Hypoxia-Triggered Transforming Immunomodulator for Cancer

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Hypoxia-Triggered Transforming Immunomodulator for Cancer Immunotherapy via Photodynamically Enhanced Antigen Presentation of Dendritic Cell Sooseok Im, Junseok Lee, Dongsik Park, Areum Park, You-Me Kim, and Won Jong Kim ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b07045 • Publication Date (Web): 18 Dec 2018 Downloaded from http://pubs.acs.org on December 19, 2018

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Hypoxia-Triggered Transforming Immunomodulator for Cancer Immunotherapy via Photodynamically Enhanced Antigen Presentation of Dendritic Cell

Sooseok Ima, Junseok Leeb, Dongsik Parkb, Areum Parkc, You-Me Kimd, and Won Jong Kima,b,*

a

School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Jigok-ro 64, Nam-gu, Pohang 37666, Republic of Korea b

Department of Chemistry, Pohang University of Science and Technology (POSTECH), Cheongam-ro 77, Nam-gu, Pohang 37673, Republic of Korea

c

Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Jigok-ro 64, Nam-gu, Pohang 37666, Republic of Korea

d

Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Korea

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ABSTRACT

A key factor for successful cancer immunotherapy (CIT) is the extent of antigen presentation by dendritic cells (DCs) that phagocytize tumor-associated antigens (TAA) in the tumor site and migrate to tumor draining lymph nodes (TDLN), for the activation of T cells. Although various types of adjuvant delivery have been studied to enhance the activity of the DCs, poor delivery efficiency and depleted population of tumor infiltrating DCs have limited the efficacy of CIT. Herein, we report a hypoxia-responsive mesoporous silica nanocarrier (denoted as CAGE) for an enhanced CIT assisted by photodynamic therapy (PDT). In this study, CAGE was designed as a hypoxia-responsive transforming carrier to improve the intracellular uptake of nanocarriers and the delivery of adjuvants to DCs. Furthermore, PDT was exploited for the generation of immunogenic debris and recruitment of DCs in a tumor site, followed by enhanced antigen presentation. Finally, a significant inhibition of tumor growth was observed in vivo, signifying that the PDT would be a promising solution for DC-based immunotherapy.

Keywords: combinatorial immunotherapy, photodynamic therapy, hypoxia-responsive drug delivery, dendritic cell modulation, tumor-associated antigen


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Cancer immunotherapy (CIT), harnessing the immune system to induce the death of cancer cells or alter the immunosuppressive tumor microenvironment, has been extensively investigated due to its therapeutic potential.1,2 In various CIT methodology research, the enormous number of results have specified that the activity of dendritic cells (DCs) is indispensable for provocation of tumor-specific immune responses.3,4 DCs are the regulator charged with disciplining the T cells in antitumor immune response, by presenting tumor associated antigens (TAAs) as a peptide loaded on major histocompatibility complex (MHC) molecules.5 Although a number of strategies have been adopted to improve antigen presentation of DCs, including delivery of external tumor antigens and application of adjuvants, poor delivery efficiency as well as heterogeneity of tumor cells hinder desired immune responses.4-6 To solve the issues related with the random mutation of tumor cells, recent studies have been trying to harness the TAAs and immune signals generated from dying tumor cells in situ.7-10 In this way, for tumor infiltrating DCs have an increased chance to encounter the continuously mutated antigens called “neoantigens”. Among the candidate methods to induce tumor cell death, photodynamic therapy (PDT) is a spatiotemporally-controllable strategy to induce apoptosis and necrosis of tumor cells, and more importantly release TAAs.11-13 PDT exploits photodynamic effect, where reactive oxygen species (ROS) are generated from adjacent oxygen by photosensitizer (PS) under light irradiation. ROS is well known as a neutrophil chemotaxis that promotes an infiltration of neutrophils, which play a key role in DC recruitment by processing the prochemerin into chemerin via secreting granule enzymes.14-16 A single photodynamic effect can induce both tumor cell death and recruiting DCs at the laser-irradiated site, facilitating uptake of the as-generated TAAs by DCs.


