INTRODUCTION:

Breast cancer is a common cancer type, with a death rate of approximately 12.5% ¹. According to a 2022 report, breast cancer caused approximately 670,000 deaths^2,3. One of the reasons behind this high mortality is drug resistance⁴. With increase in drug resistance the mortality rate increase, also drug resistance lives behind very few treatment options⁵. Drug resistance is complex process involving several mechanisms⁶. Further, this makes cancer more complex for treatment⁷. Cisplatin (Cis) is used for treatment of different type of cancers, including breast ovarian, testicular, and lung cancer⁸.

It works by forming DNA crosslinks that disrupt replication and transcription, ultimately leading to apoptosis. Cis is a front-line drug used for breast cancer treatment⁹. In initial part of the treatment Cis shows good results but later it develops drug resistance. This creates major hurdle in Cis-based treatments¹⁰. One of the mechanisms is decreased uptake of Cis by cancer cells¹¹. Overcoming cisplatin resistance remains a major challenge in oncology, and ongoing research focuses on combination therapies, novel drug formulations, and targeted strategies to improve its effectiveness¹².

In the present research work, we have developed and characterized anti-cancer activity of Cis vs Cis-Rub in multidrug resistant breast cancer cells: MDA-MB-231 and MCF-7/Adr. Results shows that Cis-Rub increases internalization of Cis in cancer cells. Further, Cis-Rub has higher anti-cancer than Cis. This higher activity is because of increase in expression of p21 protein.

MATERIAL AND METHODS:

1. Cis-Rub Nanomicelles Formulation and Characterization:

Solvent evaporation method was employed for Cis-Rub nanomicelles preparation. Cis and Rub were mixed in 1:10 ratio (w/w) and solubilized in ethanol (1:20w/v). The prepared solution was filtered and was heated at 50^o C for solvent evaporation, until powder is obtained. The powder was stored at -20^o C until needed.

Size of nanomicelles was determined by transmission election microscope¹⁴. Briefly, Cis-Rub was dissolved in deionized water (10% w/v) and placed 400-mesh carbon-coated copper grid and visualized on a JEOL 100CX instrument. Three samples were used to calculate average particle size of particle. From each sample 5 images were used to calculate particle size.

The distribution of particle size of nanomicelles was determined using a Nano-ZS Zetasizer. Samples of Cis and Cis-Rub was dissolved in water and loaded into a folded capillary cell without introducing air bubbles and secured in the instrument holder. Their characterization followed standard protocols as outlined in the Malvern Instruments manual.

2. Cell Viability Assay:

CellTiter-Glo assay was used to determine the cell viability, as previously described¹⁵. Briefly, 4000 cells/well were grown in a 96-well plate and incubated overnight. The following day, the medium was replaced with treatment media containing 5% FBS and the respective drugs, followed by a 72-hour incubation. The concentrations used for Cis and Cis-Rub were 5, 10, 20, 30, 40, and 60 µM. After the treatment period, luminescence was measured using a Synergy HT microplate reader following the addition of the CellTiter-Glo reagent.

3. Cell Cycle Analysis:

Cell cycle analysis was done using Annexin-V/7AAD assay^16,19. Briefly, 10,000 cells were collected and washed with cold PBS. Cells were re-suspended in 95µL of PBS and 5µL staining solutions were added. The solution was incubated in dark for 20min. After, 20 min, 400µL PBS was added and mixed will. Cells were analyzed using flow cytometer (BD Curri C6 plus) at 488 and 530nm.

4. Western-Blot Method:

In brief, cells were homogenized and proteins were extracted using NP40 buffer. Equal amounts of protein (50μg/Lane) were separated on a 4–20% gradient SDS-PAGE gel. Proteins were then transferred to nitrocellulose membranes, which were blocked with 5% fat-free milk in 0.05% Tween-20 in 20mM phosphate-buffered saline (PBST, pH 7.4). Membranes were incubated overnight at 4°C with primary antibodies diluted at 1:500 or 1:5000. Further, blots were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution) and visualized using the SuperSignal West Femto substrate. GAPDH served as a loading control for total protein^15,20.

5. Cisplatin HPLC:

HPLC analysis was performed as described previously^17,18. In brief, samples were dissolved in ethanol at a 1:1(v/v) ratio to extract Cis. A 50μL aliquot of each sample was injected onto a normal-phase silica column (5μm ZORBAX Rx-SIL). The analysis was performed using an Agilent 1220 Infinity LC system equipped with an Agilent 1260 fluorescence detector (Agilent Technologies). A linear gradient elution (0–15min, 1mL per min) was applied using solvent system-1(chloroform/ethanol/o-phosphoric acid, 80:20:0.1, v/v/v) and solvent system-2 (chloroform/ethanol/ water/o-phosphoric acid, 60:34:6:0.1, v/v/v/v). Cis detected by its UV absorbance at 486nm. Quantification was performed by comparison with a standard curve.

