INTRODUCTION:

At 2.1 million cases per year and a major contributor to cancer mortality rates, breast cancer is a major health concern around the world. This particular cancer stands out as it constitutes 25.1% of all female cancers, as reported by GLOBOCAN¹. Along with radiation therapy and surgical resection, chemotherapy is the treatment of choice. Yet, there are a number of drawbacks to these methods, such as toxicity, poor tumor tissue distribution, a low resection rate, and a low surgical success rate ^2,3. The low water solubility of the powerful drug paclitaxel (PTX) limits its therapeutic utility in the treatment of advanced solid carcinomas. Nanoparticle (NP) delivery of hydrophobic PTX holds great promise, but it is challenging to achieve adequate drug loading while keeping the particles small and stable in biological media ^4,5. Researchers have been concentrating on nano-drug delivery as a potential solution. Reasons being: prolonged blood circulation, improved solubility of hydrophobic drugs, and targeted drug delivery. But along the injection route, these nanocarriers face a number of obstacles, such as non-specific uptake, mucosal barriers, and extravasation in non-target locations. Thus, it is necessary to create new therapeutic methods that address the issues. Because it is non-invasive, sidesteps primary metabolism, and keeps the dosage constant, drug delivery through the skin is an effective method. Enhanced therapeutic efficacy with less toxicity and less chemotherapy administration frequency are two further advantages ^6–8.

Iron oxide NPs (Fe3O4) are one of many NPs studied by researchers; these have shown promise as a biocompatible, non-toxic, and water-soluble material. Because of its promising uses in biomedicine, diagnostics, magnetic nanoparticles (MNPs), have garnered a lot of interest in the last several decades ^9,10. Contrarily, it was found that biocompatibility may be compromised in large aggregates of pure Fe3O4 NPs due to the high dipole-dipole interactions amongst the particles. It is not possible to employ Fe3O4NP as a medication carrier on their own because of their instability in water. Oleic acid (OA) is a frequently used surfactant for stabilizing MNPs made using traditional co-precipitation methods. OA is composed of a polar carboxylic acid head group and a non-polar hydrocarbon tail ¹¹. The biocompatibility, degradability, and low toxicity of chitosan (CS) polysaccharides have made them a popular choice for modifying the behavior of metal NPs ¹². One possible explanation for the many useful biological and biochemical characteristics of chitosan and its derivatives is the presence of reactive functional groups, such as hydroxyl (-OH) and amino (-NH2) groups ¹³. Taking into account the drawbacks of PTX, this study aimed to synthesize a Nano effective polymer-based medium that could effectively transport PTX to the breast cancer, increasing its solubility in water, bloodstream circulation time, and, finally, biocompatibility while minimizing treatment-related side effects.

Materials and Methods:

Materials:

Mylan Chemicals of Karnataka provided the PTX as a complimentary sample. Sourced from the Adichunchungiri Institute of Medical Sciences in Karnataka, MDA-MB-231 cell lines were used. Adichunchungiri Pharmacy College in Karnataka supplied all of the analytical-grade polymers, chemicals, and solvents needed for the investigation.

Preparation and Optimization of OA-MNPs:

Fe3O4 NPs were synthesized by refluxing ferrous and ferric chloride solutions at 80°C, followed by ammonium hydroxide addition to reach pH 8-10. After OA coating and a 2-hour reaction at 50°C, the MNPs were precipitated with ethanol and dried. Optimization was performed using Stat-Ease Design Expert version 13. Lipid concentration (5-15%), polymer concentration (10-20%), and surfactant amount (5-10%) were studied as independent variables. Seventeen trials were generated using a Box-Behnken design, and responses such as particle size (PS) and entrapment efficiency (EE) were assessed. The optimized OA-MNPs were selected based on minimal PS and maximized EE.

Chitosan coating and preparation of PTX-CS-OA-MNPs:

Finally, optimized OA-MNPs were coated with chitosan polymer. Using 1% acetic acid (30 mL) and NaOH to maintain pH 4.8, 0.15 g CS was solidified. Fe3O4 NPs were combined with 50 mL of CS solution at 50°C in an inert atmosphere. NPs were made by cautiously adding 40 mL ammonia to the reaction mixture after 30 minutes of stirring. Magnetic decantation and continuous water washing were performed on colloidal MNPs. After mixing 100 mg of CS-OA-MNPs with PTX, the loading technique called for 48 hours of stirring at room temperature. To eliminate the unreacted particles, the suspension was meticulously washed with an abundance of water and ethanol after being carefully collected using a magnet. After drying in a vacuum, the PTX-CS-OA-MNPs powder was finally obtained ^14–16. The development of the NF is depicted in Fig.1.