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Nonetheless, photo-dynamically induced enrichment of tumor infiltrating DCs and TAAs at the tumor site may produce elevated antigen internalization of DCs, where the maturation of DCs is also a substantial step prior to antigen presentation. After modulating the DCs so they can be recruited in a tumor site and internalize debris of tumor cells, one potential strategy that can accelerate the maturation of DCs is to expose them to toll-like receptor (TLR) agonists. CpG oligonucleotide (CpG) is known to bind TLR 9 and activate the DCs, upregulating costimulatory markers essential for antigen presentation.17-24 As PS and CpG are hydrophobic small molecules and oligonucleotides, respectively, in vivo administration may end up with frustrating outcomes due to renal clearance and enzymatic degradation.25 As a potent template for solving this problem, the nanoparticle-based delivery system (NPDS) has attempted PS and adjuvant delivery, due to implicit advantages such as a facile functionalization of multimodality, and an ability to target tumors through the enhanced permeability and retention (EPR) effect.26,27 To embody an ideal NPDS capable of eliciting the prerequisites for DC antigen presentation through photodynamic effect and adjuvant delivery, the system must be able to protect the PS and adjuvant from excretion and degradation during in vivo circulation. They need to also be internalized efficiently by the cells at the target site. Although modification of poly(ethylene glycol) (PEG) is an exclusive strategy for stabilization of NPs against the environment of a blood stream, there is a dilemma that modified PEG also hinder the interaction of NP with target cell surface.28,29 In this regard, tumor microenvironment-specific detachment of PEG would be a promising strategy. Several delivery systems have exploited a shedable PEG layer that can be cleaved by tumor microenvironment conditions such as pH,30 redox potential,31 enzymes,32,33 and oxygen concentration.34 In these conditions, the hypoxia possesses great potential as a tumor-specific stimulus due to its rarity in normal tissues.35,36 Interestingly, recent 4 ACS Paragon Plus Environment

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PDT approaches have focused on utilization of hypoxia-sensitive molecules with PS to induce instantaneous consumption of oxygen through photodynamic effect, facilitating a delicate drug release behavior and increasing the efficacy of the drugs.37-40 Therefore, co-delivery of PS and hypoxia-sensitive moiety could be synergistically applied to antitumor therapy by taking advantages of both the therapeutic effect from PDT and the inducement of hypoxia-dependent functionality. In this study, we have defined the prerequisites for antigen presentation of DCs as following: 1) Infiltration of DCs into tumor site, 2) Internalization of tumor antigens by DCs, and 3) maturation of DCs into ready-to-antigen present status. Accordingly, we have developed a hypoxia-responsive PS/adjuvant co-delivery system, denoted as chlorin e6 (Ce6)-dopedazobenzene-glycol chitosan (GC)-PEG mesoporous silica nanoparticle (CAGE), to enable modulation of the activities of DC and boost antigen presentation (Figure 1). Ce6 is a biocompatible PS with high quantum yield which is feasible for conjugation due to its carboxylic acid residues.41-43 The surface of Ce6-doped mesoporous silica nanoparticle (CAP) was decorated with GC and PEG via azobenzene linker (Azo linker), a hypoxia-responsive labile linker.44-47 Negatively charged CpG was loaded onto the surface of CAGE by electrostatic interaction with GC. The Azo linker could be cleaved under intrinsic tumor hypoxia (oxygen level below 2%)32,34 or abrupt consumption of local oxygen induced by photodynamic effect, leading to both detachment of PEG for the tumor tissue-specific retention of MSNs and release of CpG/GC complexes. Here, we have confirmed multifunctionality of CAGE as a PDT agent and enhanced the delivery ability utilizing hypoxia-responsive transformation in vitro, integrating DC modulation and anti-tumoral effect. Due to the photodynamic effect of PS and delivery of CpG, the population and maturation ratio of tumor infiltrating DCs could be significantly elevated. We believe that an improved activity of DCs, 5 ACS Paragon Plus Environment

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combined with the generation of tumor debris by photodynamic effect, will increase the antigen presentation of DCs, leading to an effective inhibition of tumor growth in vivo.


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Figure 1. A schematic illustration of the preparation and application of the CAGE complex. A) A synthetic scheme of CAGE complex. Ce6 is conjugated within the pore of CAP. The surface of CAP is decorated by Azo linker for further conjugation with GC and PEG. CpG is loaded via electrostatic interaction with GC. Under hypoxic conditions, the cleavage of the Azo linker causes detachment of PEG and a release of the CpG/GC complex from CAGE. B) Mode of action of CAGE in vivo. (1) After systemic administration, CAGE is protected from immune cells or enzymes in blood stream by the shielding effect of surface PEG. (2) Nano-sized complex easily enters into tumor region through the leaky vasculature of tumor tissue. (3) Once CAGE reaches a hypoxic region within tumor tissue, Azo linkers are cleaved, and PEG and CpG/GC complex are released from CAGE, leading to delivery of CpG for DC activation. (4) Under spatiotemporally controlled laser irradiation, ROS is generated, and simultaneously recruits DCs to release the tumor associated antigens (TAA) from tumor cells. (5) Released TAAs are internalized by recruited DCs, and CpG/GC complex activates DC for maturation. (6) Finally, increased level of TAA presentation enhances the antitumor effect.


