Beyond Chemistry: Investigating the Physical, Pharmacological, and Computational Aspects of Polyoxometalate Integrated Solid Lipid Nanoparticles for Cancer Treatment

Introduction

Polyoxometalates (POMs) are a fascinating group of inorganic clusters consisting of early transition metals, such as vanadium (Na₆[H₂V₁₀O₂₈]•18H₂O), molybdenum [NH₃Pri]6[Mo₇O₂₄]•3H₂O (PM-8), and tungsten (NH₄)₁₄[NaP₅W₃₀O₁₁₀]•31H₂O) (P₅W₃₀), that are linked together by oxygen atoms.¹ Polyoxometalates (POMs) like P5W30 are metal-oxide clusters with many uses in fields as diverse as medicine, materials research, and catalysis. Because of its exceptional structural and electrical characteristics, which provide substantial benefits for medicinal and drug delivery applications. These intricate structures, which have a resemblance to small molecular cages, have unique chemical, physical, and pharmacological properties.² Polyoxometalates (POMs) have become very prospective competitors in the realm of medicinal chemistry because of their favorable pharmacological characteristics, especially in relation to their potential use in anticancer treatments.^3,4

Polyoxometalates (POMs) are known to play a crucial role in fighting cancer by triggering apoptosis, which is the controlled death of cancer cells.⁵ Polyoxometalates (POMs) have the ability to engage with cellular constituents and interfere with crucial processes that are vital for the survival of cancer cells, resulting in their targeted eradication.^6,7 Moreover, the redox characteristics of POMs allow them to regulate the levels of oxidative stress in cancer cells, so adding to their deadly effects.⁸ Furthermore, Polyoxometalates (POMs) have shown promise as agents that limit angiogenesis, which is the process of forming new blood vessels that are essential for the development of tumors.⁹ Their ability to inhibit the growth of blood vessels, known as anti-angiogenic property, highlights their effectiveness in slowing down the advancement of cancer.¹⁰

Although polyoxometalates (POMs) show potential as anticancer agents, their transformation into effective pharmaceuticals is hindered by various constraints.¹¹ A significant obstacle is their restricted solubility in water-based settings, which may hinder their capacity to be absorbed by the body and their effectiveness in treating medical conditions.¹²

The integration of water-insoluble metal complexes into solid lipid nanoparticles (SLNs) has considerable potential for therapeutic applications, particularly in the field of drug delivery.¹³ Several metal complexes with significant therapeutic qualities, such as anticancer or antibacterial medicines, often encounter the drawback of low solubility, which restricts their practical use in clinical settings.¹⁴ SLNs provide a smart solution by using a biocompatible lipid-based structure to enclose hydrophobic metal complexes, hence improving their solubility and stability.¹⁵ This formulation enhances medication bioavailability and enables precise and prolonged release, hence increasing therapeutic effectiveness.¹⁶ Moreover, SLNs can enhance precise distribution to certain tissues or cells, hence reducing the occurrence of systemic adverse effects.¹³ The use of SLNs containing water-insoluble metal complexes has significant implications in the development of safer and more efficient therapies for a range of ailments.¹⁷

Beeswax, a lipid obtained from honeybees, is important in solid lipid nanoparticles (SLNs) because it is biocompatible and has minimal toxicity.¹⁸ The incorporation of the substance improves the stability and controlled release characteristics of solid lipid nanoparticles (SLNs), leading to extended circulation of the medication and enhanced therapeutic results. The versatile nature of beeswax, acting as a lipid matrix, facilitates the encapsulation of hydrophobic medicines, hence playing a crucial role in the creation of effective and secure drug delivery systems.¹⁹ Furthermore, its sustainable and renewable characteristics are in line with the increasing focus on green formulations in the field of pharmaceuticals.²⁰

The process entails constructing three-dimensional structures of the molecules, optimizing energy, and producing numerous binding positions. This approach is crucial in the field of drug development as it allows for the identification of possible drug candidates, comprehension of binding processes, and assistance in the design of drugs based on their structure. Molecular docking is a valuable tool in the field of cancer treatment using polyoxometalate (POM) integrated solid lipid nanoparticles. It aids in improving drug delivery, ensuring precise targeting, and gaining a better understanding of the mechanisms behind the anticancer effects of POMs. This, in turn, contributes to the advancement of nanomedicine therapies that are more effective.

This work included the preparation and characterization of solid lipid nanoparticles (SLNs) containing P₅W₃₀. The P₅W₃₀ was chosen as the typical polyoxymethylene (POM) in our investigation because of its outstanding stability, solubility, and capacity to interact with biological systems in a regulated way. Due to its structural features, it is highly suitable for being included in solid lipid nanoparticles (SLNs) to achieve targeted distribution and enhance therapeutic effectiveness. The physical and in vitro pharmacological activities on HeLa cells of these P₅W₃₀ incorporated SLNs were assessed. Eventually, the obtained results were validated by experimentation on animals. Furthermore, in silico simulations which aids in improving drug delivery, ensuring precise targeting, and gaining a better understanding of the mechanisms behind the anticancer effects of POMs. This, in turn, contributes to the advancement of nanomedicine therapies that are more effective.

