Biopolymer hydrogel matrices for the stabilization and in-vitro delivery of anti-cancer polyoxometalate [CoW11O39(CpTi)]7−

Abstract In the present study, the polyoxometalate (POM), [CoW11O39(CpTi)]7−, known for its significant anti-cancer activity and relatively low toxicity compared to established organic drugs such as CP and 5FU, is investigated. However, it is acknowledged that POMs inherently exhibit toxicity to living cells due to the presence of heavy and toxic metal ions within their core structures and their limited selectivity toward biological targets. Consequently, a methodology to mitigate this toxicity is sought by encapsulating these POMs within hydrogel matrices, with the intention to facilitate slow, sustained release and enhanced target specificity. In this study, the hydrogel of the well-researched anti-cancer POM, K6H[CoW11O39(CpTi)] (hereafter abbreviated as CoW11CpTi), is synthesized through the electrostatic interaction of the positively charged carboxymethyl chitosan (CMC), carboxymethyl cellulose (CMCell), and gelatin, which serve as hydrogelators. Subsequent characterization of the resultant CMC-CoW11CpTi, CMCell-CoW11CpTi, and gelatin-CoW11CpTi gels is performed utilizing Fourier Transform Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry/Thermogravimetric Analysis (DSC/TGA), Electron Spin Resonance (ESR) Spectroscopy, Scanning Electron Microscopy (SEM), and Tgel analysis. Among these, the gelatin-CoW11CpTi gel demonstrates the highest stability, retaining its structural integrity for in excess of 10 days. In addition, cytotoxicity assays are conducted on an assortment of cell lines including NRK52e (Rattus norvegicus kidney cell line), MDA-MB-231 (human breast adenocarcinoma), and MCF-7 (human breast epithelial adenocarcinoma). It is observed that the release of POM from the hydrogel matrices under physiological pH conditions exhibits a slow and sustained profile. This study therefore offers a promising approach for the effective delivery of POMs in cancer therapy. Graphical Abstract HIGHLIGHTS Anti-cancer polyoxometalate (POM) and its encapsulation in hydrogel is optimized. Stabilization and release study of the POM from gel matrix is explored. Suppression of toxicity is evaluated using in-vitro MTT assay. Gelatin gel can be used to design and produce novel drug delivery system.