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RESULTS

Preparation and characterization of Ce6-doped and azobenzene-GC/PEG-modified MSN (CAGE) First, we synthesized the Ce6-doped and amine-modified MSNs (CAP) by following our previously reported methodology with slight modifications (Scheme S1).48,49 Addition of Ce6conjugated organosilane (Ce6-TES) during cetrimonium bromide (CTAB) stabilization stepenabled hydrophobic interaction between the chlorin part of Ce6 and the carbon chain of CTAB, to locate the Ce6 molecule inside of MSN pore.50,51 The mesoporous structure with an average size of 90 nm was observed utilizing transmission electron microscopy (Figure 2A). Then, the surface of CAP was modified with an Azo linker (CAzo), followed by the conjugation of GC and amine-terminated methoxy poly(ethylene glycol) (PEG-NH2) to obtain a final product, CAGE (Figure 2B, Scheme S2). The amount of initial surface amine and was determined by fluorescamine assay (Figure S1). Hydrodynamic size of the carrier was slightly increased after the decoration of polymers on the surface (Figure 2C). The modification of Azo linker was confirmed by UV/Vis spectroscopy and FT-IR spectroscopy (Figure S2, S3). After synthesis of CAzo, absorbance at 300-380 nm was observed as a shoulder peak, 52-54 in agreement with FT-IR spectra represented by characteristic peak of N=N bond at 1424, 1602, and 1658 cm-1.5456

In the case of CAGE, the peak at 2890 cm-1 originated form –CH2 stretching vibration of

PEG was observed.57-59 After each surface modification step, the change in surface charge of MSNs was determined by zeta potential measurements, implying successful decoration of the Azo linker, GC, and PEG (Figure 2D, S4A). Specifically, the negative zeta potential obtained from carboxylic acid group of CAzo became positive after the introduction of GC, verifying successful chemical modification of GC. Then, in sequent conjugation of GC and PEG, the value of the positive zeta potential was decreased, compared to GC-decorated MSN. As a 8 ACS Paragon Plus Environment

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hypoxic-non-responsive control, disuccinimidyl suberate was utilized instead of Azo linker to obtain CUGE control. The amount of conjugated Ce6, GC, and PEG were quantified by thermogravimetric analysis (TGA) and measured to 3.2 %, 5.3%, and 3.6% (w/w) (Table S1), respectively. In addition, the loading amount of Ce6 was confirmed by UV spectrometry (Figure 2E). After the synthesis of CAGE, negatively charged oligomer, CpG was loaded onto the surface of CAGE through electrostatic interaction, in order to obtain the CAGE/CpG complex. The loading efficiency of CpG was evaluated by agarose gel electrophoresis. As shown in Figure 2F, CAGE successfully formed a complex with CpG, implying that the carrier could be utilized for adjuvant delivery and DC activation.

Figure 2. Properties and loading efficiency of delivery carriers. A-C) TEM image of A) CAP and B) CAGE and C) their hydrodynamic size. The scale bars in TEM images represent 100 nm. D) Alteration of zeta potential after each modification. E-F) Qualification of loading of E) Ce6 and F) CpG. 9 ACS Paragon Plus Environment

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Photodynamic tumor cell death and release of tumor-associated antigens induced by CAGE in vitro Intensive generation of ROS under light irradiation is a prerequisite for the PDT agent. Therefore, the ability of CAGE to generate a singlet oxygen was evaluated by the singlet oxygen sensor green (SOSG) (Figure 3A). The increased fluorescent intensity of SOSG at the time of irradiation was observed, followed by a negligible amount of fluorescent intensity when CAGE was incubated in the dark for 60 min. These results accentuated the potential of CAGE as a template for the generation of ROS to induce both PDT for tumors and promote immune cell recruitment. In order to evaluate the biocompatibility and photo-induced cytotoxicity of carriers, the viability of B16.F1 (murine melanoma cell line) cells treated with CAGE or CUGE was measured by MTT assay (Figure 3B). Without laser irradiation, none of the samples exhibited cytotoxicity, confirming good biocompatibility of carriers. However, the viability of cells treated with CAGE and CUGE decreased significantly after laser irradiation, implying potential for CAGE and CUGE to serve as photodynamic agents. In addition, since CpG has no cytotoxic effect against tumor cells by itself, there was no significant difference in viability with or without CpG.60 Next, we investigated the release of tumor proteins utilizing the photodynamic effects of CAGE to determine whether the nanocarrier could provide internal antigens that could be processed under the laser irradiation for DCs. This is a critical step in determining the potential for high cancer-immunotherapeutic effect. To monitor the release of TAA from tumor cells, we used carboxyfluorescein succinimidyl ester (CFSE) as an indicator (Figure 3C). CFSE is a membrane-permeable fluorescent dye, but once it is internalized into cytoplasm, it covalently binds to the amine residues of intracellular proteins and cannot escape from the cell. Therefore, 10 ACS Paragon Plus Environment

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the detection of CFSE from the cell culture supernatant after laser irradiation infers that intracellular proteins are released by photodynamic effect of CAGE. CFSE-prestained B16.F1 cells were treated with PBS, bare MSN, or CAGE, and the fluorescence intensity of the supernatant was measured after laser irradiation. As demonstrated in Figure 3D, the supernatant from the group treated only by only CAGE showed a significant increase in fluorescent intensity under laser irradiation, in comparison to the intensity of the other groups. This verifies the potential for CAGE to act as a PDT agent, inducing the release of TAA towards the microenvironment.


