity, resistance to premature degradation, and good mechanical properties, including elasticity and tensile strength. These characteristics contribute to the hydrogel’s resilience under physiological conditions. Furthermore, the composition exhibits a reduced gelation time and controllable gelation behavior, making it highly suitable for biomedical applications.

[0043] The temperature-responsive in-situ gelling composition according to the present invention comprises an amphiphilic triblockcopolymer of polyoxyethylene-polyoxypropylene- polyoxyethylene (PEO-PPO-PEO) which are also known as poloxamers and commercially known by the Pluronic® trademark. The critical micelle concentration (CMC) of poloxamers decreases with temperature, making them suitable for forming micelles and subsequently gels at higher temperatures. Poloxamer 407 (Pluronic F-127) is one of the more commonly used poloxamers, comprising 70% PEO and 30% PPO. It is a nonionic surfactant with good water solubility, low toxicity, and excellent drug-release properties. Poloxamer gel formulations exhibit a thermally reversible behavior, transitioning from a liquid state at room temperature to a gel state at body temperature.

[0044] The present invention formulates PEO-PPO-PEO-based in-situ compositions that offer versatile platforms for drug delivery and other applications due to their unique properties, including controlled drug release, biocompatibility, and responsiveness to environmental conditions such as pH and temperature.

[0045] The water soluble hydrophilic polymer in the temperature-responsive in-situ gelling composition is selected from the group of consisting of chitosan, hyaluronic acid (HA), polyvinylalcohol or polyethylene glycol, carboxymethyl cellulose, alginate, pectin, carrageenan, gelatin, poly(acrylic acid), xanthan gum, hydroxypropyl methylcellulose, poly(N- isopropylacrylamide), collagen or combinations thereof. Preferably the hydrophilic polymer is chosen from polyvinylalcohol, chitosan or hyaluronic acid or combinations thereof.

[0046] The lipidic agent(s) in the temperature-responsive in-situ gelling composition is / are for example selected from the group consisting of phospholipids, sterols, glycerides, fatty acids or esters and derivatives, fatty alcohols, cholesterol and derivatives, or a combination of more than one lipidic agent. Preferably the lipidic agent(s) is / are selected from the group consisting of phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, glycerolphosphate, fatty acids esters such as ethoxylated and non-ethoxylated sorbitan fatty acid esters or cholesterol or combinations thereof. The lipidic agent may also be selected from ethoxylated or non-ethoxylated sorbitan fatty acid esters such as polyoxyethylene sorbitan monooleate or sorbitane monostearate. Both polyoxyethylene sorbitan monooleate and sorbitan monostearate are considered lipidic agents because of their fatty acid components. The temperature-responsive in-situ gelling composition according to the present invention may further comprise at least a non-ionic surfactant chosen from an amphiphilic non-ionic surfactant such as chosen from TWEEN 20, TWEEN 40, TWEEN 60, TWEEN 80, SPAN 20, SPAN 40, SPAN 60, SPAN 80, Brij 35, and Poloxamer 188. The incorporation of the non-ionic surfactants enhances the arrangement of amphiphilic structures in the micelle formation, making it more conducive for encapsulating hydrophilic APIs.

[0047] The temperature-responsive in-situ gelling composition according to the present invention preferably comprises between 10-30 w / v% of the amphiphilic triblockcopolymer based on the total weight of the composition. More preferably it comprises between 15-25 w / v% of the amphiphilic triblockcopolymer based on the total weight of the composition.

[0048] The hydrophilic polymer in the temperature-responsive in-situ gelling composition is preferably present in an amount between 0.5-5 w / v% based on the total weight of the composition. More preferably the hydrophilic polymer is present in an amount between 0.8- 3.5 w / v% based on the total weight of the composition

[0049] The temperature-responsive in-situ gelling composition preferably comprises the lipidic agent(s) in an amount between 0.1-5 w / v% based on the total weight of the composition, preferably between 0.5-3.5 w / v% based on the total weight of the composition.

[0050] The non-ionic surfactant is preferably present in an amount between 0-5 v / v% based on the total weight of the composition, preferably between 1-3 w / w% based on the total weight of the composition.

[0051] The temperature-responsive in-situ gelling composition according to the present invention preferably comprises a composition comprising the amphiphilic triblockcopolymer of polyoxyethylene-polyoxypropylene- polyoxyethylene (PEO-PPO-PEO) such as poloxamer, combined with a hydrophilic polymer such as for example polyvinyl alcohol (PVA) and lipidic agents, specifically chosen from phosphatidylcholine, cholesterol, and PEGylated phospholipid or combinations thereof. These lipidic agents introduce significant hydrophobic and hydrophilic interactions into the hydrogel matrix, enhancing its mechanical and structural properties. It seems that from a thermodynamic perspective, adding lipidic agents like phosphatidylcholine and cholesterol reduces the enthalpy of mixing in the hydrogel by promoting favorable hydrophobic interactions, especially with the PPO blocks of poloxamer. Phosphatidylcholine’s amphiphilic nature — characterized by hydrophilic heads and hydrophobic tails — facilitates the formation of organized structures such as bilayers or liposomes within the aqueous gel environment, as seen in Figure 1 and Figure 2 (TEM and AFM images). These vesicular structures strengthen the gel by adding localized order, further reducing entropy due to the structured alignment of lipids and polymer components. These vesicles play a critical role in the composition according to the present invention, particularly for encapsulating hydrophilic and hydrophobic drugs or radiopaque agents. The vesicular structure allows for controlled and targeted drug delivery, as the lipid bilayers can trap and protect the active ingredients either within the core or the bilayer of the vesicle. Meanwhile, the outer hydrophilic surface of the vesicles ensures compatibility with the surrounding aqueous medium, enhancing the formulation's biocompatibility and functionality in drug delivery applications.

[0052] In the present invention it has been found that adding phosphatidylcholine, PEGylated phospholipid, and cholesterol to a poloxamer-PVA composition significantly alters the thermodynamics of the system by enhancing hydrophobic interactions, reducing entropy, and lowering the LOST. Phosphatidylcholine interacts with both the hydrophilic (PVA) and hydrophobic (PPO) blocks, while cholesterol rigidities the structures formed, creating stronger gels with enhanced mechanical properties. This results in a composition that can form robust, stable gels at physiological temperatures.