Introduction
Polyoxometalates (POMs), characterized as discrete early transition metal-oxide clusters, have been in focus since the synthesis and report of the first heteropolyoxometalate, [(NH 4 ) 3 PMo 12 O 40 .nH 2 O], by Berzelius in 1826. [1,2]ossessing exceptional geometrical morphologies, POMs facilitate the incorporation of an array of elements into their frameworks, yielding a myriad of structures and emergent properties. [3]They have found potential applications in diverse fields such as chemical analysis, electrochemistry, photochemistry, catalysis, nuclear waste treatment, biomedicine, and materials science. [4]Nonetheless, the toxicity attributed to the presence of heavy metal ion cores and the lack of selectivity toward biomolecules or biological processes pose significant drawbacks for POMs.
In vitro, POMs have exhibited remarkable anti-viral, [5] anti-tumoral, [6][7][8] anti-diabetic, [9] anti-Alzheimer, [1,8 ] and anti-bacterial [8,10] properties; however, in vivo, their toxicity due to the presence of heavy metals has been a concern. [11]o date, no POM has reached the stage of clinical drug development, with several studies documenting toxic side effects. [12,13]However, certain in vivo studies indicate that POMs can be administered without acute toxic effects. [12]he margin between a therapeutic drug and a toxic substance is narrow, with minor derivatizations significantly altering the properties of the compounds, [14] underscoring the necessity for continued research on POMs in medicine.
Employing biopolymers such as carbohydrates and proteins in the preparation of POM nanocomposites or as hydrogelators has emerged as a viable strategy to mitigate the toxicity of biomedical POMs.Carbohydrate and protein hydrogelators have been utilized for the fabrication of hydrogels, thin films, scaffolds, or nanoparticles encompassing POMs, enabling their application in biological systems. [15]Concurrently, investigations into structural modifications of biomedicinal POMs and the self-assembly of POMs for the formation of more ordered structures are ongoing. [12]Recent literature has emphasized the potential of biopolymeric nano-composites of POMs, [12,[16][17][18] and collaboration between chemists and biologists is expected to propel the development of biomedical applications of POMs, opening new avenues in molecular biology, diagnosis, drug delivery, and therapy.
In the current study, a potent anti-cancer POM was selected for encapsulation, stabilization, and release studies.[20] The cyclopentadienyl titanium-containing POM has been investigated for its in vivo and in vitro anti-tumor activity and compared with well-known antitumor organotitanium compounds. [20,21]or practical application, it is imperative to deliver this potent POM molecule safely to the target, ensuring slow and sustained release with minimized toxicity.Encapsulating the POM in a hydrogel matrix presents a possible solution. [22]ydrogels have been extensively studied for drug delivery applications due to their biocompatibility, attributed to their high-water content and physiochemical similarity to native extracellular matrices.Hydrogel biodegradability or dissolution can be engineered to be influenced by enzymatic conditions (e.g., pH), temperature, or electric fields, [22,23] although degradation is not always desirable, depending on the timescale and location of the drug delivery device.Additionally, hydrogels are deformable and able to conform to the surfaces to which they are applied. [24]While our previous investigation, [22] have illuminated the potential of POMs encapsulated in specific hydrogel matrices, the present study ventures further.Here, we not only explore a different POM entity with its inherent characteristics but also juxtapose the performance of various hydrogel systems-each with distinct physicochemical properties.Additionally, unlike the preliminary studies, we extend our analysis to include biological activity assessments using cell lines.This approach provides a comprehensive understanding of the biocompatibility and therapeutic efficacy of these novel POM-hydrogel systems.
To elaborate, in current investigation, two carbohydrate biopolymers, namely carboxymethyl chitosan (CMC) and carboxymethyl cellulose (CMCell), alongside one protein biopolymer, gelatin, have been chosen for the encapsulation of the aforementioned potent anti-cancer POM.These three biopolymers have been extensively researched as drug delivery vehicles. [25]This study aims to meticulously characterize the synthesized hydrogels containing CoW 11 CpTi through various analytical techniques and evaluate their potential in anti-cancer drug delivery.The exploration of biopolymerbased hydrogels as a matrix for the encapsulation of POMs represents a crucial step in harnessing the therapeutic potential of POMs while addressing the challenges associated with their toxicity and selectivity.Such advancements may pave the way for the development of novel, effective, and safe POM-based therapeutics.

Synthesis of K 6 H[CoW 11 O 39 (CpTi)].13H 2 O
(CoW 11 CpTi) CoW 11 CpTi was synthesized according to the methodology developed earlier. [26]Initially, a solution comprising Cp 2 TiCl 2 (1.245 g, 5 mmol), acetylacetone (6 mmol), and deionized water (80 mL) was subjected to agitation at ambient temperature for approximately 4 h, forming a transparent, scarlet-colored solution.Subsequently, the pH of a sodium tungstate dihydrate solution (18.2 g, 0.055 mol in 100 mL of water) was adjusted to 6.3 using glacial acetic acid.A cobalt acetate solution (1.2 g, 5 mmol in 20 mL of water) was incrementally introduced to the Cp 2 TiCl 2 solution at a rate of one drop per second under continuous agitation.The resultant solution was combined with the acetylacetone solution, heated to boiling, and hot-filtered.Anhydrous KCl was added to the filtrate until precipitation ceased.The blue precipitates were recrystallized from hot water.The theoretical yield, calculated based on the quantity of Na 2 WO 4 .2H 2 O used, was estimated at approximately 30%.

Synthesis of carboxymethyl chitosan (CMC)
The synthesis of CMC followed the protocol reported earlier. [27]Chitosan (5.0 g) was subjected to alkalization at 50 � C for 1 h with sodium hydroxide (6.75 g) in a solvent mixture of deionized water and isopropanol (1:4, 50 mL).Monochloroacetic acid (7.5 g) dissolved in 10 mL of isopropanol was incrementally added to the reaction mixture over 30 min at 50 � C.After 4 h, the reaction mixture was quenched with ethanol (70%, 100 mL).CMC was isolated by filtration, washed with ethanol (70-94%) to remove residual salt and water, and lyophilized for additional analyses.The yield of CMC was found to be 87.00%.

