Self-immolative micelle compositions, methods and uses thereof

Abstract

The present disclosure relates to a hydrogel composition comprising micelles incorporated into a pharmaceutical carrier, wherein the micelles are present in an amount ranging from 1% (w / w) to 50% (w / w) based on the total weight of the hydrogel composition. The micelles comprise a mixture of micelles including micelles having a hydrophobic core containing a hydrophobic vitamin and a hydrophobic active ingredient, and micelles having an outer shell comprising a vitamin E derivative and / or a vitamin E-polymer composite and a hydrophilic active ingredient, and / or micelles comprising both a hydrophobic core and an outer shell. The hydrophilic active ingredient is covalently linked to the vitamin E derivative and / or the vitamin E-polymer composite by a disulfide linkage cleavable in the presence of glutathione. The hydrogel composition enables controlled delivery of hydrophobic and hydrophilic active ingredients and is suitable for therapeutic applications, including cancer treatment.

Description

D E S C R I P T I O NSELF-IMMOLATIVE MICELLE COMPOSITIONS, METHODS AND USES THEREOFTECHNICAL FIELD

[0001] The present disclosure relates to the field of pharmaceutical and biomedical compositions for controlled drug delivery. In particular, the disclosure concerns hydrogel compositions comprising micelles incorporated into a pharmaceutical carrier, wherein the micelles comprise a hydrophobic core containing a hydrophobic vitamin and a hydrophobic active ingredient, and / or an outer shell comprising a vitamin E derivative and / or a vitamin E-polymer composite and a hydrophilic active ingredient. The disclosure further relates to redox-responsive micellar systems in which a hydrophilic active ingredient is covalently linked via a disulfide linkage cleavable in the presence of glutathione. Such hydrogel compositions are suitable for medical and veterinary applications, including the treatment of cancer.

[0002] The present disclosure relates to the field of pharmaceutical carriers, particularly a self-immolative micelle as a drug delivery system for the treatment of tumours, namely solid cancer, in particular in the treatment of pancreatic ductal adenocarcinoma (PDAC) and its metastases. The present disclosures the use of the micelle and a pharmaceutical composition, namely hydrogel composition.BACKGROUND

[0003] The introduction of immunotherapy and targeted therapies has significantly improved the outcomes of many patient populations across several solid and hematologic tumors; however, these have shown limited clinical benefit in pancreatic cancer.

[0004] Pancreatic cancer was the third highest cause of cancer-related death in 2021. Such discrepancy is largely attributed to a late diagnosis, conferring a rate of 52% distant dissemination at diagnosis and a dismal 5-year survival rate of only 3%.

[0005] The number of cases of pancreatic cancer are estimated to increase in the next decades. By 2025, it is estimated that the number of new cases will increase by 9829, and by 2040 by 37,824.

[0006] Pancreatic cancer is one of the most lethal malignancies. It is estimated that the number of deaths will rise to 141,653 by 2025 and to 169,389 by 2040, marking an increase of more than 37,255 deaths compared to the numbers in 2020. Pancreatic ductal adenocarcinoma (PDAC), a type of pancreatic cancer, remains one of the most lethal malignancies, owing to its aggressive nature and limited responsiveness to conventional chemotherapy.

[0007] Systemic chemotherapy remains the mainstay in both first-line (IL) and second line and beyond (2L+) for patients with locally advanced and metastatic PDAC without actionable genomic alterations.Gemcitabine (GEM) with nanoparticle albumin-bound paclitaxel (PTX), with or without cisplatin, or fluoropyrimidine regimens are preferred regimens in both IL and 2L+ settings for patients with good to intermediate performance status (PS) are associated with limited benefit and high systemic toxicity, with no durable responses. Thus, patients inevitably progress and have limited access to high value 2L+ therapies, contributing to the low 5-year survival and highlighting the remarkable need to develop more effective therapeutic strategies.

[0008] Current treatments, such as GEM and PTX, suffer from poor pharmacokinetics and nonspecific toxicity, leading to suboptimal clinical outcomes. The need for more effective therapies is urgent, especially given the high mortality rate associated with PDAC.

[0009] The outlook for PDAC has remained poor, despite advancements in cancer therapeutics. The complexity of the tumor microenvironment, coupled with the strong desmoplastic reaction, has historically rendered systemic chemotherapy regimens only modestly effective.

[0010] The dense desmoplasic pancreatic tumor microenvironment (TME) has been shown to play an important role in the failure of most therapeutic strategies, promoting a particular low drug accumulation at the tumor site and for a short period of time.

[0011] Nanomedicines are revolutionizing the standardized means to deliver therapeutic cargo. While systemic chemotherapy and radiotherapy are the main conventional therapies in oncology, the follow up problems are mostly only for chemotherapy and include inadequate drug bioavailability, cellular selectivity, and development of multidrug resistance, causes that contribute to the limited efficacy and high systemic toxicity of chemotherapy. Nanomedicine has demonstrated numerically higher efficacy and safety compared to standard systemic free drug chemotherapy by overcoming some of these challenges, namely i) avoiding side and off-target effects by active biomarker targeting or enhanced permeability and retention (EPR) at the site of interest; ii) enhancing intracellular uptake in cancer and metastatic cells; iii) improving controlled payload pharmacokinetics and pharmacodynamics; and iv) protecting the encapsulated payload from degradation or rapid clearance.

[0012] While limited, approved nanoparticle-based therapies for PDAC, highlight the importance of leveraging nanosystems to improve efficacy and safety of standard chemotherapies.

[0013] Onivyde (liposomal irinotecan) is approved for use in combination with other drugs for the treatment of metastatic pancreatic cancer after disease progression following GEM-based regimens. Onivyde encapsulates irinotecan, a topoisomerase inhibitor, in a pegylated liposome, which improves the delivery of the active drug to the tumor site and reduces the associated side effects. This nanodelivery system enhances the therapeutic index of irinotecan, making it more effective in killing cancer cells, and minimizing damage to healthy tissues.

[0014] Another approved nanoparticle-based therapy for PDAC is Abraxane (nanoparticle-bound albumin-PTX), which has received approval for the treatment of metastatic breast cancer, non-small-cell lung cancer, and pancreatic cancer. Abraxane uses albumin-bound nanoparticles to deliver PTX more efficiently to cancer cells. This nanoparticle-based formulation improves drug solubility and bioavailability, leading to better clinical outcomes compared to traditional formulations of paclitaxel.

[0015] NanoTherm therapy represents a different approach to cancer treatment by using iron oxide nanoparticles for the thermal ablation of tumors. This therapy is particularly utilized for pancreatic cancer, among others. Under the influence of an external magnetic field, the iron oxide nanoparticles generate localized heat, which ablates the tumor cells. This method offers a non-invasive treatment option that can precisely target and destroy cancerous tissues while sparing surrounding healthy tissue.

[0016] These advancements highlight the potential of nanotechnology in improving cancer treatment outcomes. However, there remains a need for further innovation to enhance the specificity, efficacy, and safety of these therapies, particularly for difficult-to-treat cancers like metastatic pancreatic cancer.

[0017] There is a need for new therapies that effectively modulate intratumoral and metastatic signalling pathways, addressing chemoresistance at a molecular level, and additionally improve the efficacy and safety of current guideline-recommended regimens. These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRIPTION

[0018] The present disclosure provides a hydrogel composition comprising micelles incorporated into a suitable pharmaceutical carrier. The micelles are present in an amount ranging from 1% (w / w) to 50% (w / w), preferably from 2% (w / w) to 20% (w / w), more preferably from 3% (w / w) to 10% (w / w), based on the total weight of the hydrogel composition.

[0019] According to the disclosure, the micelles are present as a mixture of micelles. In one embodiment, the micelles comprise a hydrophobic core containing a hydrophobic vitamin and a hydrophobic active ingredient. In another embodiment, the micelles comprise an outer shell containing a vitamin E derivative and / or a vitamin E-polymer composite and a hydrophilic active ingredient. In a further embodiment, the micelles comprise both a hydrophobic core and an outer shell, wherein the hydrophobic core contains a hydrophobic vitamin and a hydrophobic active ingredient, and wherein the outer shell comprises a vitamin E derivative and / or a vitamin E-polymer composite and a hydrophilic active ingredient.

[0020] The hydrophilic active ingredient is covalently linked to the vitamin E derivative and / or the vitamin E-polymer composite by a disulfide linkage. The disulfide linkage is cleavable in the presence of glutathione, enabling redox-responsive release of the hydrophilic active ingredient under reducing conditions.

[0021] The micelles are incorporated into the pharmaceutical carrier in the form of a hydrogel matrix, providing localized and sustained delivery of therapeutic agents. The hydrogel composition enables coordinated delivery of hydrophobic and hydrophilic active ingredients from a single pharmaceutical system and is suitable for medical and veterinary applications, including the treatment of cancer.

