Macrophage Membrane-Camouflaged shRNA and Doxorubicin: A pH-Dependent Release System for Melanoma Chemo-Immunotherapy - PubMed (original) (raw)

Macrophage Membrane-Camouflaged shRNA and Doxorubicin: A pH-Dependent Release System for Melanoma Chemo-Immunotherapy

Chengli Yang et al. Research (Wash D C). 2022.

Abstract

Improving the efficacy of melanoma treatment remains an important global challenge. Here, we combined chemotherapy with protein tyrosine phosphatase nonreceptor type 2(Ptpn2) based immunotherapy in an effort to address this challenge. Short-hairpin RNA (shRNA) targeting Ptpn2 was coencapsulated with doxorubicin (DOX) in the cell membrane of M1 macrophages (M1HD@RPR). The prepared nanoparticles (NPs) were effectively phagocytosed by B16F10 cells and M1 macrophages, but not by M0 macrophages. Hence, NP evasion from the reticuloendothelial system (RES) was improved and NP enrichment in tumor sites increased. M1HD@RPR can directly kill tumor cells and stimulate immunogenic cell death (ICD) by DOX and downregulate Ptpn2. It can promote M1 macrophage polarization and dendritic cell maturation and increase the proportion of CD8+ T cells. M1HD@RPR killed and inhibited the growth of primary melanoma and lung metastatic tumor cells without harming the surrounding tissue. These findings establish M1HD@RPR as a safe multifunctional nanoparticle capable of effectively combining chemotherapy and gene immunotherapies against melanoma.

Copyright © 2022 Chengli Yang et al.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1

Schematic illustration of the M1HD@RPR construction and its application in combined cancer therapy.

Figure 2

Figure 2

Characterization of nanoparticles. (a) and (b) Size distribution, zeta potential (inset graph), and morphology of HD@RPR and M1HD@RPR (inset image). (c) Protein profiles in the M1, HD@RPR, and M1HD@RPR determined via SDS-PAGE assay. (d) DNA fragment migration by agarose gel electrophoresis. Lane 1, DNA ladder; lane 2, shRNA-Ptpn2 plasmid; lane 3-6: RR, RPR, HD@RPR, M1HD@RPR. (e) DOX-release profiles in the presence different pH values.

Figure 3

Figure 3

Cellular behavioral studies. (a) Flow cytometry analysis of B16F10 cellular uptake of Free DOX, HD@RPR, M1HD@RPR at 0.5 h, 1 h, and 2 h. (b) Distribution of DOX uptake investigated by cytoskeleton labeled with FITC-phalloidin, (scale bar: 20 _μ_m). (c) Study on penetration ability of nanoparticles into 3D tumor spheroids. (d) Uptake of free DOX, HD@RPR, and M1HD@RPR by macrophages (scale bar: 20 _μ_m).

Figure 4

Figure 4

Cellular pharmacodynamics. (a) and (b) Ptpn2 protein expression in B16F10 cells determined by western blotting after different treatments.(c) Immunofluorescence detection of CRT and HMGB1 expressed on B16F10 cell (scale bar: 20 _μ_m).(d) and (e) Apoptosis of B16F10 cells treated with different formulations for 12 h.

Figure 5

Figure 5

Study on cellular immunity mechanism. (a) and (b) Representative flow cytometry plots of CD80+ CD86+ in each group. (c) Immunofluorescence results of M1 macrophages induced by nanoparticles. a, PBS; b, Blank NPs; c, M1; d, OHA@RPR; e, Free DOX; f, HD@RPR; g, M1HD@RPR (scale: 20 _μ_m). (d) and (e) Flow cytometry results of M1 induced by nanoparticles. a, PBS; b, Blank NPs; c, M1; d, OHA@RPR; e, Free DOX; f, HD@RPR; g, M1HD@RPR.

Figure 6

Figure 6

Tissue distribution of DOX after intravenous administration of (a) Free DOX (b) HD@RPR, and (c) M1HD@RPR. (d) DOX concentration in tumor tissues of each group. (e) and (f) CLSM images and its semiquantitative results of nanoparticles distribution in B16F10 tumor sections 24 h postinjection (scale bar: 200 _μ_m).

Figure 7

Figure 7

Chemo-immunotherapy effect for melanoma. (a) Scheme of treatment for primary tumor (n = 5). (b) Images of the tumors collected from various groups of mice after treatments. (c) Tumor volume in B16F10 tumor-bearing mice treated with different groups (n = 5). (d) Images of the tumors' weight collected from various groups of mice at the end of the treatments (n = 3). (e) Body weight of mice bearing B16F10 tumors after various treatments. (f) Scheme of treatment for pulmonary metastasis (n = 4). (g) and (h) Typical photographs and H&E staining of B16F10 metastatic foci for mice after different treatments, scale bar: 2000 _μ_m.

Figure 8

Figure 8

(a) Histological images of TUNEL, Ki67, Ptpn2, and CRT expression in tumor sections of different treatment groups (scale bar: 200 _μ_m). (b) to (d) mRNA expression of Ptpn2, CRT, and HMGB1 were detected by qPCR after treatment.

Figure 9

Figure 9

Study on immune mechanism. (a) to (c) The population of CD8+ T cells, CD11c+CD86+ cells, and F4/80+ CD86+ cells in the tumor. (d) to (f) The population of CD8+ T cells, CD11c+ CD86+ cells, and F4/80+ CD86+ cells in the blood.(g) Immunofluorescence and IHC analysis of CD3+ T cells (red) and CD86 in the tumor collected after the mice were subjected to different treatments (scale bar 200 _μ_m).

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