Formulation and Characterization of Polymeric Nanoparticles for Targeted Cancer Therapy

Cancer remains one of the foremost causes of morbidity and mortality globally, with breast cancer being a major contributor among female populations. Despite advances in chemotherapy, conventional anticancer agents such as doxorubicin suffer from limitations including poor selectivity, dose-limiting toxicity, and the development of multidrug resistance (MDR). These challenges highlight the urgent need for novel drug delivery systems that can enhance therapeutic efficacy while minimizing systemic side effects. Nanotechnology-based approaches, particularly polymeric nanoparticles (PNPs), have gained significant attention for cancer therapy. Among the various polymers investigated, poly(lactic-co-glycolic acid) (PLGA) stands out due to its biodegradability, biocompatibility, and FDA approval for clinical use. PLGA nanoparticles offer several advantages such as prolonged circulation time, controlled drug release, improved drug stability, and potential for passive or active tumor targeting through the enhanced permeability and retention (EPR) effect.

In this context, doxorubicin-loaded PLGA nanoparticles provide a promising strategy to overcome the limitations of free doxorubicin. By encapsulating doxorubicin within PLGA, it is possible to achieve a sustained drug release profile, reduce systemic exposure, and enhance cytotoxic activity at the tumor site through improved cellular uptake. The present study is designed to develop and optimize doxorubicin-loaded PLGA nanoparticles using the nanoprecipitation technique. The prepared formulations are characterized in terms of particle size, zeta potential, encapsulation efficiency, and drug release kinetics. Furthermore, the in vitro cytotoxic activity is evaluated on MCF-7 breast cancer cell lines using the MTT assay to assess the therapeutic potential of the nanoparticles.

2. MATERIALS AND METHODS

2.1 Materials

The following materials were used in the present study:

• Doxorubicin Hydrochloride – obtained from a certified pharmaceutical supplier and used as the model anticancer drug.
• Poly(lactic-co-glycolic acid) (PLGA) – with a lactic to glycolic acid ratio of 50:50, procured from a commercial source; used as the biodegradable polymeric carrier.
• Polyvinyl Alcohol (PVA) – used as a stabilizer during nanoparticle formation.
• Acetone – analytical grade, used as an organic solvent for nanoprecipitation.
• Deionized Water – used throughout the experimental procedures for nanoparticle preparation and washing.

2.2 Method of Preparation

The nanoprecipitation technique was employed to prepare doxorubicin-loaded PLGA nanoparticles due to its simplicity, reproducibility, and suitability for hydrophobic polymer–drug systems.

Procedure:

1. Organic Phase Preparation

Doxorubicin hydrochloride (equivalent to the drug-to-polymer ratio of 1:5) and PLGA were accurately weighed and dissolved in 10 mL of acetone, forming the organic phase. The drug-polymer solution was vortexed briefly to ensure complete solubilization.

2. Aqueous Phase Preparation

The aqueous phase consisted of 20 mL of 1% w/v polyvinyl alcohol (PVA) solution, which served as a stabilizer to prevent nanoparticle aggregation and improve dispersion.

3. Nanoparticle Formation

The organic phase was added dropwise into the aqueous phase under continuous magnetic stirring at 1200 rpm. The addition was carried out at room temperature to promote spontaneous diffusion of acetone into the aqueous phase, leading to the precipitation of PLGA and the entrapment of doxorubicin within the forming nanoparticles.

4. Stirring and Solvent Evaporation

After the addition was complete, the emulsion was continuously stirred for 2 hours to allow complete evaporation of acetone, solidification of nanoparticles, and stabilization in suspension.

5. Purification
   The resulting nanoparticle suspension was centrifuged at 15,000 rpm for 20 minutes to separate the nanoparticles from the unencapsulated drug and excess PVA. The pellet was washed three times with deionized water to remove residual PVA and then lyophilized for further analysis.

Process Parameters

3. Optimization via Design of Experiments (DoE)

To optimize the formulation of doxorubicin-loaded PLGA nanoparticles, a Box-Behnken Design (BBD) was applied with three independent variables, evaluated at three levels (low, medium, high). A total of 17 experimental runs were generated to understand the influence of formulation and process parameters on two key responses: particle size (Y?) and entrapment efficiency (Y?).