Materials and Methods

Materials

Beeswax (BW), Soy lecithin (SL), Tween 80, Polyoxotungstate ((NH₄)₁₄[NaP₅W₃₀O₁₁₀]•31H₂O)) (P₅W₃₀), Sulforhodamine B (SRB) dye, MTT dye (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), Tris base (pH 10.5), 4.6–Diamidino–2–phenylindole dihydrochloride (DAPI), Phosphate-buffered saline (PBS) tablets, Ethylenediaminetetraacetic acid (EDTA), Low melting point agarose (LMPA), Sodium hydroxide (NaOH), Sodium chloride (NaCl), Annexin–V–FITC, Trypsin, staining kit, Chloroform, Lactose, Fetal bovine serum (FBS), Ethanol, Trichloroacetic acid (TCA), Dulbecco’s modified Eagle’s medium (DMEM), Dimethyl sulfoxide (DMSO), Dialysis membrane (MWCO = 10K Da), Triton X–100 and phosphate-buffer tablets (pH 7.4) were acquired from Sigma Aldrich (St. Louis, MO, USA). These compounds were utilized without further purification. Furthermore, the Vero cell line (kidney tissue derived from a normal, adult African green monkey, CCL-81) and HeLa cells were acquired from ATCC, Japan).

Methods

Preparation of SLNs

The preparation of solid lipid nanoparticles (SLNs) was conducted using the microemulsion approach as described in a recent study.²¹ The designated quantities (see Table 1) of a blend consisting of BW, SL, and POM (P₅W₃₀) were liquefied using a water bath heated to a temperature of 90°C. Tween 80 was combined with deionized water at a temperature of 90°C and a rotational speed of 2000 rpm for a duration of 2 min, and then introduced into the molten lipid mixture kept at 90°C. The resultant optically clear system was disseminated at a speed of 24,000 rpm within a time frame of 5 min utilizing a rotor-stator (Ultra-Turrax, IKA T18 basic; Staufen, Germany). Ultimately, the microemulsion was slowly distributed in water chilled to a temperature of 2–4°C, with a volume ratio of 1:10, while stirring at a speed of 3000 rpm.

Table 1 Experimental Conditions for Preparation of Solid Lipid Nanoparticles (SLNs)

Physical Characterization

FTIR Spectroscopy

The BW, P₅W₃₀, and P₅W₃₀-BW-SLN spectra were acquired through the utilization of the KBr disc method.²² To create the KBr disc, a sample was combined with 2% KBr powder. The Nicolet Avatar 370 FTIR spectrophotometer was utilized for collecting an FTIR spectrum encompassing the frequency range of 4000 to 400 cm⁻¹.

DSC Studies

To assess the physical integrity of the P₅W₃₀ in SLNs, thermal analysis was conducted on BW, P₅W₃₀, and P₅W₃₀-BW-SLNs.²³ The heating rate was adjusted to 10°C/min while maintaining a constant flow of nitrogen (2 mL/min) to prevent oxidation. The DSC scan for each sample was recorded using a DSC 214 Polyma-NETZSCH (GmbH Germany) instrument, by heating it up to 350°C. An emptied aluminum pan served as a reference.

Percent Entrapment and Loading Efficiency

The drug loading efficiency (LE) along with drug encapsulation effectiveness (EE) of P₅W₃₀ in BW-SLNs were determined by centrifuging the colloidal samples, containing a 2g BW-SLNs (equal to 30.76 mg P₅W₃₀), using a Hettich EBA 200S centrifuge at a speed of 18,000 rpm (39,846×g) for 15 min at a temperature of 25°C. The unentrapped P₅W₃₀ in the supernatant obtained by centrifuging the SLNs was quantified using UV-visible spectroscopy at a specific wavelength of 241 nm (Shimadzu UV 1900i, Tokyo, Japan).²⁴ Prior to evaluating the drug concentration, we conducted UV-visible spectroscopy on the dispersion of Solid Lipid Nanoparticles (SLNs) to quantify the absorbance associated with the SLNs. The absorbance result was subtracted from the total absorbance obtained in the supernatant comprising both the drug and solid lipid nanoparticles (SLNs).

To calculate the percentage of P₅W₃₀ that is enclosed inside nanoparticles, the difference between the total amount of P₅W₃₀ used and the amount of unbound P₅W₃₀ found in the aqueous phase of the supernatant was divided by the total amount of P₅W₃₀ used. The LE (%) was computed by dividing the difference by the total weight of SLNs using the given calculations.

Hydrodynamic Dimension and Zeta Potential Measurement

To ascertain the hydrodynamic size of P₅W₃₀-BW-SLNs, the nanoparticles underwent several dispersion techniques using purified water. The particle sizes and zeta potential were analyzed using the Malvern Zetasizer Nano ZS (Cambridge, UK).²⁵

Drug Release and Kinetics

The objective of this study is to determine kinetic models utilizing a pre-existing approach to analyze the release pattern of P₅W₃₀ from BW-SLNs.²² To summarize, a 2g of P₅W₃₀-BW-SLNs were combined with 5 mL of phosphate-buffered saline at a pH of 7.4. Afterward, the mixture was transferred into a dialysis bag with a molecular weight cut off (MWCO) of 10K Da. The dispersion containing the membrane pocket was then submerged in a solution of 500 mL of phosphate-buffered saline with a pH of 7.4. The system rotated at a frequency of 75 rpm at a temperature of 37°C. The release of P₅W₃₀ was estimated on UV-visible spectrophotometer (Shimadzu UV 1900i, Tokyo, Japan) operated at a wavelength of 241 nm. Mathematical representations using release kinetics were employed to P₅W₃₀ to verify the mechanism by which P₅W₃₀ was discharged from BW-SLNs. The implementation of these models was carried out utilizing the DDsolver program.