Preparation of hydrogels
CMC (33.3 mg/mL), CMCell (110 mg/mL), and gelatin (66.6 mg/mL) biopolymers were individually dissolved in distilled water, and the pH of each solution was adjusted to 7.4, 8.0, and 9.1, respectively, via the addition of diluted HCl.CoW 11 CpTi was incorporated into each biopolymer solution to achieve loadings ranging from 10 to 50% (w/w).The hydrogels formed immediately for CMC and CMCell, while gelatin required 10-15 min of stirring at room temperature.For stability and biological assessments, hydrogels containing 50% CoW 11 CpTi were utilized.

Fourier transform infrared (FT-IR) spectroscopy
FT-IR spectra of the xerogels (desiccated hydrogels) were acquired employing a ThermoScientific Nicolet 6700 instrument and the KBr pellet technique.This analysis enabled evaluation of the stability of POM within the hydrogel matrix.

Thermogravimetric analysis (TGA)
TGA of CoW 11 CpTi-containing hydrogels was executed using Mettler-Toledo Stare System software to ascertain alterations in cross-linking attributable to POM incorporation.The temperature ranged from room temperature to 900 � C, with a consistent heating rate of 10 � C per minute.

Scanning electron microscopy (SEM)
SEM analysis using a JEOL instrument (Model no.JSM-5610LV) was conducted on xerogels to assess the uniform distribution of CoW 11 CpTi and examine surface morphology.

Gel transition temperature (T gel ) studies
To evaluate the gel transition temperature, 3 mL hydrogel samples with POM loadings from 10 to 50% (w/w) were prepared in 15 mL test tubes.A 358 mg glass ball was placed on the surface of the gel, which was subjected to linear temperature increases in a water bath.The temperature at which the glass ball reached the bottom of the test tube was recorded as T gel .

Electron spin resonance (ESR) spectroscopy
ESR spectra of CoW 11 CpTi and its hydrogel matrices were recorded to assess the stability of CoW 11 CpTi within the hydrogels, providing insights into the interactions and structural integrity.

POM release studies
Following ESR analysis, the release of POM from the gelatin matrix was examined.A hydrogel sample (1 g, 50% POMloaded) was prepared, to which 15 mL of phosphate-buffered saline (pH 7.4) was added.The mixture was gently stirred, and 0.5 mL aliquots were collected at 10-minute intervals for UV-Vis-NIR analysis (Shimadzu UV 3600) to monitor POM release.

Cytotoxicity assessment (MTT assay)
The cytotoxicity of CoW 11 CpTi-hydrogel was evaluated through the MTT assay using NRK52e, MDA-MB-231, and MCF-7 cell lines.This colorimetric assay is based on the ability of viable cells to reduce MTT to formazan crystals.Cells were harvested during the log phase and plated in 96well plates at a density of approximately 10,000 cells per well.Following overnight incubation at 37 � C, cells were exposed to CoW 11 CpTi-hydrogel at concentrations ranging from 0.017 to 1.15 mM for 24 h.Cell viability was determined by measuring optical absorbance at 570 nm using a Biotek ELISA plate reader.
In conclusion, this study provides a comprehensive investigation of the synthesis and characterization of hydrogels containing CoW 11 CpTi, a potent anti-cancer polyoxometalate.Through various analytical techniques, the structural integrity, stability, and release profiles of POMs within hydrogel matrices were examined.These findings contribute valuable insights toward the development of polyoxometalate-based therapeutic systems for cancer treatment.

Characterization using FT-IR
In the FT-IR spectrum of CoW 11 CpTi, displayed as spectrum A in Figure 1a-c, a single sharp absorption peak at 1620 cm-1 was observed, which corresponds to the C-C stretch of an g5-C 5 H 5 ligand bonded to Ti.Additionally, four characteristic peaks in the range of 800 to 911 cm -1 , corresponding to (W-Od), (W-Ob-W), (W-Oc-W), and (X-Oa) bonds, were identified.These frequencies suggest that CoW 11 CpTi retains the fundamental Keggin structure.The same absorption peak, along with other bands, was observed in the FT-IR spectra of xerogels (dried gels) shown as spectrum B in Figure 1a-c.Spectrum B represents the xerogel and shows additional peaks characteristic of both CoW 11 CpTi and hydrogelators, such as CMC, CMCell, and gelatin.The spectra indicate that CoW 11 CpTi remains stable under gelling conditions (pH 7.4 at room temperature) and after xerogel formation (drying at 50 � C).This stability is further supported by the minor changes in the vibrational frequencies of CoW 11 CpTi in the range of 800 to 911 cm -1 in the FT-IR spectrum of the xerogels.These changes are attributed to the electrostatic and non-covalent interactions between CoW 11 CpTi and the biopolymers.