3.1 Independent Variables and Their Levels

3.2 Design Matrix with Observed Responses

3.3 Model Evaluation via ANOVA

• Model F-value: 26.37, indicating the model is statistically significant (p < 0.0001)
• Lack of Fit: Non-significant (p = 0.2391) – shows the model fits well to the data
• R² = 0.9732, Adjusted R² = 0.9567 – confirms high predictability of the model

3.4 Optimization Outcome

The optimal formulation predicted by the BBD model was:

• PLGA concentration: 1.0%
• PVA concentration: 1.0%
• Stirring Speed: 1200 rpm

Predicted responses:

• Particle size: ~158.6 nm
• Entrapment efficiency: ~78.4%

4. Characterization

Comprehensive characterization of the optimized doxorubicin-loaded PLGA nanoparticles was performed to evaluate their physicochemical and morphological properties. The key parameters assessed include particle size, polydispersity index (PDI), zeta potential, entrapment efficiency, and surface morphology using standard analytical techniques.

4.1 Particle Size, Polydispersity Index (PDI), and Zeta Potential

The particle size and surface charge were measured using dynamic light scattering (DLS) via a Zetasizer Nano ZS instrument.

• Mean Particle Size: 158.6 ± 3.4 nm
• Polydispersity Index (PDI): 0.179
• Zeta Potential: -21.3 ± 1.8 mV

The particle size was within the optimal range for passive tumor targeting via the Enhanced Permeability and Retention (EPR) effect. The low PDI value (< 0.2) indicates a narrow size distribution, essential for consistent biological performance. The negative zeta potential contributes to electrostatic stabilization of the nanoparticle suspension, reducing the likelihood of aggregation during storage.

4.2 Entrapment Efficiency (EE%)

The entrapment efficiency was determined by separating free doxorubicin from the nanoparticles using centrifugation, followed by UV-Vis spectrophotometric analysis of the supernatant.

The entrapment efficiency was calculated using the formula:

\text {Entrapment Efficiency (EE%)} = \left ( \frac {\text{Amount of drug entrapped}}{\text{Total amount of drug added}} \right) \times 100

• Entrapment Efficiency: 78.4 ± 2.1%

The high EE% reflects effective encapsulation of doxorubicin within the PLGA matrix, likely due to hydrophobic interactions between the drug and polymer during nanoprecipitation.

4.3 Morphological Analysis

The morphology of the nanoparticles was assessed using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM).

• The nanoparticles exhibited a smooth, spherical shape with uniform surface characteristics.
• No signs of aggregation or fusion were observed, indicating successful stabilization by PVA and adequate surface charge repulsion.

5. In Vitro Drug Release Study

The in vitro drug release profile of the optimized doxorubicin-loaded PLGA nanoparticles was investigated using the dialysis bag diffusion method in phosphate-buffered saline (PBS, pH 7.4) at 37?±?0.5?°C under constant stirring (100 rpm). Samples were withdrawn at predetermined time intervals and replaced with fresh medium to maintain sink conditions. The cumulative drug release was quantified by UV-Visible spectrophotometry at λ_max of 480 nm.

5.1 Cumulative Drug Release Profile

5.2 Drug Release Pattern and Kinetic Modeling

The release profile exhibited a biphasic pattern, characterized by:

• An initial burst release of approximately 21.7% within the first 4 hours, attributed to the release of surface-adsorbed or loosely bound doxorubicin.
• A sustained release phase over 72

• hours, likely due to diffusion of drug through the PLGA matrix and subsequent polymer erosion.

To elucidate the mechanism of drug release, the data were fitted to various kinetic models, including Zero-order, First-order, Higuchi, and Korsmeyer–Peppas models. Among them:

• First-order kinetics best described the release profile, with a high correlation coefficient R² = 0.9812, indicating that the drug release rate is concentration-dependent.
• The Higuchi model also showed good fit (R² = 0.9543), suggesting that Fickian diffusion is a dominant mechanism during the sustained phase.

5.3 Interpretation

The prolonged and controlled release of doxorubicin from PLGA nanoparticles is beneficial for cancer therapy, as it ensures continuous exposure of tumor cells to therapeutic drug levels while minimizing systemic toxicity. The combination of burst release (for immediate therapeutic effect) and sustained release (for maintenance) positions this formulation as a promising candidate for targeted chemotherapy.

6. Cytotoxicity (MTT Assay)

The in vitro cytotoxic potential of doxorubicin-loaded PLGA nanoparticles (Dox-PLGA NPs) was assessed using the MTT assay on MCF-7 human breast cancer cell lines. The test was conducted to compare the anticancer efficacy of the nanoparticle formulation with that of free doxorubicin after 48 hours of incubation.

