Doxorubicin‐loaded nanoparticle coated with endothelial cells‐derived exosomes for immunogenic chemotherapy of glioblastoma

Abstract Treatments of glioblastoma (GBM) have not been very effective, largely due to the inefficiency of drugs in penetrating the blood brain barrier (BBB). In this study, we investigated the potential of exosome‐coated doxorubicin (DOX)‐loaded nanoparticles (ENPDOX) in BBB penetration, inducing immunogenic cell death (ICD) and promoting survival of GBM‐bearing mice. DOX‐loaded nanoparticles (NPDOX) were coated with exosomes prepared from mouse brain endothelial bEnd.3 cells. ENPDOX cellular uptake was examined. Penetration of ENPDOX through the BBB was tested in an in vitro transwell system and a GBM mouse model. The effects of ENPDOX in inducing apoptosis and ICD were assessed. Finally, the efficacy of ENPDOX in the treatment of GBM‐bearing mice was assessed. ENPDOX was taken up by bEnd.3 cells and could penetrate the BBB both in vitro and in vivo. In vitro, ENDDOX induced apoptosis and ICD of glioma GL261 cells. Systemic administration of ENPDOX resulted in maturation of dendritic cells, activation of cytotoxic cells, altered production of cytokines, suppressed proliferation and increased apoptosis of GBM cells in vivo and prolonged survival of GBM‐bearing mice. Our findings indicate that ENPDOX may be a potent therapeutic strategy for GBM which warrants further investigation in clinical application.


| INTRODUCTION
Glioblastoma (GBM) is a highly aggressive brain tumor with an extremely poor prognosis and a small rate (4%-5%) or 5-year survival. 1 Current treatments of GBM include surgeries, radiotherapy, and/or chemotherapy. These treatments not only cause severe side effects, but also only slightly improve the overall median survival (only 15 months) and 5-year survival rate. 2 Although many therapeutic strategies targeting have been developed, their application in the clinic for treatment GBM has been largely impeded due to the lack of safe and efficient drug delivery system that delivers drugs to tumor location. 3 Recent research findings suggest that, in various cancer types including GBM, human immune response has significant potential in promoting immune mediated tumor eradication and improving long term survival. 4 Recent studies have shown that anthracyclines, such as doxorubicin (DOX), not only induce apoptosis of tumor cells, but also immunogenic cell death (ICD). 5 ICD is a special type of cell death that elicits immune responses, leading to maturation of dendritic cells and activation specific T cells, an outcome is that largely preferred in anticancer therapy. 6 DOX treatment leads to translocation of calreticulin (CRT) from the endoplasmic reticulum to the plasma membrane surface, ATP secretion, and release of the nonhistone chromation protein high-mobility group box 1 (HMGB1). These events are characteristics of ICD, the highly sought for goal in cancer therapy. DOX has been shown to cause cytotoxicity in various tumor cells and is currently used as treatment for different cancers. When delivered locally, DOX is able to improve the survival of rats bearing malignant intracranial glioma. 7 Additionally, interstitial administration of DOX in patients with recurring malignant glioma who underwent repeated surgeries resulted in significant improvement with one patient showing no tumor recurrence following 2 months of treatment. 8 However, it is not clear whether this route of drug delivery is safe and DOX was administered by insertion of a catheter into the location of removed tumor following surgery. Therefore, we aimed to explore a systemic drug delivery method that allows penetration of DOX through the blood brain barrier (BBB) with minimal invasiveness and that targets specifically to tumor cells.
Recent discovery of exosome-mediated drug delivery has attracted great attention in the fields of neurological disorders, whose treatments are largely impeded by the inefficiency of drugs in penetrating the BBB, a natural barrier that separates the central nervous system from the peripheral circulation. Exosomes are membrane-wrapped extracellular microvesicles with diameters of 40-200 nm that are secreted by most cell types. Exosomes contain multiple nucleic acids and proteins and play important roles in cellular communication. The membranes of exosomes have various membrane proteins with specific functions and may be involved in targeting exosomes to brain vascular endothelial cells and in penetration of the BBB. Exosomes have been shown to have great therapeutic potential in the treatment of various diseases, such as cancer, chronic obstructive pulmonary disease, and Alzheimer's disease. [9][10][11] Recently, exosomes have been extensively studied as a vehicle for drug delivery. Exosomes are able to cross the BBB and have been use to carry specific siRNAs and mediate gene knock down in the brain. 12 Exosome-coated drug loading nanoparticles have been developed and its potential in treating breast cancer has been explored. 13 Importantly, this drug-loading method has been shown to successfully deliver DOX in breast cancer cells leading potent apoptosis of these cells and suppression of tumor growth. 13 Although the effectiveness of DOX for treating GBM is largely impacted by its inefficiency in BBB penetration, it has been shown in vitro that DOX can effectively induce death of GBM cells and DOX-coated nanoparticles can penetrate a model of BBB composed by a monolayer of Madin-Darby canine kidney transfected with multidrug resistant protein 1. 14 Therefore, in this study, we took advantage of the exosome-mediated drug delivery system and explored the potential of exosome-coated DOX-loaded nanoparticles in penetrating the BBB, in inducing apoptosis and ICD of tumor cells and in the survival of GBM-bearing mice.