Thermo gravimetric analysis
Using a TGA/DSC instrument, the water content, loss of water, and stability of the gel system were investigated.A water content of approximately 96% was observed in the CMC gel (Figure 2a) with water loss occurring up to 180 � C. In the CMCell gel (Figure 2b), a water content of about 94% was observed, with water loss occurring up to 220 � C; the water loss was slower, indicative of electrostatic and non-covalent interactions between the POM, gelator, and water.In contrast, the gelatin gel (Figure 2c) had a water content of around 96%, with water loss occurring up to 160 � C. For both CMC and gelatin gels, a sharp endothermic thermogram (red in color) indicates that water loss is faster compared to the CMCell gel, as evidenced by the broader thermogram in Figure 2b.The CMCell gel was found to be the strongest hydrogel matrix, followed by the CMC gel, and the gelatin gel matrix was the weakest with CoW 11 CpTi.The interactions between the anionic POM and the cationic hydrogelators are dependent on the extent of cross-linking, as other parameters such as the concentration of POM and polymer remained constant, and all experiments were performed at physiological pH 7.4.It can be inferred that carbohydrate gelators retain water and POM more efficiently compared to the protein gelator.
The Differential Scanning Calorimetric (DSC) data for CMC and gelatin gel (Figure 2a, c, red line) showed a narrow and sharp endothermic peak, indicating fast and spontaneous water evaporation from the gel matrix.In contrast, the CMCell hydrogel (Figure 2b, red line) displayed a broader endothermic peak, suggesting comparatively slow water evaporation from the matrix.
The characterization of CoW 11 CpTi-Hydrogels provides critical insights into the potential applications of these hydrogels in drug delivery systems.The FT-IR analysis indicates that the incorporation of CoW 11 CpTi into the hydrogel matrix does not compromise the structural integrity of the compound, which is essential for its biological activity.Furthermore, the thermo-gravimetric analysis demonstrates the ability of the hydrogel matrix to retain water, which is a crucial characteristic for sustained release applications.The CMCell hydrogel, in particular, exhibits promising properties in terms of stability and water retention, making it a suitable candidate for further development as a drug delivery system for CoW 11 CpTi.

Analysis of gelatin-CoW 11 CpTi xerogel using SEM
The encapsulation and stability of the POM in the gelatin gel were investigated using FT-IR and TGA/DSC analysis.Additionally, the degree of cross-linking with the polymer was evaluated through T gel analysis.To assess the uniform distribution of the anti-cancer POM within the gelatin gel matrix, the xerogel was examined using Scanning Electron Microscopy (SEM).The SEM image of the gelatin-POM hydrogel (xerogels) confirmed the homogeneous distribution of the encapsulated POM throughout the hydrogel matrix (Figure 3).Moreover, no noticeable differences were observed in the morphological pattern of the gel, which further verifies the compatibility between the POM and the gelatin matrix.Achieving a homogeneous distribution of the POM across the gel matrix is crucial for uniform drug delivery applications.

T gel study
T gel analysis provides insights into the extent of physicochemical interactions between the hydrogelators and crosslinkers used in the system.The T gel values of the three gels displayed different trends as the % loading of POM increased from 10 to 50% (Figure 4a-c).In the case of CMC hydrogel (Figure 4a), an abrupt increase in T gel from 30 to 55 � C was observed as the % loading of POM increased from 10 to 20%.Beyond 20%, T gel increased gradually from 55 to 75 � C. For the CMCell hydrogel (Figure 4b), T gel exhibited a sudden rise from 35 to 65 � C when the % loading of POM increased from 10 to 30%.Subsequently, it remained constant at 65 � C between 30 and 40% POM loading, and then experienced another sudden increase above 95 � C after reaching 40% POM loading.These observations indicate that the gels did not exhibit a steady and constant response to temperature variations at different % POM loadings.
In contrast, the gelatin hydrogel (Figure 4c) displayed the expected behavior.As the % loading of CoW 11 CpTi increased from 10 to 50%, T gel gradually increased from 35 to 45 � C, which closely aligns with the physiological temperature of 37 � C.This behavior is desirable for drug delivery applications.Given the constant response of the gelatin gel toward temperature and the gel formation occurring under physiological pH (7.4) conditions, this gel was selected for further investigation of its anti-cancer properties and in-vitro drug release studies.