[0053] In the present invention it has been found that the vesicular structures or lipid vesicles accelerate gelation, as evidenced by reduced gelation time in experiments. The thermodynamic interactions of lipidic vesicles with the poloxamer and PVA matrix lower the LOST, facilitating gel formation at lower temperatures. Rheological analysis shows that lipid vesicles improve the system's rigidity and elasticity, strengthening the gel network's resistance to mechanical stress. Texture analysis further corroborates these effects, showing that the poloxamer-PVA-lipidic agent(s) combination enhances hardness and reduces deformation, indicating that a more robust gel with higher fracture resistance is formed.

[0054] The hydrogel formulation of poloxamer and the hydrophilic polymer enhanced by lipidic vesicles, qualifies as a nanocomposite whereby the poloxamer-hydrophilic polymer provides the bulk structure and sol-gel transition behavior, whereby the lipidic vesicles serve as nanoscale reinforcing agents, enhancing mechanical strength and stability through strong hydrophobic and hydrophilic interactions.

[0055] By acting as nanoscale structures within the polymer network, these lipidic vesicles adjust critical characteristics such as strength, elasticity, and cohesiveness, aligning with nanocomposite principles where dispersed nanostructures within a matrix significantly enhance the material's overall properties. This innovative formulation offers a highly tunable, robust platform for biomedical applications requiring controlled mechanical and release characteristics.

[0056] Also possible is that the temperature-responsive in-situ gelling composition according to the invention comprises the amphiphilic triblockcopolymer of polyoxyethylene- polyoxypropylene- polyoxyethylene (PEO-PPO-PEO) such as poloxamer, whereby the hydrophilic polymer is chosen from hyaluronic acid, polyvinyl alcohol, chitosan or combinations thereof whereby the lipidic agent is selected from ethoxylated or nonethoxylated sorbitan fatty acid esters such as polyoxyethylene sorbitan monooleate or sorbitane monostearate. Both polyoxyethylene sorbitan monooleate and sorbitan monostearate are considered lipidic agents because of their fatty acid components. These surfactants are lipid-like in nature.

[0057] Another possibility is that the temperature-responsive in-situ gelling composition comprises the amphiphilic triblockcopolymer of polyoxyethylene-polyoxypropylene- polyoxyethylene (PEO-PPO-PEO) such as poloxamer whereby the hydrophilic polymer is chitosan and the lipidic agent is glyceryl phosphate. Chitosan contributes to the stability and viscosity of the gel. Glyceryl phosphate will enhance gel formation and drug delivery.

[0058] The present invention also relates to a temperature-responsive in-situ gelling formulation comprising the temperature-responsive in-situ gelling composition according to the present invention and a bioactive agent selected from a pharmaceutical agent or a radio opaque agent.

[0059] The formulation can be administered in a liquid state, such as by injection or topical application, to a patient requiring targeted therapy. After administration, non-covalent crosslinking within the formulation can be induced, resulting in the formation of a hydrogel. One embodiment of this invention involves providing the hydrogel-forming composition, administering it to the target site, and allowing it to form a hydrogel at body temperature. This thermosensitive injectable hydrogel is highly adaptable and suited for intratumoral injection, trans-tumor-arterial injection, subcutaneous injection, nasal delivery, oral delivery, ocular and ophthalmic treatments, transdermal and topical applications, wound healing, intraperitoneal injections, and gene therapy. It also finds applications in tissue engineering, colon-specific drug delivery, and spinal or joint therapy. The formulation’s versatility as a hydrogel system offers localized, sustained, and controlled release of therapeutic agents across a wide range of medical applications.

[0060] In another aspect, the invention provides a drug delivery system, comprising a thermosensitive hydrogel composition of the present invention and a bioactive agent.

[0061] In another aspect, the invention provides a method for treating or alleviating one or more symptoms of a disease in a subject, comprising administering to a subject in need thereof a drug delivery system of the present invention. Typically the bioactive agent can be any substance that can be released from the composition to treat an undesirable physiological condition. Preferably the bioactive agent may be selected from a pharmaceutical agent or a radio opaque agent.

[0062] The present invention accommodates a broad spectrum of pharmaceutical agents, allowing for localized treatment to reduce systemic exposure and associated side effects. Pharmaceutical agent suitable for use within this formulation include wound healing compounds, anticancer drugs, radionuclides, gene therapy agents, hormones, nutraceuticals, antibiotics, anti-inflammatory agents, antiviral agents, and antibacterial compounds, as well as combinations of these agents.

[0063] A significant advantage of the present invention is its capacity to deliver pharmaceutical agents or a radio opaque agents directly to target sites, thereby minimizing systemic distribution that could lead to adverse effects. This localized delivery is particularly advantageous for substances with potential toxicity, which can be administered safely at specific sites, such as toxins for targeted cancer therapy.

[0064] Examples of antibiotics that can be used within the present invention are selected from the group of tetracycline, minocycline, doxycycline, ofloxacin, levofloxacin, ciprofloxacin, clarithromycin, erythromycin, cefaclor, cefotaxim, imipenem, penicillin, gentamycin, streptomycin, and vancomycin, as well as related derivatives or mixtures. For anti-cancer applications, this bioactive agents can be chosen of for example methotrexate, carboplatin, taxol, cisplatin, 5-fluorouracil, doxorubicin, etoposide, paclitaxel, docetaxel, camptothecin, cytosine arabinoside, and similar compounds, which are ideal for controlled, localized release in tumor treatments. Examples of anti-inflammatory agents are indometacin, ibuprofen, ketoprofen, piroxicam, flurbiprofen, and diclofenac which can be effectively delivered for localized inflammation reduction. Additionally, corticosteroids such as dexamethasone and triamcinolone acetonide can be incorporated for enhanced antiinflammatory effects in therapeutic applications that benefit from sustained, site-specific corticosteroid delivery.

[0065] Additionally, antiviral agents, may include acyclovir and ribavirin, provide targeted antiviral effects when used within this delivery system. Examples of antibacterial agents, are ketoconazole, itraconazole, fluconazole, amphotericin-B, and griseofulvin, which are suitable for managing localized infections within the hydrogel formulation of the present invention.

[0066] Beyond traditional therapeutic agents, the formulation can be expanded to include regenerative medicine agents, such as stem cell factors, growth factors, and tissue repair promoters. These additions extend the potential applications of the invention to advanced wound care, tissue engineering, and regenerative therapies, making the system highly versatile for a range of medical needs.

