Elsevier

Biomaterials

Volume 244, June 2020, 119964
Biomaterials

Engineering antigen as photosensitiser nanocarrier to facilitate ROS triggered immune cascade for photodynamic immunotherapy

https://doi.org/10.1016/j.biomaterials.2020.119964Get rights and content

Abstract

Despite of the documented immunogenic cell death (ICD) and antigen cross-presentation (AC) in photodynamic therapy (PDT), the overall immune efficacy is rather limited. This study aims to expand the immune potential of PDT by spatially packaging antigen as photosensitiser nanocarrier to trigger efficient immune cascade for photodynamic immunotherapy. The package of ovalbumin antigen (OVA) into sub-100 nm nano-assembly is realized by driving intermolecular disulfide network between OVA molecules. OVA nanoparticles loading photosensitiser Ce6 (ON) are subsequently coated with B16-OVA cancer cell membrane, resulting in membrane cloaked ON (MON). Importantly, laser irradiation generated ROS significantly potentiates OVA antigen cross-presentation efficiency. Whilst, MON is endowed with homophilic targeting towards tumor due to cancer cell membrane coating. In treating B16-OVA tumor-bearing mice, MON effectively triggers the immune cascade, completely eliminates the tumor under laser irradiation and provokes a long-term antitumor immune memory effect. Conversely, a marginal effect is found if substituting OVA for bovine serum protein (BSA) in nanoparticle design or using MON to treat non-OVA expressing tumor. The antigen nanocarrier design promises to complement conventional PDT by boosting immune cascade, thereby leading to unique photodynamic immunotherapy.

Graphical abstract

Antigen is spatially packaged as photosensitiser nanocarrier to construct an immunogenic and photocytoxic nanoplatform (MON) that is able to amplify the immune response during the PDT. By successfully eliciting abundances of CD8+ T cells, MON completely ablates the B16-OVA tumor and provokes a long-term antitumor immune memory effect, which powers the photodynamic therapy into an immunotherapy.

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Introduction

Photodynamic therapy (PDT) is a clinically approved approach that can exert the selective cytotoxic activity toward malignant tumor cells upon exogenous light [1,2]. It encompasses the advantages of minimally systemic toxicity for localized light irradiation and low resistance occurrence with repeated treatments [3,4]. In the implement of PDT, photosensitizers can be excited by visible light in the presence of oxygen to form reactive oxygen species (ROS), which possess cytotoxic properties for inducing cell apoptosis or necrosis, microvasculature shutdown and immune response [5,6]. During the past 30 years, PDT has been exploited increasingly in the treatment of a variety of solid tumors, including brain, skin, breast, cervix and ovarian etc. [[7], [8], [9], [10]].

Recently, it has been well documented that the immune response usually accompanied within the PDT process [11,12]. Among those PDT concurrent immune responses, ICD is described as a death process that can trigger immune response and complement the PDT effect [[13], [14], [15], [16], [17]]. Evidence has also been accumulated that PDT generated ROS can alter the tumor microenvironment and recruit inflammatory and immune mediators, favourable to prime immune response [11,18]. During the priming process, tumor-associated antigens originating from tumor debris are released and captured by antigen-presenting cells (APCs) to consequently activate tumor specific CD8+ T cells [[19], [20], [21]]. Damage-associated molecular patterns (DAMPs) released during PDT process act as immunoadjuvant to stimulate the APCs maturation [22,23]. Another benefit is the recent discovery of ROS capable of improving antigen cross-presentation (AC) efficiency necessary for CD8+ T cells generation [24]. Mechanistic study showed ROS could mediate alkalization of phagosomal pH due to the consumption of protons intracellularly, which prevents antigen degradation by the acidic lysosomal proteases [25,26]. Such a behavior facilitates the processing of antigen by major histocompatibility complex class I (MHC-I) in APCs, and subsequently enhances CD8+ T cell responses for cancer immunotherapy [27,28].

