Stimuli-responsive magnetic silica-poly-lactic-co-glycolic acid hybrid nanoparticles for targeted cancer chemo-immunotherapy

Abstract

Chemotherapy and immunotherapy are two important modalities in cancer management. However, due to multiple reasons, a monotherapy is only partially effective. Hence, if used concurrently in targeted and stimuli-responsive manner, it could have been superior therapeutically. To facilitate co-delivery of chemotherapeutic and immunotherapeutic agent to the target cancer cells, engineered nanoparticles, i.e., a pH-responsive polymer PLGA-coated magnetic silica nanoparticles (Fe₃O₄-SiO₂-PLGA-PDA-PTX-siRNA NPs) encapsulating paclitaxel (PTX) and siRNA against programmed cell death ligand-1 (PD-L1) are synthesized and characterized. Developed nanoparticles demonstrated pH-sensitive sustained drug release up to 10 days. In vitro 4T1 cell line studies showed efficient cellular uptake, PD-L1 gene downregulation, and apoptosis. Further, in vivo efficacy studies carried out in the mice model demonstrated a significant reduction of tumor growth following treatment with dual-Fe₃O₄-SiO₂-PLGA-PDA-PTX-siRNA NPs as compared with monotherapy with Fe₃O₄-SiO₂-PLGA-PDA-PTX NPs. The high therapeutic efficacy observed with dual-Fe₃O₄-SiO₂-PLGA-PDA-PTX-siRNA NPs was mainly due to the cytotoxic effect of PTX combined with targeted silencing of the gene of interest, i.e., PD-L1, which in turn improve CD8⁺ T cell-mediated cancer cell death as evident with increased proliferation of CD8⁺ T cells in co-culture experiments. Thereby, dual-Fe₃O₄-SiO₂-PLGA-PDA-PTX-siRNA NPs may have a promising anti-cancer treatment potential against breast cancer; however, the beneficial effects of dual loading of PTX + PD-L1 siRNA may be corroborated against other cancer models such as lung and colorectal cancer models as well as in clinical trials.

Graphical Abstract

Nanoparticles targeting to the tumor site, pH-specific release of PTX and PD-L1 siRNA by dual-loaded fabricated nanoparticles within tumor cells for enhanced chemo-immunotherapy.

Supplementary methods

Synthesis of pH-responsive PLGA-coated core-shell magnetic silica nanoparticle

In brief, iron oxide nanoparticles (Fe₃O₄) were prepared by co-precipitation of iron (II) and iron (III) chloride salts under N2 atmosphere using 2M NaOH [26]. Initially, the iron salts were dissolved in the 50 ml of water in the molar ratio of 2:1, then NaOH was added slowly and the reaction temperature was raised to 70 °C. After 3h, the reaction was cooled down and the Fe₃O₄ was collected with the help of magnet and vacuum dried.

In the second step, core-shell magnetic silica nanoparticles were prepared by modified sol-gel homogenous co-precipitation process [27]. Fe₃O₄ NPs were dispersed in a mixture of ethanol and water (80:20). Tetra ethyl ortho silicate (TEOS), 0.1 ml (0.1% w/v) was added to the magnetic nanoparticles and stirred for 10 min at room temperature. Then, 2M NaOH (1 ml) was added slowly and reaction mixture was stirred for 6 h at room temperature. The core-shell magnetic silica nanoparticles (Fe₃O₄-SiO₂ NPs) were separated using an external magnet, washed with deoxygenated distilled water and vacuum dried. To achieve amine functionalization, Fe₃O₄-SiO₂ NPs were dispersed in pure ethanol and 2 mmol, 3-aminopropyl triethoxysilane (APTES) was added to it, refluxed at 70 °C, washed with ethanol two-three times and vacuum dried.

In the third step, PLGA-coated Fe₃O₄-SiO₂ nanoparticles were prepared by double emulsion solvent evaporation method [28]. Poly lactic co-glycolic acid (PLGA) was dissolved in dichloromethane (DCM). Fe₃O₄-SiO₂ NPs were dispersed in 0.5 ml of 1% PVA solution and added to PLGA solution with probe sonication (30% amplitude, 20 duty cycles, 2 min) to form primary emulsion (W₁/O). The ratio of Fe₃O₄-SiO₂ nanoparticles to PLGA was optimized as 1:1. Thus formed primary emulsion was then added to 30 ml of 2% PVA solution with sonication (30% amplitude, 20 duty cycles, 3 min) to form double emulsion (W₁/O/W₂) and continuously stirred for 5 h to evaporate DCM. After 5 h, PLGA-coated nanoparticles designated as Fe₃O₄-SiO₂-PLGA were collected using external magnet, washed and dried.

In the fourth step, in order to fabricate pH-sensitive release system, PLGA-coated Fe₃O₄-SiO₂ nanoparticles (100 mg) were dispersed in 10 ml of dopamine hydrochloride solution in Tris-HCl buffer (10 mmol/L, pH 8.5) and continuous stirred for 6 h at room temperature [29]. After 6 h, poly dopamine (PDA) capped nanoparticles designated as Fe₃O₄-SiO₂-PLGA-PDA NPs/bare NPs were separated with magnet, washed with water, lyophilized and stored for further use.