[0067] Radiopaque agents, also known as contrast agents, are substances that help enhance the contrast of images in radiographic studies, such as X-rays and CT scans. They work by absorbing X-rays, making certain areas of the body appear white or lighter on radiographs. Examples of commonly used radiopaque agents are Iodine-Based Contrast Agents such as lohexol (e.g., Omnipaque), lopamidol (e.g., Isovue), loversol (e.g., Optiray), lodixanol (e.g., Visipaque) or Barium-Based Contrast Agents such as Barium Sulfate or Gadolinium-Based Contrast Agents such as Gadopentetate dimeglumine (e.g., Magnevist), Gadobutrol (e.g., Gadovist), Gadoteridol (e.g., ProHance).

[0068] The present invention in particular relates to a vascular embolizing agent comprising the temperature-responsive in-situ gelling composition according to the invention or the temperature-responsive in-situ gelling formulation according to the present invention. A preferred temperature-responsive in-situ gelling composition may comprise poloxamer 407, PVA, phosphatidylcholine, PEGylated phospholipids, and cholesterol. Preferably the vascular embolizing agent comprising the temperature-responsive in-situ gelling composition and a hemostatic agent. Hemostatic agents are substances used to promote blood clotting and control bleeding. They can be used in surgery, trauma care, and emergency situations to help manage bleeding. Examples of hemostatic agents are fibrin sealants, thrombin, gelatin sponges, oxidized cellulose, chitosan, kaolin, tranexamic acid and microporous polysaccharide hemospheres.

[0069] The present invention also relates to a radiotherapy-enhancing system that uses the temperature-responsive, in-situ gelling composition of the present invention for targeted, controlled release of radiosensitizing agents. Upon administration, the liquid composition forms a stable gel at body temperature, creating a localized reservoir for agents that increase tumor sensitivity to radiation. This setup ensures a sustained presence of radiosensitizing agents at the target site, reducing exposure to healthy tissue and maximizing radiotherapy effectiveness. With flexibility for various delivery routes — including trans-arterial and intratumoral — the system enables precise placement and steady agent release in conjunction with radiation therapy sessions. The inclusion of lipid vesicles like phosphatidylcholine and PEGylated phospholipids in the composition of the present invention enhances the hydrogel's stability, forming a nanocomposite that improves strength and controlled release, optimizing the therapeutic outcome in radiation oncology.

[0070] The present invention also relates to a drug delivery system comprising the temperature-responsive in-situ gelling formulation according to the present invention. Reference will now be made in detail to the following more specific embodiments of the present invention. However, the following examples and comparative examples are illustrative only, and the scope of the present invention is not limited thereto.

[0071] A first embodiment of the present invention considers a composition of an in-situ hydrogel preferably comprising triblock copolymers PEG-PPO-PEG, hyaluronic acid, phosphatidylcholine and cholesterol. The composition for example comprises 20 w / v% triblock copolymers PEG-PPO-PEG, 1 w / v% hyaluronic acid, 0.5 w / v% phosphatidylcholine and 0.5 w / v% cholesterol. Triblock copolymers serve as the major gelling agent in the composition. Fluctuations in temperature may induce a rearrangement of hydrophilic and hydrophobic groups, leading to either the formation or deformation of micelles. These micelles, when formed, subsequently alter the positioning and concentration of the aqueous solvent such as water, thereby facilitating the formation of a gel.

[0072] Phosphatidylcholine enhances the stability of the composition and aids in lipid- based drug delivery. Cholesterol improves the structural integrity and fluidity of the gel. Hyaluronic acid hydrates the gel and promotes tissue compatibility. This embodiment will form an in-situ gel when combining the listed components which has generalized use in medical and pharmaceutical applications such as localized treatments and controlled drug delivery. In the process of micelle formation, thermodynamic principles dictate that a specific number of amphiphilic units assemble to create a micelle structure, minimizing thermodynamic energy. In compositions with diverse amphiphilic materials, mixed micelles may be produced, each with distinct energy levels and unit quantities. Consequently, the number of micelles generated per unit of gel volume may vary, influencing the arrangement of water molecules and impacting the timing and extent of gelation.

[0073] Figure 1 demonstrates the formation of the micelles also called the micellar vesicles in the gel state for a second embodiment of the present invention which considers a composition of an in-situ hydrogel comprising poloxamer 407, chitosan with a molecular weight in the range of for example 50,000 to 150,000 Da and glyceryl phosphate. Preferably the composition comprises 20 w / v% poloxamer 407, 1 w / v% chitosan and 15 v / v% glyceryl phosphate. Poloxamer 407 serves as the primary gelling agent of the composition. Chitosan contributes to the stability and viscosity of the gel. The micellar vesicles can be used for the encapsulating active pharmaceutical ingredients (APIs) and have the capacity to encapsulate both hydrophilic and lipophilic APIs. The encapsulation can also extend to other polymeric matrices present in the composition such as chitosan. Glyceryl phosphate enhances gel formation and drug delivery. This embodiment will form an in-situ gel when combining the above mentioned components which have a generalized use in medical and pharmaceutical applications such as localized treatments and controlled drug delivery.

[0074] A third embodiment of the present invention considers a composition of an in-situ hydrogel comprising poloxamer 407, chitosan with a molecular weight for example in the range of 50,000 to 150,000 Da, glycerophosphate, and sodium tripolyphosphate (TPP). Preferably the composition comprises 20 w / v% poloxamer 407, 1 w / v% chitosan with a molecular weight for example in the range of 50,000 to 150,000 Da, 14.5 v / v% glycerophosphate, and 0.5 w / v% sodium tripolyphosphate. The poloxamer 407 serves as the primary gelling agent of the formulation. Chitosan contributes to the stability and viscosity of the gel. Glycerophosphate enhances gel formation and drug delivery. In the presence of chitosan in the composition, aside from micelle formation, another mechanism promotes gel formation. Chitosan, when in proximity to negative ions like TPP or glycerophosphate, undergoes a gelation process. Elevated temperatures enhance the interactions between chitosan chains and negative ions, resulting in the formation of nanogel and facilitating macroscopic gelation. When poloxamer forms a gel in the presence of chitosan, it increases the concentration of chitosan and ions, intensifying interactions. This collaborative effect between poloxamer and chitosan enhances the gelation process, yielding a more uniform and enduring gel.

[0075] Figure 3 shows atomic force microscopic (AFM) images of the spheroid nanogels formed through embodiment 3. The image of the gel surface reveals a rough, structured topography that is indicative of micellar organization within the gel. The raised areas visible in the image likely represent aggregates of micelles formed by the amphiphilic poloxamer, along with the lipid components incorporated into the composition. This surface roughness is consistent with micelle formation, which occurs when poloxamers undergo phase separation above their Lower Critical Solution Temperature (LCST), leading to the self-assembly of micelles.