| Design of exosome-coated DOX-loading nanoparticles for GBM treatment
To enhance the delivery of DOX to tumor area across the BBB in patients with GBM and induce antitumor immune response, we developed a drug delivery system by coating DOX-loaded nanoparticles with exosomes isolated from bEnd.3 cells (a murine brain endothelial cell line) (Figure 1) according to a previous method. 13 Briefly, PEG-PLA nanoparticles loading DOX (NP DOX ) were prepared through an emulsion method. 15 Exosomes were harvested from endothelial cell cultured and their contents were emptied. The NP DOX were then packaged into the empty exosomes to form the exosome-coated DOX-loaded nanoparticles (ENP DOX ). Following intravenous administration, ENP DOX is expected to penetrate the BBB, accumulate at the GBM site and induce apoptosis and ICD, resulting in maturation of dendritic cells and infiltration of cytotoxic T lymphocytes (right panel, Figure 1).

| ENP DOX was taken up by endothelial cells in vitro
We first analyzed the characteristics of ENP DOX . The morphology of the nanoparticles was examined by TEM (Figure 2 We then determined the uptake of nanoparticles by mouse endothelial bEnd.3 cells. DOX fluorescent intensity was measured by flow cytometry analysis (Figure 2(e)). We found that cells treated with ENP DOX showed significant increase in DOX fluorescent intensity ( Figure 2(f)). We also examined DOX uptake by confocal imaging and found that DOX was more efficiently taken up by bEnd.3 cells ( Figure 2(g)) compared to DOX or NP DOX treatment.

| ENP DOX induces apoptosis and ICD of murine glioma cells in vitro
To assess the capability of ENP DOX in penetrating the BBB, we first utilized an in vitro model of the BBB with a transwell system. 16 In this culture system, bEnd.3 cells were cultured in the upper compartment and were treated with NP DOX and ENP DOX , respectively, and GL261 cells were cultured in the lower compartment (Figure 3(a)). Examination of DOX signal intensity by flow cytometry (Figure 3 Consistently, significantly more ATP was released from cells treated with ENP DOX compared to other treatments. These results suggest that ENP DOX induced a higher level of ICD among GB261 cells compared to DOX or NP DOX treatment.

| Systemic administration of END DOX leads to efficient DOX accumulation in tumor tissue in a mouse model of GBM
To determine whether END DOX administered systemically could penetrate the BBB and accumulate in the tumor tissue of the brain, we established an orthotopic GBM xenograft model by stereotactical implantation of GL261 cells into the brain as described previously. 17 GBM-bearing mice were injected with DOX, NP DOX , or END DOX intra-   To determine the effect of ENP DOX on survival, GBM-bearing mice were treated with DOX, NP DOX , or ENP DOX and their survival duration was recorded (Figure 6(b)). Kaplan-Meier survival analysis showed that both DOX and NP DOX treatments slightly extended the survival of GBM-bearing mice compared to control mice. Importantly, mice treated with ENP DOX survived significantly longer compared to that treated with DOX and NP DOX . The BBB has been a great obstacle in the treatment of various neurological disorders, including GBM, with only small molecules being able to penetrate through the BBB. Most available drugs are unable to transition from the blood circulation to the brain, making systemic administration impossible for treating brain diseases. 18 The objective of drug development in the GBM treatment is to search for drugs with assistant from drug delivery systems to penetrate the BBB and target glioma cells. Several strategies have been exploited for delivering drugs to brain including viral vectors, nanoparticles, and brain permeability enhancers, with each method having its advantages and limitation, such as safety concerns and toxicity. Recently, exosomes have been actively studies as a promising vector for drug delivery in various cancer types. 19 For example, exosomes derived from macrophages have been specifically engineered to deliver drugs that can target tumors, leading to inhibition of tumor growth and increases apoptosis of tumor cells. 13 In a zebrafish brain cancer models, exosomes are used for delivery of anticancer drug which suppressed markers of tumor growth. 20 In our study, we purified exosomes derived from murine brain have been used as a carrier of siRNA which promoted efficient systemic delivery of siRNA to brain regions. 21 The previous studies together with ours suggest that exosome can be used to target drugs to the brain through noninvasive systemic administration.
Previously, engineered exosomes derived from macrophages have been used for coating nanoparticles loading DOX and have been shown to induce potent apoptosis of breast cancer cells and inhibition of tumor growth. 13 In addition to inducing apoptosis, DOX is also an inducer of ICD and this property has been utilized in the studies of cancer therapy for antitumor immunity. DOX treatment leads to immuomodulation following death of tumor cells in a mouse model of neuroblastoma. 22 After confirming efficient uptake of DOX delivered through ENP DOX by glioma cells, our study focused on investigating the efficacy of this anticancer drug in tumor suppression in order to determine its potential in the treatment of GBM. We showed that ENP DOX induced apoptosis and ICD of glioma cells in vitro with increased CRT surface exposure and increased release of HMGB1 and ATP. Consistently, END DOX -induced dendritic cell maturation and cytotoxic T lymphocyte activation in GBM-bearing mice. Our study not only confirmed the previous findings of DOX in tumor suppression by inducing apoptosis and ICD, but also successfully achieved penetration of DOX through the BBB using the exosome-mediated drug delivery system. The finding that systemic administration of ENP DOX significantly prolonged the survival of GBM-bearing mice is of great clinical importance for the treatment of GBM since GBM is the deadliest brain cancer and patients with GBM have an extremely short average survival with a 5-year survival rate of approximately 5%. 23 Nanomedicines in cancer field have been designed to target tumors with an intention to reduce side effects. 24 DOX-loading nanoparticles (Doxil) has been approved by FDA for treating various cancers including ovarian cancer, HIV-associated Kaposi's sarcoma, and other cancers. 24 Some side effects were reported such as skin toxicity with less frequent cases with mild cardiotoxicity and hepatotoxicity. 25 In this study, we have not investigated the impact of on ENP DOX healthy cells. Survival analysis of mice bearing GBM treated with ENP DOX showed that ENP DOX significantly prolonged the survival of the mice, suggesting that ENP DOX effectively suppressed GBM growth without causing any devastating side effects. However, detailed analysis of the side effects will be carried out in the future studies for clinical application.
In summary, our study revealed a potent drug delivery system by coating DOX-loaded nanoparticles with brain endothelial cell derived exosomes to facilitate penetration of anticancer drugs across the BBB and target the GBM. We have shown that ENP DOX induced apoptosis and ICD both in vitro and in vivo, and it significantly prolonged the survival of GBM-bearing mice. Our important findings warrant further investigation of this system in clinical application.