Electron spin resonance (ESR) analysis
The paramagnetic cobalt (II) ion present in the structure of the POM, CoW 11 CpTi, makes it sensitive to Electron Spin Resonance (ESR).To evaluate its stability in the gelatin hydrogel under physiological pH conditions (pH 7.4), ESR analysis was performed.The ESR spectrum of CoW 11 CpTi has been previously reported. [26]In this study, the ESR tetrahedron. [26]The second signal was a weak and unassigned hyperfine signal with g ¼ 2.09. [26]Additionally, the ESR spectrum of the gelatin gel containing CoW 11 CpTi (Figure 5) was recorded, revealing an asymmetric signal with g ¼ 4.2 and another hyperfine signal at g ¼ 2.0, which matched the signals of the pristine CoW 11 CpTi reported earlier. [26]The slight shift in the ESR signals observed in the gelatin gel could be attributed to the non-covalent interactions between the POM and the hydrogelator, gelatin.ESR analysis provides valuable insights into the stability of CoW 11 CpTi in the hydrogel matrix and further corroborates the results obtained from FT-IR analysis, which also supports the stability of the POM in the hydrogel matrix.Hence, this study confirms that the anti-cancer POM, CoW 11 CpTi, remains stable in the gelatin gel for up to 10 days.

Sustained release of POM
Visual examination of the samples (Figure S1, A, B, C, supporting information) clearly indicated that the POM remained stable in the gelatin gel compared to the CMC and CMCell gels.The FT-IR and ESR analyses further supported the stability study and confirmed that the POM exhibited enhanced stability (more than 10 days) in the gelatin gel.Consequently, release studies were conducted exclusively with the POM-gelatin matrix at 37 � C and pH 7.4.
Figure 6 illustrates the release studies of the POM from the gelatin hydrogel matrix at regular time intervals.The release of CoW 11 CpTi was investigated using UV-Vis analysis by monitoring the absorption maxima (kmax) at 590 nm.The release of the POM took approximately 55-60 min (�1 h) from the gelatin hydrogel matrix, with the maximum release achieved after 1 h.

In-vitro cytotoxicity study
For the in-vitro cytotoxicity studies, as described in Section 2.6, the POM, CoW 11 CpTi, was encapsulated in CMC,  CMCell, and gelatin hydrogels at various concentrations and then incubated at 37 � C for further investigation.Carcinogenic cell lines, including NRK52e, MDA-MB-231, and MCF-7, were utilized as model cell lines to assess the anti-cancer activities of the POM encapsulated gel matrix.The IC50 value of the POM and various POM-gel formulations was calculated using the standard MTT assay.The IC50 value of the bare POM ranged from 0.368 to 0.410 mM (Table 1) for these three cell lines.Upon encapsulation in the three gels, a loss of activity (increase in IC50 value) was observed in the case of CMC (Figure 7c) and CMCell gels (Figure 7b).The percent cell viability of the bare CoW 11 CpTi at the highest concentration, 1.15 mM, was observed to be 27% (Figure 7d), while in the case of encapsulated CMC-CoW 11 CpTi (Figure 7c) and CMCell-CoW 11 CpTi (Figure 7b), it increased to 60 and 40%, respectively.These results indicate that the CMC and CMCell gel matrices are not suitable for anti-cancer activities and even diminish the anti-cancer activity of the bare POM, CoW 11 CpTi.Conversely, in the case of the gelatin gel (Figure 7a), the anti-cancer activity remained constant, suggesting no loss in the anti-cancer activity of the gelatin-POM formulation.The minimum effective concentration of the gelatin-POM gel (Table 1, row 1), compared to the bare CoW 11 CpTi (Table 1, row 4), indicates that the biopolymer gelatin enhances the anti-cancer activity of the POM.