[0076] The incorporation of lipids, such as phosphatidylcholine and cholesterol, appears to further reinforce the micelle packing, contributing to the distinct, rough texture observed on the gel surface. This topographical feature indicates a highly organized internal structure, where micelles form well-defined domains. This organization could be beneficial for the mechanical stability of the gel, enhancing its resilience and structural integrity, especially under physiological conditions like those present in biomedical applications.

[0077] The well-formed micelle structures, as observed in the AFM image, also suggest enhanced drug loading and controlled release properties. The micelles provide additional space within their hydrophobic cores for encapsulating hydrophobic drugs or radiopaque agents, while the organized structure of the gel could help sustain the release of these agents over time.

[0078] Figure 4 illustrates the impact of 5-Fluorouracil (5FU) and the in-situ forming gel loaded with 5-Fll on the viability of HEPG2 cells at various concentrations after 24 and 48 hours. Data are presented as the average ± standard deviation (n = 3), with significance denoted by #p-value < 0.05, analyzed using a two-way ANOVA, specifically referring to the formulation described in embodiment 3.

[0079] A fourth embodiment of the present invention considers a composition of an in-situ hydrogel comprising poloxamer 407, chitosan with a molecular weight for example in the range of 50,000 to 150,000 Da, glycerophosphate, and tween 80 (polysorbate 80). Preferably the composition comprises 20 w / v% poloxamer 407, 1 w / v% chitosan, 14 v / v% glycerophosphate, and 1 v / v% tween 80 (polysorbate 80). Poloxamer 407 serves as the primary gelling agent of the composition. Chitosan contributes to the stability and viscosity of the gel. Glycerophosphate enhances gel formation and drug delivery. The physicochemical characteristics of the formed micelles can be modified by incorporating additional components, allowing them to encapsulate various drugs with diverse properties. The incorporation of Tween 80 enhances the arrangement of amphiphilic structures in micelle formation, making it more conducive for encapsulating hydrophilic APIs. This embodiment will form an in-situ gel when combining the above listed components which has generalized use in medical and pharmaceutical applications such as localized treatments and controlled drug delivery.

[0080] A fifth embodiment of the present invention considers a composition of an in-situ hydrogel comprising poloxamer 407, chitosan with a molecular weight for example in the range of 50,000 to 150,000 Da, glycerophosphate, and span 60 (sorbitan monostearate). Preferably the composition comprises 20 w / v% poloxamer 407, 1 w / v% chitosan of with a molecular weight for example in the range of 50,000 to 150,000 Da, 14 v / v% glycerolphosphate, and 1 v / v% span 60 (sorbitan monostearate). Poloxamer 407 serves as the primary gelling agent of the composition. Chitosan contributes to the stability and viscosity of the gel. Glyceryl phosphate enhances gel formation and extended drug delivery. Span 60 and lipids increase the ability to entrap lipophilic APIs. This embodiment will form an in-situ gel when combining the above listed components which have a generalized use in medical and pharmaceutical applications such as localized treatments and controlled drug delivery.

[0081] A sixth embodiment of the present invention considers a composition of an in-situ hydrogel comprising poloxamer 407, chitosan with a molecular weight for example in the range of 50,000 to 150,000 Da, glycerophosphate, span 60, and lecithin. Preferably the composition comprises 20 w / v% poloxamer 407, 1 w / v% chitosan, 14 v / v% glycero phosphate, 0.5 v / v% span 60, and 0.5 v / v% lecithin. Poloxamer 407 serves as the primary gelling agent of the formulation. Chitosan contributes to the stability and viscosity of the gel. While chitosan exhibits a quicker degradation rate compared to hyaluronic acid, it conversely has a slower dispersion rate in aqueous solutions. Glyceryl phosphate enhances gel formation and drug delivery. Incorporating lipid agents further enhances the gel's uniformity. The introduction of lipids or Span enhances the hydrophobic properties of the formed micelles, resulting in a more consistent gel formation. This, in turn, can facilitate a more prolonged and controlled release of the APIs, as the API needs to traverse a lipid barrier to access the gel structure before diffusing out of this network. This embodiment will form an in- situ hydrogel when combining the above listed components which have a generalized use in medical and pharmaceutical applications such as localized treatments and controlled drug delivery.

[0082] A seventh embodiment of the present invention considers a composition of an in- situ gel comprising poloxamer 407, chitosan with a molecular weight for example in the range of 50,000 to 150,000 Da, glycerophosphate, tween 80, and lecithin. Preferably the composition comprises 20 w / v% poloxamer 407, 1 w / v% chitosan of a medium molecular weight, 14 v / v% glycerophosphate, 0.5 v / v% tween 80, and 0.5 v / v% lecithin. Poloxamer 407 serves as the primary gelling agent of the composition. Chitosan contributes to the stability and viscosity of the hydrogel. Glycerophosphate enhances gel formation and drug delivery. Owing to the positively charged NH groups, chitosan imparts bio adhesive properties to the resulting hydrogel. In regions containing divalent ions like calcium and magnesium, chitosan can effectively substitute these ions. These ions are abundant in intercellular junctions, and chitosan can bind to these sites. In areas characterized by slow blood flow rates, formulations containing chitosan significantly enhance their attachment to endothelial vessel linings. This embodiment will form an in-situ hydrogel when combining the above listed components which have a generalized use in medical and pharmaceutical applications such as localized treatments and controlled drug delivery. Figure 5a through 5c present data comparing the drug concentration at the brain and plasma, and the brain-to-plasma concentration ratio (Kp) between free drug application as well as a methotrexate-gel, niosomal methotrexate, and a niosomal methotrexate gel of the formulation of embodiment 7 loaded by methotrexate at t = 60 min. Initially, the methotrexate-loaded niosomal carrier was formulated using Span 60 and cholesterol. Subsequently, this niosomal system was incorporated into the formulation of this specific embodiment.

[0083] An eight embodiment of the present invention considers a composition of an in-situ hydrogel comprising poloxamer 407, hyaluronic acid, span 60, and lecithin. Preferably the composition comprises 20 w / v% poloxamer 407, 1 w / v% hyaluronic acid, 0.5 v / v% span 60, and 0.5 v / v% lecithin. Poloxamer 407 serves as the primary gelling agent of the composition. Hyaluronic acid contributes to this formulation’s viscoelastic properties. Span 60 serves a supporting role in the formulation influencing stability and performance. Lecithin contributes to the formation of microemulsion in the gel state to incorporate lipophilic APIs. This embodiment will form an in-situ hydrogel when combining the above listed components which have a generalized use in medical and pharmaceutical applications such as localized treatments and controlled drug delivery.

