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. 2020 Feb 18;117(7):3687-3692.
doi: 10.1073/pnas.1917891117. Epub 2020 Feb 6.

Transdermal cold atmospheric plasma-mediated immune checkpoint blockade therapy

Guojun Chen  1   2   3 , Zhitong Chen  4 , Di Wen  1   2   3 , Zejun Wang  1   2   3 , Hongjun Li  1   2   3 , Yi Zeng  1   2   3 , Gianpietro Dotti  5 , Richard E Wirz  6 , Zhen Gu  7   2   3   8
Affiliations

Transdermal cold atmospheric plasma-mediated immune checkpoint blockade therapy

Guojun Chen et al. Proc Natl Acad Sci U S A. .

Abstract

Despite the promise of immune checkpoint blockade (ICB) therapy against cancer, challenges associated with low objective response rates and severe systemic side effects still remain and limit its clinical applications. Here, we described a cold atmospheric plasma (CAP)-mediated ICB therapy integrated with microneedles (MN) for the transdermal delivery of ICB. We found that a hollow-structured MN (hMN) patch facilitates the transportation of CAP through the skin, causing tumor cell death. The release of tumor-associated antigens then promotes the maturation of dendritic cells in the tumor-draining lymph nodes, subsequently initiating T cell-mediated immune response. Anti-programmed death-ligand 1 antibody (aPDL1), an immune checkpoint inhibitor, released from the MN patch further augments the antitumor immunity. Our findings indicate that the proposed transdermal combined CAP and ICB therapy can inhibit the tumor growth of both primary tumors and distant tumors, prolonging the survival of tumor-bearing mice.

Keywords: cancer immunotherapy; cold atmospheric plasma; drug delivery; immune checkpoint blockade; microneedle.

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Conflict of interest statement

Competing interest statement: G.C., Z.C., R.E.W., and Z.G. have applied for patents related to this study.

Figures

Fig. 1.
Fig. 1.
Illustration of the transdermal cold atmospheric plasma (CAP)-mediated immune checkpoint blockade (ICB) therapy. Schematic of the transdermal combination of CAP and ICB therapy assisted by the polymeric hollow-structured microneedle patch loaded with aPDL1. Nomenclature: DC, dendritic cell; TAA, tumor-associated antigen; CTL, cytotoxic T lymphocyte; TCR, T cell receptor; MHC, major histocompatibility complex; PVP, polyvinylpyrrolidone; PVA, polyvinyl alcohol; ROS, reactive oxygen species; RNS, reactive nitrogen species; AC, alternating current.
Fig. 2.
Fig. 2.
Characterization of the hollow-structured microneedle (hMN) patch. (A) Representative SEM images of the hMN patch from views of needle side and base side. (B) A 3D CLSM image of hMN patch (rhodamine loaded). (C) Penetration test of the CAP through hMN patch. (Scale bar, 1 cm.) The CAP through the hMN included reactive species and reflection. Representative OES spectra of the CAP (D) above hMN patch and (E) penetrating through the hMN patch.
Fig. 3.
Fig. 3.
Combination of CAP and hMN-aPDL1 inhibits B16F10 melanoma growth in vivo. (A) Schematic of B16F10 melanoma-bearing mice treated either with the hMN path (Upper image) or hMN/CAP (Lower image). (B) Quantification and (C) representative flow cytometry plots of DC maturation in vivo in the tumor-draining lymph nodes. Cells in the tumor-draining lymph nodes were collected 3 d after the treatments (G1: untreated; G2: CAP; G3: sMN/CAP; and G4: hMN/CAP) for assessment by flow cytometry. (D) Schematic of the treatment schedule. (E) In vivo tumor bioluminescence of the untreated mice and mice treated with CAP, sMN/CAP, hMN/CAP, hMN-aPDL1, and hMN-aPDL1/CAP (aPDL1: 200 μg; CAP treatment: 4 min). Four representative mice per treatment group are shown. (F) Individual and (G) average tumor growth kinetics in experimental groups (n = 7). Growth curves were stopped when the first mouse of the corresponding group died. Data are presented as mean ± SEM. (H) Kaplan-Meier survival curves for treated and control mice (n = 7). Statistical significance was calculated via the log-rank (Mantel-Cox) test. *P < 0.05; **P < 0.01; ***P < 0.001. Intratumoral (I) CD3+ T cells, (J) CD8+ T cells, and (K) CD4+ T cells in the B16F10 tumor detected 3 d after treatments (n = 4). Data are presented as mean ± SEM. Statistical significance was calculated via one-way ANOVA with a Tukey post hoc test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 4.
Fig. 4.
CAP and hMN-aPDL1 inhibit distant tumor growth. (A) Schematic of the treatment schedule. Tumors on the right side were designated as “primary tumor” and were treated with hMN-aPDL1/CAP, and tumors on the left side were designated as “metastatic tumor” and were not treated (n = 7). (B) In vivo tumor bioluminescence images of the untreated mice and treated mice. Three representative mice per treatment group are shown. (C) Left and right tumor growth curves and (D) tumor weights of the mice untreated and treated with hMN-aPDL1/CAP. Intratumoral (E) CD3+ T cells, (F) CD8+ T cells, and (G) CD4+ T cells in the B16F10 tumor detected 3 d posttreatment (n = 4). Data are presented as mean ± SEM. Statistical significance was calculated via one-way ANOVA with a Tukey post hoc test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001.
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