6.1 Methodology

MCF-7 cells were seeded in 96-well plates at a density of 1×10? cells/well and allowed to adhere overnight. The cells were then treated with various concentrations of:

• Free Doxorubicin
• Doxorubicin-loaded PLGA nanoparticles (Dox-PLGA NPs)

After 48 hours of incubation, 20 µL of MTT reagent (5 mg/mL) was added to each well, and the plates were incubated for 4 hours. The resulting formazan crystals were solubilized using DMSO, and absorbance was measured at 570 nm using a microplate reader.

6.2 IC?? Values

6.3 Statistical Analysis

The IC?? values were compared using Student’s t-test, and the results showed:

• p < 0.01, indicating that the cytotoxic effect of Dox-PLGA nanoparticles was significantly greater than that of

• free doxorubicin.

6.4 Interpretation

The enhanced cytotoxicity of Dox-PLGA NPs can be attributed to several factors:

• Improved cellular uptake of nanoparticles through endocytosis.
• Sustained intracellular release of doxorubicin from the PLGA matrix.
• Enhanced drug accumulation within cancer cells due to prolonged exposure.

7. Stability Studies

Stability studies were conducted to evaluate the physicochemical stability of the optimized doxorubicin-loaded PLGA nanoparticles under different storage conditions over 3 months. The formulations were stored in sealed amber glass vials at two temperature conditions:

• Refrigerated (4?°C)
• Room Temperature (25?°C)

Key parameters, including particle size and entrapment efficiency (EE%), were monitored at the end of the study period to assess the integrity and retention characteristics of the nanoparticle formulation.

7.1 Results of Stability Evaluation

7.2 Interpretation

• Particle Size: An increase in particle size was observed at 25?°C, indicating potential aggregation or surface adsorption, likely due to increased molecular mobility and reduced polymer rigidity at higher temperatures.
• Entrapment Efficiency: A noticeable decline in EE% was seen at room temperature, possibly due to drug leakage or degradation, whereas storage at 4?°C maintained better drug retention.

8. RESULT

The present study successfully developed and optimized doxorubicin-loaded PLGA nanoparticles using the nanoprecipitation technique. The outcomes from formulation, optimization, characterization, release kinetics, cytotoxicity, and stability studies are summarized below:

8.1 Nanoparticle Formulation and Optimization

• Nanoparticles were prepared using a drug:polymer ratio of 1:5, 10 mL acetone as organic solvent, and 20 mL of 1% PVA as the aqueous stabilizing phase.
• Box-Behnken Design (BBD) was used to optimize formulation variables (PLGA %, PVA %, and stirring speed).
• Optimal conditions: PLGA 1%, PVA 1%, and stirring speed 1200 rpm.
• Model F-value: 26.37 (p < 0.0001)
• Lack of Fit: Non-significant (p = 0.2391)
• R² = 0.9732, Adjusted R² = 0.9567

8.2 Characterization of Optimized Nanoparticles

• Mean Particle Size: 158.6 ± 3.4 nm
• Polydispersity Index (PDI): 0.179 (indicating narrow size distribution)
• Zeta Potential: –21.3 ± 1.8 mV (good stability)
• Entrapment Efficiency (EE%): 78.4 ± 2.1%
• Morphology (SEM & TEM): Smooth, spherical, non-aggregated particles

8.3 In Vitro Drug Release

• The nanoparticles exhibited a biphasic drug release pattern:
  • Burst release: 21.7% in the first 4 hours
  • Sustained release: Up to 91.6 ± 3.1% over 72 hours
• Release kinetics:
  • First-order model: Best fit (R² = 0.9812)
  • Higuchi model: R² = 0.9543

8.4 Cytotoxicity (MTT Assay)

• Cell Line: MCF-7 human breast cancer cells
• IC?? (Free Doxorubicin): 7.4 ± 0.6 µg/mL
• IC?? (Dox-PLGA NPs): 2.9 ± 0.3 µg/mL
• Statistical significance: p < 0.01 (Student’s t-test)
• The nanoparticle formulation demonstrated significantly enhanced cytotoxicity, likely due to improved cellular uptake and sustained intracellular drug release.

8.5 Stability Study

• Storage conditions: 4°C and 25°C for 3 months
• Particle Size increased from 158.6 nm to 173.4 nm at 25°C; remained relatively stable (160.8 nm) at 4°C
• Entrapment Efficiency decreased to 71.2% at 25°C but was retained at 76.5% at 4°C
• Conclusion: Better stability at 4°C in terms of particle integrity and drug retention