[0084] FORMULATION EVALUATION TESTS

[0085] Gelation Time Test

[0086] The primary criterion for the suitability of a formulation is the gelation time of the samples. After formulating, the samples were placed at room temperature for two minutes in a resting state. Then, they were transferred to a 37°C water bath, and the gelation time was measured from the moment the formulation was placed in the 37°C bath until complete gelation. This process was repeated three times.

[0087] Gelation Temperature Evaluation

[0088] The effect of temperature on gelation time was investigated by placing a gel forming composition at temperatures of 25°C, 37°C, 45°C, and 60°C, and measuring the gelation.

[0089] Drug Release Test

[0090] To assess the drug release profile, formulations containing hydrogel and free drugs were examined. The drug release profile from the in-situ gel formulation was evaluated using a Franz cell.

[0091] Morphological Characteristics Examination

[0092] The morphology of the in-situ gel composition in solution state was investigated using an Atomic Force Microscope (AFM). To perform this, a portion of the composition was placed on a solid substrate. After drying, the substrate was subjected to imaging using the AFM. pH and Conductivity Evaluation of the composition

[0093] After preparing the desired composition, pH and conductivity measurements were taken using a pH meter on the first, second, and third days, as well as the first, second, and third weeks, and one month after formulation preparation.

[0094] Stability Assessment of a Formulation Based on Gelation Capability in Solution State to assess the gelation time of a target pharmaceutical formulation, its ability to convert to gel and the time required for this conversion were measured on days 1, 2, and 3, as well as in the first, second, and third weeks, and one month after formulation preparation. During these time intervals, the formulations were kept in solution state at a temperature of 2-8 degrees Celsius.

[0095] MEASUREMENT METHODS

[0096] Shear strength measured via Rheology: Brookfield, R / S Plus™, S. Nr 303,217, USA

[0097] Texture Analysis: TexturePro CT V1.5 Build, UK

[0098] AFM-JPK, NanoWizard® II, Germany

[0099] TEM: Zeiss, EM 10C, Germany

[0100] All mechanical properties were measured with TexturePro CT Software V1.5.

[0101] • Hardness (mN): Hardness is measured by compressing the hydrogel sample with a probe and recording the maximum force applied before deformation. This is typically done with a texture analyzer in compression mode.

[0102] • Deformation at Hardness (mm): This refers to the distance (or depth) the probe moves into the sample at the point of maximum hardness, also recorded by a texture analyzer during the compression test.

[0103] • Hardness Work (mJ): Calculated as the area under the force-deformation curve up to the hardness point. It quantifies the energy required to reach maximum hardness.

[0104] • Recoverable Deformation (mm): After a compression, the sample is allowed to recover, and the remaining deformation is measured. This is often done by releasing the load after reaching a specific deformation and measuring how much the sample “springs back.”

[0105] • Recoverable Work (mJ): The work (energy) the sample recovers after deformation is also recorded as the area under the recovery curve of the force-deformation plot.

[0106] • Total Work (mJ): The entire area under the force-deformation curve during both compression and recovery phases, showing the total energy involved in deformation and recovery.

[0107] • Load at Target (mN): The force at a specific, predefined deformation (target) point, measured to determine sample resistance.

[0108] • Deformation at Target (mm): The actual distance deformed at a specified load, giving insight into material flexibility. • Adhesive Force (mN): Measured as the maximum force required to detach a probe from the hydrogel’s surface, reflecting surface adhesiveness. A texture analyzer in pull-off mode is used.

[0109] • Adhesiveness (mJ): The total energy required to overcome the adhesive force between the probe and sample, calculated as the area under the force-distance curve during the probe’s withdrawal.

[0110] • Resilience: The ratio of recoverable energy to total energy during deformation. It is calculated by dividing the recoverable work by the work done to reach hardness.

[0111] • Stringiness Length (mm): This is the maximum distance the probe can stretch the sample before it detaches, recorded during the probe’s withdrawal from a sticky or viscoelastic sample.

[0112] • Stringiness Work Done (mJ): The total work done to stretch the sample to its maximum stringiness length, as observed from the force-distance curve.

[0113] • Quantity of Fractures: Recorded as the number of noticeable drops in force within a specified deformation interval, indicating the number of fractures or breaks within the sample structure.

[0114] • Fracturability (mN): Measured as the force at which the sample first fractures or breaks under compression, often identified by a sudden drop in the force curve.

[0115] • Fracture Load Drop Off (mN): The difference between the fracture load and the lowest point following it, reflecting the sharpness of the fracture event.

[0116] • Fracture Work Done (mJ): The energy consumed until the fracture point, as recorded under the force-deformation curve.

[0117] • Fracture Deformation (mm): The deformation at the point of fracture, providing insight into material brittleness.

[0118] • Cohesiveness: Calculated as the ratio of the work done during the second compression cycle to the work done in the first, indicating the material’s ability to hold together.

[0119] • Springiness (mm): The recovery distance after a compressive load is released, showing the extent to which the material returns to its original form.

[0120] • Springiness Index: The ratio of springiness to the initial deformation, offering a normalized measure of elasticity. EXAMPLES

[0121] EXAMPLE 1

[0122] Gelation time was measured of different formulations whereby the compositions a) and b) are comparatives while formulation c) is a formulation according to the invention.

[0123] Composition a) comprises 20 w / v% Poloxamer 407

[0124] Composition b) comprises 20 w / v% Poloxamer 407 and 2 w / v% polyvinylalcohol Mw 72000

[0125] Composition c) comprises: 20 w / v% Poloxamer 407 + 2 w / v% polyvinylalcohol (PVA) Mw 72000 and a lipidic agents comprising:

[0126] • 1.5 w / v% Hydrogenated Phosphatidylcholine

[0127] • 1 w / v% Cholesterol

[0128] • 0.125 w / v% LIPOID PE 18:0 / 18:0 - PEG 2000 (MPEG-2000-DSPE)

[0129] After formulating, the samples were placed at room temperature for two minutes in a resting state. Then, they were transferred to a 37°C water bath, whereby the gelation time was measured from the moment the formulation was placed in the 37°C bath until complete gelation. This process was repeated three times. In table 1 the results are given.