Synthesis of PTX-CS-OA-MNPs loaded transdermal patch:

The evaporation casting method was used to create transdermal patches of MNPs. Using different ratios of HPMC and EC polymers, MNPs transdermal patch formulae were created. A magnetic stirrer was used to dissolve HPMC in 15 ml of a 1:1 combination of dichloromethane and ethanol. Two mL of chloroform was used to dissolve the EC and menthol. After that, a magnetic stirrer was used to homogenize the HPMC solution after adding the EC and menthol solutions. Then, PEG400 was included into the concoction. To make the dope solution, 10 mg of PTX-CS-OA-MNPs were added to the mixture and stirred with a magnetic stirrer for 10 minutes. The 10 mL solution was subsequently transferred to a plate and dried in an oven at 30°C for 24 hours. After that, the patches that came out of it were put in a desiccator to be studied further ^17,18.

Characterization of PTX-CS-OA-MNPs:

PS, polydispersity index (PDI), zeta potential (ZP), morphology and molecular characterization. After the NPs solution was sonicated, it was analyzed via Nanotrac to find the PS, PDI and ZP for.

Figure 1. Diagrammatic representation of the PTX-CS-OA-MNPs preparation process

ZP of the optimized MNPs was determined using a zeta sizer. The surface morphology was examined with a scanning electron microscope (SEM), Hitachi S4700; Tokyo, Japan, Hitachi Scientific Ltd Supra 35 VP, Zeiss, Germany, operating at 1.00 kV, and a transmission electron microscope (TEM) 2100, manufactured by JEO, Tokyo, Japan, operating at 160 kV. PTX-CS-OA-MNPs were analyzed using Fourier-transform infrared (FTIR) spectroscopy ^19,20.

Drug loading and %EE:

Following the procedures described in an earlier research, the EE and drug loading capacity (LC) of PTX-CS-OA-MNPs were determined. A UV-Vis spectrophotometry (UV-Vis) system operating at 277 nm was used to quantify the amount of PTX. To find the EE, one had to multiply the total amount of medication added by 100%, then divide the ratio of the drug contained within the NP by that amount. Furthermore, The LC was determined by multiplying the ratio of the PTX encapsulated by the total quantity of drug used by 100% ²¹.

Characterization of PTX-CS-OA-MNPs loaded transdermal patch:

The NP-based patches loaded with PTX were assessed for consistency, thickness, weight uniformity, folding endurance, and moisture uptake. Patch thickness was measured at three locations, and weight uniformity was determined by weighing ten randomly selected patches. Folding endurance was evaluated by folding a patch repeatedly until it broke. For moisture uptake, patches were placed in a desiccator with AlCl3 for three days, then reweighed. Drug encapsulation efficiency (%EE) was determined by placing the PTX-loaded patch in phosphate buffer, sonicating for one hour, and analyzing the solution using UV-Vis spectroscopy at 277 nm ^22,23.

Wf - Wi

% Moisture uptake= ------------- × 100

Where Wf is the end weight of the patch and Wi is its initial weight.

In-vitro drug release:

The drug release from PTX-CS-MNPs, PTX-dispersion, and PTX-CS-OA-MNPs transdermal patches at pH 7.4 was assessed using the dialysis membrane method. The study was conducted in a shaking water bath at 37°C and 100 rpm. Aliquots (0.5 ml) were taken at intervals (0.5–48h) and replaced with fresh PBS. The absorbance of the samples was measured using a UV-Vis system at 277 nm, with a blank patch used to correct for background absorbance, determining the cumulative drug release percentage.

Ex-vivo skin permeation study:

Full-thickness pig ear pinna skin was used in a Franz diffusion cell study with PBS (pH 7.4) at 37 ± 1°C. PTX-dispersion, PTX-CS-MNPs, and PTX-CS-OA-MNPs-loaded patches were tested. The patch covered 1 cm², and the receptor compartment had 0.77 cm² diffusion surface. To maintain sink conditions, 2 ml samples were periodically removed and replaced with PBS. After filtration and ethanol dilution, drug release was analyzed using a UV-Vis spectrophotometer at 227 nm.