[0022] Surprisingly, the hydrogel composition of the present disclosure provides a markedly enhanced and durable antitumour effect compared with conventional micellar or hydrogel-based delivery systems. In particular, the incorporation of a defined amount of vitamin-based, redox-responsive micelles into a hydrogel carrier, wherein the micelles comprise a hydrophobic vitamin in the core and a hydrophilic active ingredient covalently linked via a glutathione-cleavable disulfide linkage, results in a coordinated and efficient delivery of hydrophobic and hydrophilic active ingredients. This specific combination leads to a therapeutic efficacy that is significantly superior to that achieved with the free active ingredients or with micellar systems not embedded in a hydrogel matrix. Such an effect could not be derived from the prior art, which neither discloses nor suggests that the claimed micelle composition, micelle loading, and redox-responsive linkage, when combined within a hydrogel carrier, would result in the observed improvement in antitumour activity. The achieved effect is therefore unexpected and supports the presence of an inventive step.

[0023] Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies due to its aggressive nature and limited responsiveness to conventional chemotherapy. This limited responsiveness is attributed to poor drug stability, nonspecific toxicity, and inadequate accumulation at the tumor site, which results in suboptimal drug delivery and limited therapeutic efficacy These factors result in low therapeutic efficacy, high systemic toxicity, and poor patient prognosis. Consequently, there is an urgent need for treatments that can enhance drug delivery, improve drug stability, and reduce toxicity, thereby improving patient outcomes and survival rates.

[0024] The present solution relates to a hydrogel composition comprising micelles for the delivery of active ingredients, in particular for therapeutic applications. The micelles comprise a hydrophobic core including a hydrophobic vitamin and / or micelles a hydrophobic active ingredient, and an outer shell comprising a vitamin E derivative and / or a vitamin E-polymer composite. A hydrophilic active ingredient is covalently linked to the vitamin E derivative and / or vitamin E-polymer composite via a disulfide linkage, such that the hydrophilic active ingredient is releasable under reducing conditions, in particular in the presence of glutathione.

[0025] The micelles are incorporated into a pharmaceutical hydrogel matrix and are present in a defined amount relative to the total weight of the hydrogel composition. The hydrogel matrix provides structural support and enables sustained local retention of the micelles, while the micellar architecture allows controlled release of both hydrophobic and hydrophilic active ingredients. The disulfide linkage facilitates intracellular release of the hydrophilic active ingredient in response to a reducing environment.

[0026] In certain embodiments, the hydrogel comprises one or more polymeric matrix materials, such as hyaluronic acid or other biocompatible polymers. The micelles may have a defined particle size and polydispersity and may be configured for the delivery of a hydrophobic active ingredient and a hydrophilic active ingredient. In particular embodiments, the active ingredients comprise anticancer agents.

[0027] The hydrogel composition of the present disclosure is suitable for use in medicine or veterinary medicine, in particular for the treatment of tumours, including solid cancers. The disclosure further relates to methods for producing the hydrogel composition, comprising the formation of the micelles and their incorporation into the hydrogel matrix.

[0028] The present disclosure provides a solution to this need by introducing a novel hydrogel composition comprising a self-immolative micelle comprising D-a-tocopheryl polyethylene glycol succinate. The composition of the present disclosure surprisingly enhances the targeted and controlled delivery of active ingredients, while significantly improving their efficacy and stability, while minimizing systemic toxicity.

[0029] An aspect of the present disclosure relates to a hydrogel composition comprising a micelle and a suitable pharmaceutical carrier, wherein the micelle comprises:a hydrophobic core;an outer shell, wherein the outer shell comprises D-a-tocopheryl polyethylene glycol succinate and; a hydrophilic active ingredient;wherein the hydrophilic active ingredient is linked to the D-a-tocopheryl polyethylene glycol succinate by a site-selective stable linkage (disulfide linkage) suitable to release the hydrophilic active ingredient in the presence of glutathione; in order to minimize cytidine deaminase (CDA) metabolization and the systemic exposure. Glutathione is typically overexpressed in tumor cells, reducing cytidine deaminase (CDA)-mediated drug metabolism and systemic exposure while enabling the selective release of the drug within tumor cells. The present disclosure enables controlled delivery due to the site-selective stable linkage and self-immolative mechanism, which significantly improves the efficacy of the active ingredients. The combination of the simultaneous delivery of PTX and GEM described herein and incorporation of the micelles into an hydrogel forming the hydrogel composition of the present disclosure results in a pronounced and durable antitumor effect. The composition of the present disclosure significantly reduced cell metabolic activity compared to the free drugs, enabling 50-fold and 100-fold lower pharmaceutical ingredient concentrations to achieve comparable or greater reductions to free GEM and PTX, respectively. Additionally, the present disclosure modulates intratumoral and metastatic signalling pathways effectively, thus addressing chemoresistance at a molecular level.

[0030] Surprisingly, the composition of the present disclosure changes the pharmacokinetics and pharmacodynamics of the active ingredients, thereby modulating the active ingredients interactions withkey intratumoral and metastatic genomic signalling pathways more effectively, maximizing therapeutic outcomes and reducing off-target toxicities, namely with the use of lower active ingredient dosage for obtaining the desired therapeutical effect, enabling reductions of 50-fold to 100-fold in GEM and PTX concentrations. Another unexpected effect is the precise modulation of oncogenic pathways, suggesting a more pronounced and durable targeted antitumor effect.

[0031] The present disclosure addresses the development of a novel self-immolative micelle composition carrying active ingredients for treating PDAC and its metastases.

[0032] The present disclosure significantly reduces pancreatic cancer primary tumor growth and metastatic spread, presenting an unexpected 50 to 100-fold increase in activity when delivered by the micelle incorporated in the hydrogel, forming the hydrogel composition and compared to the free active ingredients.

[0033] The present disclosure effectively enables targeted and controlled delivery conferred by the self-immolative and site-selective stable linkage while enhancing active ingredients efficacy. Thus, the use of the composition of the present disclosure for the delivery of active ingredients altered the pharmacokinetics and pharmacodynamics of the said active ingredients, thereby modulating their interactions with key intratumoral and metastatic genomic signalling pathways more effectively, maximizing therapeutic outcomes and at the same time, reducing off-target toxicities. Another unexpected effect is the precise modulation of oncogenic pathways, suggesting a more pronounced and durable targeted antitumor effect.

[0034] The present disclosure addresses the unmet need for more effective therapies in pancreatic cancer treatment by overcoming the limitations of current treatments, such as poor drug stability and nonspecific toxicity. The result is a more efficient and specific antitumor activity.

[0035] An aspect of the present disclosure relates to a hydrogel composition comprising micelles, wherein the amount of the micelle ranges from 1% (w / w) - 50% (w / w) based on the total weight of the hydrogel composition;wherein the micelles are selected from the group consisting of:micelles comprising a hydrophobic core containing a hydrophobic vitamin and a hydrophobic active ingredient;micelles comprising an outer shell containing a vitamin E derivative and / or a vitamin E-polymer composite, and a hydrophilic active ingredient;micelles comprising both a hydrophobic core comprising a hydrophobic vitamin and a hydrophobic active ingredient; and an outer shell, wherein the outer shell comprises a vitamin E derivative and / or a vitamin E - polymer composite; and a hydrophilic active ingredient;wherein the hydrophilic active ingredient is linked to the vitamin E derivative and / or a vitamin E - polymer composite by a disulfide linkage; andwherein the disulfide linkage is cleavable in the presence of glutathione.

[0036] The term "hydrogel composition" refers to a formulation comprising a hydrogel matrix and micelles dispersed or embedded within the hydrogel matrix, optionally together with one or more pharmaceutically acceptable excipients.

[0037] The term "micelle" or "micellar structure" refers to a self-assembled nanostructure formed by amphiphilic molecules in an aqueous environment, comprising a hydrophobic core and a hydrophilic or amphiphilic outer shell. In the present invention, the micelle is configured to encapsulate or associate with active ingredients.

[0038] The term "hydrophobic core" refers to the inner region of a micelle formed predominantly by hydrophobic components, including at least one hydrophobic vitamin and at least one hydrophobic active ingredient.

[0039] The term "outer shell" refers to the region of the micelle surrounding the hydrophobic core and interfacing with the aqueous environment. In the present invention, the outer shell comprises a vitamin E derivative and / or a vitamin E-polymer composite and is capable of bearing a hydrophilic active ingredient.

[0040] The term "vitamin E derivative" refers to a compound derived from vitamin E, including tocopherols, tocotrienols, and chemically modified forms thereof, such as esterified or polymer-conjugated vitamin E derivatives.

[0041] The term "vitamin E-polymer composite" refers to a conjugate or association between a vitamin E derivative and a polymer, formed via covalent or non-covalent interactions, and capable of participating in micelle formation.

[0042] The term "hydrophobic vitamin" refers to a vitamin or vitamin derivative that exhibits limited solubility in aqueous media and preferentially associates with the hydrophobic core of a micelle, such as vitamin E, vitamin D, or derivatives thereof.