[0130] Table 1

[0131] From table 1 it can be seen that the gelation times of these formulations vary significantly whereby the composition c) according to the present invention performs best. The progressive decrease in gelation time from Poloxamer alone (23 seconds) to Poloxamer + PVA (12 seconds) to Poloxamer + PVA + Lipids (7 seconds) reflects the increasing complexity and efficiency of a network formation. The introduction of PVA accelerates gelation by enhancing hydrogen bonding and network cross-linking, while the addition of lipids amplifies hydrophobic interactions, further reducing the gelation time and yielding a more structured, faster-gelling system. EXAMPLE 2

[0132] In order to achieve precise control over the gelation behavior at 37°C, three studies were examined whereby the effects of water, surfactants, and organic solvents on gelation time of composition c) as described in Example 1 was measured.

[0133] Each additive demonstrated a significant influence on the composition's gelation kinetics, with specific mechanisms contributing to delayed or inhibited gelation. Results are given in Table 2.

[0134] Table 2

[0135] Table 2 clearly demonstrates that water, surfactants, and organic solvents can be used to regulate or even inhibit gelation in a Poloxamer, PVA, and lipidic based composition. Water increases gelation time in a concentration-dependent manner, while surfactants delay gelation through mechanisms related to hydrophilicity and steric hindrance. Organic solvents, on the other hand, can prevent gelation altogether. These insights provide valuable strategies for controlling gelation behavior in formulations designed for biomedical applications. EXAMPLE 3 pH and conductivity were measured on the compositions a), b) and c) as disclosed in example 1.

[0136] Results are given in table 3.

[0137] Table 3

[0138] All compositions show pH values between 6.24 and 6.56, which fall within the acceptable range for biocompatibility. Human biological systems generally tolerate pH between 6.5 and 7.5 without significant irritation or adverse reactions, especially for applications like drug delivery and tissue interaction.

[0139] Conductivity: Higher negative conductivity values, particularly in the in situ hydrogel formulation, indicate better ionic interaction and potentially improved bioactivity. This is favorable in biomedical applications, as it suggests the formulation can maintain stability and function within the ionic environment of the human body.

[0140] Overall, the formulations show promising pH and conductivity profiles suitable for biomedical applications, particularly in environments where precise control over gelation and tissue interaction is crucial, such as in embolization and drug delivery systems.

[0141] EXAMPLE 4

[0142] A hydrogel formulation comprising composition c) as described in examples 1 and 2 was prepared and injected in a comparative study involving the tails of two mice and the jugular vein of one rat.

[0143] The CT scans image demonstrate the radiopacity and in situ gel formation of the injected formulation as shown in Figure 6

[0144] The images show clear radiopacity, indicating that the formulation contains a radiopaque agent which enhances visibility under CT imaging. This is crucial for embolization and localized drug delivery applications where the injected material must be monitored in realtime for proper localization.

[0145] The high radiopacity observed in these images suggests that the formulation is well-suited for medical applications requiring image-guided intervention, such as tumor embolization or vascular occlusion.

[0146] The scans also indicate successful in situ gel formation following injection. The presence of distinct areas with higher radiodensity and gel-like consistency suggests that the formulation transitions from a liquid to a gel phase upon exposure to physiological conditions.

[0147] The injections show radiopaque material distributed along the injection pathway. The radiopacity is well-defined, and the gel appears to remain localized, without significant migration, which is a positive indication for localized embolization.

[0148] The CT scans confirm that the formulation demonstrates strong radiopacity and effective in situ gel formation, essential properties for clinical applications such as embolization and targeted drug delivery. The formulation's performance in both small vessels and the larger veins highlights its versatility for different anatomical sites.

[0149] EXAMPLE 5

[0150] Texture analysis was performed on the compositions a), b) and c) as described in example 1.

[0151] Several mechanical properties were measured in different cycles. Results are give in table 4.

[0152] Table 4

[0153] Mechanical properties Composition a) Composition b) Composition c)

[0154] Poloxamer Poloxamer + Poloxamer + PVA

[0155] PVA + Lipids

[0156] Hardness (mN) cycle 1 98 97 99 cycle 2 112 96 99

[0157] Deformation at hardness (mm) 7.36 4.61 2.41

[0158] Hardness work (mJ) cycle 1 0.23 0.31 0.15 cycle 2 0.02 0.26 0.13

[0159] Recoverable deformation (mm) cycle 1 0.14 0.00 0.1 1 cycle 2 0.17 0.19 0.05

[0160] Recoverable work (mJ) cycle 1 0.00 0.01 0.00 cycle 2 0.00 0.00 0.00

[0161] Total work (mJ) cycle 1 0.23 0.32 0.15 cycle 2 0.02 0.26 0.14

[0162] Load at target (mN) 98 97 99

[0163] Deformation at target (mm) 7.36 4.61 2.41

[0164] Adhesive force (mN) 190 79 112

[0165] Adhesiveness (mJ) 0.45 0.29 0.22

[0166] Resilience 0.01 0.02 0.00

[0167] Stringiness length (mm) 3.84 1.43 0.60

[0168] Stringiness work done (mJ) 0.28 0.10 0.06

[0169] Quantity of fractures 16 9 2

[0170] Fracturability (mN) 3 41 49

[0171] Fracture load drop off (mN) 6 30 19

[0172] Fracture work done (mJ) 0.00 0.00 0.00

[0173] Fracture deformation (mm) 0.08 0.18 0.19

[0174] Cohesiveness 0.08 0.83 0.86

[0175] Springiness (mm) 7.67 4.44 2.23

[0176] Springiness index 1.04 0.96 0.93

[0177] The following observations can be derived from the results in table 4.

[0178] Hardness was measured and a small increase in hardness is observed when phosphatidylcholine and cholesterol are added to the composition. Poloxamer alone shows comparable hardness to the Poloxamer + PVA and Poloxamer + PVA + Lipids composition. The slight increase in hardness for the Poloxamer + PVA + Lipids system (99 mN) compared to Poloxamer (98 mN) suggests that the lipids marginally strengthen the gel matrix, likely due to hydrophobic interactions between the PPO block of Poloxamer and the lipid components.

[0179] Deformation at Hardness (mm) shows that the Poloxamer alone exhibits significantly higher deformation under hardness, indicating that it is more flexible. The addition of PVA reduces deformation, and the introduction of lipids further decreases the deformation, indicating a stiffer and more resistant structure in the lipid-containing composition. The rigidity likely arises from cholesterol's stiffening effect and cross-linking provided by phosphatidylcholine

[0180] The hardness work, or energy required to reach hardness, decreases when lipids are added to the Poloxamer + PVA composition. The Poloxamer + PVA composition shows the highest energy requirement, indicating that it is harder to deform initially. In contrast, the addition of lipids lowers the energy required to reach hardness, which can be attributed to the more efficient stabilization of the gel by the lipid components.

