Nanoplatelets modified with RVG for targeted delivery of miR-375 and temozolomide to enhance gliomas therapy

Abstract

Gliomas are one of the most frequent primary brain tumors and pose a serious threat to people's lives and health. Platelets, a crucial component of blood, have been applied as drug delivery carriers for disease diagnosis and treatment. In this study, we designed engineered nanoplatelets for targeted delivery of therapeutic miR-375 and temozolomide (TMZ, a first-line glioma treatment agent) to enhance glioma therapy. Nanoplatelets were prepared through mild ultrasound, TMZ and miR-375 were co-loaded through ultrasound and electrostatic interactions, respectively, to combine chemotherapy with gene therapy against glioma. To improve the blood brain barrier (BBB) crossing efficiency and glioma targeting ability, the nanoplatelets were modified with central nervous system-specific rabies viral glycoprotein peptide (RVG) through thiol-maleimide click reaction. The RVG modified nanoplatelets co-loaded TMZ and miR-375 (NR/TMZ/miR-375) not only inherited the good stability and remarkable biocompatibility of platelets, but also promoted the cellular uptake and penetration of glioma tissues, and effectively induced cell apoptosis to enhance the therapeutic effect of drugs. In vivo studies showed that NR/TMZ/miR-375 significantly increased the circulation time of TMZ, and exhibited superior combined antitumor effects. In summary, this multifunctional 'natural' nanodrug delivery system provides a potent, scalable, and safety approach for platelet-based combined cancer chemotherapy and gene therapy.

Keywords: Gliomas; MiR-375; Nanoplatelets; TMZ; Targeted drug delivery.

Fig. 1

Schematic illustration of synthetic procedure of engineered nanoplatelets and gene therapy combined with chemotherapy against glioma

Fig. 2

Characterization of nanoplatelets. (A) TEM images of NPs. Scale bar: 500 nm (left), 100 nm (right). (B) DLS particle size distribution of NPs. (C) TEM images of NR/TMZ/miR-375. Scale bar: 500 nm (left), 100 nm (right) (D) DLS particle size distribution of NR/TMZ/miR-375. (E) CLSM images of nanoplatelets (DiI) modified with RVG (FITC). (F) Zeta potential analysis of nanoplatelets modified with various concentrations of PEI. The results are shown as mean ± standard deviation, n = 3. (G) Results of agarose gel electrophoresis analysis of nanoplatelets incubated with various concentrations of miR-375

Fig. 3

Drug loading properties and stability of nanoplatelets. (A) DLE and (B)EE of TMZ in nanoplatelets incubated with different concentrations of TMZ. (C) Zeta potential of NP and (D) NR/TMZ/miR-375. (E) Stability study of NP and NR/TMZ/miR-375 in PBS (pH 7.4, n = 3) within 7 days. (F) Kinetics of cumulative release of TMZ from NP/TMZ and NR/TMZ/miR-375 in PBS (pH 7.4, n = 3)

Fig. 4

In vitro cellular uptake and penetration evaluation of nanoplatelets. The cellular uptake of (A) NP, (B) NR and (C) NR/TMZ/miR-375 by U87 cells at different time points was observed by CLSM (scale bars 50 μm). (D) Schematic illustrates of in vitro penetration evaluation (left), distribution of NP, NR and NR/TMZ/miR-375 in glioma organoids (scale bars 50 μm)

Fig. 5

(A) Quantitative statistical results of cellular uptake. (B) The effects of different nanoparticles on the cell viability of U87 and SH-SY5Y cells for 24 h and 48 h. (C) The effect of engineered nanoplatelets on the migration of U87 and SY5Y cells and (D) the statistical results of the relative migration distance ratio; (E) Flow cytometry results of U87 and SY5Y cells treated with different groups and (F) statistical results of apoptotic rate. (1: control, 2: NP, 3: NP/miR-375, 4: NR/miR-375, 5: NR/TMZ, 6: NR/TMZ/miR-375; The results are shown as mean ± standard deviation, n = 3, *P < 0.05, **P < 0.01 was statistically significant)

Fig. 6

Western blot studies of U87 cells treated with nanoplatelets. (A) The expression level of miR-375 target protein was detected by Western blot. (B, C) Relative quantitative statistical results of YAP1 and CTGF protein expression. (D) Expression levels of apoptosis-related proteins in different experimental groups. (E-G) Relative quantitative statistical results of cleaved Caspase3, Bax, and Bcl-2 protein expression. (H) miR-375 gene expression in U87 and SH-SY5Y (I) cells. (1: control, 2: NP/NC, 3: NP/miR-375, 4: NR/miR-375, 5: NR/TMZ, 6: NR/TMZ/miR-375; The results are shown as mean ± standard deviation, n = 3, *P < 0.05, **P < 0.01 was statistically significant)

Fig. 7

In vivo targeting of engineered nanoplatelets. (A) Accumulation of nanoplatelets in subcutaneous glioma model of mice and (B) quantitative statistical analysis of tumors; (C) Accumulation of nanoplatelets in the organs of mice with subcutaneous tumor and (D) quantitative statistical analysis; (E) Accumulation of nanoplatelets in orthotopic glioma model of mice and (F) quantitative statistical analysis of tumors; (G) Accumulation of nanoplatelets in the organs of mice with orthotopic glioma model and (H) quantitative statistical analysis; (The results were shown as mean ± standard deviation, n = 3; *P < 0.05, **P < 0.01 was statistically significant)

Fig. 8

In vivo antitumor study. (A) Schematic diagram of the in vivo antitumor study of nanoplatelets. (B) Fluorescence imaging of orthotopic gliomas in vivo. (C) TMZ plasma concentrations after intravenous injection of free TMZ and NR/TMZ/miR-375 in mice. (D) Statistical analysis of tumor fluorescence imaging results. (E) Statistical results of body weight changes in mice during treatment. (F) Survival curve of orthotopic glioma mice. (G) Biosafety evaluation of major organs (The results were shown as mean ± standard deviation, n = 5; ^**p < 0.01, ^***p < 0.001 was statistically significant)