[0043] The term "active ingredient" refers to a biologically or pharmacologically active compound capable of producing a therapeutic, prophylactic, or diagnostic effect. In the present invention, the active ingredient may be hydrophobic or hydrophilic.

[0044] The term "hydrophobic active ingredient" refers to an active ingredient having limited solubility in aqueous media and capable of being incorporated into the hydrophobic core of a micelle.

[0045] The term "hydrophilic active ingredient" refers to an active ingredient that is soluble or dispersible in aqueous media and capable of being associated with or covalently linked to the outer shell of a micelle.

[0046] The term "disulfide linkage" refers to a covalent bond between two sulfur atoms that is cleavable under reducing conditions. In the present invention, the disulfide linkage enables release of the hydrophilic active ingredient in a reducing environment.

[0047] The term "reducing conditions" refers to an environment capable of cleaving disulfide linkages, including intracellular environments containing reducing agents such as glutathione.

[0048] The term "glutathione" refers to a naturally occurring intracellular tripeptide that acts as a reducing agent and is present at elevated concentrations in many cancer cells.

[0049] The term "pharmaceutically acceptable" refers to components, carriers, excipients, or formulations that are suitable for administration to a subject without causing unacceptable toxicity or adverse effects.

[0050] The term "treatment" refers to the therapeutic management of a disease or condition, including alleviation of one or more symptoms, reduction of disease progression, or eradication of the disease.

[0051] The term "tumour" refers to an abnormal growth of cells, including benign and malignant neoplasms, and in particular solid cancers.

[0052] In an embodiment, the amount of the micelle ranges from 2% (w / w) - 20%(w / w); preferably 3%(w / w) - 10%(w / w).

[0053] In an embodiment, the suitable pharmaceutical carrier is a polymer selected from a list consisting of: hyaluronic acid, agarose, alginate, chitosan, collagen, gelatin, fibrin, starch, cellulose, polyvinyl alcohol, polyethylene glycol, polyacrylamide, poly(acrylic acid), poly(N-isopropylacrylamide), poly(hydroxyethyl methacrylate), poly(methyl methacrylate), poly(ethylene oxide), nanocomposite hydrogel, hydrogelmetal composite, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), hydrogel-ceramic composite, hydrogel-lipid composite, silk fibroin, soy protein, or mixtures thereof; preferably the polymer is hyaluronic acid.

[0054] In an embodiment, the vitamin E derivative and / or a vitamin E - polymer composite may be linked by one side to the outer shell through a covalent bond and by the other side to the hydrophilic active ingredient by a site-selective stable self-immolative linkage (disulfide linkage) represented by the formula:wherein:Ri is a vitamin;R2 is oxygen;Rs is a hydrophilic active ingredient.

[0055] In an embodiment, the hydrophobic core may be formed by vitamin E or vitamin D, vitamin E derivatives, vitamin D derivatives, or mixtures thereof.

[0056] In an embodiment, the vitamin E derivative may be selected from a group consisting of: a-tocopherol, p-tocopherol, y-tocopherol, 6-tocopherol, a-tocotrienol, p-tocotrienol, y-tocotrienol, 6-tocotrienol, a-tocopheryl acetate, a-tocopheryl nicotinate, a-tocopheryl phosphate, a-tocopheryl polyethylene glycol succinate, a-tocopheryl linoleate, or tocophersolan, or mixtures thereof; preferably a-tocopheryl succinate.

[0057] In an embodiment, the vitamin D derivative may be selected from a group consisting of: calcitriol, calcifediol, cholecalciferol, ergocalciferol, alfacalcidol, doxercalciferol, paricalcitol, calcipotriol, tacalcitol, falecalcitriol, eldecalcitol, or mixtures thereof.

[0058] In an embodiment, the polymer of the vitamin E - polymer composite may be selected from a list consisting of: polyethylene glycol, polylactic acid, polyvinyl alcohol, polycaprolactone, or mixtures thereof.

[0059] In an embodiment, the vitamin E - polymer composite may be D-a-tocopheryl polyethylene glycol succinate.

[0060] In an embodiment, the hydrophobic core may comprise a hydrophobic active ingredient.

[0061] In an embodiment, the hydrophobic active ingredient:micelle weight ratio ranges from 1:20 to 1:100; preferably 1:20 to 1:50; more preferably 1:30.

[0062] In an embodiment, the vitamin E - polymer composite:hydrophilic active ingredient weight ratio ranges from 2:6 to 4:6; preferably 3:5 to 5:7; more preferably 4:6.

[0063] In an embodiment, the pharmaceutical amount of the hydrophobic active ingredient may be inferior to 0.5 pM; preferably from 0.002 pM to 0.3 pM; more preferably 0.003 pM to 0.2 pM.

[0064] In an embodiment, the pharmaceutical amount of the hydrophilic active ingredient may be inferior to 8 pM; preferably from 0.01 pM to 7.5 pM; more preferably 0.015 pM to 7 pM.

[0065] In an embodiment, the hydrophobic active ingredient may be a chemotherapeutic agent.

[0066] In an embodiment, the hydrophilic active ingredient may be a chemotherapeutic agent.

[0067] In an embodiment, the chemotherapeutic agent may be selected from a list consisting of: alkylating agents, anthracyclines, cytoskeletal disruptors, epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, monoclonal antibodies, antibody-drug conjugates, nucleotide analogs, precursor analogs, peptide antibiotics, platinum-based agents, retinoids, vinca alkaloids, cytokines, anti-metabolites, and vinca alkaloids derivatives, or other cytotoxics.

[0068] In an embodiment, the hydrophobic active ingredient may be paclitaxel (PTX).

[0069] In an embodiment, the hydrophilic active ingredient may be gemcitabine (GEM).

[0070] In an embodiment, the micelle of the present disclosure may have a spherical morphology.

[0071] In an embodiment, the average particle size of the micelle of the present disclosure may range from 100 nm - 140 nm; preferably 110 nm - 130 nm; more preferably 120 nm.

[0072] In an embodiment, the polydispersity index of the micelle of the present disclosure may range from 0.3 to 0.2; preferably 0.25 to 0.1; more preferably 0.1.

[0073] In an embodiment, the encapsulation efficiency of the hydrophobic active ingredient may range from 50% to 100%; preferably 60% to 95%; more preferably 70% to 90%.

[0074] An aspect of the present disclosure relates to the use of the hydrogel composition described in the present disclosure in medicine or veterinary.

[0075] In an embodiment, the hydrogel composition of the present disclosure may be used in the treatment of tumours.

[0076] In an embodiment, the hydrogel composition of the present disclosure may be used in the treatment of cancer; preferably in the treatment of a solid cancer; preferably pancreatic cancer, resulting in significant retardation of tumour growth and reduction in metastatic spread.

[0077] A further aspect of the present disclosure relates to a hydrogel composition comprising the micelle of the present disclosure for enhanced therapeutic delivery and efficacy.

[0078] Another aspect of the present disclosure relates to a method of producing the hydrogel composition, comprising the steps of:providing a vitamin E - polymer composite or vitamin E derivative, a disulfide linker, an activated para-nitrophenol carbonate, and a hydrophilic active ingredient;modifying the vitamin E - polymer composite or vitamin E derivative to include the disulfide linker; adding the activated para-nitrophenol carbonate to the vitamin E - polymer composite or vitamin E derivative with the disulfide linker and forming a carbamate linkage (a redox-cleavable conjugate); purifying the carbamate linkage between the vitamin E - polymer composite or vitamin E derivative and the hydrophilic active ingredient;inducing self-assembly of micelles by adding the organic phase to an aqueous phase under stirring and removing the organic solvent, thereby forming micelles comprising an outer shell containing the vitamin E derivative and the hydrophilic active ingredient;incorporating a hydrophobic active ingredient into the organic phase prior to self-assembly to form micelles comprising a hydrophobic core containing the hydrophobic vitamin and the hydrophobic active ingredient,incorporating the micelles into a hydrogel matrix by mixing the micelles with one or more polymer;thereby obtaining the hydrogel composition.

[0079] In an embodiment, the method of production further comprising the addition of a hydrophobic active ingredient in the organic phase during the self-assembly step of the micelle.

[0080] In an embodiment, the vitamin E-polymer composite and the vitamin E derivative are used at a weight ratio of 4:6, and wherein the hydrophobic active ingredient is added at a weight ratio of about 1:30 relative to the total micelle-forming material.

[0081] In an embodiment, the method may comprise self-assembling of the micelle at a temperature interval of 15 °C to 35 °C; preferably 20 °C to 30 °C; more preferably 22°C to 28°C in a buffer solution selected from the group consisting of: Tris(hydroxymethyl)aminomethane and hydrochloric acid buffer, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer, 3-(N-morpholino)propanesulfonic acid buffer, citrate buffer, glycine buffer, Tris and ethylenediaminetetraacetic acid buffer, borate buffer, or 2-(N-morpholino)ethanesulfonic acid buffer; preferably phosphate-buffered saline.

