Design-of-Experiment-Assisted Fabrication of Biodegradable Polymeric Nanoparticles: In Vitro Characterization, Biological Activity, and In Vivo Assessment

[Image: see text] Berberine (BER) is an alkaloid obtained from berberis plant having broad biological activities including anticancer. BER-encapsulated alginate (ALG)/chitosan (CHS) nanoparticles (BER–ALG/CHS-NPs) were developed for long-acting improved treatment in breast cancer. The surface of the NPs was activated by a conjugation reaction, and thereafter, the BER–ALG/CHS-NP surface was grafted with folic acid (BER–ALG/CHS-NPs–F) for specific targeting in breast cancer. BER–ALG/CHS-NPs–F was optimized by applying the Box–Behnken design using Expert design software. Moreover, formulations are extensively evaluated in vitro for biopharmaceutical performances and tested for cell viability, cellular uptake, and antioxidant activity. The comparative pharmacokinetic study of formulation and free BER was carried out in animals for estimation of bioavailability. The particle size recorded for the diluted sample using a Malvern Zetasizer was 240 ± 5.6 nm. The ζ-potential and the predicted % entrapment efficiency versus (vs) observed were +18 mV and 83.25 ± 2.3% vs 85 ± 3.5%. The high % drug release from the NPs was recorded. The analytical studies executed using infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction expressed safe combinations of the components in the formulation and physical state of the drug revealed to be amorphous in the formulation. Cytotoxicity testing demonstrated that the formulation effectively lowered the cell viability and IC(50) of the tested cell line in comparison to a raw drug. The cellular uptake of BER–ALG/CHS-NPs–F was 5.5-fold higher than that of BER–suspension. The antioxidant capacities of BER–ALG/CHS-NPs–F vs BER–suspension by the DPPH assay were measured to be 62.3 ± 2.5% vs 30 ± 6%, indicating good radical scavenging power of folate-conjugated NPs. The developed formulation showed a 4.4-fold improved oral bioavailability compared to BER–suspension. The hemolytic assay intimated <2% destruction of erythrocytes by the developed formulation. The observed experimental characterization results such as cytotoxicity, cellular uptake, antioxidant activity, and improved absorption suggested the effectiveness of BER–ALG/CHS-NPs–F toward breast cancer.