Discussion
Polyoxometalates (POMs) are discrete metal-oxide clusters that exhibit diverse structures and properties. [28]They have shown potential applications in biomedicine, such as anticancer, anti-viral, anti-diabetic, and anti-bacterial activities. [29]owever, POMs also have drawbacks such as toxicity, instability, and lack of selectivity. [16]Therefore, there is a need to develop strategies to overcome these challenges and enhance the therapeutic potential of POMs.
One of the strategies is to encapsulate POMs within hydrogel matrices, which can provide protection, stabilization, and controlled release of POMs.Hydrogels are threedimensional cross-linked polymer networks that can absorb and retain large amounts of water or biological fluids. [16]hey have been widely used as drug delivery systems due to their biocompatibility, biodegradability, and ability to respond to various stimuli such as pH, temperature, or electric fields.However, most hydrogels have low mechanical strength and stability, which limit their applications in vivo.Several studies have reported the synthesis and characterization of POM-hydrogel composites using various biopolymers such as chitosan, [30] amino acids, [31] or nanoparticles. [32]hese composites have shown improved stability, reduced toxicity, and enhanced interactions with biological targets compared to pure POMs. [16]However, the optimal conditions for POM encapsulation and release are still under investigation.POM-hydrogels have potential applications in cancer therapy, as they can deliver POMs to tumor sites with controlled release and reduced toxicity. [33]POMs have shown anti-cancer activity by inducing apoptosis, inhibiting angiogenesis, interfering with DNA replication, and modulating signal transduction pathways in cancer cells. [34]However, POMs also have drawbacks such as poor solubility, low stability, and high toxicity in biological systems. [17]Therefore, encapsulating POMs within hydrogels can overcome these challenges and enhance their therapeutic efficacy.For example, Guo et al. reported that gelatin-POM hybrid nanoparticles exhibited higher anti-cancer activity and lower toxicity than free POMs against human breast cancer cells. [14]imilarly, in current study we demonstrated that gelatin-POM hydrogels showed sustained release and improved anti-cancer activity of POMs against various cancer cell lines.These studies suggest that POM-hydrogels can be promising candidates for novel anti-cancer drug delivery systems.Furthermore, the novelty of this work is underscored by the optimized encapsulation efficiency, detailed comparative analysis of three hydrogel systems, and advanced investigation into the stability and release kinetics of POMs within these hydrogels.Notably, the in-vitro toxicity analysis using the MTT assay extends the understanding of biocompatibility in these systems.Concerning the challenge of POM toxicity post-release, the sustained and controlled release properties of these hydrogel matrices are emphasized.They potentially reduce the exposure of healthy tissues to POMs, thereby addressing the critical issue of minimizing toxicity in non-targeted areas.This aspect of controlled release, coupled with the potential for targeted delivery, forms a significant advancement over existing literature and contributes novel insights into the field of drug delivery and cancer therapy.
][9]11,35] However, we also acknowledge the challenges associated with POMs, such as their toxicity due to the presence of heavy metal ions, their instability under physiological conditions, and their lack of selectivity toward biological targets.So, we propose a strategy to overcome these challenges by encapsulating POMs within hydrogel matrices, which are biocompatible, biodegradable, and capable of controlled drug release.We selected a specific POM, CoW 11 CpTi, which has been reported to have significant anti-cancer activity and relatively low toxicity compared to established organic drugs such as CP and 5FU. [28]We synthesize three hydrogels containing CoW 11 CpTi using different biopolymers as hydrogelators: carboxymethyl chitosan (CMC), carboxymethyl cellulose (CMCell), and gelatin.They choose these biopolymers because they have been extensively researched as drug delivery vehicles. [16]We characterized the hydrogels using various analytical techniques, such as Fourier Transform Infrared Spectroscopy (FT-IR), Thermogravimetric Analysis (TGA), Scanning Electron Microscopy (SEM), Gel Transition Temperature (T gel ) analysis, and Electron Spin Resonance (ESR) Spectroscopy.We then evaluated the stability, morphology, cross-linking, distribution, and release of CoW 11 CpTi within the hydrogel matrices.We then conducted cytotoxicity assays using three cell lines: NRK52e (Rattus norvegicus kidney cell line), MDA-MB-231 (human breast adenocarcinoma), and MCF-7 (human breast epithelial adenocarcinoma).The anti-cancer activity and toxicity of CoW 11 CpTi alone and encapsulated in the hydrogels were lastly compared.
Release studies of CoW 11 CpTi from the gelatin hydrogel matrix at physiological pH (7.4) and temperature (37 � C) were conducted by the authors using UV-Vis analysis.It was observed that the release of CoW 11 CpTi was slow and sustained, with the maximum release being reached after 1 h.A comparison was made between the results obtained by the authors and those reported in other studies involving the release of POMs from different hydrogel matrices, including carboxymethyl chitosan, mesoporous silica nanoparticles, and poly (ethylene glycol).It was noted that the release of POMs is dependent on various factors such as pH, temperature, redox potential, enzymatic activity of the environment, and the interactions between the POMs and hydrogelators.Furthermore, in-vitro cytotoxicity studies were performed using three cell lines: NRK52e, MDA-MB-231, and MCF-7.The cell viability and the IC50 values of CoW 11 CpTi, both alone and encapsulated in the three hydrogels, were measured.It was found that anti-cancer activity was exhibited by CoW 11 CpTi against all three cell lines, though its toxicity was reduced when encapsulated in the gelatin hydrogel.In addition, a comparison was drawn between the findings of the authors and those of other studies which reported on the anti-cancer activity and toxicity of POMs and their hybrid materials, such as curcumin-POM, copper-POM, and titanium-POM.It was suggested that the encapsulation of POMs in suitable biopolymers has the potential to enhance their selectivity and efficacy toward cancer cells, while simultaneously minimizing their adverse effects on normal cells.
