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. 2020 Feb 10;10(7):3325-3339.
doi: 10.7150/thno.41228. eCollection 2020.

Bioinspired tumor-homing nanoplatform for co-delivery of paclitaxel and siRNA-E7 to HPV-related cervical malignancies for synergistic therapy

Cong Xu  1   2 , Wan Liu  1   2 , Yuan Hu  1   2 , Weiping Li  1   2 , Wen Di  1   2
Affiliations

Bioinspired tumor-homing nanoplatform for co-delivery of paclitaxel and siRNA-E7 to HPV-related cervical malignancies for synergistic therapy

Cong Xu et al. Theranostics. .

Abstract

Because of the complexity of cancer, a combination of chemotherapy and gene therapy is an emerging treatment modality. To realize the full potential of this strategy, a smart, highly biocompatible nanosystem that enables the precise co-delivery of small-molecule anticancer drugs and small interfering RNA (siRNA) is urgently needed. This study aimed to improve the therapeutic effect against cervical cancer by using cancer cell membrane-camouflaged nanoparticles for simultaneous delivery of paclitaxel (PTX) and siRNA targeting E7. Methods: By camouflaging HeLa cell membranes onto siRNA/PTX co-loaded (lactic-co-glycolic acid) (PLGA) nanoparticles, a biomimetic dual-drug delivery system (Si/PNPs@HeLa) was developed to simultaneously deliver PTX and siRNA targeting E7. After evaluating the physicochemical characteristics as well as their cell uptake and biodistribution behavior, studies on the RNA interference efficiency and antitumor ability of Si/PNPs@HeLa in vitro and in vivo were further carried out. Results: The Si/PNPs@HeLa was capable of delivering PTX and siRNA simultaneously to HeLa cells both in vitro and in vivo. Moreover, benefiting from the recognition and adhesion molecules on the surface of HeLa cells, Si/PNPs@HeLa exhibited an improved immune escape ability and an increased tumor region accumulation (3-fold higher than bare nanoparticles). As a result, an excellent synergistic anti-tumor effect was observed in the HeLa tumor-bearing mice, with tumor volume inhibiting rates of 83.6% and no side effects in major organs. The mechanistic studies confirmed that E7 knockdown sensitized HeLa cells to PTX chemotherapy, mainly by inhibiting PTX-induced AKT pathway activation. Conclusion: Si/PNPs@HeLa, by integrating immune escape and tumor-homing ability, can serve as an efficient dual-drug delivery system to achieve precise treatment of cervical cancer through chemo-gene combined therapy.