[0082] In an embodiment, the method of production of the hydrogel composition of the present disclosure may further comprise the addition of a hydrophobic active ingredient in the organic phase during the self-assembly step of the micelle.

[0083] In an embodiment, the self-assembly of the micelle may occur at a temperature interval of 15 °C to 35 °C; preferably 20 °C to 30 °C; more preferably 22°C to 28°C.

[0084] In an embodiment, the self-assembly of the micelle may occur in a buffer solution.

[0085] In an embodiment, the buffer solution may be selected from the group consisting of: Tris(hydroxymethyl)aminomethane and hydrochloric acid buffer, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer, 3-( / V-morpholino)propanesulfonic acid buffer, citrate buffer, glycine buffer, Tris and ethylenediaminetetraacetic acid buffer, borate buffer, or 2-( / V-morpholino)ethanesulfonic acid buffer; preferably phosphate-buffered saline.BRIEF DESCRIPTION OF THE DRAWINGS

[0086] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0087] Figure 1: Production and characterisation of preferred embodiments of the micelle of the present disclosure containing paclitaxel (micelle@PTX) or gemcitabine (micelle@GEM). a) Schematic representation of vitamin-E-based micelles for triggerable multimodal pancreatic ductal adenocarcinoma (PDAC) nanotherapy, b) Schematic representation of main micellar components, c) i) Chemical construction of the main micellar components, including tocopheryl succinate (TOS), TOS modified to include a short disulfide linker (TOS-SSOH), and GEM carbamate (TOS-SS-GEM). ii) liquid chromatography-high resolution mass spectrometry (LC-HRMS), iii) ultraviolet-visible spectroscopy (UV-vis), and iv) carbon nuclear magnetic resonance (C-NMR) spectra of the GEM product site-specifically modified at the -NH2 moiety, d) i-ii) scanning electron microscopy (SEM) images of micelle@GEM. Scale: d-i) 500 nm, d-ii) 50 nm. e) i) hydrodynamic size by dynamic light scattering (DLS), ii) polydispersity index, N = 3 independent experiments, mean ± standard deviation (SD), * p < 0.05, two-tailed unpaired t-test. and iii) charge by zeta potential of micelle@PTX and micelle@GEM. f) LC-HRMS study of TOS-SS-GEM and micelle@GEM at different glutathione (GSH) concentrations and pH. g) Plasma metabolism by cytidine deaminase (CDA). h) Profile of release of agents (Rhodamine or PTX) from the produced micelles. N = 3 independent experiments, mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, ordinary ANOVA test with Tukey's multiple comparisons test.

[0088] Figure 2: Representation of in vitro analysis of preferred embodiments of the prodrug micelle of the present disclosure in pancreatic cancer cells, a. Heatmap displaying drug release kinetics from micelles gemcitabine (GEM), b. Heatmap depicting the concentration-dependent release of PTX from micelles over a period of 72 h, demonstrating controlled release at therapeutic levels c. Bar graph illustrating the enhanced cytotoxicity of micelle-carried PTX and GEM (micelle@PTX+GEM) compared to free drugs on pancreatic cancer cells, with significant reductions in cell viability at various drug concentrations at 72 hours after NPs incubation IC50 (pm): GEM=0.035, micelle@GEM=0.015, PTX=0.185, and micelle@PTX=0.003. IC10 (pm): GEM=0.01, micelle@GEM=0.0035, PTX=0.04, and micelle@PTX=0.0007. d. i) Bar chart showing the percentage of NP internalisation over time, indicating rapid uptake by pancreatic cancer cells sustained over 72 h, N = 3 independent experiments, mean ± SD, * p < 0.05, ** p < 0.01, Kruskal-Wallis test with Dunn's multiple comparison test, ii) Bar chart of the mean fluorescence intensity quantifying the retention of rhodamine-labelled NPs (NP-Rho) inside the cells, corroborating prolonged intracellular presence, e. Fluorescence microscopy images (i and ii.) Uptake of rhodamine-labelled NPs by pancreatic cancer cells (rhodamine-labelled NPs: red, nuclei: blue), confirming the internalisation observed in quantitative assays. Scale bar: 10 pm (e-i) and 2 pm (e-ii). f. Bar graph summarising the cell cycle distribution of pancreatic cancer cells after treatment with free GEM, linker-modified micelles (micelle@linker), and micelles loaded with GEM (micelle@GEM), indicating a possible G1 phase cell cycle arrest by micelle@GEM. N = 3 independent experiments, mean ± SD, * p < 0.05, vs. control, *** p < 0.001 vs. control, *** p < 0.0001 vs. control; # p < 0.05 vs. micelle@linker, ### p < 0.001 vs. micelle@linker, #### p < 0.0001 vs. micelle@linker, ordinary ANOVA test with Tukey's multiple comparisons test.

[0089] Figure 3: Representation of the evaluation of the biodistribution of preferred embodiments of the nanoparticle-based micelle delivery systems of the present disclosure in live animals, a. Sequential fluorescence imaging series illustrating the in vivo biodistribution of rhodamine-labelled micelles after intraperitoneal (IP) injection over a 24-hour period. The images compare the signal intensity from noinjection to 24 h post-injection, highlighting the systemic circulation and localisation of the micelles, b. Violin plot quantifying the fluorescence intensity of the rhodamine signal from the micelles at various time points after hydrogel injection, providing a statistical distribution of the biodistribution data over time, c. Bar graph depicting the accumulation of rhodamine-labelled micelles in major organs at the 24-hour time point. This graph shows the relative uptake of micelles by organs such as the liver, spleen, pancreas, heart, lungs, and kidneys, which is essential for evaluating the specificity and potential off-target effects of the nanocarrier system, which affect therapeutic outcomes, depending on the balance between achieving effective drug concentrations at the tumour and metastatic sites (in this case, pancreas, liver, and spleen) and minimising exposure to critical organs to avoid toxicity.

[0090] Figure 4: Representation of the evaluation of the efficacy of preferred embodiments of the nanoparticle-based micelle delivery systems hydrogel of the present disclosure in a preclinical model of pancreatic cancer, a. Quantitative analysis of tumour growth as measured by the bioluminescent signal intensity from the tumours in mice. The bar chart shows signal quantification at various time points posttreatment, with significant growth inhibition observed in the hyd + micelle@PTX+GEM group. Statistical analysis was performed using a one-way analysis of variance (Brown-Forsythe) test, ****p < 0.0001, ***P < 0.001, *P < 0.05). b. Kaplan-Meier survival curve illustrating the survival probability of mice following treatment with different hydrogel formulations. The graph highlights the improved survival outcome in the group treated with a combination of PTX and GEM carried in micelles. Statistical analysis was performed using the log-rank (Mantel-Cox) test (*P < 0.05). c. Ex vivo bioluminescence imaging of organs extracted from treated mice was performed to assess metastatic spread. The images show the presence or absence of metastases in the lungs, heart, pancreas, spleen, liver, and the primary tumour location in the abdominal wall. N = 5, mean ± SD, * p < 0.05, vs. control, two-way ANOVA test with Tukey's multiple comparisons test.

[0091] Figure 5: Body weight monitoring of mice subjected to preferred embodiments of the present disclosure over time. Boxplot representing body weight changes in mice over 19 days following the injection of various formulations: empty hydrogel, hyd-conjugated with naked micelles, hyd-conjugated micelles with PTX, hyd-conjugated micelles with GEM, a combination of hyd-conjugated micelles with PTX and GEM, and free combination of PTX and GEM. Weight trends serve as an indirect measure of the tolerability and systemic toxicity of different therapeutic regimens tested in a peritoneal pancreatic carcinomatosis mouse model.

[0092] Figure 6: Representation of the evaluation of the genetic response of primary and metastatic pancreatic tumours to preferred embodiments of the pro-drug micelle of the present disclosure and the impact on gene expression profiles, a. Multimodal imaging showing the localisation of primary and metastatic pancreatic tumours. The left image employs live bioluminescence imaging scanning to highlightareas of high metabolic activity indicative of tumour sites, whereas the right image displays the respective organ-specific distribution of metastases.DETAILED DESCRIPTION

[0093] The present disclosure relates to a hydrogel composition comprising micelles for the delivery of active ingredients. Namely, a hydrogel composition comprising micelles incorporated into a pharmaceutical carrier, wherein the micelles are present in an amount ranging from 1% (w / w) to 50% (w / w) based on the total weight of the hydrogel composition. The micelles comprise a mixture of micelles including micelles having a hydrophobic core containing a hydrophobic vitamin and a hydrophobic active ingredient, and micelles having an outer shell comprising a vitamin E derivative and / or a vitamin E-polymer composite and a hydrophilic active ingredient, and / or micelles comprising both a hydrophobic core and an outer shell. The hydrophilic active ingredient is covalently linked to the vitamin E derivative and / or the vitamin E-polymer composite by a disulfide linkage cleavable in the presence of glutathione. The hydrogel composition enables controlled delivery of hydrophobic and hydrophilic active ingredients and is suitable for therapeutic applications (medical or veterinary use), including cancer treatment, in particular solid cancer, preferably pancreatic cancer.