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Figure 3. Photo-responsive A) generation of singlet oxygen and B) cytotoxicity. C) Schematic illustration of tumor protein release experiment and D) the release profile of tumor proteins induced by nanocarrier-mediated PDT, which was investigated by fluorescence of CFSE.

Hypoxia-responsive behavior of CAGE The modification of GC and PEG on the surface of CAGE was designed to be shedable in a hypoxic microenvironment, altering the physicochemical properties of the carrier and improving its delivery efficiency. The release behavior of CpG/GC complex from CAGE under hypoxic conditions was investigated in vitro by the designed experiment (Scheme S3).9 FITClabeled CpG was loaded onto the two types of nanocarrier, hypoxia-responsive CAGE and nonresponsive CUGE, and they were exposed to conditions mimicking biological hypoxia or normoxia. After exposure to the predetermined oxygen conditions, the solutions were centrifuged to measure fluorescent intensity of FITC-labeled CpG in supernatant and pellet (Figure 4A-B, S5). As shown in Figure 4A, approximately 70% of loaded CpG was released from CAGE through the cleavage of an Azo linker in hypoxic conditions, while less than 10% of CpG was released and most of the CpG was detected in the pellet with CAGE at normoxia. However, in the case of CUGE, a hypoxia non-responsive control carrier, there was no significant difference in the amount of released CpG between hypoxic and normoxic conditions. Interestingly, the CpG/GC complex structure was found through transmission electron microscopic (TEM) imaging at the supernatant of hypoxia-exposed CAGE (Figure 4B). This may be the result of hypoxia-responsive cleavage of the linker, followed by spontaneous complexation between released GC and CpG by electrostatic interaction. The segregated CpG/GC complex would be more flexible than a chemically conjugated form if placed onto a nanocarrier surface.61 Softer nanoparticles are known to have advantageous 12 ACS Paragon Plus Environment

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circulation and infiltration after intravenous injection, because the deformability of nanoparticles have major impacts on their mobility in the blood stream.62-64 Therefore, we hypothesized that the release of the CpG/GC complex would influence the proportion of CpG that reaches the tumor draining lymph nodes (TDLN) in vivo. In order to observe the released CpG/GC complex from CAGE entering the TDLN under hypoxic conditions of a solid tumor, fluorescence labeled-CpG was loaded on the CAGE and injected intravenously into a tumorbearing mouse. Ex vivo images of dissected TDLN were obtained at 96 h after injection. As demonstrated in Figure 4C, the lymph nodes of the group treated with CAGE exhibited significantly increased fluorescence intensity compared to that of free CpG-treated or CUGEtreated group (Figure 4D). The difference of the delivery efficiency between CAGE and CUGE could be explained using either of the following two reasons. First, as mentioned above, the separation of the CpG/GC complex under hypoxic conditions and within the solid tumor would enhance the mobility of the complex. Second, in a tumor microenvironment, the cleavage of PEG would benefit the internalization of the complex into DCs whose destination is TDLN. In addition, PEG-modified nanoparticles are known to escape rapidly from lymph nodes. For instance, PEG on the surface of CUGE was preserved even after penetrating through the tumor, resulting in an insignificant interaction between nanoparticles and an extracellular matrix of lymph nodes leading to facile escape. Taken together, an increased entrance of CpG into TDLN could be explained by an improved mobility of the released CpG/GC complex and an effective uptake of CpG by DCs. The DCs then migrated into TDLN as a result of the PEG cleavage under solid tumor intrinsic hypoxia.


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Figure 4. A) Hypoxia-responsive CpG ODN release from CAGE. CpG ODN was labeled with FITC, and the amount of released CpG ODN was investigated by measuring fluorescence intensity of FITC-labeled CpG in supernatant. B) CpG/GC complex in the supernatant observed by TEM. C) Accumulation of CpG in TDLNs after i.v. injection of CAGE/CpG complex in the tumor-bearing mouse model. Fluorescence intensity from flamma-774 labeled CpG in dissected inguinal node was observed by IVIS. D) Quantitative assay of CpG ODN accumulated in TDLN after i.v. injection of CAGE/CpG complex


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The influence of the hypoxia-responsive GC/PEG detachment on the cellular internalization of the Ce6-doped MSN was observed by monitoring the fluorescence of Ce6 inside a nanocarrier using confocal microscopy (Figure 5A). Under hypoxic conditions, a strong fluorescence signal was observed in the cytoplasm after treatment with CAGE, while much lower fluorescence was observed at normal oxygen level. To investigate whether the increased internalization resulted from hypoxia-induced PEG detachment, hypoxia-non-responsive CUGE was also treated with the melanoma cells under a hypoxic and a normoxic environment. The results showed that CUGE was rarely integrated into the cells regardless of the oxygen level, demonstrating that the shielding effect of PEG on the surface of the nanocarrier is a crucial parameter that lowers internalization of the nanocarriers (Figure S6B). To quantitatively study the enhancement of cellular internalization at hypoxic conditions, the CAGE-treated cells were analyzed using flow cytometry. In accordance with the pre-described confocal microscopic results, approximately 4.5-fold higher intensity was detected in hypoxic conditions than in normoxic conditions (Figure 5B-C). Overall, the results show that cleavage of PEG from CAGE is a crucial factor for improvement of cellular internalization of nanocarrier.