Stability study:

Stability of PTX-CS-OA-MNPs and PTX-CS-OA-MNPs loaded patches was evaluated using ICH Guidelines at 40±0.5°C and 75±5% humidity. Samples were analyzed at 0, 1, 3, and 6 months for physical appearance, PS, PDI, ZP, %EE, and drug content.

In-vitro cell line study and cellular uptake:

Cytotoxicity of PTX-CS-OA-MNPs and pure PTX on MDA-MB-231 cancer cells was assessed using the MTT assay. Cells were incubated with MTT, and formazan crystals were dissolved in DMSO, with absorbance measured at 570 nm. For cellular uptake, MDA-MB-231 cells were seeded in 96-well plates and exposed to 1% test compounds (PTX or PTX-CS-OA-MNPs). After one day of treatment, cells were fixed with 4% PFA and imaged using a fluorescence microscope.

Animal study:

The animal study was approved by CPCSEA and IAEC (permission No: SACCP-IAEC/22-01/54). Female Swiss albino mice (20-30g) were housed at 25 ± 1°C with 55 ± 5% humidity, in polypropylene pens with unrestricted access to food and water.

Skin irritation test:

The day before the experiment, mice had their dorsal hair removed. Six mice were divided into three groups: Group I (control), Group II (PTX-CS-OA-MNPs patches), and Group III (0.8% formalin). Treatment was applied daily for six days. Erythema was visually graded from 0 (no redness) to 4 (intense redness).

In-vivo pharmacodynamic study

On day 0, MDA-MB-231 cells were subcutaneously injected to induce tumors in mice. Once tumors reached 80-90 mm³ by day 21, animals were divided into four groups: untreated, oral PTX-loaded MNPs (20 mg/kg), and PTX-CS-OA-MNPs loaded transdermal patch (equivalent PTX dose). Tumor size was measured daily from day 22 to 31. Tumor volumes were calculated using the formula: volume = 0.5 × a × b. Statistical significance was determined by one-way ANOVA, with P < 0.05 indicating significant differences in tumor volume.

Results and discussion:

Preparation and Optimization of OA-MNPs:

A three-level, 2-factor Box-Behnken design (BBD) optimized OA-MNPs using Design-Expert software, demonstrating that the quadratic model provided the best fit (p < 0.05). The PNP9 batch, with 15% w/v OA, 15% w/v CS, and 5% surfactant, yielded optimal particle size (PS) of 77 nm and encapsulation efficiency (EE) of 85%. Analysis of variance (ANOVA) indicated high precision for the quadratic model, with signal-to-noise ratios of 14.38 for PS and 14.79 for % EE. The developed formulation achieved a low polydispersity index and demonstrated reliability, as shown in contour plots (Figure 2) and experimental comparisons (Figure 3).

Effect of A, B and C on PS:

To illustrate the complex relationship between several elements and their effect on PS, the highly regarded Design-Expert® Software provides the polynomial Eq. (1). The polynomial equation indicates that PS directly affects the concentration of total lipids (A). The PS of OA-MNPs increased with higher total lipid concentration (Figure 3c). Increased polymer and surfactant concentrations decreased PS. Higher surfactant concentrations may cause smaller emulsion droplets by reducing interfacial tension between the aqueous and lipid phases. Increased surfactant concentration effectively stabilizes NPs by creating a surface barrier, preventing smaller particles from aggregating into larger ones.

PS = +84.40 – 7.37 * A + 4.13B – 2.50C + 1.25 *

AB + 13.00 * AC + 3.50BC – 4.83 *A² + 10.17 *

B² + 4.43 * C²----------------------------(1)

Effect of A, B and C on EE:

Experimental runs showed an EE in a range of 45% to 85%. A greater EE estimate is ideal for MNPs to stack more drugs and reach the target site. The model graphs showed that increasing lipid concentration from 5% to 15% gradually increased EE. The hydrophobic properties of OA may promote drug encapsulation in lipids due to greater lipid concentrations. Increased in B and C concentration decreased EE. An elevated surfactant content in the external phase may cause the PTX to move from the internal to the exterior phase of the medium. This higher partition is due to improved drug solubility in the exterior aqueous phase, allowing for greater dispersion and dissolution. Figure 3d shows the effect of these independent variables on EE. Polynomial equations 2 demonstrate that EE are influenced by A, B and C.

