Elsevier

Journal of Controlled Release

Volume 285, 10 September 2018, Pages 67-80
Journal of Controlled Release

Targeting of drug-loaded nanoparticles to tumor sites increases cell death and release of danger signals

https://doi.org/10.1016/j.jconrel.2018.07.007Get rights and content

Abstract

Innovative strategies fighting cancer by inducing anti-tumor responses from the immune system are urgently needed. Amongst others, chemotherapeutics from the class of the anthracyclines have been shown to induce death of cancer cells with immunogenic features and triggering immunogenic anti-tumor responses. However, the patient's immune system often becomes severely impaired by the unspecific action of these cytotoxic drugs in systemic chemotherapy, preventing any effective immune reactions. To reduce systemic side effects and to accumulate the drug exclusively in the tumor region, we developed an iron oxide nanoparticle-based system for the magnetically targeted delivery of mitoxantrone to the tumor, which has previously proven its long-term therapeutic efficacy in tumor bearing rabbits. Here, we show in vitro that superparamagnetic iron oxide nanoparticles (SPIONs), loaded with the chemotherapeutic drug mitoxantrone, are able to induce cell death with immunogenic features and concomitant maturation of dendritic cells, comparable to the free drug, whereas unloaded nanoparticles are very biocompatible. We conclude that the targeted delivery of mitoxantrone to the tumor region might be a promising possibility to selectively modulate the tumor microenvironment and to locally stimulate immune responses against the tumor. Thus, SPIONs can be used as a platform to specifically bring immunogenic cell death inducers (e.g. mitoxantrone, hypericin, doxorubicin) to the tumor region by sparing the immune system from their toxic effects.

Introduction

Accumulations of mutations in proto-oncogenes and tumor suppressor genes can lead to the emergence of cancer. Immune effector cells recognize and eliminate mutated tumor cells, referred to as immune surveillance. Due to tumor heterogeneity, less immunogenic tumor cells may escape and during this Darwinian selection process, tumor cell variants with lower immunogenicity and higher resistance emerge (cancer immunoediting) [1]. Beside surgery, chemotherapy and radiotherapy are still standard treatments of cancer. In the last years also immune therapy has developed as additional treatment regimen, which shall increase the strength of immune responses against the tumor by either stimulating activities of the immune system or block signals that are produced by cancer cells to suppress immune responses. Therapeutic antibodies against tumor antigens, stimulatory cytokines and immune checkpoint inhibition have shown clinical activity in many different types of cancer. Also, several other experimental immunotherapies are under development.

Additionally, several “conservative” tumor treatment strategies have been shown to induce an immunogenic form of cell death in the tumor, making the tumor “visible” for the immune system. These treatments include radiation, hyperthermia, photodynamic therapy and chemotherapy with anthracyclins and their derivatives [[2], [3], [4], [5], [6], [7], [8]]. Immunogenic cell death (ICD) is characterized by alterations of the cell surface and the release of Damage-associated molecular patterns (DAMPs) in a timely regulated fashion [9,10]. Initially, calreticulin as well as heat shock proteins HSP70 and HSP90 (from Endoplasmic Reticulum) are exposed on the plasma membrane of dying cells [11]. In later phases of cell death, adenosine triphosphate (ATP) is actively secreted by autophagy [12] and after loss of plasma membrane integrity, high-mobility group protein B1 (HMGB1) and ATP are passively leaking out of cells with ruptured plasma membranes [13]. Altogether, these signals act as endogenous adjuvants and promote the recruitment of antigen presenting cells and foster them to take up dead cell-derived material priming an adaptive immune response [2,14].

Despite several cytotoxic agents have shown the ability to induce ICD, systemic treatments (e.g. chemotherapy) are accompanied by severe side effects, in particular destruction of the immune system [15]. To deliver drugs precisely to the desired place and sparing healthy tissues, nanoparticles serving as carriers for therapeutics have come into focus. Passive approaches of enriching nanoparticles in the tumor region utilizing the leaky vasculature of tumors (enhanced permeability and retention effect, EPR) [16] have been exploited with ICD inducers previously. To enrich oxaliplatin or doxorubicin in pancreatic cancer Zhao et al. developed a nanocarrier-based drug delivery system for intravenous injection which showed beneficial outcomes in pancreatic cancer xenografts [17]. Going one step further Lu et al. added indoximod into oxaliplatin containing nanovesicles, a small molecule interfering with immune suppressive indoleamine 2,3-dioxygenase pathway, increasing the efficacy in murine pancreas cancer [18]. Furthermore, mouse EL4 lymphoma were successfully treated with HPMA copolymer-bound doxorubicin by induction of ICD [19]. Also a modulation of apoptosis and alteration of the inflammatory milieu of MDA-MB-231 cells by doxorubicin-hyaluron conjugated SPIONs has been proven [20].