[0181] Recoverable Deformation (mm): Poloxamer shows a higher initial recoverable deformation compared to the other compositions. This means that Poloxamer alone has more elastic properties than the formulations with PVA and lipids. The lipid-containing composition exhibits some elastic behavior, but it is less flexible than Poloxamer alone, suggesting that the addition of lipids creates a stiffer, more structured gel.

[0182] The total work required to deform the gel is lowest in the Poloxamer + PVA + Lipids composition, indicating a more rigid network that requires less energy to deform. Poloxamer alone requires slightly less energy than the Poloxamer + PVA composition, showing that PVA alone adds to the gel’s resistance to deformation but that lipids counteract this by forming a more structured and cohesive gel.

[0183] Adhesive Force and Adhesiveness (mN / mJ):The addition of lipids, specifically phosphatidylcholine, enhances the composition's adhesive properties, which is valuable in biomedical applications where adherence to biological tissues is critical.

[0184] Table 3 shows that

[0185] • Poloxamer alone shows a high adhesive force of 190 mN, reflecting strong surface interactions. However, this strong interaction may not be ideal in certain biomedical applications, as it could limit the controlled adhesion needed for specific applications. • In the Poloxamer + PVA composition PVA significantly reduces the adhesive force to 79 mN, likely due to PVA’s hydrophilic nature, which interferes with the poloxamer’s natural surface interactions. While this reduction may make the composition less sticky, it may also reduce its ability to adhere effectively to target tissues.

[0186] When lipids are added to the Poloxamer + PVA composition:

[0187] • The adhesive force increases to 112 mN, balancing between effective adhesion and controlled detachment. This increase is due to the interactions between the hydrophilic head of phosphatidylcholine and the PEO blocks of poloxamer, which improve surface affinity without overwhelming stickiness.

[0188] • Phosphatidylcholine’s amphiphilic nature also contributes to better integration within biological environments. Its hydrophilic head binds well with poloxamer’s PEO segments, enhancing adhesion, while the hydrophobic tails improve compatibility with cell membranes.

[0189] This optimized adhesive force makes the composition according to the present invention more effective for applications like localized drug delivery, tissue scaffolding, and wound healing, where strong yet manageable adhesion is essential for stable placement on biological tissues. Additionally, these lipid interactions contribute to a stable, biocompatible interface, making the formulation highly suitable for medical use.

[0190] Resilience: Poloxamer alone shows some resilience, meaning it can recover slightly after deformation. Adding PVA increases resilience, but adding lipids to the system reduces resilience to zero, reflecting the stiffer and less elastic nature of the lipid-containing hydrogel.

[0191] Stringiness (Length and Work Done): Poloxamer alone exhibits greater stringiness, indicating more flexible and stretchable chains. As expected, the addition of PVA significantly reduces stringiness, and lipids further reduce this property, showing a more cohesive and less deformable structure.

[0192] Fracturability (mN): The fractureability increases significantly in the PVA and lipid- containing compositions compared to Poloxamer alone. This shows that the PVA and lipids make the gel more resistant to breaking, enhancing its toughness and making it more fracture-resistant.

[0193] Cohesiveness: Poloxamer alone is far less cohesive than the compositions with PVA and lipids. The increase in cohesiveness with the addition of PVA and further enhancement with the addition of lipids suggests a more interconnected network, providing greater structural integrity and resistance to breakdown. Springiness and Springiness Index were measured on the compositions a), b) and c). In biomedical applications, the combination of reduced springiness with improved stability due to lipids is suitable if the formulation needs to maintain its shape under physiological conditions, like tissue engineering, wound healing, or controlled drug release, where steady placement and slow, controlled deformation are advantageous.

[0194] EXAMPLE 6

[0195] A rheological comparison has been performed of three compositions F1 , F2 and F3 which are similar to compositions a), b), c) as disclosed in example 1.

[0196] The composition F3 which comprises poloxamer and PVA and lipidic agents demonstrates superior rheological properties, including high yield stress, great resistance to shear, and better structural stability. The addition of phosphatidylcholine and cholesterol makes the gel stronger and more resilient, ideal for applications where mechanical strength and controlled release are critical

[0197] Flow Curve Analysis (Tau vs. Shear Rate) (Figure 7)

[0198] • F1 comprising only Poloxamer: Flow curve shows the lowest shear stress values among the three formulations. This indicates that pure poloxamer has the weakest gel structure and requires less stress to initiate flow. The flow behavior is more Newtonian, with a lower resistance to deformation.

[0199] • F2 comprising Poloxamer + PVA: Adding PVA increases the shear stress values, indicating a more robust network that requires more force to deform. PVA adds crosslinking and enhances the gel strength, making it more resistant to flow compared to pure poloxamer.

[0200] • F3 comprising Poloxamer + PVA + Lipidic agents comprising: o 1.5 w / v% Hydrogenated Phosphatidylcholine o 1 w / v% Cholesterol o 0.125 w / v% LIPOID PE 18:0 / 18:0 - PEG 2000 (MPEG-2000-DSPE)

[0201] The composition F3 containing the lipidic agents (phosphatidylcholine and cholesterol) show the highest shear stress values. This means that the gel structure is much stronger due to the lipids' contribution to forming more rigid micelles and vesicle structures. The lipids interact with both the poloxamer and PVA, further stabilizing the network and increasing the gel strength significantly. Viscosity Curve Analysis (Viscosity vs. Shear Rate) as given in Figure 8

[0202] • F1 comprising only Poloxamer: The viscosity of pure poloxamer decreases rapidly with increasing shear rate, showing typical shear-thinning behavior. This indicates that the gel is easily deformed under stress, and its structure breaks down more quickly.

[0203] • F2 comprising Poloxamer + PVA: The viscosity decreases less rapidly than in F1, showing a more stable gel under shear. The addition of PVA increases the gel’s resistance to flow, but the system is still shear-thinning.

[0204] • F3 comprising Poloxamer + PVA + Lipidic agents comprising: o 1.5 w / v% Hydrogenated Phosphatidylcholine o 1 w / v% Cholesterol o 0.125 w / v% LIPOID PE 18:0 / 18:0 - PEG 2000 (MPEG-2000-DSPE)

[0205] This formulation shows the highest viscosity values overall, and the viscosity decreases at a slower rate with increasing shear. This suggests a pseudo-plastic behavior, meaning the gel is more resistant to flow and deformation, maintaining a more consistent structure even under shear. The lipids help to reinforce the gel, slowing down the breakdown of the structure under stress.