Table 1. Box-Behnken experimental runs for optimized PTX loaded MNPs+76.20 + 6.12 * A – 4.00 * B + 2.63 * C – 3.50 * ab – 10.75 * AC – 11.50 * BC + 8.03 *A² – 9.22 * B² – 9.48 * C² -----(2)

Run	Factor 1 A: Lipid concentration (% v/v)	Factor 2 B: Polymer concentration (%w/v)	Factor 3 C: Surfactant concentration (%w/v)	Response 1 Particle size (nm)	Response 2 Entrapment efficiency (%)
1	10	15	7.5	81	70
2	5	15	5	107	58
3	5	20	7.5	100	68
4	10	10	10	89	78
5	10	20	5	102	60
6	5	10	7.5	95	67
7	10	15	7.5	81	75
8	15	15	10	87	70
9	15	15	5	77	85
10	10	15	7.5	88	80
11	10	15	7.5	81	77
12	15	20	7.5	87	76
13	10	20	10	105	45
14	15	10	7.5	81	83
15	5	15	10	75	82
16	10	10	5	100	45
17	10	15	7.5	90	76

Table 2. ANOVA tables for particle size and entrapment efficiency

Best suit Models	SD		R²	Adjusted R²	Predicted R²	Adequate precision	P-value	Remark
Response 1: Particle size
Quadratic	3.69		0.9536	0.8939	0.8976	14.38	0.0007	Significant
Response 2: Entrapment efficiency of PTX
Quadratic	3.91		0.9591	0.9064	0.7248	14.79	0.0005	Significant
		Actual values			Point prediction values
PS (nm)		75±2.23			77.27			Model suggested
EE of PTX (%)		85.92±0.11			86.08

Evaluation of PTX-CS-OA-MNPs loaded transdermal patch

The formulated patch was clearly visible as being uniformly smooth and clear based on the patch's physical appearance. Both PTX-MNPs and PTX-MNPs based patch seemed physically to be transparent, smooth, and flexible. Thickening, weight uniformity, folding durability, and moisture uptake investigations were also conducted on the PTX-CS-OA-MNPs loaded patch. As a percentage of weight, the patch contained 87±2% of the medication. The present research' findings fell within the acceptable range (Table 3).

In-vitro drug release:

The release of PTX from PTX-CS-MNPs, PTX-dispersion and PTX-CS-OA-MNPs’s transdermal patch was examined at pH 7.4, 37˚C. The study found sustained release of PTX-CS-MNPs and PTX-CS-OA-MNPs loaded patches. Figure 6A indicates for PTX-CS-MNPs, roughly 57.88% of the PTX was released in 24 h and about 80.47% of the entrapped PTX was released after 48 h. The PTX-CS-OA-MNPs’s patch released 68.55% of the medication in 12 h, 78.99% after 24 h and 94.87% after 48 h. Hydrogen bonding between CS and OA contributes to the drug's delayed and sustained release. All entrapped drug (approximately 90% w/v) discharged after 48 hours. In the first 24 hours, only 20.29% of the PTX was released and after 48h and 24.99% of PTX was released after 48h from PTX-dispersion, PTX release was lower than PTX-CS-MNPs and MNPs loaded patch due to low solubility and permeability in water, allowing only tiny molecules to permeate past the surface.

Kinetics of drug release from MNPs and patch:

Utilizing kinetic models such as zero-order, first-order, Higuchi, Korsmeyer Peppas, and Hixon-Crowell, the release processes of PTX from nanoparticles and patches were examined. Although the patch adheres to Higuchi release kinetics (R2:0.900), the drug release from nanoparticles followed a first-order kinetic model (R2:0.910). With exponent values of 0.55 in the first-order kinetic model and 0.65 in the Higuchi release kinetics (0.45 n), non-Fickian transport is indicated.

Ex- vivo skin permeation studies:

Figure 6B compares the permeability of PTX-dispersion, PTX-CS-MNPs, and PTX-CS-OA-MNPs loaded patch. Formulations with PTX-dispersion, PTX-CS-MNPs, and PTX-CS-OA-MNPs loaded patch showed drug release of 47%, 81% and 95.06% respectively after 48h. This study utilized OA as a permeation enhancer, comparing patches with and without it to assess its effectiveness. OA improves skin penetration by increasing the fluidity of the stratum corneum lipid-protein layer. An analysis of the patch with and without permeation enhancer suggests that PTX-CS-OA-MNPs-based patches may be a non-invasive medication delivery solution for transdermal use in many disorders. There was a substantial difference in penetration between PTX-MNPs, PTX-CS-MNPs, and PTX-CS-OA-MNPs loaded patch.