Despite of the abovementioned encouraging immune responses, PDT is far from being perceived as an effective immunotherapy for eliminating tumor or metastatic tumor for the limited effectiveness of immune response [29]. This is partially evidenced by the fact that PDT induced tumor lysates produce little immunotherapy effect [30]. To potentiate immunotherapy effect, PDT has to resort to integrate other complementary therapies to achieve synergistic therapy. Incorporation of immune checkpoint inhibitor is frequently employed, for example the inhibitor of indoleamine 2, 3-dioxygenase (IDO)[14], PD-1/PD-L1 [31] and CTLA-4 [32]. However, a prerequisite for checkpoint immunotherapy relies on the pre-establishment of adaptive immune response, i.e., the existence of CD8+ T cell. Additionally, the high cost and side effect of the combination also complicate the application in clinic [33].

To amplify the antitumor immune response during the PDT, this study focuses on spatially packaging antigen ovalbumin (OVA) as photosensitiser Ce6 nanocarrier to construct an immunogenic and photocytoxic nanoparticle. The Ce6 equipped antigen nanoplatform is expectedly to synergistically integrate the benefit of ROS mediated ICD and AC effects[25,26], and consequently augment the immune response. Onto the antigen nanocarrier, the particle surface will be further camouflaged with the cancer cell membrane to facilitate targeting efficiency towards tumor site by homophilic targeting [[34], [35], [36], [37]]. In this fashion, the camouflaging nanoparticles should be able to selectively accumulate in tumor site and exert a photocytoxic effect to produce tumor-associated antigens under laser irradiation. The produced tumor-associated antigens as well as MON were captured by APCs in situ then migrating to tumor draining lymph nodes (dLNs). The spatial antigen nanopackage and the colocalization with photosensitiser by structural organization are expected to cooperatively improve antigen cross-presentation. B16-OVA tumor bearing mice (expressing ovalbumin) are then employed to evaluate the efficacy of antitumor immune cascade leading to photodynamic immunotherapy. The role of the amplified immune effect will be discriminated from photodynamic effect by using non-OVA expressing tumor bearing mice as the control.

Section snippets

Materials

The photosensitiser (PS) Chlorin e6 was obtained from J&K Scientific, Ltd. Fetal bovine serum (FBS), RPMI Media 1640 (RPMI 1640), penicillin-streptomycin and trypsin were supplied by Gibco Invitrogen. 4, 6-diamidino-2-phenylindole (DAPI), WST-1, prestained color protein ladder, 1 × EDTA-free protease inhibitor and 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) were obtained from Beyotime Institute of Biotechnology. Paraformaldehyde (4%) was obtained from DingGuo Chang Sheng Biotech. Ovalbumin

OVA antigen nanoparticle synthesis, Ce6 loading, cancer cell membrane camouflaging and structural characterization

The widely employed model antigen, OVA[39], was chosen in this study. We attempted to assemble antigens into nanoparticle by avoiding using any other conventional nanocarrier, to purposefully maximize the content of antigen. One OVA molecule contains four free thiols which lays the basis for spatially packaging OVA antigens themselves into nanoparticles with disulfide bond network between OVA molecules. However, a pretreatment of OVA solution by SDS is imperative since most of those free thiol

Conclusions

In this study, we have developed an integrative approach to achieve photodynamic immunotherapy by ROS triggered antitumor immune cascade. In this approach, OVA was nano-packaged by establishing an intermolecular disulfide bond network between antigen, which was employed as photosensitiser nanocarrier and subsequently coated with cancer cell membrane. The membrane coated OVA nanoparticle loading Ce6 (MON) exhibited the advantages of homophilic targeting, prolonged circulation time, promoting

Author statement

Huaiji Wang and Yongyong Li conceived the project and designed the experiments. Huaiji Wang performed most of the experiments and statistical analyses of the data. Kun Wang, Lianghua He and Ying Liu helped with animal experiment. Kun Wang assisted with data interpretation of immune assays. Huaiji Wang wrote the original manuscript, with edits by Yongyong Li and Haiqing Dong, and reviewed by all authors. Yongyong Li and Haiqing Dong supervised the whole project.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was financially supported by research grants from the National Natural Science Foundation of China (NSFC51773154, 31771090, 51473124 and 81571801), Shanghai Natural Science Foundation (17ZR1432100), the Fundamental Research Funds for the Central Universities (22120180062) and Young Hundred-Talent Program of Tongji University.

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