[0094] Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, owing to its aggressive nature and limited responsiveness to conventional chemotherapy. The present disclosure is a novel self-immolative micelle hydrogel composition for the delivery of hydrophilic and hydrophobic active ingredients, namely paclitaxel (PTX) and gemcitabine (GEM), two frontline chemotherapeutic agents, to target PDAC and its metastases.

[0095] In an aspect of the present disclosure, the micelle composition, which is a tailor-made vitamin E derivative micelle designed to encapsulate lipophilic payloads, such as PTX, and / or append and protect hydrophilic PDAC mainstay drug GEM. GEM serum instability and off-target effects were bypassed by surface linkage to the micelle system via a responsive stable linker which allows near quantitative and chemically intact GEM release inside cancer cells through a glutathione (GSH) stimulus. The active ingredients when delivered by the micelle composition of the present disclosure presented a 50 to 100-fold increase in chemotherapeutic activity, comparing to the free active ingredients.

[0096] Through longitudinal bioluminescence imaging, a significant retardation of primary tumor growth was observed and a reduction in metastatic spread in the micelle@GEM and micelle@PTX+GEM groups compared to free drug formulations.

[0097] Gene expression analysis of treated tumours revealed substantial modulation of oncogenic pathways, including phosphatidylinositol 3-kinase (PI3K)-Akt (PI3K-Akt) and mitogen-activated protein kinase (MAPK) signalling, suggesting targeted action at the molecular level. The integrated analysisprovided by these multi-faceted bioinformatics approaches underscores the robust alterations in gene expression prompted by micelle-carried chemotherapeutics. The data suggest that the delivery of PTX and GEM within these micelle compositions altered the pharmacokinetics and pharmacodynamics of these agents, thereby modulating their interactions with cellular targets. This led to a more pronounced and targeted antitumor effect, and at the same time, mitigated the off-target toxicities often associated with these drugs.Precise stimulus-responsive tocopherol-tailored prodrug micelles

[0098] In an embodiment of the present disclosure for better results, the micellewas tailored to accommodate site-selective stable linkage of GEM on the nanoparticle (NP) surface. This selectivity protected the drug from cytidine deaminase (CDA) metabolization, the main degradation pathway for GEM, and allow GSH-mediated reversibility to selectively deliver GEM via a self-immolative chemical mechanism. Tocopheryl succinate (TOS) was generated in quantitative yield from a-tocopherol and modified to include a short disulfide linker (TOS-SSOH). Subsequent formation of activated paranitrophenol carbonate allowed installation of GEM and purification to afford homogeneous and pure GEM carbamate (TOS-SS-GEM). Product purity and selectivity was confirmed by liquid chromatography-high resolution mass spectrometry (LC-HRMS), nuclear magnetic resonance (NMR), and the bathochromic shift in ultraviolet-visible spectroscopy (UV-Vis) absorbance in TOS-SS-GEM compared to GEM (Fig. lc ii-iv), which supported chemical acylation of the cytidine scaffold. Precise N-chemoselective engineering was pursued as O-linked side products suffer a quick hydrolysis to liberate GEM while leaving the cytidine moiety from CDA metabolism.

[0099] In an embodiment of the present disclosure for better results, the micelle formulation tolerated high levels of surface modification and / or encapsulation and was optimized by varying TPGS (D-a-tocopheryl polyethylene glycol 1000 succinate) / TOS-SS-GEM ratios under self-assembly conditions (in PBS, 25°C). This resulted in spherical micelles ± 120 nm (±20) with low polydispersity at an optimal weight ratio of 4:6 TPGS / TOS-SS-GEM (Fig. ld-e). Encapsulation of hydrophobic payloads can be achieved by simple addition of the payload in the organic phase during the self-assembly process. The system tolerated up to a 1:30 payload / NP weight ratio. Alternatively, GEM-naked micelles were synthesized using TPGS / TOS-SS-OH in a similar 4:6 weight ratio and 1:30 PTX / NP weight ratio.

[0100] In an embodiment of the present disclosure, micelle@GEM systems employing N-linked TOS-SS-GEM protected the payload from metabolic degradation and were highly stable while showing a near-quantitative stimulus-release of GEM in GSH-rich environments. To demonstrate this LC-HRMS studies of TOS-SS-GEM and micelle@GEM over time were carried in different environments ranging in it GSH concentration (0-10mM). A less than 20% degradation was observed after 120h in PBS pH 7.4, 37°C, whereas near-quantitative GSH-dependant release of GEM was observed for high GSH levels often observed in cancer cells (approx. 99% GEM release; 72h in PBS pH 7.4, 37°C, 10 mM GSH). Importantly,discrete GEM release was observed during the same time range for GSH concentration within healthy cell values (Fig. If), suggesting a potential for tumor specific release of GEM and manageable safety profile due to low and slow GEM release under healthy cellular conditions. Additionally, incubation of TOS-SS-GEM with CDA afforded negligible metabolite formation compared to free GEM (<5% and ~99% after 90 minutes, respectively) (Fig. lg). This demonstrates that GEM is protected from plasma CDA metabolism when linked to the micelle, enabling the delivery of high concentrations of unmetabolized GEM to tumour cells, while minimizing systemic exposure, thereby maximizing therapeutic efficacy. The release of PTX from micelle@PTX was evaluated using the dialysis diffusion method and quantified by LC-HRMS, showing high stability (<20% release after 120h, pH 7.4, 37°C, Fig. lh).In vitro mechanisms of enhanced anticancer efficacyIn an embodiment of the present disclosure, BxPC-3 cells, a pancreatic cancer cell line derived from human pancreatic adenocarcinoma were used to collectively assess the controlled release, cellular uptake, cytotoxic impact, migration inhibition, and cell cycle modulation by the micelles.In an embodiment of the present disclosure, the metabolic activity of cancer cells was measured after treatment with micelle@GEM or micelle@PTX, and then compared to their respective free drugs (Fig. 2a-c). Micelle@GEM and micelle@PTX significantly reduced cell metabolic activity compared to the free drugs, enabling 50-fold and 100-fold lower drug concentrations to achieve comparable or greater reductions to free GEM and PTX, respectively. Similarly, the inhibitory concentration for the PTX / GEM combination demonstrated that drug delivery via the nanoparticles enhances cytotoxicity, due to improved cellular uptake and sustained intracellular drug release (Fig. 2c). Concerning cellular uptake and retention, it was observed the internalization and retention of rhodamine-labelled NPs (NP-Rho) in pancreatic cancer cells (Fig. 2di-ii). The data indicated that NPs are not only rapidly internalized, but also retained within the cells over a 72-hour period. This sustained retention is essential for continuous drug action inside the cancer cells and contribute to the increased cytotoxicity observed in Fig. 2c. Confocal microscopy images (Fig. 2e) visually confirm the cellular uptake of NP-micelles, with a clear distinction between the cells with internalized NP-Rho, highlighting the NPs' ability to penetrate and reside within the cancer cells, which correlates with the quantitative data in Fig. 2d. To understand if the effects of the drugs on the cell cycle were altered when using the micelles as a delivery vehicle, cells were incubated with half maximal inhibitory concentration ( IC5o) doses of each system. GEM is known to cause S phase arrest, whereas PTX arrests the cell cycle during the G2 / M phase in various cancer cells, including pancreatic cells. Micelle@GEM-treated cells showed an increase in G1 phase indicative of G1 arrest was more pronounced (approximately 20% more cells in Gl) compared to the control free GEM (Fig. 2f). In an embodiment of the present disclosure, collectively, these assays and mechanisms indicate that NP-micelles are a superior alternative to free drugs, underpinning their enhanced antitumor activity. By enhancing drug delivery and retention, exerting potent cytotoxic effects, potentially inhibiting cellmigration, and inducing cell cycle arrest, NP-micelles emerge as a potent modality for improving the therapeutic index of chemotherapeutic agents in vivo.Mapping prodrug micelle biodistribution