Transmission Electron Microscopic Assessment

The morphology of P₅W₃₀-BW-SLNs (10 µL) was analyzed through the technique of transmission electron microscopy (TEM; JEM-2100; JEOL; Tokyo, Japan; 200 kV). Before imaging, the samples were treated using 2% uranyl acetate solution (w/v) to create a negative stain. The samples were then dried for 20 min. Afterward, the desiccated samples were positioned on a copper grid coated with carbon with an aperture size of 300.²⁶

Scanning Electron Microscopy (SEM)

The SEM investigation was conducted using the Hitachi S–4700 (Hitachi, Ltd. Tokyo Japan). SEM apparatus, with an acceleration voltage ranging from 10 to 20 kV. The P₅W₃₀-BW-SLNs were dispersed using ethanol and immediately applied onto pristine silicon wafers. The samples underwent sputter-coating with gold to facilitate conduction.²⁷

Pharmacological Assessment

Cytotoxicity and Cell Viability Assays

An SRB investigation was used to compare and assess the inhibitory effects on cell growth of pure P₅W30 and P₅W₃₀-BW-SLNs on the HeLa cell line.²⁸ A total of 10,000 cells were distributed evenly in 96-well plates and provided 24 hr to multiply. The distinct wells were subjected to different quantities of both P₅W₃₀ and P₅W₃₀-BW-SLNs. After the cells were incubated, they were washed with PBS, allowed to dry naturally, and then fixed with 40% chilled trichloroacetic acid (TCA). The cells were treated with a 0.4% w/v solution of SRB dye for a duration of 30 min. The cells were then exposed to a 100 µL solution incorporating tris-base (10 mm, pH 10.5). The ELx808TM microplate reader (BioTek instruments Vermont, USA) was used to get readings at a wavelength of 565 nm. The IC₅₀ value (µg/mL) was determined using Prism 5.0 software (GraphPad Software; California, USA).

To assess the effect of P₅W₃₀ and P₅W₃₀-BW-SLNs on the survival of Vero cell line (a healthy adult African green monkey’s kidneys, CCL-81, ATCC, Japan), a previously established MTT test was used with slight modifications.²⁹ All wells were seeded with 10,000 Vero cells and allowed to incubate for 24 hr for growth and proliferation. The cells were then subjected to varying doses of P₅W₃₀ and P₅W₃₀-BW-SLNs. The duration of the treatment was extended for 48 hr. Following the treatment time, the cells were subjected to MTT and incubated in a light-restricted environment at a constant temperature of 37°C for a duration of 3–4 hr. As incubation time ended, the dark purple crystals of formazan emerged and were dispersed in isopropanol. The ELx808TM (BioTek instruments Vermont, USA), was utilized to get measurements at a wavelength of 565 nm.

DAPI Staining

A sterile chamber slide with two wells was used for HeLa cells, with a density of 1×10⁴ cells per well. The cultivated cells were treated with pure P₅W₃₀ (IC₉₀ = 30.87 µg/mL) and P₅W₃₀-BW-SLNs (IC₉₀ = 14.72 µg/mL) for a duration of 24 hr. The cells were subjected to a 0.1% solution of Triton X-100. Subsequently, the cells were subjected to staining with DAPI (10 µg/mL) and maintained in a light-restricted environment for a duration of 10 minutes. The remaining DAPI that was not absorbed was removed by rinsing with PBS many times. The fluorescent microscope (Nikon Eclipse Japan) used for observation of HeLa cells. The wavelengths that were used for the emission and excitation phases were, respectively, 461 nm and 358 nm.³⁰

Genotoxicity Assessment (Comet Assay)

The comet test, a well utilized technique for evaluating genotoxicity, was employed to quantify the level of double-strand DNA (dsDNA) damage.^30,31 HeLa cells (1×10⁴ cells/well) were treated with pure P₅W₃₀ (IC₉₀ = 30.87 µg/mL) and P₅W₃₀-BW-SLNs (IC₉₀ = 14.72 µg/mL) for a duration of 24 hr. Before being put onto comet slides, the cultivated cells were treated with 1% LMPA. To dissolve the cells, the slides were submerged in a pH-10 lysis solution. The lysis solution included 0.1% v/v Triton-X 100, 2% v/v DMSO, 10 mm Trizma X, and 100 mm EDTA. In the time-course investigation, samples and a buffer solution containing 1 mm EDTA and 300 mm NaOH at a pH of 13 were placed in horizontally oriented electrophoretic-agarose chambers. An alkaline buffer was used to unwind the DNA. We allowed the slides to dry completely after rinsing them with methanol. The comet DNA was examined using CaspLab 1.2.3b2 software.

The Analysis of Flow Cytometry

The HeLa cell line (1×10⁴) underwent to a 24-hour exposure with pure P₅W₃₀ (IC₉₀ = 30.87 µg/mL) and P₅W₃₀-BW-SLNs (IC₉₀ = 14.72 µg/mL). The cells underwent trypsinization for 5 min at a temperature of 37 °C, using a mixture of trypsin and EDTA. To reduce the incidence of cell groupings the cell medium for cultivation was gradually introduced. The cells were collected in a binding solution (100 µL) for 15 minutes after exposure to a 500 mm concentration of H₂O₂. A fluorescent marker called annexin-V FITC with propidium iodide (PI) was used throughout this incubation. For fluorescence-triggered sorting of cells (FACS), a suitable FL2-A channel was used. The emission frequency of propidium iodide (PI) was 600 nm and that of annexin-V FITC was 545 nm. The CytoFLEX equipment (Beckman-Coulter Life Sciences, CA, USA) was used to evaluate 10,000 harvested cells in a single cycle. The data were presented using a cell cycle grading plot.³²

Animal Studies

The study used adult female albino BALB/C mice with an average weight of 30 g. The mice were housed in the animal facility located at Bahauddin Zakariya University (60800), Multan-Pakistan. The mice were raised in a controlled setting with a constant temperature of 25°C ± 3°C. They were subjected to a light-dark cycle of 12 hr and were given unrestricted access to both food and water. A steel-mesh cage provided enough space for five albino female mice, alleviating their anxieties about crowding. Following the recommendations of the institution’s ethical committee (256/PEC/2023), the animals were housed and cared for humanely. Each of the four groups consisted of five mice selected at random. Female mice were injected with HeLa cells (1×10⁴) intraperitoneally (i.p). Ten days were allotted for the tumor to achieve a volume of 50–100 mm³. The volume of a tumor in mice may be estimated without causing damage or intrusion by using calipers to measure the exterior dimensions of the tumor. This technique entails the measurement of the tumor’s length (L) and width (W), followed using a formula to approximate its volume. The formula that is most often used is:^22,33,34

The various mice groups and the therapies they received are listed in Table 2.