Keywords: cancer cell membrane camouflage; cervical cancer; homotypic targeting; paclitaxel; siRNA-E7.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Scheme 1
Scheme 1
Co-delivery of paclitaxel and siRNA-E7 using a bioinspired tumor-homing nanoplatform to synergistically treat HPV-associated cervical malignancies.
Figure 1
Figure 1
Characterization of Si/PNPs@HeLa. (A) Preparation procedure of Si/PNPs@HeLa. Size distribution (B) and ζ potential (C) of Si/PNPs (blue) and Si/PNPs@HeLa (red). (D) TEM image of Si/PNPs (left) and Si/PNPs@HeLa (right). Scale bar, 100 nm. (E) SDS-PAGE analysis of proteins in the lysate of HeLa membrane and Si/PNPs@HeLa. (F) TEM image of Si/PNPs@HeLa stained with extracellular-domain specific AuNPs-AS1411. Scale bar, 100 nm. (G) Intracellular co-localization of the HeLa membrane shell (visualized with green DiO dyes) and the SiNPs core (visualized with red Cy5-siRNA dyes).
Figure 2
Figure 2
Validating co-delivery capability, homologous targeting property, and stealth ability of Si/PNPs@HeLa in vitro. (A) CLSM images of HeLa cells incubated with Si/PNPs@HeLa. Coumarin-6 (green), representing the location of PTX, and siRNA labeled by Cy5 (red) were co-loaded into the nanoparticles. Scale bar, 50 µm. (B) Fluorescence microscopy images of HeLa cells, Ect1 cells, LO2 cells, and RAW264.7 cells after incubation with Cy5 labeled SiNPs or SiNPs@HeLa for 3 h. Scale bar, 100 µm. The nucleus was stained with DAPI (blue); the siRNA loaded into the NPs was labeled with Cy5 (red). MFI values (C) and flow cytometric histogram (D) of each cell line treated with Cy5 labeled SiNPs or SiNPs@HeLa for 3 h. Untreated cells were used as isotype controls. Data are shown as mean ± SD (n=3). **p < 0.01, ***p <0.001.
Figure 3
Figure 3
Gene silencing efficacy and cytotoxicity evaluation. Quantitative RT-PCR (A) and western blot (B) determination of E7 levels in HeLa cells treated with different siRNA-E7-based formulations at a constant siRNA concentration of 100 nM. Free siRNA-E7 and formulations containing scrambled siRNA were used as negative controls, lipofectamine 2000 was used as a positive control. (C) Cell viability of HeLa cells when treated with various formulations at indicated PTX concentrations and fixed siRNA concentrations (100 nM) for 48 h. (D) Flow cytometry analysis of HeLa cell apoptosis induced by SiNPs, free PTX, PNPs, PNPs@HeLa, NC/PNP@HeLa, Si/PNPs, and Si/PNPs@HeLa at a PTX concentration of 10 nM and an siRNA concentration of 100 nM for 48 h. (E) Corresponding statistics on the proportion of apoptotic cells. (F) Alteration of the pathways in the HeLa cells when treated for indicated times with 10 nM PTX. (G) The effects of Si/PNPs and Si/PNPs@HeLa on PTX-induced alteration of pathways in the HeLa cells, as detected by western blotting. Data are given as the mean ± SD (n=3). *p < 0.05, **p, < 0.01, ***p <0.001.
Figure 4
Figure 4
Biodistribution and pharmacokinetics of NPs and NPs@HeLa. (A) Whole body fluorescence images of HeLa subcutaneous xenograft mice at 2, 8, 24, and 48 h after injection of free DiR, DiR-NPs, and DiR-NPs@HeLa (at DiR dose of 0.4 mg/kg). The tumor area is circled in red. (B) Representative ex vivo images of tumor (Tu), heart (H), liver (Li), spleen (Sp), lung (Lu), and kidney (Ki) at 48 h post-injection of free DiR, DiR-NPs or DiR-NPs@HeLa. (C) The corresponding average radiation efficiency of resected tumors and major organs. (D) Representative fluorescence microscopy images of tumor cryosections 24 h after intravenous injection with dual-labeled NPs or NPs@HeLa. The nucleus was stained with DAPI (blue); Coumarin-6 (green), representing the location of PTX, and siRNA labeled by Cy5 (red) were co-loaded into the nanoparticles. Scale bar, 100 µm. (E) Blood circulation of Coumarin-6 loaded NPs and NPs@HeLa. Data are given as the mean ± SD (n = 3 mice per group). *p < 0.05, **p < 0.01, ***p <0.001.
Figure 5
Figure 5
In vivo antitumor effects. (A) Tumor growth profiles of HeLa tumor-bearing mice receiving intravenous injections of different formulations every 2 days, for 9 injections, at a fixed PTX dose (6 mg/kg) and siRNA dose (2 mg/kg). Tumor weight (B) and photographs of the collected tumor tissues (C) on day 29. (D) Representative images of H&E staining (up, scale bar, 60 µm) and TUNEL assays (middle, scale bar, 25 µm) of tumor tissue; immunohistochemical analysis of the expression of HPV18 E7 and Rb (down, scale bar, 60 µm) in tumor sites. (E) Statistical data of HPV18 E7 and Rb expression using the optical staining intensities by ImagePro Plus (version 6.0). Data are given as the mean ± SD (n = 5 mice per group). *p < 0.05, **p < 0.01, ***p <0.001.
Figure 6
Figure 6
Biosafety estimation of various formulations. (A) Cytotoxicity of empty NPs and empty NPs@HeLa against HeLa cells after 72 h treatment with different concentrations. (B) Effects of different formulations on body weight of HeLa subcutaneous xenograft mice. (C) Test results of white blood cells count, plasma ALT, and AST at the end of treatment. (D) Representative H&E staining histological images of major organs from mice receiving different treatment. Scale bar, 60 µm. The arrow points to liver necrosis. Data are given as the mean ± SD (n = 5 mice per group). *p < 0.05, NS indicates p > 0.05.
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