We performed various characterization techniques on the CoW 11 CpTi-containing hydrogels to evaluate their properties and performance as potential drug delivery systems.These techniques include: (i) Thermogravimetric Analysis (TGA) technique measures the mass change of a sample as a function of temperature or time under a controlled atmosphere.TGA can provide information about the thermal stability, decomposition temperature, water content, and composition of a sample. [36]We used TGA to investigate the water content, water loss, and stability of the hydrogel matrices containing CoW 11 CpTi.We found that the CMCell hydrogel exhibited the highest water retention and stability, followed by the CMC hydrogel, while the gelatin hydrogel showed the lowest water retention and stability.TGA also revealed the extent of cross-linking between the POM and the biopolymers, which influenced the water evaporation rate and the thermal behavior of the hydrogels.(ii) Scanning Electron Microscopy (SEM) technique uses a focused beam of electrons to generate high-resolution images of the surface morphology and structure of a sample.SEM can provide information about the size, shape, distribution, and homogeneity of a sample. [37]We used SEM to examine the surface morphology and distribution of CoW 11 CpTi within the gelatin hydrogel matrix and found that CoW 11 CpTi was homogeneously distributed throughout the hydrogel matrix and that no noticeable differences were observed in the morphological pattern of the gel.SEM also confirmed the compatibility between CoW 11 CpTi and gelatin, which was essential for maintaining the structural integrity and activity of CoW 11 CpTi.(iii) The gel transition temperature (T gel ) technique measures the temperature at which a gel changes from a solid-like state to a liquid-like state or vice versa.[40] T gel is a parameter that reflects the extent of cross-linking between the POM and the polymer in the hydrogel matrix.It is defined as the temperature at which the hydrogel changes from a gel state to a sol state. [38]T gel is influenced by several factors, such as the concentration and molecular weight of the polymer, the pH and ionic strength of the solution, and the presence of additives or cross-linkers. [39]T gel can be measured by observing the movement of a glass ball on the surface of the hydrogel under different temperatures. [40]We used T gel studies to evaluate the degree of cross-linking between CoW 11 CpTi and the biopolymers.We found that CMC and CMCell hydrogels showed abrupt changes in T gel as the POM loading increased, indicating that they did not exhibit a steady and constant response to temperature variations.In contrast, gelatin hydrogel showed a gradual increase in T gel as the POM loading increased, indicating that it exhibited a constant response to temperature variations.The authors also found that gelatin hydrogel had a T gel value close to the physiological temperature of 37 � C, which was desirable for drug delivery applications.(iv) POM Release Studies measures the amount and rate of drug release from a delivery system under simulated physiological conditions.POM release studies can provide information about the drug release profile, mechanism, kinetics, and efficiency of a delivery system.The authors used POM release studies to investigate the release behavior of CoW 11 CpTi from the gelatin hydrogel matrix under physiological pH (7.4) and temperature (37 � C) conditions.They found that CoW 11 CpTi was released completely from the gelatin hydrogel matrix within 60 min, exhibiting a slow and sustained release profile.The authors also found that gelatin hydrogel enhanced the anti-cancer activity of CoW 11 CpTi and reduced its toxicity to normal cells compared to bare CoW 11 CpTi.Therefore, by performing these characterization techniques, we justify our choice of biopolymers for encapsulating CoW 11 CpTi and demonstrated their potential as drug delivery systems for cancer treatment.(v) One of the objectives of this study was to evaluate the cytotoxicity of CoW 11 CpTicontaining hydrogels on different breast cancer cell lines using the MTT assay.The MTT assay is a colorimetric method that measures the metabolic activity of viable cells by their ability to reduce a yellow tetrazolium salt (MTT) to a purple formazan product. [41]This assay is widely used to assess the anti-cancer activity of various compounds and formulations on different cancer cell models. [1]In this study, three breast cancer cell lines were chosen: NRK52e, MDA-MB-231, and MCF-7.NRK52e is a normal rat kidney cell line that serves as a non-tumorigenic control. [41]MDA-MB-231 and MCF-7 are human breast cancer cell lines that represent different subtypes of breast cancer: MDA-MB-231 is a triple-negative breast cancer (TNBC) cell line that lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression, while MCF-7 is an ER-positive and HER2-negative breast cancer cell line. [41]These cell lines were selected to investigate the differential effects of CoW 11 CpTi-containing hydrogels on various molecular subtypes of breast cancer, which have different biological characteristics and clinical outcomes. [1]e conclude that gelatin is an ideal biopolymer for encapsulating, stabilizing, and delivering CoW 11 CpTi as a novel anti-cancer therapeutic agent.Gelatin is a natural protein derived from collagen, which is abundant in animal tissues.It has been widely used as a biocompatible and biodegradable hydrogelator for various biomedical applications. [32]Gelatin has several advantages as a hydrogelator, such as low cost, easy availability, high water content, good swelling behavior, and ability to form physical cross-links through hydrogen bonding and electrostatic interactions.Moreover, gelatin can be modified by chemical or enzymatic methods to introduce functional groups or improve its mechanical properties. [42]e highlight the benefits of using gelatin as a hydrogelator, including its biocompatibility, biodegradability, high water content, excellent swelling behavior, and capability to form physical cross-links with the POM.We also suggest that additional studies focusing on structural modifications of POMs and biopolymers are necessary to enhance their selectivity and efficacy in cancer therapy.We point out potential strategies such as introducing functional groups, altering the metal composition, or designing stimuli-responsive systems.In our research, we provide a comprehensive investigation into the synthesis and characterization of POM-hydrogels using a range of analytical techniques and biological assays.We demonstrate the structural integrity, stability, morphology, crosslinking, distribution, release, cytotoxicity, and anti-cancer activity of CoW 11 CpTi and its hydrogel formulations.Finally, we contribute valuable insights toward the development of polyoxometalate-based therapeutic systems for cancer treatment.We show that POM-hydrogels are capable of overcoming the challenges associated with POMs, such as toxicity, instability, and lack of selectivity, thus enhancing their therapeutic potential.