Stability study:

PTX-MNPs’s stability was assessed by physical appearance, PS, and %EE. Data from a 6month period showed minimal fluctuation in particle dimension and EE, but no change in NF color. We assessed the stability of the PTX-CS-OA-MNPs loaded patch by analyzing its physical and chemical properties. The patch was evaluated for changes in color, moisture loss (%w/w), thickness (mm), and %EE. The accelerated stability research results indicate good stability of PTX-CS-OA-MNPs and PTX-CS-OA-MNPs loaded patches.

MTT assay and cellular uptake study:

Results indicated that PTX-loaded MNPs were more effective against tumor cells compared to the free drug. The IC50 of PTX-CS-OA-MNPs was < 0.0001 μM with a 73.98% survival rate, while pure PTX had an IC50 < 0.001 μM and 98.77% survival. Cellular uptake experiments on MDA-MB 231 cells revealed greater absorption of PTX-loaded MNPs compared to pure PTX. Fluorescent microscopy images showed PTX-loaded MNPs localized effectively within cells, whereas pure PTX remained mostly in the outer layer of the cell line, confirming improved internalization (Figure 7a, 7b).

Pharmacodynamic Study:

Tumor latency period was found to be 21 days after subcutaneous injection of MBA-MB-231 cells in in vivo investigations conducted on female Swiss albino mice. As previously indicated, the tumors were randomly separated into 2 groups of animals once they reached an approximate size of 250-300 mm3 as seen on day 22. A transdermal patch containing 20 mg of PTX-CS-OA-MNPs, and an oral formulation of PTX-MNPs, were all administered to the animals (Group II and Group III). Group I animals were left without treatment (negative control group). Figure 7c showed that compared to the negative control group, mice treated with transdermal patch and PTX oral formulation had significantly smaller tumor sizes on day 33. Mice treated with PTX-transdermal patches had significantly smaller tumor sizes (P < 0.05) compared to those given PTX-MNPs oral formulations. Because of their positive disposition and widespread localization in breast cells, PTX-CS-OA-MNPs loaded transdermal patch also dominated in-vivo pharmacodynamic studies indicating maximized therapeutic effect.

Figure 7. (a) Cell-cytotoxicity assay, (b) cellular uptake study in MBA-MB-231 cell lines and (c) effect of transdermal patch on tumor volume

Conclusion:

Developing OA-MNPs for the long-term transdermal administration of PTX as a treatment for breast cancer was the primary goal of this investigation. A more effective formulation and optimization of PTX-OA-MNPs resulted in nano-sized particles with enhanced encapsulation efficiency. Optimized OA-MNPs via BBD were formulated into the transdermal patch coated with CS polymer that might improve skin and tumor permeation and deposition for local therapy. Substantially and in a pH-dependent manner, the medication was released from the nanocarrier in the in vitro release profile that could sustain the release of PTX for 48 hours. This new method successfully delivered PTX through a non-invasive route (transdermal) and ex vivo permeability research showed high drug permeability from a CS based MNPs with OA. In addition to cytotoxic action, the MTT test demonstrated that the PTX-CS-OA-MNPs exhibited a higher affinity for MBA-MB-231 cancer cells. Furthermore, it performed in vitro against MBA-MB-231 and had a greater ability for intracellular transport. There is no doubt that the designed patch has an advantage over other standard and oral formulations based on its pharmacodynamic profile. PTX loaded CS-OA-based transdermal patch was effective, safe, and did not irritate the skin in any way and hence, additional research is required to delve into the potential applications of this formulation from a standpoint.

ACKNOWLEDGMENTS:

All the authors thanks Sri Adichunchanagiri College of Pharmacy, Adichunchanagiri University, B.G.Nagar, Karnataka, India, Deanship of Scientific Research, King Faisal University, Saudi Arabia, and Vidya Siri College of Pharmacy, Off Sarjapura Road, Bangalore 560035, India for supporting to the project.

FUNDING:

This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia [Grant No. KFU253437].

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Received on 20.09.2024 Revised on 11.01.2025

Accepted on 09.03.2025 Published on 01.10.2025

Available online from October 04, 2025

Research J. Pharmacy and Technology. 2025;18(10):4727-4735.

DOI: 10.52711/0974-360X.2025.00680

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Formulation	Thickness (mm)	Uniformity of weight	Content uniformity (%)	Moisture content (%)	Moisture uptake (%)	Folding endurance	Tensile strength g/cm2
Patch	0.20±0.01	0.79±0.03	95.15±1.00	8.14±0.61	5.012±0.07	98.14±1.25	2.35±0.03