Table 2 Control and Experimental Animals Given Pure P5W30, P5W30-BW-SLNs, and Cisplatin

Each formulation’s efficacy against cancer was evaluated using the tumor inhibition rate (TIR).

In silico Method

Structures Preparation and Docking Studies

Sybyl-X1.3/SKETCH³⁵ was used to create a three-dimensional conformation of POM ((NH₄)₁₄[NaP₅W₃₀O₁₁₀]•31H₂O). To get biologically active conformation, the built POM was subjected to energy optimization using the Tripos force field with Gasteiger Hückel atomic charges.³⁶ The co-crystal structures of Cyclin-Dependent Kinase 2 (CDK2), Protein Kinase B alpha (PKB-α), and Epidermal Growth Factor Receptor (EGFR) were retrieved from the RCSB Protein Data Bank using the corresponding PDB entries: 2FVD,³⁷ 4GV1,³⁸ and 1XKK,³⁹ respectively). The protein structures were subsequently subjected to processing through the structure preparation tools integrated into the SYBYL-X 1.3 biopolymer module.⁴⁰ Energy minimization was performed utilizing the Powell algorithm, with a convergence gradient set at 0.5 kcal (mol)⁻¹ over 1000 cycles. This process included the addition of missing hydrogens, application of charges, and assignment of atom types based on the AMBER 7 FF99 force field.⁴¹ Lastly, using the Surflex-Dock module of the SYBYL-X 1.3 software package, the energy optimized bioactive conformation of POM was docked in to the active site of CDK2, PKB-α, and EGFR proteins using the same procedure and parameters as those described in our previous work.^42,43 The top twenty docked conformations were saved and investigated for their binding modes into the active site of their respective target. The Hammerhead scoring method was used to rate these potential ligand poses.^35,44

Statistical Analysis

The cell cycle analysis and genotoxicity findings were subjected to statistical analysis using a paired t-test and one-way ANOVA with a significance level set at p<0.05. GraphPad Software’s Prism 5.0 was used for the purpose of graphical depiction, whereas SPSS 9.0 (SPSS Inc. Chicago, IL, USA) was employed for statistical analysis. The findings were presented in the form of the mean standard deviation (SD).

Results and Discussion

Physical Characterization and Optimization of P₅W₃₀-BW-SLNs

Effect of Varying Surfactant Concentration

The experiment’s findings demonstrated that modifying the concentration of SL and Tween 80 had a notable impact on the creation of microemulsion. Table 3 demonstrates that the creation of microemulsion was unsuccessful in cases where a surfactant combination was not used (F1-P₅W₃₀-BW-SLNs and F3-P₅W₃₀-BW-SLNs). The microemulsion was formed when a 1:1 combination of surfactant was used (F2-P₅W₃₀-BW-SLNs). The F2-P5W30-BW-SLNs had henceforth denoted as P5W30-BW-SLNs.

Table 3 Physical Characterization of Hybrid Nanoparticles

FTIR Spectroscopy

As can be seen in Figure 1A, the spectrum that was acquired by FTIR analysis includes P₅W₃₀, P₅W₃₀-BW-SLNs, and BW. In the pure P₅W₃₀ spectra, a large and medium O–H stretching band was seen at a wavenumber of 3373.24 cm–1. The spectrum of P₅W₃₀-BW-SLNs showed a medium, broad and large peak (3380.69 cm^–1) because of O-H stretching. The FTIR spectra of BW showed one intense and large peak at 2955.77 cm^–1 and other peaks at 2847.68 cm^–1 due to stretching of O-H. Pure P₅W₃₀ spectrum represented intensified peaks at 1684.75 cm^–1 because of C=O stretch while that of P₅W₃₀-BW-SLNs showed clear peak at 1735.07 cm^–1 owing to C=O stretch.⁴⁵ Intense peak was observed in BW spectra at 1462.98 cm^–1 owing to C=C stretch. An intense peak was also found in spectra of P₅W₃₀-BW-SLNs in finger print region at 1084.65 cm^–1 which was due to C-O stretching movements while another peak with sharp edges was at 728.69 cm^–1 which may be because of bending vibrations. Pure P₅W₃₀ showed peak of 1013.83 cm^–1 because of P-O stretch. Another peak was shown at 907.60 cm^–1 in region of fingerprint due to bending type vibrations at W=O. Very strong and broad W-O-W stretch pattern at 732.42 cm^–1 was observed.⁴⁶ BW showed C-O stretch in fingerprint region at 1377.25cm^–1 and an intense peak was also noticed at 659.73 cm^–1 because of bending vibrations.

Figure 1 FTIR spectra of pure P5W30, BW, P5W30-BW-SLNs (A), DSC, Pure P5W30, P5W30-BW-SLNs (B).