[0101] In an embodiment of the present disclosure, to map and evaluate the biodistribution of prodrug micelles, temporal and spatial distribution profiles were analysed. Mice were treated with a 4% (w / v) hyaluronic acid (HA) hydrogel containing rhodamine-labelled micelles by intraperitoneal injection (500 pL / mouse). This data is crucial for the development of targeted drug delivery systems using nanocarriers like micelles, which aim to maximize therapeutic efficacy while minimizing systemic toxicity. These nanocarriers are designed for optimal delivery of chemotherapeutic agents to tumor sites. Whole-body fluorescence was measured for 24h after the injection and showed an intense signal intraperitoneally at the time of injection (Fig. 3a). The absence of fluorescence in the "no inject" control contrasted with the progressive intensity and spread of the rhodamine signal post-injection. The signal had a maximum intensity in the whole body at 0.5h after the injection and gradually decreased afterwards (Fig. 3a-b).Following 24h from the injection, the rhodamine signal was more evident in the peritoneal cavity (Fig. 3a).This series captured the dynamics of micelle movement through the body over 24 hours, highlighting the initial widespread distribution and subsequent concentration in specific areas, likely indicative of tumor sites and organs of clearance. This means that the 4% (w / v) HA hydrogel enabled the retention of the micelles and that these remained longer in the abdominal cavity. Individual organ assessment showed greater accumulation of rhodamine-labelled micelles in the liver and pancreas (Fig. 3c). Thus, the HA hydrogel scaffold represents a promising strategy for the delivery of therapeutic agents, allowing prolonged contact with the abdominal organs and reducing off-target effects. The peak accumulation at early time points suggested a rapid initial distribution, which is important to maximize tumour uptake and minimize exposure to non-target tissues.Nano-booster prodrug micelles for in vivo pancreatic cancer

[0102] In an embodiment of the present disclosure, to assess the therapeutic efficacy of the HA hydrogel (Hyd) containing drug-loaded micelles, luciferase-expressing BxPC-3 human pancreatic cancer cells were injected intraperitoneally. The proposed in vivo model resembles PDAC late-stage at diagnosis, recapitulating rapid progression, metastasis, and tumour aggressiveness. At day 21 post tumour induction (Day 0), peritoneal pancreatic carcinomatosis mouse model recapitulates key aspects of advanced pancreatic cancer. In this context, the term "primary tumors" refers specifically to the neoplastic growths located in the peritoneal cavity, modelled by the implantation of BxPC-3-Luc cells. Conversely, the term "pancreatic metastases" denotes secondary tumor growths localized in the liver, spleen, pancreas, and peritoneal membrane. After treatment with 6 different hydrogel-loaded formulations, tumor progression was assessed by evaluating bioluminescence signal and survival analysis, both correlating with the tumour burden. A single injection of hyd + micelle@GEM induced highly significant tumour regression (Fig. 4a),with duration of response for more than 10 days across all tumour loci as observed by a sustained negligible luminescence signal. Similarly, hyd + micelle@PTX and hyd + micelle@PTX+GEI\ / l inhibited tumour growth (Fig. 4a), corroborating the in vitro results, and indicating a potent in vivo antitumour effect. A single injection of hyd + drug-carrying micelles resulted in prolonged survival compared with the control groups (Fig. 4b), supporting the higher effective delivery of therapeutic agents. One hundred percent of the animals treated with hyd + drug-carrying micelles were alive during the entire experiment with a single dose of treatment; however, no animals have survived in the other groups for the exact same period (Fig.4b). At the final endpoint, the tissues and organs were assessed for the presence of pancreatic cancer metastasis. In addition to the abdominal wall, the abdominal organs, such as the liver, pancreas, and spleen, had the highest number of metastases (Fig. 4c). As previously observed via live imaging, animals treated with hyd + micelle@GEM exhibited markedly less luminescence, indicating reduction of the metastatic implants, which was closely followed by animals that received a single injection of hyd + micelle@PTX+GEM (Fig. 4c). The heatmap that quantifies the metastatic burden in different organs corroborates the findings from bioluminescence imaging, with the lowest metastatic burden observed in the combination treatment group.

[0103] In an embodiment, the hydrogel composition comprises micelles, wherein each micelle comprises a hydrophobic core comprising a hydrophobic vitamin and a hydrophobic active ingredient, and an outer shell comprising a vitamin E derivative and a hydrophilic active ingredient, wherein the hydrophilic active ingredient is linked to the vitamin E derivative by a disulfide linkage cleavable in the presence of glutathione, and wherein the micelles are incorporated into a hydrogel matrix.

[0104] In an embodiment, the micelle comprising a hydrophilic active ingredient are prepared by selfassembly of a vitamin E-polymer composite and a vitamin E derivative bearing a hydrophilic active ingredient. In particular, 12 mg of D-a-tocopheryl polyethylene glycol succinate (vitamin E-polymer composite) and 18 mg of a tocopheryl succinate derivative covalently linked to gemcitabine via a disulfide linkage are dissolved in 1.5 mL of ethanol. The resulting solution is added dropwise to 15 mL of aqueous medium under continuous stirring at 600 rpm at room temperature and stirred overnight to evaporate the organic solvent, thereby forming micelles comprising an outer shell containing the vitamin E derivative and the hydrophilic active ingredient linked by the disulfide linkage. In this embodiment, micelles comprising a hydrophobic active ingredient are prepared by self-assembly of a vitamin E-polymer composite and a vitamin E derivative forming a hydrophobic core comprising a hydrophobic vitamin, wherein the hydrophobic active ingredient is incorporated into the hydrophobic core. The vitamin E-polymer composite and the vitamin E derivative are used at a weight ratio of 4:6, and the hydrophobic active ingredient is added to the organic phase at a weight ratio of 1:30 relative to the total micelleforming material, followed by self-assembly under aqueous conditions. In this embodiment, the micelles are incorporated into a hydrogel composition by mixing the micelles with a pharmaceutical carriercomprising a hydrogel matrix. The hydrogel matrix is formed from hyaluronic acid at a concentration of 4% (w / w). The micelles comprising the hydrophobic active ingredient and the micelles comprising the hydrophilic active ingredient are dispersed within the hydrogel matrix to obtain a hydrogel composition wherein the amount of micelles is within the range defined in the claims. In this embodiment, the hydrogel composition comprises micelles dispersed within the hydrogel matrix, wherein the micelles enable controlled delivery of the hydrophobic active ingredient from the hydrophobic core and controlled release of the hydrophilic active ingredient from the outer shell upon cleavage of the disulfide linkage in the presence of glutathione.

[0105] Throughout the experiment, the body weight remained unchanged (Fig. 5), suggesting the treatment is associated with reduced toxicity or side effects.

[0106] In an embodiment of the present disclosure, the data from these experiments presented strong evidence for the potential of micelle-carried chemotherapy in treating pancreatic cancer. The use of micelles as a delivery system seems to have enhanced the efficacy of PTX and GEM, due to improved drug solubility, the EPR effect at the tumor site, and sustained release of the drugs in the tumor microenvironment. This approach overcomes the challenges of poor drug stability and nonspecific toxicity associated with conventional chemotherapy. The reduced tumor burden decreased metastatic spread, and extended survival times in the GEM alone or in the combination treatment group suggest a promising therapeutic strategy. The synergy between PTX and GEM, when delivered in a targeted fashion, has the potential to maximize therapeutic outcomes.Tumor and metastasis genetic profile in response to prodrug micelles

[0107] In an embodiment of the present disclosure, to explore the genetic profiling of primary and metastatic pancreatic cancer tumors under different treatment conditions using the tested prodrug micelles, RNA was extracted from the different primary tumors and their metastases in the spleen and liver from the peritoneal pancreatic carcinomatosis mice model. Whole-body imaging (Fig. 6) confirmed the presence of metastatic sites and primary tumors, which are crucial for understanding the spread of this type of cancer. The differential accumulation of micelles in primary versus metastatic sites raised the possibility of tailoring nanoparticle properties to enhance delivery to specific tumor niches, a critical consideration given the notorious challenges of drug delivery in PDAC due to its dense stromal barrier and immunosuppressive microenvironment. To achieve this knowledge, a comprehensive genetic analysis of primary and metastatic pancreatic cancer lesions was performed, specifically examining the impact of various micelle-based delivery systems on gene expression profiles. This analysis falls at the intersection of oncogenomics, cancer therapeutics, and nanotechnology, offering insights into the precision targeting of oncogenic pathways via advanced drug delivery mechanisms.

[0108] In an embodiment of the present disclosure, to provide a clear visualization of the transcriptomic changes induced by treatments, Volcano plots were drawn, showing that the magnitude and significanceof gene expression shifts underscore the pharmacogenomic impact of micelle-carried PTX and GEM. Notably, these plots underscored the differential pharmacogenomic response of primary versus metastatic lesions, indicating a spatial heterogeneity in therapeutic susceptibility. The differential gene expression between primary and metastatic lesions suggested that micelle-mediated delivery influence tumor behavior in a site-dependent manner, offering potential for targeted therapeutic interventions that address the heterogeneity within the tumor microenvironment.

[0109] In an embodiment of the present disclosure, to reveal the complexity of treatment responses, Venn diagrams showed substantial overlap in altered gene expression, suggesting common pathways affected by micelle-carried active ingredients. This comparative genomic approach is instrumental in determining the unique and shared pathways modulated by individual and combination treatments. The intersectional subsets suggested convergent mechanisms of action or compensatory pathways activated in response to the treatment. The unique gene expression changes induced by the combination of PTX and GEM may reflect a synergistic mechanism, potentially providing a more comprehensive blockade of oncogenic signalling pathways than either agent alone. Concerning primary tumors, the central intersection elucidates a core set of genes commonly influenced across all treatment modalities, potentially implicating fundamental processes in tumor survival and resistance. For pancreatic METs, the significant overlap between PTX and GEM treatments suggested a shared therapeutic impact on pancreatic metastatic lesions, while unique gene sets might reflect drug-specific actions. Both spleen METs and liver METs, highlight the site-specific genomic response to treatment, which is critical given the organ-specific microenvironmental influences on metastatic cancer cell behavior.