[0206] Creep Compliance Curve (J(t) vs. Time) as given in Figure 9

[0207] • F1 comprising only Poloxamer: The pure poloxamer gel shows the highest creep compliance, meaning it is the most easily deformed under constant stress. This indicates a less stable structure that gradually flows under a sustained load.

[0208] • F2 comprising Poloxamer + PVA: With the addition of PVA, the creep compliance decreases, indicating a more stable structure with greater resistance to deformation. PVA helps in forming a more robust network that withstands stress for longer periods before significant deformation occurs.

[0209] • F3 comprising Poloxamer + PVA + Lipidic agents comprising: o 1.5 w / v% Hydrogenated Phosphatidylcholine o 1 w / v% Cholesterol o 0.125 w / v% LIPOID PE 18:0 / 18:0 - PEG 2000 (MPEG-2000-DSPE) The creep compliance is the lowest in this composition, showing that the gel is highly resistant to deformation under constant stress. The inclusion of phosphatidylcholine and cholesterol creates a stronger, more resilient gel, with the lipidic agents acting as stabilizers in the gel matrix, preventing significant creep.

[0210] EXAMPLE 7

[0211] Measurement on gelation time has been performed on different compositions at different temperatures. Mixed-micelle in situ gels based on 1 w / v% chitosan, 20 w / v% poloxamer, and 1 w / v% oleic acid, 1 w / v% dipalmitin, 1 w / v% triolein, 0.5 w / v% brij 35, 0.5 w / v% span 20, or 0.5 w / v% capryol PGMC were individually placed in water baths at 37, 45, and 60°C to assess the impact of temperature on gelation time. Each formulation was freshly prepared and divided into three 5mL beaker. Then, each beaker was transformed into a water bath with temperature of 37, 45 or 60°C. The sol-gel transition temperature was determined using the visual tube inversion method. Duration of time between placing the beaker in the water bath and the time at which the formulation stopped flowing was assumed as a gelation time.

[0212] From Figure 10, the gelation time of various mixed-micelle in situ gel formulations decreased with increasing temperature. This illustrates that irrespective of lipid or surfactant type, MIGs retain thermoresponsive behaviour akin to the in situ gel.

[0213] EXAMPLE 8

[0214] In vitro drug release test. (Figure 11)

[0215] Doxorubicin-loaded in situ gel based on the below composition, were placed at microtubes (1mL in each microtube) and incubated at 37° C to form a hydrogel. The vials were incubated in a shaker incubator (LSI-3016R, Lab Tech, Thailand) at 37 °C and shaken at 80 rpm. After 30 minutes, 2 mL PBS (pH=7.4) was added to each tubes. Every three microtubes were assumed for a specified day. The amount of doxorubicin 0.2 w / v% was tested by UV spectroscopy ( X =480 nm). The composition comprising 20 w / v% poloxamer, 0.5 w / v% hyaluronic acid, 0.5 w / v% cholesterol, 0.5 w / v% lecithin and 0.2 w / v% Doxorubicin showed a sustained drug release profile of 70% drug release within 35 days.

[0216] EXAMPLE 9

[0217] Conductivity (Adwa, AD1000, Hungary) of poloxamer-based in situ gel, poloxamer and PVA- based in situ gel, and poloxamer, PVA and lipids-based in situ gel were assessed triplicate. The gels are comprising the compositions as described in example 1. Results are given in Table 5.

[0218] Table 5

Claims

AMENDED CLAIMS received by the International Bureau on 07 April 2025 (07.04.2025) Temperature-responsive in-situ gelling composition comprising : a) an amphiphilic triblockcopolymer of polyoxyethylene- polyoxypropylene- polyoxyethylene (PEO-PPO-PEO) b) a water soluble hydrophilic polymer selected from the group consisting of chitosan, hyaluronic acid (HA) and / or polyvinylalcohol, polyethylene glycol, carboxymethyl cellulose, alginate, pectin, carrageenan, gelatin, poly(acrylic acid), xanthan gum, hydroxypropyl methylcellulose, poly(N-isopropylacrylamide) or combinations thereof. c) whereby the composition further comprises at least a lipidic agent selected from the group consisting of phospholipids or derivatives, sterols, fatty alcohols, cholesterol and derivatives, or a combination of more than one lipidic agent. Temperature-responsive in-situ gelling composition according to Claim 1 whereby the lipidic agent is selected from the group consisting of phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, glycerolphosphate or cholesterol and combinations thereof. Temperature-responsive in-situ gelling composition according to any one of the claims 1-2 whereby the amphiphilic triblockcopolymer is present in an amount from 10-30 w / v% based on the total weight of the composition. Temperature-responsive in-situ gelling composition according to any one of the claims 1-3 whereby the hydrophilic polymer is present in an amount from 0.5-5w / v% based on the total weight of the composition. Temperature-responsive in-situ gelling composition according to any one of the claims 1-4 whereby the lipidic agent is present in an amount from 1-5 w / v% based on the total weight of the composition, preferably between 0,1-1 w / v% based on the total weight of the composition. Temperature-responsive in-situ gelling composition according to any one of the claims 1-5 whereby the hydrophilic polymer is polyvinylalcohol, chitosan or hyaluronic acid or combinations thereof. Temperature-responsive in-situ gelling composition according to any one of the claims 1- 6 whereby the hydrophilic polymer is polyvinylalcohol and the lipidic agent is selected from a combination of cholesterol and hydrogenated phosphatidylcholine. Temperature-responsive in-situ gelling composition according to any one of the claims 1- 6 whereby the hydrophilic polymer is hyaluronic acid and the lipidic agent is selected from phosphatidylcholine or cholesterol and combinations thereof.AMENDED SHEET (ARTICLE 19)Temperature-responsive in-situ gelling composition according to any one of the claims 1- 6 whereby the hydrophilic polymer is chitosan and the lipidic agent is glycerophosphate. Temperature-responsive in-situ gelling formulation comprising the composition according to any one of the claims 1-9 and a bioactive agent selected from a pharmaceutical agent or a radio opaque agent.Temperature responsive in situ gelling composition according to any one of the claims 1-9 or formulation according to claim 10 for use as a medicament.Temperature responsive in situ gelling composition according to any one of the claims 1-9 or formulation according to claim 10 for the manufacturing of a medicament for therapeutic application as a vascular embolizing agent.Drug delivery system comprising the temperature-responsive in-situ gelling formulation according to claim 10.Temperature responsive in situ gelling composition according to any one of the claims 1-9 or formulation according to claim 10 for the manufacturing of a medicament for therapeutic application as radiotherapy-enhancing system.AMENDED SHEET (ARTICLE 19)

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