RESULTS AND DISCUSSION:

1. Characterization of Cis-Rub nanomicelles:

Solubility plays very important role in the cellular bioavailability. To improve this bioavailability Cis was encapsulated with Rub. Cis-Rub nanomicelles was soluble generating a clear medium at concentration of 50 mg/ml. Particle size was determined using TEM, which show that Cis-Rub nanomicelles has diameter 32nm. The particle size distribution was calculated using DLS analysis. DLS analysis shows that most particles have size of ~32nm. This shows that most particles are uniformed size and improves solubility of Cis.

Fig. 1. Characterization of Cis-Rub nanomicelles: (A) Graphical representation of Cis-Rub nanomicelles formation (B) TEM image of Cis-Rub nanomicelles (C) particle size distribution of Cis-Rub nanomicelles

2. Cis-Rub nanomicelles improves Cis uptake in cancer cells:

Uptake of Cis by MCF-7/Adr were studied using HPLC. MCF-7/Adr cells were treated with Cis or Cis-Rub for 0, 30, 60 120, and 180mins and Cis was extracted from the cells and samples were analyzed. The results shows that, with increase in time Cis uptake was increased for both Cis and Cis-Rub. At 180min Cis-Rub reached max concentration of 311µM, which is 4-fold more for Cis. While Cis reached max concentration of 182µM at 120 min.

Fig.2. Uptake of Cis-Rub nanomicelles vs Cis in MCF-7/Adr cells (A) HPLC curve of Cis (B) Std curve of HPLC (C) Uptake of Cis by MCF-7/Adr cells at different time points

3. Cis-Rub nanomicelles increases anti-cancer activity of Cis:

Anti-cancer activity of Cis vs Cis-Rub was accessed using CellTiter-Glo cell viability assay. We have used multi drug resistant breast cancer cells: MCF-7/Adr and MDA-MB231. Cells were treated with Cis vs Cis-rub. Our results shows that IC50 of Cis vs Cis-Rub in MCF-7/Adr and MDA-MB231 was 32 vs 12µm and 50 vs 23 µm, respectively. There results shows that Cis-Rub nanomicelles are more potent than Cis. Cis-Rub nanomicelles has better anticancer activity and can be a better option for cis for treatment of drug-resistant breast cancer.

Fig. 3. Anticancer activity of Cis-Rub nanomicelles vs Cis. (A) IC50 of Cis-Rub nanomicelles vs Cis in MCF-7/Adr and MDA-MB-231 cells (B) Cell viability curve of Cis-Rub nanomicelles vs Cis in MCF-7/Adr cells (C) Cell viability curve of Cis-Rub nanomicelles vs Cis in MDA-MB-231 cells

4. Cis-Rub nanomicelles induces apoptosis and upregulates p21:

Apoptosis is important for cancer treatment. Most successful cancer drug induce apoptosis. Apoptosis was accessed using 7AAD/Annexin-V assay. The results shows that when MCF-7 cells treated with Cis vs Cis-Rub shows early apoptotic cells in 10.17% vs 42.17% cells while late apoptotic cells were 0.69% vs 11.96% respectively. We also performed a western blot analysis of p21 to understand the mechanism, as p21 is involved in the apoptosis. The p21/GAPDH expression for Cis vs Cis-Rub was 0.32 vs 0.72. This shows that expression of p21 was increased after Cis-Rub treatment compared with Cis.

Fig.4. Mechanistic and apoptosis study. (A) p21 expression study using western blotting (B) Expression of p21/GAPDH (C) Apoptosis study in MCF-7/Adr using flowcytometry (D) Percentage of cells in live, early apoptotic and late apoptotic stages

CONCLUSION:

In here, we have developed Cis-Rub nanomicelles as novel therapy for treatment of multi drug resistant breast cancer cells. Synthesized Cis-Rub nanomicelles had means size of ~32nm with uniform distribution. The in-vitro update study showed that update of Cis-Rub was higher with longer time compared with Cis. This shows that update of Cis was increased in MCF-7/Adr cells. Anti-cancer activity shows that Cis-Rub has better potency compared with Cis. To understand the mechanism, we have studied the apoptosis in MCF-7/Adr cells. Cis-Rub induce apoptosis in cells by increasing the upregulation of p21. In conclusion, these results shows that Cis-Rub provides better option for treatment of multi drug resistant breast cancer cells.

DECLARATION OF INTEREST:

None.

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Received on 28.04.2025 Revised on 11.08.2025

Accepted on 17.11.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2117-2120.

DOI: 10.52711/0974-360X.2026.00304

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