Differential Scanning Calorimetry (DSC) Analysis

DSC provides data on drug temperature behavior, structural changes, crystallinity, and excipient interactions.⁴⁷ The physical characteristics of a polymer can be characterized using differential scanning calorimetry. Measurement of melting points and crystallization temperatures, enthalpy and entropy changes carried out by using DSC. This technique can also be used to characterize the glass transition and other phenomena that indicate either a transition in heating capability and latent thermal heat energy.⁴⁸ In DSC thermogram (Figure 1B), the P₅W₃₀ thermogram presented two endothermic shifts. There were several high points, including the glass transition temperature (Tg = 35 °C) and melting temperature (Tm =70 °C). Material showed glass transition peak at 40 °C and melting point peak at 75 °C. As the temperature rises, material starts to decompose at 165 °C.⁴⁹ A sharp peak was observed showing decomposition at 190 °C.⁵⁰ When P₅W₃₀ was loaded into SLNs, two endothermic shifts were seen on DSC thermogram. It was examined that the glass transition point starts at 40 °C while its sharp peak was observed at 60 °C. The distinctive peak reflecting the melting point of the substance had vanished, suggesting the transition of pure P₅W₃₀ from its crystal state to amorphous form. However, the material starts to melt at 120 °C. After that no decomposition occurs because of incorporation of P₅W₃₀ into solid lipid nanoparticles which implies stability of the lipid nanocarriers system and its ability to withstand high temperature. It also suggests better holding capacity and suitability of SLNs for P₅W₃₀. Both the scans show glass transition temperature below 50 °C. The material when heated above temperature of glass transition had released the stress that have been generated in the material. At transition temperature (Tg), the structure of the molecule changes from being rigid to flexible. The material underwent a transition from a crystalline state to an amorphous state.

Percent Entrapment and Loading Efficiency

As shown in Table 3, the synthesized P₅W₃₀-BW-SLNs had remarkable entrapment and loading efficiency, with values of around 88.55 ± 4.27% and 20.74 ± 6.39% respectively. The data suggested that the SLN formulation was capable of encapsulating P₅W₃₀ in an efficient manner. The effectiveness of entrapment inside SLN is highly reliant on the intrinsic properties of the drug as well as the nature of the link between the medication and the SLN components involved.⁵¹ While simultaneously increasing the water solubility of the contained ingredient, SLNs made from BW-SL and Tween 80 were able to protect the constituent from environmental risks. Prior research has verified the notable capacity of BW-SLNs to encapsulate diverse active compounds. The experiments demonstrated that the nanocarrier had a much higher rate of optimal encapsulation.^21,52–54 The enhanced entrapment efficacy of P₅W₃₀ incorporated BW-SLNs aligns with the findings obtained from our research.

Hydrodynamic Dimension and Zeta Potential Measurement

When determining particle sizes, this is crucial to consider two main factors. The first need is that particles must have a size large enough to hinder their excretion via the kidneys.⁵⁵ The second condition is that the particles have a small size to avoid being engulfed by phagocytosis and then eliminated by the system of reticuloendothelial cells (RES).⁵⁶ Current research suggests that biomolecules weighing more than 40 kDa, together with nanostructures varying in dimension from 10 to 500 nm, may pass through the network of capillaries and concentrate in the interstitial region of tumors. This process aids in the passive targeting of these substances.⁵⁷ The human body is more effective at removing particles larger than 200 nm.⁵⁸ A satisfactory hydrodynamic dimension of about 160.5 ± 8.61 nm and an appropriate polydispersity index (PDI) of 0.3814 ± 0.096 were shown by the findings of the dynamic light scattering (DLS) experiment, as shown in Figure 2A. Nanomedicines’ size is crucial because it affects how well their nano-formulation mimics bioactive substances, which in turn affects how well they provide therapeutic effects.⁵⁹ The hydrodynamic dimension, which measures the dispersion, biological absorption, and robustness of nanoparticles in water-based environments.⁶⁰

Figure 2 The physiochemical characterization of P5W30-BW-SLNs was examined by two methods: Measurement of the hydrodynamic diameter of the P5W30-BW-SLNs (A) and dissolution profile of P5W30-BW-SLNs at a pH of 7.4 (B). The data represents a particle size distribution. The peak of the curve is around −0.32 cm2/Vs which indicates the average mobility (C). Zeta potential measurement of P5W30-BW-SLNs. This measurement indicates a strong negative surface charge, suggesting a high degree of stability for the SLNs (D).

The charge, stability, and dispersion capacity of P₅W₃₀-BW-SLNs were evaluated using the measurement of the zeta potential, that served as an indication of their stability. The tendency for colloidal dispersion to join and form bigger aggregates is indicated by lower zeta potential values, while higher values imply better equilibrium within the system.^61–63 The zeta potential measurements of P₅W₃₀-BW-SLNs show a negative zeta potential of between −0.6 mV (C) and −60 mV (Figure 2C and 2D). These results show that the surface of the P₅W₃₀-BW-SLNs has a net negative charge. This negative zeta potential is most likely due to the presence of negatively charged functional groups on the surface of solid lipid nanoparticles (SLNs), such as carboxyl (-COOH) and phosphate (-PO₄) groups. The presence of these charges promotes interactions with the surrounding media, resulting in the creation of a hydration layer around the SLNs. This hydration layer is critical for stabilizing the SLNs and preventing aggregations.