[0110] In an embodiment of the present disclosure, these findings prompt a reconsideration of combination chemotherapies, where the role of drug formulation and delivery method becomes as critical as the agents chosen. The overall implications of this analysis highlight the transformative potential of nanoparticle technology in PDAC treatment. The ability of micelles to alter gene expression profiles suggests a paradigm, where the efficacy of chemotherapy can be enhanced beyond the broad action of the drugs themselves. These insights into gene expression modulations offer a roadmap for further investigation into specific pathways, with the aim of developing more effective, personalized therapeutic strategies. The data emphasizes the need to consider the micelles formulation as an integral component of the therapeutic regimen, with the potential to alter the pharmacokinetics and pharmacodynamics of chemotherapeutic agents, thereby redefining the standard of care for patients with PDAC.Modulation of genomic pancreatic cancer signalling pathways in primary tumor and metastasis

[0111] In an embodiment of the present disclosure, to provide a detailed transcriptomic analysis of primary and metastatic pancreatic cancer lesions treated with different drug formulations carried within micelles, it is essential to understand the molecular dynamics induced by nanocarrier-mediated drug delivery and its implications for targeted cancer therapy. Using gene ontology (GO) analysis fordifferentially expressed genes, categorized by biological processes, cellular components, and molecular functions allows for the classification of genes into hierarchies based on their associated biological processes and functions, offering a macroscopic view of the cellular activities impacted by the treatments. This analysis is key for identifying the overarching biological themes and cellular machineries that are perturbed by the treatment, potentially unveiling new targets for therapeutic intervention. The GO heatmap offered a granular view of the modulation of critical signalling pathways in response to treatment, showing how micelle drug delivery reprograms the cancer cell transcriptome. It was observed that all prodrug micelles significantly alter GO pathways, mainly within pancreas METs and primary tumor, when compared to free drugs. This is clear when observing a higher incidence of up-regulated processes for micelle@PTX and Hyd + micelle@PTX / GEM in both primary tumors and pancreatic METs. This upregulation suggests that the drug formulations are actively influencing key cellular mechanisms, potentially leading to increased stress responses, apoptosis, or immune activation within the tumor microenvironment. The capacity of these treatments to modulate gene expression significantly can be attributed to the enhanced cellular uptake and sustained release properties of the micelles, ensuring that the chemotherapeutic agents maintain prolonged contact with their intracellular targets, thus amplifying their therapeutic effects. On the contrary, a higher incidence to down-regulate processes was observed when using Hyd + micelle@GEM in pancreas METs and up-regulated in primary tumors. Here, the upregulation could indicate a suppression of genes associated with metastatic progression, angiogenesis, or other pro-survival pathways. Interestingly, the same treatment upregulates processes in primary tumors, which might reflect a context-dependent response where primary tumor cells and metastatic cells exhibit differential sensitivity to GEM when delivered via micelles.

[0112] In an embodiment of the present disclosure, in order to display the enrichment of the different oncogenic pathways and processes, including signalling, transcriptional misregulation, ribosome biogenesis, and others across the primary tumor and metastatic sites, circular bar plots of KEGG analysis are shown. Each concentric ring represents a specific treatment condition, and the length of the bar correlates with the enrichment significance of the connected pathways. This analysis helps visualize the extent to which each pathway is influenced under different treatment conditions, revealing critical insights into how micelle drug delivery can modulate cancer pathophysiology. It was observed a broader impact of the prodrug micelles on the tumour microenvironment, affecting processes such as cell adhesion, autophagy, and oxidative stress response, which are crucial for tumour growth and metastasis.

[0113] In an embodiment of the present disclosure, to explore deeper into the molecular pathways of pancreatic cancer's response to treatment, gene differential analysis clustering was performed. The data suggested differential gene expression profiles in response to treatments with PTX and GEM, either as a single agent or in combination, carried by micelles. These transcriptomic changes are not visible with the same free drugs. This is significant as it indicates that micelles not only enhanced drug solubility anddelivery efficiency but also altered the drug's interaction with cellular targets, thus affecting gene expression uniquely. The heatmap elucidates the expression patterns of genes within pivotal signalling axes such as PI3K-Akt, MAPK, and vascular endothelial growth factor (VEGF). These pathways are cornerstones in tumoral survival, proliferation, and angiogenesis, and their dysregulation is often implicated in pancreatic cancer resistance to conventional therapies. The PI3K-Akt pathway is a key regulator of cell survival and proliferation, often hyperactivated in pancreatic cancer. Aberrations in this pathway can lead to enhanced growth, survival, and metabolism of cancer cells, and are associated with resistance to chemotherapy. A transcriptomic analysis revealed significant modulation of this pathway by micelle@PTX and micelle@GEM, suggesting that the micelles can enhance the drugs' inhibitory effects on this pathway, which results in reduced cell viability and increased apoptosis of the pancreatic cancer cells. Additionally, the MAPK pathway plays a critical role in the transduction of extracellular signals to cellular responses, including proliferation, differentiation, and migration. Its involvement in pancreatic cancer has been linked to aggressive tumour behavior and metastasis. The treatment-specific changes in gene expression within the MAPK pathway depicted in the heatmap are indicative of the effectiveness of the micelle-based formulations in curtailing these processes. This is especially relevant for metastatic disease, where inhibiting MAPK signalling can significantly impact tumor spread and patient outcomes. This activation is in accordance with previous studies, showing that displayed upregulation of genes that regulate networks involved in KRAS activation (1,2). The VEGF signalling pathway is vital in angiogenesis, which is essential for tumor growth and metastasis by providing the necessary blood supply. Pancreatic tumours often exhibit high levels of VEGF, promoting a robust angiogenic network (3,4). The observed expression patterns suggested that the delivery of PTX and GEM by the micelle can affect this angiogenic signalling, potentially leading to reduced neovascularization and, consequently, limited tumor nourishment and growth. By affecting these pathways, the micelles could not only impede primary tumor growth but also target the metastatic cascade at various stages, from local invasion to distant colonization. This corroborates the in vivo efficacy results. This targeted molecular disruption, combined with the advantages of micelle delivery, such as improved drug stability and reduced systemic toxicity, mark a significant advancement in the therapeutic strategy against pancreatic cancer, recalibrating critical signalling networks that are often hijacked by pancreatic cancer cells.

[0114] This project has received funding from the Funda ao para a Ciencia e Tecnologia research and innovation programme under grant agreement PTDC / BTM-MAT / 4738 / 2020 and PTDC / BTM-MAT / 4738 / 2020.

[0115] This project has also received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme under grant agreement ERC-StG- 2019-848325.

[0116] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0117] Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first numerical value in a series of two or more numerical values, the term "at least," "greater than" or "greater than or equal to" applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0118] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. "Detecting the presence of" can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0119] As used herein, the term "about" a number refers to that number plus or minus 10% of that number. The term "about" a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[0120] As used herein, the terms "pharmaceutically acceptable" and "cosmetically acceptable" are used interchangeably and refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. More specifically, pharmaceutically acceptable refers to a material, compound, or composition which is suitable for use in contact with the skin, scalp, or hair. Pharmaceutically acceptable materials are known to those of ordinary skill in the art.

[0121] As used herein, the terms "treatment" or "treating" are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject atrisk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0122] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0123] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0124] The above-described embodiments are combinable.

[0125] The following claims further set out particular embodiments of the disclosure.References1. Cao, L. et al. Proteogenomic characterization of pancreatic ductal adenocarcinoma. Cell. 184, 5031-5052.e5026 (2021).2. Bailey, P. et al. Genomic analyses identify molecular subtypes of pancreatic cancer. Nature. 531, 47-52 (2016).3. Kim, D.K.; Jeong, J., Lee, D.S. et al. PD-Ll-directed PIGF / VEGF blockade synergizes with chemotherapy by targeting CD141+cancer-associated fibroblasts in pancreatic cancer. Nature Communications. 13, 6292 (2022). https: / / doi.org / 10.1038 / s41467-022-33991-64. Hosein, A.N.; Brekken, R.A.; & Maitra, A. Pancreatic cancer stroma: an update on therapeutic targeting strategies. Nature Reviews Gastroenterology & Hepatology. 17, 487-505 (2020).