| Preparation and characterization of exosomecoated DOX-loaded nanoparticles
DOX-loaded nanoparticles were prepared using an emulsion method as described previously. 15 Briefly, PEG-PLA and DOX solution in dimethyl sulfoxide (DMSO) and chloroform was emulsified in water for an oil-in-water emulsion. Chloroform was then removed by evaporation and DMSO was removed by dialyzing the resulting production against water. Free DOX was removed by centrifugation.
Nanoparticles encapsulating DOX were denoted as NP DOX .
Exosomes derived from endothelial cells were isolated with the contents removed according to a previous study. 13  The morphology and diameter of NP DOX and ENP DOX were determined by transmission electron microscopy. Zeta potential was measured through dynamic light scattering. 13

| In vitro BBB model
The transwell system was adopted to study penetration of ENP DOX through the BBB in vitro as described previously. 16 Briefly, the murine

| Flow cytometry
To determine the uptake of DOX by mouse brain endothelial bEnd.3 cells, bEnd.3 cells were incubated with free DOX, NP DOX , or ENP DOX with a concentration of DOX at 0.5 μg/mL for 4 h and the fluorescent intensity of DOX was assessed through a flow cytometer. Similarly, cellular uptake of DOX in GL261 glioma cells following transwell culture was also assessed by flow cytometry.
To determine apoptosis of GL261 cells following transwell culture, GL261 cells were stained with Annexin-V and propidium iodide for 15 min according to manufacturer's instruction and were subjected to flow cytometry.
To determine surface exposure of CRT, GL261 cells following transwell culture were stained with propidium iodide and FITC-labeled anti-CRT antibody and CRT positive cells were counted through flow cytometry.
Mature dendritic cells were determined by the ratio of CD80 + CD86 + cells gated in CD45 + CD11b + CD11c + population in deep cervical lymph nodes and cytotoxic T cells were determined by ratio of CD8 + cells among CD45 + tumor-infiltrating lymphocytes in tumor tissue as described previously. 26  To determine uptake of DOX in the tumor tissue, orthotopic GBM xenograft mice were established and were treated with intravenous injection of DOX, NP DOX , or ENP DOX . One day after indicated treatment, tumors were isolated, sectioned, stained with DAPI, and DOX accumulation in the tumor tissue were assessed by CLSM.

| ELISA
ATP release from GL261wells following transwell culture was determined by ELISA using a chemiluminescent ATP Determination Kit according to manufacturer's instruction (Invitrogen™, cat# A22066).
Cytokines produced from the tumors of mice treated with intravenous DOX, NP DOX , or ENP DOX were examined using ELISA assay kits following manufacturer's instruction.