Table 3 displays the zeta potential of −32.57 ± 6.44 mV, suggesting a negative charge on the particles. The probable cause of this is the presence of the anionic surfactant Tween 80. The presence of negative charge on the SLNs may contribute to the prevention of aggregation and enhancement of their stability in solution. Beeswax is a substance that lacks polarity, while Tween 80 is a surfactant that has polarity. When used in the microemulsion technique, it is probable that the Tween 80 molecules will envelop the beeswax particles, resulting in the formation of a hydrophilic layer. A negative zeta potential often signifies a high level of electrostatic repulsion among the particles. This repulsion serves to inhibit the aggregation of particles, which is crucial for maintaining the stability and efficacy of the SLNs.⁶⁴

Drug Kinetics and Release

The dissolution profiles presented in Figure 2B provided a visual representation of pure P₅W₃₀ and P₅W₃₀-BW-SLN particles. After a period of one hour, the medium showed that pure P₅W₃₀ had completely dissolved in it. Because of the low quantity of data points, it was not possible to create a kinetic model. The rapid degradation of P₅W₃₀ might likely be ascribed to its very little biological half-life, that has been approximated to be less than one hour.⁶⁵ Nevertheless, the release kinetics approach was used to get a more thorough comprehension of the drug delivery system of P₅W₃₀-BW-SLNs. The investigation primarily aimed to analyze the release characteristics by using several kinetic models, such as the zero-order, first order, Peppas, and Higuchi equation. The R² coefficient served as a quantitative measure to assess the kinematics and release characteristics of P₅W₃₀ from BW-SLNs.⁶⁶

Table 3 had presented the R² data obtained from zero-order (0.9862) analysis, suggesting that the concentration of the medication does not have an impact on its release at a certain time. This study’s kinetic model showed controlled drug release from SLNs. The Korsmeyer-Peppas R² value (0.9876, n = 1.083) demonstrated that the release of P₅W₃₀ from hybrid nanosponges occurred by a non-fickian super case II type erosion and expansion mechanism. Complex polymeric systems that include the dispersion or dissolution of drugs inside a polymer matrix, or the chemical bonding of drugs to the polymer, often demonstrate non-Fickian super-case II release. The release process is influenced by several factors, including polymer erosion, expansion, relaxation, and drug dissolution or dispersion within the polymer matrix.⁶⁷

Transmission Electron Microscopic Assessment

The surface characteristics of P5W30-BW-SLNs were assessed using transmission electron microscopy (TEM). Figure 3A demonstrates the nanoparticles’ spherical form and their smooth, unbroken borders, as seen in respect to a reference scale line measuring 200 nm. The results of our investigation are consistent with previous research that has investigated the surface characteristics of SLNs.^68,69

Figure 3 The representation of transmission electron microscopy image (A), and scanning electron microscopy image (B) of P5W30-BW-SLNs.

SEM Analysis

Utilizing scanning electron microscopy (SEM), the examination of solid lipid nanoparticles (SLNs) derived from beeswax and soy lecithin offers significant understanding of the structure and surface properties of these nanoparticles.⁷⁰ The scanning electron microscopy (SEM) pictures provide a detailed representation of the unique characteristics of the solid lipid nanoparticles (SLNs), displaying their precise dimensions, morphology, and surface structure Figure 3B. Beeswax, which acts as a lipid matrix, and soy lecithin, which contributes to the amphiphilic nature of the nanoparticles, have important functions in influencing the observed structural characteristics using SEM.

Pharmacological Assessment

Cytotoxicity and Cell Viability Assays

The SRB test was used to assess the cytotoxic effects of pure P₅W₃₀ and P₅W₃₀-BW-SLNs on HeLa cells. The results of this evaluation were shown in Figure 4A. Over the course of 24 h, HeLa cells were subjected to varying concentrations of pure P₅W₃₀ and P₅W₃₀-BW-SLNs. According to the data shown in Table 3, the IC₅₀ values for pure P₅W₃₀ and P₅W₃₀-BW-SLNs were found to be 7.93 ± 5.08 and 3.02 ± 2.14 (µg/mL), respectively. The anticancer properties of pure P₅W₃₀ were investigated in a prior study, which used a more than one cancer cell lines, namely MCF-7 and HeLa cells, to determine the anticancer properties of the compound in both its pure form and when it was incorporated into nanoparticles.^71–73 The P₅W₃₀-BW-SLNs were much more effective than pure P₅W₃₀ in inducing cytotoxic activity might be due to enhanced solubility and stability as compared to the unbound molecule. This may lead to increased medication concentrations at the specific location of interest (cancer cells) and enhance the efficacy of cell destruction. Furthermore, cancer cells have a higher degree of uptake efficiency for nanoparticles in comparison to unbound medicines. This is mostly attributed to the nanoparticle’s dimensions and surface properties, which may enhance the cancer cell membranes contact. Finally, nanoparticles may be designed to selectively gather in tumor tissues because of the increased permeability and retention (EPR) effect. Consequently, the SLNs could efficiently transport P₅W₃₀ to cancerous cells while reducing the exposure to healthy cells.^74–76

Figure 4 The pharmacological characterization assesses the proportion of HeLa and Vero cells that undergo cell death (A) and viability (B), respectively, when treated with purified P5W30 and P5W30-BW-SLNs.

The SRB assay measures the rate at which cells proliferate and, alternatively, the reduction in viable cells caused by metabolic processes that initiate necrosis or apoptosis.⁷⁷ The investigation shown in Figure 4B demonstrated a significant decrease in the viability of Vero cells, which was directly proportional to the quantity of the substance (ranging from 0 to 50 μg/mL). It is possible that the quick solubility of pure P₅W₃₀ might facilitate its absorption into the tight connections of healthy cells, which would bring about a reduction in the viability of the cells. While the P₅W₃₀-BW-SLNs released pure P₅W₃₀ in a regulated way, which is the reason why the nano-formulation was able to provide greater cell viability.