Claims

C L A I M S1. Hydrogel composition comprising micelles,wherein the amount of the micelle ranges from 1% (w / w) - 50% (w / w) based on the total weight of the hydrogel composition;wherein the micelles area mixture of micelles comprising:micelles comprising a hydrophobic core containing a hydrophobic vitamin and a hydrophobic active ingredient, andmicelles comprising an outer shell containing a vitamin E derivative and / or a vitamin E- polymer composite and a hydrophilic active ingredient; and / ormicelles comprising a hydrophobic core and an outer shell, wherein the hydrophobic core comprises a hydrophobic vitamin and a hydrophobic active ingredient, wherein the outer shell comprises a vitamin E derivative and / or a vitamin E - polymer composite and a hydrophilic active ingredient;wherein the hydrophilic active ingredient is linked to the vitamin E derivative and / or a vitamin E - polymer composite by a disulfide linkage; and wherein the disulfide linkage is cleavable in the presence of glutathione.

2. Hydrogel composition according to the previous claim, wherein the amount of the micelle ranges from 2% (w / w) - 20%(w / w); preferably 3%(w / w) - 10%(w / w).

3. Hydrogel composition according to any of the previous claims, wherein the hydrogel comprises at least one polymer matrix material selected from: hyaluronic acid, agarose, alginate, chitosan, collagen, gelatin, fibrin, starch, cellulose, polyvinyl alcohol, polyethylene glycol, polyacrylamide, poly(acrylic acid), poly(N-isopropylacrylamide), poly(hydroxyethyl methacrylate), poly(methyl methacrylate), poly(ethylene oxide), nanocomposite hydrogel, hydrogel-metal composite, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), hydrogel-ceramic composite, hydrogel-lipid composite, silk fibroin, soy protein, or mixtures thereof; preferably the polymer is hyaluronic acid.

4. Hydrogel composition according to any of the previous claims, wherein the vitamin E derivative and / or a vitamin E - polymer composite is linked by one side to the outer shell through a covalent bond and by the other side to the hydrophilic active ingredient by a disulfide linkage represented by the formula:<wherein:Ri is a vitamin;R2 is oxygen;Rs is a hydrophilic active ingredient.

5. Hydrogel composition according to any of the previous claims, wherein the hydrophobic core is formed by vitamin E or vitamin D, vitamin E derivatives, vitamin D derivatives, or mixtures thereof; preferably the vitamin E derivative is selected from a group consisting of: a-tocopherol, p-tocopherol, y-tocopherol, 6-tocopherol, a-tocotrienol, p-tocotrienol, y-tocotrienol, 6-tocotrienol, a-tocopheryl acetate, a-tocopheryl nicotinate, a-tocopheryl phosphate, a-tocopheryl polyethylene glycol succinate, a-tocopheryl linoleate, or tocophersolan, or mixtures thereof; preferably a-tocopheryl succinate; and / or the vitamin D derivative is selected from a group consisting of: calcitriol, calcifediol, cholecalciferol, ergocalciferol, alfacalcidol, doxercalciferol, paricalcitol, calcipotriol, tacalcitol, falecalcitriol, eldecalcitol, or mixtures thereof.

6. Hydrogel composition according to any of the previous claims, wherein the polymer of the vitamin E - polymer composite is selected from a list consisting of: polyethylene glycol, polylactic acid, polyvinyl alcohol, polycaprolactone, or mixtures thereof; preferably the vitamin E - polymer composite is D-a- tocopheryl polyethylene glycol succinate.

7. Hydrogel composition according to the previous claims, wherein the hydrophobic core comprises a hydrophobic active ingredient; preferably the hydrophobic active ingredient:micelle weight ratio ranges from 1:20 to 1:100; preferably 1:20 to 1:50; more preferably 1:30.

8. Hydrogel composition according to any of the previous claims, wherein the vitamin E - polymer composite:hydrophilic active ingredient weight ratio ranges from 2:6 to 4:6; preferably 3:5 to 5:7; more preferably 4:6.

9. Hydrogel composition according to any of the previous claims, wherein the pharmaceutical amount of the hydrophobic active ingredient may be inferior to 0.5 pM; preferably from 0.002 pM to 0.3 pM; more preferably 0.003 pM to 0.2 pM.

10. Hydrogel composition according to any of the previous claims, wherein the pharmaceutical amount of the hydrophobic active ingredient may be inferior to 8 pM; preferably from 0.01 pM to 7.5 pM; more preferably 0.015 pM to 7 pM.

11. Hydrogel composition according to any of the previous claims, wherein the hydrophobic active ingredient or the hydrophilic active ingredient is a chemotherapeutic agent; preferably the chemotherapeutic agent is selected from a list consisting of: alkylating agents, anthracyclines, cytoskeletal disruptors, epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, monoclonal antibodies, antibody-drug conjugates, nucleotide analogs, precursor analogs, peptide antibiotics, platinum-based agents, retinoids, vinca alkaloids, cytokines, anti-metabolites, and vinca alkaloids derivatives, or other cytotoxics; preferably wherein the hydrophobic active ingredient is paclitaxel and the hydrophilic active ingredient is gemcitabine.

12. Hydrogel composition according to any of the previous claims, wherein the micelles have a spherical morphology, wherein the average particle size ranges from 100 nm - 140 nm; preferably 110 nm - 130 nm; more preferably 120 nm.

13. Hydrogel composition according to any of the previous claims, wherein the micelles have a polydispersity index that ranges from 0.3 to 0.2; preferably 0.25 to 0.1; more preferably 0.1.

14. Hydrogel composition according to any of the previous claims, wherein the encapsulation efficiency of the hydrophobic active ingredient is from 50% to 100%; preferably 60% to 95%; more preferably 70% to 90%.

15. Hydrogel composition according to any of the previous claims 1-14, for use in medicine or veterinary.

16. Hydrogel composition for use according to the previous claim, in the treatment of tumours.

17. Hydrogel composition for use according to any of the previous claim 15-16, in the treatment of a solid cancer; preferably pancreatic cancer; more preferably pancreatic cancer and its metastases.

18. Use of a hydrogel composition according to any of the previous claims for the manufacture of a medicament for the treatment of cancer.

19. A method for treating cancer in a subject in need thereof, comprising administering to the subject a hydrogel according to any of the previous claims.

20. Pharmaceutical composition comprising the hydrogel according to any of the previous claims 1-19 in a therapeutical amount; preferably consisting of: powders, tablets, capsules, syrups, creams, ointments, gels, lotions, suspensions, emulsions, patches, inhalers, nebulizers, suppositories, enemas, eye drops, ophthalmic ointments, nasal sprays, or nasal drops.

21. Method of producing the hydrogel composition described in any of the claims 1-19, comprising the steps of:providing a vitamin E - polymer composite or vitamin E derivative, a disulfide linker, an activated para-nitrophenol carbonate, and a hydrophilic active ingredient;modifying the vitamin E - polymer composite or vitamin E derivative to include the disulfide linker; adding the activated para-nitrophenol carbonate to the vitamin E - polymer composite or vitamin E derivative with the disulfide linker and forming a carbamate linkage;purifying the carbamate linkage between the vitamin E - polymer composite or vitamin E derivative and the hydrophilic active ingredient;inducing self-assembly of micelles by adding the organic phase to an aqueous phase under stirring and removing the organic solvent, thereby forming micelles comprising an outer shell containing the vitamin E derivative and the hydrophilic active ingredient;incorporating a hydrophobic active ingredient into the organic phase prior to self-assembly to form micelles comprising a hydrophobic core containing the hydrophobic vitamin and the hydrophobic active ingredient,incorporating the micelles into a hydrogel matrix by mixing the micelles with one or more polymer; thereby obtaining the hydrogel composition.

22. Method according to the previous claim, further comprising the addition of a hydrophobic active ingredient in the organic phase during the self-assembly step of the micelle.

23. Method according to any of the previous claims 21-22, wherein the vitamin E-polymer composite and the vitamin E derivative are used at a weight ratio of 4:6, and wherein the hydrophobic active ingredient is added at a weight ratio of about 1:30 relative to the total micelle-forming material.

24. Method according to any of the previous claims 21-23, wherein the micelles are incorporated into the hydrogel matrix in an amount of 2% (w / w) to 20% (w / w), based on the total weight of the hydrogel composition.

25. Method according to any of the previous claims 21-24, wherein the hydrogel matrix is a hyaluronic acid hydrogel matrix; preferably wherein the hyaluronic acid is present at a concentration of about 3 - 10% (w / w); more preferably 4-5 (w / w).

6. Method according to any of the previous claims 21-25, comprising self-assembling of the micelle at a temperature interval of 15 °C to 35 °C; preferably 20 °C to 30 °C; more preferably 22°C to 28°C in a buffer solution selected from the group consisting of: Tris(hydroxymethyl)aminomethane and hydrochloric acid buffer, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer, 3-( / V-morpholino)propanesulfonic acid buffer, citrate buffer, glycine buffer, Tris and ethylenediaminetetraacetic acid buffer, borate buffer, or 2-( / V-morpholino)ethanesulfonic acid buffer; preferably phosphate-buffered saline.

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