Although loading drugs onto nanoparticles can increase their systemic circulation times and accumulation in the tumor region via the EPR effect, only a fraction of the nanoparticles end up in the tumor whereas the majority of the administered nanoparticles are known to accumulate in other organs, in particular the liver, spleen, and lungs [16]. Superparamagnetic iron oxide nanoparticles (SPIONs) as drug carriers for application in magnetic drug targeting (MDT) might be a promising solution to overcome this challenge. Thereby, the SPIONs are loaded with a (chemo-)therapeutic agent, applied to the tumor supplying vascular system and enriched in the tumor region by application of an external magnetic field [[21], [22], [23]]. We and others have shown successful targeting and improved anti-tumor efficacy of chemotherapeutic drugs (doxorubicin, mitoxantrone, MTO) administering SPIONs in the presence of a magnetic field [21,[24], [25], [26]]. With MTO-loaded lauric acid coated SPIONs we showed that after MDT the amount of MTO in the tumor can be dramatically increased compared to systemic application (50–60% versus 1%) while the immune system is preserved from cytotoxic effects [21,27]. Rabbits with induced squamous cell carcinomas at the leg treated with these particles in a MDT regimen showed a progressive and continuous tumor regression up to complete tumor disappearance after several weeks, indicating rather an immunological process than a simple immediate killing of the tumor cells [21]. Importantly, for immune mediated effects, the integrity of the immune system is a prerequisite, which will be improved by proper targeting of the cytotoxic drug. With this study, we aimed to investigate if SPIONs can serve as magnetically controllable drug carrier for ICD inducing compounds. Optimized SPIONs, stabilized with lauric acid and an albumin protein corona [[28], [29], [30]], loaded with MTO as bona fide inducer of ICD [4] were targeted to the desired place in vitro in dynamic flow systems mimicking blood circulation. MTO loaded SPIONs thereby induced a similar cell death pattern, release of danger signals and concomitant maturation of dendritic cells as the free drug. Thus, we propose that SPIONs are a promising platform to deliver (ICD) drugs to the desired place without compromising the immune system, which is urgently needed for an immune system-mediated therapeutic long-term success.

Section snippets

Synthesis of nanoparticles and loading with MTO

Lauric acid (LA) and human serum albumin (HSA) hybrid coated magnetite nanoparticles were in-house synthesized in the Section of Experimental Oncology and Nanomedicine (SEON) in Erlangen as described previously [30]. In brief, after co-precipitation and in-situ coating with LA, nanoparticles were covered by a protein corona of HSA. The resulting nanoparticles were then loaded with the chemotherapeutic drug MTO (TEVA Pharma, Ulm, Germany) by mixing. MTO strongly binds to the HSA corona of the

Characterization of SPION and SPIONMTO

Synthesis and physicochemical characterization of SPIONs stabilized with a protein corona of HSA and loaded with MTO were described in detail previously and physicochemical features are summarized in Table 1 [30]. Absence of microbial and endotoxin contaminations of the nanoparticles was ensured before use in cell culture experiments. Incubation of the nanoparticles on agar plates for 72 h showed no bacterial colonies in the negative control (water) and the nanoparticle samples, whereas in the

Discussion

In summary, we showed that the cell death phenotype induced by SPIONMTO is very similar to that induced by the free drug. Like free MTO, used as chemotherapeutic agent in the treatment of advanced breast carcinoma, acute myeloid leukemia, and non-Hodgkin's lymphoma [[45], [46], [47]], SPIONMTO induced a cell cycle arrest in G2 phase, with following DNA degradation, stop of proliferation and induction of apoptosis indicated by phosphatidylserine exposition (Fig. 3, Fig. 4, Fig. 5). After

Conclusion

In sum, we showed that SPIONMTO induced a similar cell death phenotype as the free drug, which is known as a bona fide ICD inducer. Moreover, we previously observed tumor remissions in tumor-bearing rabbits after magnetic drug targeting with MTO-loaded nanoparticles with a slightly different formulation [21]. Based on these findings we conclude that SPIONMTO might serve as a valuable tool to selectively modulate the tumor microenvironment and to stimulate immune responses against the tumor in

Funding

The work was supported by the ELAN Program of the Medical Faculty of the Friedrich-Alexander Universität Erlangen-Nürnberg, the Bavarian State Ministry for the Environment and Consumer Protection, FUMIN Bridge Funding appropriations, and the Manfred Roth Foundation, Fürth, Germany.

Author contributions

C.J., M.A. and B.F. designed the experiments; M.A., L.E., L.M., B.W. performed the in vitro experiments, M.A., RP.F., and M.P. performed fluorescence microscopy, M.A., C.A. and C.J. wrote the manuscript. The present work was performed in fulfillment of the requirements for obtaining the degree “Dr. med. dent.” (Magdalena Alev and Laura Mühleisen).

Competing interests

The authors declare no competing interests.

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