DAPI Staining

We utilized a qualitative test to get an understanding of the process by which POMs cause cell death. Upon treatment with P₅W₃₀ cells, the HeLA cells exhibited condensed nuclei and collapsed spherical cells, as seen in Figure 5A. The presence of apoptotic bodies inside Figure 5B confirmed the process of cell death when P₅W₃₀ was encapsulated within BW-SLNs. The findings of our study were consistent with those of earlier research.⁷¹

Figure 5 Representation of apoptotic cell death through DAPI staining: Pure P5W30 (A) and P5W30-BW-SLNs (B). The arrows (white color) indicate apoptotic bodies in HeLa cells.

Genotoxicity Analysis

Utilizing the comet test allowed for the confirmation of the existence of DNA damage. Quantifying tail DNA as a fraction of total DNA content is a crucial technique used to verify the existence of DNA damage. Other strategies that were utilized include TM and OTM. The terminal segment of the genetic material (DNA) molecule had responsible for the detection of DNA damage. The experiment utilized pure P₅W₃₀, P₅W₃₀-BW-SLNs and H₂O₂ as seen in Figure 6A–6C respectively.

Figure 6 The comet assay demonstrates the DNA damage in HeLa cells exposed to purified P5W30 (A), P5W30-BW-SLNs (B), and H2O2 (C).

The estimation of DNA damage might be accomplished by assessing the difference in content of DNA among the head and tail of the comet. Table 4 demonstrated that the use of P₅W₃₀-BW-SLNs resulted in a significant rise in damaged DNA of HeLa cells, in comparison to the usage of pure P₅W₃₀. The quantification of DNA damage may be accomplished by analyzing the disparity in the amount of DNA between the head and tail parts of the comet.^33,34

Table 4 An Examination of Various Aspects Pertaining to the Comet Assay on the HeLa Cell Line Utilizing Purified P5W30 and P5W30-BW-SLNs

The Analysis of Flow Cytometry

The flow cytometry method was used to get more understanding of the phases of cell cycle arrest, mitochondrial function and apoptosis.⁷⁸

The flow cytometry experiment confirmed the quantitative aspect of the previously subjective findings on cancer cell death. Figure 7A shows that in the absence of medication (control group), a higher proportion of HeLa cells (86.2 ± 3.19%) remained in the G0/G1 phase, whereas only a tiny fraction of cells was observed in the sub-G1 phase (0.17 ± 0.045%). The cells demonstrated an elevated rate of cellular apoptosis when exposed to pure P₅W₃₀, as shown by an augmentation in sub-G1 (4.97 ± 1.72%) levels and a subsequent reduction in the proportion of cells during the G0/G1 (77.4 ± 5.38%) phase, illustrated in Figure 7B histograms. The exposure concerning HeLa cells with P₅W₃₀-BW-SLNs resulted in increased cell death, as shown by a greater percentage of sub-G1 cells (11.6 ± 3.04%) with a lower proportion of G0/G1 cells (65.7 ± 5.89%) (Figure 7C). Figure 7D displayed the distribution of cells in different stages of the cell cycle after being treated with P₅W₃₀-BW-SLNs, pure P₅W₃₀, or a control. The current research’s findings corroborated those of a previous study that found that hydrolysates of fenugreek protein inhibited the proliferation of colorectal cancer cells. The hydrolysates have shown the ability to impede cell proliferation by augmenting intrinsic apoptosis.⁷⁹

Figure 7 Flow cytometry analysis of HeLa cells treated with control (A), pure P5W30 (B), and P5W30-BW-SLNs (C). The percentage of cells treated throughout the cell cycle (D) with pure P5W30, P5W30-BW-SLNs, or a control. (Results show a significant difference (p<0.05).

Animal Studies

For validating the data gained through in vitro experiments, the study used female albino mice as respondents in an in vivo study. Figure 8A and Table 5 displayed the survival rates of mice that were fed various dosages of cisplatin (30 µg/kg), pure P₅W₃₀ (30.87 µg/kg), and P₅W₃₀-BW-SLNs (14.72 µg/kg). A comparison was made between the findings and the standard that had been set for cisplatin effectiveness. When compared to the group that was treated alone with pure P₅W₃₀ plus the conventional anticancer medication cisplatin, the group that was given P₅W₃₀-BW-SLNs showed a higher survival rate.⁸⁰

Table 5 Percent Survival Data of Cancerous Mice

Figure 8 The in vivo longevity rate (A) and the % reduction in tumor weight (B) of pure P5W30, and P5W30-BW-SLNs treated with HeLa cells. Statistical significance was determined using ANOVA, with a p-value of <0.05 indicating significant differences between the treatment groups.

The present research included the analysis of the percentage of malignant mice that survived under various treatment conditions over a period of 10 days. The treatments consisted of Cisplatin, Pure P₅W₃₀, P₅W₃₀-BW-SLNs, BW-SLNs, and a control group that did not receive any therapy. On the first day of the trial (Day 0), all groups showed complete survival. During the following days, the group that received Cisplatin treatment had a complete survival rate. However, on Day 2, there was a decline to 88.89% which continued until Day 5. On the other hand, the Pure P₅W₃₀ group showed complete survival until Day 8, but then had a decrease to 48.61% on Day 10. The P₅W₃₀-BW-SLNs administered group exhibited 87.5% survival rate until Day 4, after which a progressive decrease was observed, reaching 72.91% on Day 10. Significantly, Group 1 and Group 2 demonstrated 100% survival over the whole duration of the investigation. The findings indicated that P₅W₃₀-BW-SLNs had a positive impact on prolonging life as compared to P₅W₃₀ and cisplatin (survival percent at day-10th 48.61 and 35.56 respectively) (p<0.05). These results emphasize the possible influence of various therapies on the survival rates of mice with cancer and justify the need for more research on the mechanisms and efficacy of each therapeutic option in this experimental setting.