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Figure 5. Hypoxia-responsive internalization of CAGE into cells under hypoxic or normoxic conditions analyzed by A) confocal fluorescence microscopy (Blue: nucleus, Red: Ce6) and BC) flow cytometry. PEG detachment under in vitro hypoxic conditions resulted in an enhanced internalization of CAGE.


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Alteration of dendritic cell by CAGE-mediated PDT and adjuvant delivery in vivo So far, we have demonstrated the multi-functionality of CAGE including photodynamically induced TAA release and enhanced delivery efficiency of CpG after segregation under hypoxic conditions. Following the performance of CAGE in vitro, we examined whether the population, maturation, and antigen presentation ability of DCs in tumor tissue would be enhanced after systemic administration of CAGE in vivo. The tumor-infiltrating DCs were analyzed using flow cytometry to confirm the effect of the photodynamic treatment and hypoxia-mediated CpG delivery on the activity of DCs. As aforementioned, CAGE is intended to perform critical functions: 1) the hypoxia-responsive cleavage of the Azo linker would facilitate the efficient delivery and uptake of PS and adjuvant to cells in tumor sites and lymph nodes; 2) the photodynamic effect of Ce6-conjugated CAGE would generate ROS under laser irradiation, resulting in the creation of both a cytotoxic effect on tumor cells releasing tumor proteins and the migration of DCs to the irradiated site; 3) the release of CpG/GC complexes from the nanocarrier would increase the chance to encounter and differentiate the DCs more prone to antigen presenting states. Therefore, we have expected that if all of these functions of the nanocarriers work successfully, the recruited DCs capture tumor debris and present antigens efficiently (Figure 6). The ability of CAGE to recruit DCs utilizing a photodynamic effect and activate the DCs by delivered CpG were estimated by measuring the number of DCs (CD11c+ MHC II+) and activated DCs (CD80+ CD86+) in tumor tissue, respectively. The flow cytometry results confirmed that the tumor treated with CAGE and laser irradiation showed an increase in the CD11c+ MHC II+ DC population regardless of CpG delivery(Figure 6A, 6D). We confirm our hypothesis that the CAGE-mediated PDT is crucial factor for DC recruitment as opposed to the delivery of CpG. In the case of DC activation, the CD80+ CD86+ DC population was only 17 ACS Paragon Plus Environment

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enhanced in CAGE/CpG complex treated groups, regardless of laser irradiation (Figure 6B, 6E). In other words, CpG should be delivered to elicit upregulated DC activation. Interestingly, the results from CAGE/CpG complex-treated group not subjected to laser irradiation, which do not generate ROS for DC recruiting, but can deliver CpG in hypoxia-responsive manner, confirmed our hypothesis by showing a similar CD11c+ MHC II+ DC population with the PBS treated group but with highly activated DC portion. In contrast, when CAGE without CpG was injected and laser-irradiated, CD80+ CD86+ DC population shown normal portion despite the highly recruited DC, also confirming that CpG delivery and photodynamic effect have major effect on maturation and recruitment respectively. Interestingly, the tumors treated with free Ce6/CpG and CUGE/CpG exhibited the lowest amount of recruited and activated DCs. In the case of free Ce6/CpG group, free Ce6 has a negative charge and a small size, which is susceptible to renal clearance and readily degraded by DNase during circulation. In contrast, although PEG-modified CUGE could protect the cargo from rapid excretion and enzymes, its stealth-effect reduced the interaction with targeted tumor tissue, causing a decrease in the retention and uptake of nanocarriers, as revealed by the relatively low recruitment and activation of DCs. The maturation procedure makes DCs able to express abundant MHC molecules and able to present antigens in peptide form and loaded onto the MHC molecules. Also, photodynamically induced tumor cell death could assist DC antigen presentation by providing the damage associated molecular patterns, including TAA. Thus, the extent of antigen presentation was evaluated using flow cytometry to analyze tumor antigen-loaded MHC I molecule to confirm whether the antigen presentation of DCs are practically elevated by multifunctionality of CAGE. As a model study, an ovalbumin (OVA)-expressing melanoma cell line, B16.Mo5, was exploited to analyze the population of OVA-presenting DCs (Figure 6C, 6F). As we predicted, 18 ACS Paragon Plus Environment

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the DCs from the tumor treated with CAGE/CpG and laser irradiation exhibited the highest OVA presentation, indicating that all of 1) hypoxia-responsible transformation, 2) photodynamic effect, and 3) adjuvant delivery are necessary for an increase of antigen presenting process.

