Hybrid Cell Membrane-Functionalized Biomimetic Nanoparticles for Targeted Therapy of Osteosarcoma

Figures & data

Figure 1 Preparation and characterization of PLGA@[143B-RAW] NPs. (A) Preparation of paclitaxel (PTX)-loaded PLGA nanoparticles with 143B-RAW hybrid membrane coating (PTX-PLGA@[143B-RAW] NPs). (B) Membrane extraction process by gradient centrifugation. (C) The particle size and zeta potential of PLGA nanoparticles. (D) The particle size of the membrane-coated nanoparticles prepared by the two methods. (I) co-extrusion; (II) ultrasonication. (E) TEM images of (I) bare nanoparticles and (II) hybrid membrane coated nanoparticles. (F) Protein distribution of cell membrane. (I) PLGA nanoparticles; (II) 143B cell membrane; (III) RAW264.7 cell membrane; (IV) Hybrid membrane; (V) PLGA@143B NPs; (VI) PLGA@RAW NPs; (VII) PLGA@[143B-RAW] NPs. (G) Comparison of protein distribution of cell membrane coated nanoparticles prepared by different methods. (I) PLGA nanoparticles; (II) PLGA@143B NPs by co-extrusion; (III) PLGA@RAW NPs by co-extrusion; (IV) PLGA@[143B-RAW] NPs by co-extrusion; (V) PLGA@143B NPs by ultrasonication; (VI) PLGA@RAW NPs by ultrasonication; (VII) PLGA@[143B-RAW] NPs by ultrasonication. (H) Average size of membrane coated nanoparticles over 8 days in PBS. (I) PLGA@143B NPs; (II) PLGA@RAW NPs; (III) PLGA@[143B-RAW] NPs. (I) Western blot analysis of cell membrane proteins. (I) PLGA nanoparticles; (II) 143B cell membrane; (III) RAW264.7 cell membrane; (IV) Hybrid membrane; (V) PLGA@143B NPs; (VI) PLGA@RAW NPs; (VII) PLGA@[143B-RAW] NPs. Characteristic 143B membrane marker was MMP-2, and characteristic RAW264.7 membrane marker was integrin α4. (Na⁺-K⁺-ATPase was used as a reference protein) (J) PTX release from (I) PLGA NPs at pH 7.4; (II) PLGA@[143B-RAW] NPs at pH 7.4; (III) PLGA NPs at pH 5.3; (IV) PLGA@[143B-RAW] NPs at pH 5.3.

Figure 2 The uptake of PLGA@[143B-RAW] NPs. (A) 143B cells uptake of different nanoparticles. (B) 143B cells uptake of PLGA@[143B-RAW] NPs at different times. Nucleus was labeled by DAPI (Blue). PLGA NPs was labeled by Dil (Red). (C) Semi-quantitative analysis of 143B cells uptake of Dil stained nanoparticles. (D) Semi-quantitative analysis of 143B cells uptake of PLGA@[143B-RAW] NPs at different times. (I) PLGA NPs; (II) PLGA@143B NPs; (III) PLGA@RAW NPs; (IV) PLGA@[143B-RAW] NPs. Scale bar=50 μm. Each point represents the mean ± SD, ****P < 0.0001.

Figure 3 The cytotoxicity of PTX-PLGA@[143B-RAW] NPs in vitro. (A) Effects of different preparations on the viability of 143B cells. (I) PLGA-PTX NPs; (II) PTX-PLGA@143B NPs; (III) PTX-PLGA@RAW NPs; (IV) PTX-PLGA@[143B-RAW] NPs. (B) Apoptosis of 143B cells induced by different preparations. (I) Free PTX; (II) PLGA-PTX NPs; (III) PTX-PLGA@143B NPs; (IV) PTX-PLGA@RAW NPs; (V) PTX-PLGA@[143B-RAW] NPs. Each point represents the mean ± SD, **P < 0.01.

Figure 4 Chemotaxis of different nanoparticles on HUVEC cells. (A) The fluorescence co-localization of different preparations and HUVEC cells. The nucleus was labeled by DAPI (Blue). PLGA NPs were labeled by Dil (Red). (B) Semi-quantitative analysis of chemotaxis of HUVEC cells by different nanoparticles. The inflammatory factor TNF-α provided the inflammatory environment. (I) PLGA NPs; (II) PLGA@143B NPs; (III) PLGA@RAW NPs; (IV) PLGA@[143B-RAW] NPs. Scale bar=50 μm. Each point represents the mean ± SD, ****P < 0.0001.

Figure 5 In vivo biodistribution of PLGA@[143B-RAW] NPs. (A) In vivo biodistribution of different formulations intravenously administered to 143B xenograft mice at different times. (B) Ex vivo images of major organs and tumors from 143B xenograft mice 24 h after intravenous injection of the five formulations. (C) Fluorescence intensity of the five formulations at tumors and major organs 24 h after intravenous injection. (I) DIR; (II) PLGA NPs; (III) PLGA@RAW NPs; (IV) PLGA@143B NPs; (V) PLGA@[143B-RAW] NPs.

Figure 6 Therapeutic efficacy of PTX-PLGA@[143B-RAW] NPs against 143B tumors. (A) Absolute and (B) relative 143B tumor growth in mice during the experimental period. (C) Excised tumors at the end of experiments. (D) Tumor weight at the end of experiments. (I) PBS; (II) Free PTX; (III) PLGA-PTX NPs; (IV) PTX-PLGA@RAW NPs; (V) PTX-PLGA@143B NPs; (VI) PTX-PLGA@[143B-RAW] NPs. N = 5, each point represents the mean ± SD, *P < 0.05, **P < 0.01.

Figure 7 Histopathological confirmation of efficient therapeutic efficacy of PTX-PLGA@[143B-RAW] NPs against 143B tumor. H&E and TUNEL-stained tumors of mice after treatment. (I) PBS; (II) Free PTX; (III) PLGA-PTX NPs; (IV) PTX-PLGA@RAW NPs; (V) PTX-PLGA@143B NPs; (VI) PTX-PLGA@[143B-RAW] NPs. Scale bar = 200 μm.

Figure 8 Safety and biocompatibility evaluation of PTX-PLGA@[143B-RAW] NPs. (A) Absolute and (B) relative body weight change of tumor-bearing mice during the experimental period, n = 5. (C) H&E-stained hearts, livers, spleens, lungs and kidneys of mice after treatment. Scale bar = 200 μm. Plasma levels of (D) ALT, (E) AST, (F) BUN, (G) Cr in tumor-bearing mice two days after treatment for four times with an interval of two days, n = 5. (I) PBS; (II) Free PTX; (III) PLGA-PTX NPs; (IV) PTX-PLGA@RAW NPs; (V) PTX-PLGA@143B NPs; (VI) PTX-PLGA@[143B-RAW] NPs. Each point represents the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.