Conclusion
In conclusion, it has been shown by us that the biopolymers such as CMC, CMCell and gelatin can be used as potential, safe and easy delivery vehicles for biomedicinal POMs.The toxicity of anti-cancer POM has been reduced after encapsulation in gelatin gel and the activity has been improved as compared to bare POM.Stability is also a key role in these conditions as POMs may not be stable in the presence of hydrogelators.However, CoW 11 CpTi is found to be stable up to 10 (Ten) or more days in gelatin hydrogels by the FT-IR and ESR analysis.The release parameters have been monitored by us and it has been found that CoW 11 CpTi can be released completely from gelatin gel up to 60 min.The other derivatives of gelatin or combination of gelatin with other biopolymers and their hydrogels are being further investigated by us to encapsulate, stabilize and release the POM, CoW 11 CpTi, so that this POM can be released for long duration in various practical applications.The anti-cancer POM, CoW 11 CpTi is reported to be toxic for normal cells by Wang et al. [20] and in this piece of work we have proved that, just simple encapsulation of this molecule in supramolecular hydrogels of gelatin by means of electrostatic interactions between the two reduces the toxicity and stabilizes the POM for more than 10 (Ten) days.CMC-CoW 11 CpTi was found to be less toxic as compared to CMCell-CoW 11 CpTi by the MTT assay, but CoW 11 CpTi is unstable in these gels.So, we conclude that, POMs and their hydrogels can be fabricated by easy methods and could be characterized by sophisticated techniques but stability in gelling conditions remains the major challenge.These Hydrogels will be explored in depth & utilized for futuristic applications as "INORGANIC MEDICINES" in coming years.

Table 1 .
Calculated IC 50 value of gels on three different cell lines.