Figure 6. Recruiting, activation, and antigen presentation of DC by CAGE/CpG complex. A) DC recruitment profile induced by CAGE under laser irradiation. DC recruitment was evaluated by counting CD11c+ MHC II+ DC in tumor tissue. B) DC activation profile induced by CpG delivery using CAGE. DC activation was evaluated by counting CD80+ CD86+ DC in tumor tissue. C) Antigen presentation of DC. Antigen presentation of DC was evaluated by


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counting OVA peptide (SIINFEKL) loaded MHC I+ DC in tumor tissue. D-F) Corresponding quantification of D) recruited, E) mature, and F) OVA-presenting DC population.

In vivo tumor growth inhibition by immune-functional nanocarrier The in vivo tumor growth inhibition experiment was performed to identify functionalities including tumor protein release by photodynamic effect, hypoxia-responsive PEG detachment, and resultant enhanced cellular internalization for antitumor therapeutic effect. Tumor volume was monitored in a B16.F1-bearing mouse allograft model after treatment with nanocarriers. In order to induce the photodynamic effect, we used a 660 nm laser (200 mW/cm2) for irradiation after 24 h of the intravenous administration of the various nanocarriers. As shown in Figure 7A-B, the CAGE/CpG-treated group exhibited significant and long-lasting inhibition of tumor growth under the laser irradiation. In contrast, the group with CAGE/CpG administered without laser irradiation showed significantly lower tumor regression. This result emphasizes that the photodynamic effect of mediated alteration of tumor microenvironment immensely contributed to tumor growth suppression, even when including the release of the tumor proteins and recruitment of various immune cells. Applying laser irradiation after the injection of CAGE without CpG derived poor therapeutic effect, identifying the importance of the activation of DCs and resulting T cells. Comparison of tumor growth rate between CAGE/CpG and CUGE/CpG-treated mice strongly suggests that the hypoxia-responsive cleavage of PEG and release of CpG/GC complex from CAGE elicited more efficient antitumor responses. Mice treated with free Ce6/CpG showed little tumor growth inhibition, resulting in an absence of ability for the carrier to protect the molecules from clearance. Histological assay also proved the successful anticancer effect of CAGE/CpG complex with laser irradiation (Figure 7C). In addition, while there was no significant change in body weight, 20 ACS Paragon Plus Environment

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the CAGE/CpG group had a 100% survival rate for 28 days after tumor inoculation (Figure 7D-E). Taken together the cooperation of PDT and CIT, resultant from the administration of CAGE as a CpG carrier, has produced the practical therapeutic antitumor effect.


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Figure 7. In vivo inhibition of tumor growth by nanocarriers. A) In vivo tumor growth curve, B) representative tumor images, and C) their hematoxylin and eosin

stained image of tumor

tissues after treatment with each sample (V: Viable, N: Necrosis) D) body weight and E) survival curve mice (n=6)

DISCUSSION

For decades, CIT has drawn much attention due to its practical strategy and high efficiency to overcome the drawbacks of conventional anti-cancer treatments. It has been generally understood that successful CIT requires an enhancement of immune system to induce a cytotoxic effect against tumor cells. This includes causing DCs to: 1) infiltrate into tumor site, 2) uptake TAAs, 3) undergo maturation, and finally 4) migrate into TDLN to present tumor antigen to T cells. In this study, we have focused on nanoparticle-based PDT to realize all these criteria in a single strategy. Our precisely designed multifunctional immunomodulator, CAGE/CpG complex, could play several roles to exploit a tumor-specific microenvironment. Surface of CAGE was PEGylated to enhance in vivo stability after administration, and a nano-sized carrier accumulated in the tumor region, due to the EPR effect of tumor. Subsequently, surface PEG and CpG/GC complexes were easily detached under a hypoxic tumor environment. Detachment of polymers from nanocarriers could enhance both the uptake of CAPs, proven by fluorescence microscopy and flow cytometry in vitro, and the migration of delivered CpG into TDLNs, observed from ex vivo fluorescence image of TDLNs. Moreover, photodynamic effect of the CAGE/CpG complex could generate ROS for recruiting DC and killing cancer cells, closely followed by a release of tumor proteins. In special, we have speculated that ROS23 ACS Paragon Plus Environment

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induced influx of neutrophil and its enzymatic actions are the key steps for the enrichment of DC at tumor site, though the precise further study is needed for this phenomenon in mechanistic aspect, which would make way to the consummate combinatorial immunotherapy. After all, the flow cytometry assay demonstrated recruitment, activation, and tumor antigen presentation of DC, serial prerequisites for successful CIT. The antitumor effect of the CAGE/CpG complex was incomparable in vivo against other control groups, a result of photodynamically driven cancer immunotherapy.