In Figure 8B, the group that received P₅W₃₀-BW-SLNs therapy observed a more significant reduction in tumor weight % compared to the group which obtained pure P₅W₃₀ alone, on the 10th day after administration. It has been found that cisplatin is a chemotherapeutic drug that is often employed. This substance is known to interrupt the process of DNA replication in cells that are quickly growing, such as cancer cells. This was evidenced by the considerable 3.687 ± 0.375% reduction in tumor weight, which proved its efficiency in inhibiting the formation of tumors. The administration of P₅W₃₀ resulted in a reduction of 2.113 ± 0.402% in the weight of the tumor, which indicates that it had a moderate influence on suppressing the development of the tumor in comparison to cisplatin, which generated a decrease of 3.687 ± 0.375%. The tumor weight reduction of 2.967 ± 0.543% indicates that the effects of P₅W₃₀-BW-SLNs are stronger to those of purified P₅W₃₀. The potential utilization of nanoparticles to enhance the delivery and localization of therapeutic drugs to the site of the tumor could result in improved efficacy and reduced adverse effects. Prior research has shown evidence for the benefits of nanoparticles in enabling regulated and precise delivery of medications, hence improving the efficacy of therapy in different cancer treatment regimens.⁸¹

Molecular Docking Studies

Molecular docking is among the most employed approaches to unveil the binding mechanisms of ligands towards various molecular targets.^82–87 Utilizing an in-silico approach can yield valuable insights into the structural and functional relationships within ligand-protein systems, aiding our comprehension of the molecular mechanisms that impede the rapid growth of cancerous cell lines. Through both in-vivo and in-vitro analyses (Figure 9), a noticeable reduction in growth was observed in the HeLA cell line upon continuous exposure to POM. To gain a comprehensive understanding, an extensive literature survey was conducted to identify three pivotal proteins CDK2, PKB-α, and EGFR that regulate distinct signaling pathways in HeLA cells.^88,89 Subsequently, molecular docking was employed to unveil the mechanism underpinning the anti-proliferative potential of POM.

Figure 9 Docking generated complexes of POM bonded to its corresponding molecular target CDK2 (hydrophobic, light blue), PCG (green, yellow) and EGFR (slate, purple): (A and E) CDK2-POM, (C and F) POM-PKB-α, (D and G) EGFR-POM. H-bonds are displayed in Orange dotted lines.

Leveraging the cumulative score (cScore), optimal ligand poses within protein active sites were identified. Docking scores (cScore) for inhibitor POM binding to CDK2, PKB-α, and EGFR are 68.21, 47.93, and 44.15, respectively. These scores suggest POM forms the most robust complex with CDK2, though it efficiently binds to PKB-α and EGFR. Overall, the docking scores indicate commendable binding affinity for all three proteins, with a slightly higher affinity for CDK2. Detailed docking results, including scores, are presented in Table 6. To comprehend docking score variations, top-ranked docking complexes were saved and visually examined (Figure 9A–9G). Figure 9A illustrates substantial conformational changes in inhibitor POM, adopting a complex shape post energy minimization. Despite POM’s intricate structure with numerous oxygen atoms, it establishes several H-bond contacts with nearby residues in all protein-ligand complexes, supporting its higher docking scores. In the CDK2-bonded system, POM deeply penetrates the active site between N- and C-terminal domains, interacting with residues such as E8, I10, V18, E12, T14, F80, E81, F82, L83, Q85, D86, Q131, E162, and V164 (Figure 9B). Conserved H-bond interactions with L83 in the hinge region are crucial, with POM forming additional bonds with Q85 and D86 in the solvent-exposed region. hese residues have been consistently highlighted in previous publications, demonstrating their crucial role in facilitating the binding of potent CDK2 inhibitors.^42,43,90 For the PKB-bonded system, POM occupies the ligand binding cavity, but its large structure prevents reaching the hinge region. Consequently, POM establishes maximum interactions in the solvent-exposed region, forming at least six H-bonds with residues T195, M227, K276, N279, and T319 (Figure 1C). In the EGFR-bonded system, only a couple of H-bond interactions are observed. The binding cavity in the EGFR-POM bonded system includes V702, K721, T766, C773, L775, D813, A815, D831, and A816. Similar to the POM-PKB complex, in the EGFR-inhibitor complex, POM remains near the solvent-exposed region and does not exploit hinge residues. For more better understanding the 2D interaction diagram of inhibitor-protein complex is displayed in Figure 9E–9G In conclusion, these findings explain the enhanced binding affinity of POM towards CDK2. The computational results strongly correlate with experimental findings, providing a plausible explanation for POM’s substantial chemopreventive efficacy against HeLa cell lines.

Table 6 Surflex Scores of Docked Ligand POM in the Binding Site of Proteins CDK2, PKB-α and EGFR

Conclusion

The incorporation of polyoxometalates into solid lipid nanoparticles is a multidimensional method that has the potential to have positive consequences for the treatment of cancer. The study’s physical characterization reveals these nanoparticles’ structural features, revealing their stability and biological connections. Pharmacological tests show polyoxometalate-loaded nanoparticles’ cancer therapy effectiveness and therapeutic potential. Computational studies help anticipate molecular interactions and optimize nanoparticle design for anticancer effects. The findings demonstrate polyoxometalate-integrated solid lipid nanoparticles’ potential as a multifunctional cancer therapy platform, bridging chemistry, pharmacology, and computational sciences to develop novel and effective treatments.