CONCLUSION

In summary, we developed an enhanced PS/adjuvant delivery system using a hypoxiaresponsive nanocarrier and demonstrated its functions in the tumor-associated immunological microenvironment. An improved internalization of nanocarrier was accomplished by creating a hypoxia-shedable PEG modification, while enhancing retention and tumor-specific uptake of the delivered PS and adjuvant. Hypoxia-responsive release of CpG/GC facilitated the uptake by DCs while photodynamic effect of PS exhibited various functions including antitumor effect, generation of tumor peptides as tumor-associated antigens, and recruitment of DCs. Furthermore, tumor growth was successfully inhibited by treatment of CAGE/CpG under irradiation of red laser, indicating that the proposed CAGE/CpG has a potential for acting as an antitumor agent by utilizing photodynamic effect assisted cancer immunotherapy.


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EXPERIMENTAL SECTION

Materials. The chemicals without specific mention were obtained from Sigma Aldrich (St. Louis, MO). Ce6 and Azobenezene-4,4’-dicarboxylic acid were purchased from Frontier Scientific Inc. (Logan, UT, USA) and Tokyo Chemical Industry (Tokyo, Japan), respectively. CpGs were obtained from InvivoGen (San Diego, USA). The fluorescent dye labeled CpGs were purchased from Bioneer Corp. (Daejeon, South Korea). The PEG–NH2 (Mw = 5 kDa) was obtained from SunBio, Inc. (Daejeon, Korea). Flamma-774 was obtained from Bioacts (Incheon, Korea). Instrumental methods. TEM imaging was obtained with a TEM (JEM-2200FS with image CS-corrector, JEOL) and imaged using Gatan Digital Microscope. UV-vis spectrometer (UV 2550, Shimadzu) and a spectrofluorophotometer (RF-5301 PC, Shimadzu) were used for measurement of UV-vis spectra and fluorescence spectra, respectively. Both of hydrodynamic size and zeta potential were estimated using a Zetasizer instruments (Nano S90 and Nano Z, Malvern). Olympus FV-1000 and OLYMPUS FLUOVIEW ver. 1.7 Viewer software were used for confocal laser scanning microscopy (CLSM). Davinch Western Imaging System (Davinch-K, Younghwa Science, Korea) were used to obtain monochrome images in gel retardation experiment. The dissected lymph nodes were analyzed by a small-animal in vivo imaging system at Pohang Technopark Biotech Center (Califer Lifescience, Hopkinton, MA). For the internalization studies, fluorescence images were obtained by Eclipse Ti-E fluorescence microscopy (Nikon) equipped with Coolsnap MYO CCD camera (Photometrics) and analyzed with NIS-Elements Advanced Research software (Nikon,Tokyo). Using FACS Calibur (Becton Dickinson) and BD Cell Quest software (Becton Dickinson), flow cytometry data were acquired, respectively. For the flow cytometry analysis of recruitment, maturation, and antigen


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presentation of DCs, the data were analyzed with an LSRFortessa (BD) and analyzed with FlowJo software (Tree Star) Cell culture and animal model. The B16.F1, a murine melanoma cell line, was purchased from American type culture collection (ATCC).

MO5, an OVA-transfected clone derived

from a B16 melanoma, was donated by Dr.You-Me Kim (POSTECH, Pohang). Both cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone), 100 U/mL penicillin (Hyclone) and 100 μg/mL streptomycin. All the cell lines were cultured in 5% CO2 humidified incubator at 37 °C. Female C57BL/6 mice were received from POSTECH Biotech Center. All animal experiments in this study were performed in accordance with the relevant guidelines and regulations that approved by the POSTECH Biotech Center Ethics Committee and all methods. Synthesis of Chlorin e6-doped Aminopropyl-modified MSNs (CAP). CAPs were synthesized following the reported method with several modifications.37 Briefly, at first, 52.5 mg of Ce6 was added in 5 mL of anhydrous DMF and covalently conjugated to equivalent amount of APTES using EDC/NHS chemistry. 200 mg of CTAB was dissolved in 96 mL of water and 700 μL of 2 M NaOH solution was added. The mixture was incubated for 30 min at 80 oC. After incubation, 1 mL of TEOS, 700 μL of prepared Ce6-APTES, and equivalent amount of EDC/NHS were added under vigorous stirring. After 30 min, 117 μL of APTES was added for aminopropyl modification onto MSN surface and the mixture was stirred at 80 oC for additional 90min. The mixture was centrifuged at 13000 rpm for 20 min at RT. Subsequently the dark green pellet washed with water and methanol. The product material was further treated to remove the surfactant in the mesoporous structure. 1 g of product material was dispersed in 100 mL of methanol and 100 uL of concentrated HCl was added. Reaction mixture was heated at 60 oC for 18 h. The mixture was centrifuged to isolate CAPs, washed with water and methanol for drying under high vacuum for 24 h. The synthesized CAPs were characterized by 26 ACS Paragon Plus Environment

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using TEM and Zetasizer. The amine group on the MSN surface was quantified by fluorescamine assay for further coupling reaction of GC/PEG and Azo DCA linker using EDC/NHS chemistry. Synthesis of hypoxia-responsive GC/PEG modified CAP (CAGE). For the synthesis of CAGE, as prepared CAP (100 mg) was dispersed in 30 mL of DMSO/pyridine (4:1) and covalently conjugated with 10 mg of azobenzene-4,4’ dicarboxylic acid (Azo DCA) using EDC/NHS chemistry. The product material was centrifuged and washed with DMSO/pyridine solution, 5 M NaCl, PBS, water, and methanol to get rid of unreacted Azo DCA and dried under high vacuum. Subsequently, 50 mg of Azo DCA-conjugated CAP, 50 mg of GC, and 50 mg of 5k mPEG-amine was dispersed into 50 mL of DW and reacted via EDC/NHS chemistry again. The product material was centrifuged and washed with PBS, 5 M NaCl, DW, and methanol and dried under high vacuum. For the synthesis of hypoxia-nonresponsive GC/PEG modified CAP (CUGE), GC and 5k mPEG-amine was conjugated onto surface of CAP using disuccinimidyl suberate via NHS ester reaction instead of Azo DCA. Gel Electrophoresis. CpG Loading capacity of CAGE was evaluated by gel retardation assay. CAGE/CpG complexes were prepared by mixing CpG solution and CAGE solution under vortexing. The complex solution was incubated for 30 min at 4 oC. Each solution with different N/P raito was loaded in comb wells of a ethidium bromide (EtBr, 0.5 μg/mL) containing 2% (w/v) agarose gel in 0.5 TAE (Tris-acetate-EDTA) buffer. Loaded complexes were electrophoresed through the gel at 100 V for 20 min. Detection of singlet oxygen. Generated singlet oxygen from CAGE was measured using SOSG as according to the protocol provided form manufacturer. Briefly, 1 mg/mL CAGE was dispersed in PBS and 1 μM SOSG was added. 660 nm laser was irradiated on the suspension 27 ACS Paragon Plus Environment

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at a power density of 100 mW/cm2. After laser irradiation, the fluorescence at 530 nm at different time points was measured. Control experiment was operated under dark condition. Cell viability test and tumor protein release test in vitro. Cytotoxicity of the various condition of nanocarrier was evaluated by the MTT assay. B16.Mo5 cells were seeded on 96 well plate with an initial density of 8000 cells/well and incubated for overnight. CAGE and CUGE containing final concentration of 0 to 4 μg/mL Ce6 was treated and incubated for 4 h. After incubation, laser irradiation was applied to the treated cells using the 660 nm laser at 150 mW/cm2 power density for 15 min and further incubated for 24 h. After washing with DPBS, the cells were treated with MTT solution (500 μg/mL) and incubated for 4 h in incubator. Resulting purple crystal was dissolved in DMSO for the measurement of absorbance at 570 nm. To demonstrate tumor protein release induced by photodynamic effect, the cells were stained with CFSE following manufacturer’s protocol and seeded in 96 well plate same as above. After laser irradiation, the fluorescent intensity (517 nm) of CFSE labelled protein in cell culture supernatant were measured by a spectrofluorophotometer. The relative percentages of the supernatant from totally lysed CFSE stained cells were used to represent 100% of fluorescent intensity of CFSE labelled protein. Hypoxia sensitivity in vitro. A hypoxia-responsive cleavage of Azo DCA linker in CAGE was measured by monitoring the fluorescence intensity of FITC-labelled CpG after centrifugation following exposure to hypoxia condition. Hypoxia condition was produced by using N2-purged solution containing rat liver microsomes and NADPH as reported.34, 44-45 CAGE and CUGE was suspended in phosphate buffer at concentration of 0.1 mg/mL and incubated with 0.5 mg/mL rat liver microsome and 50 μM NADPH for 2 h in 37 oC with gentle


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agitation. The reaction mixture was centrifuged at 13,000 rpm for 20 min and the pellet and supernatant collected separately for fluorescent intensity evaluation and TEM observation. TDLN imaging ex vivo. C57Bl/6 mice, which bearing B16.F1 tumor volume of ~200 mm3 at day 0 in the right flank, were randomly divided into each groups (n = 4). To each group of mice, PBS, Flamma-774 labelled CpG, CUGE/Flamma-774-CpG complex, and CAGE/Flamma774-CpG complex samples were injected intravenously (2 mg/kg Ce6 and 0.5 mg/kg CpG). At day 4, all mice were sacrificed and inguinal lymph node at right flank from each mouse were dissected and the fluorescence intensity from the lymph nodes were analyzed by an IVIS. Intracellular uptake of nanoconstruct in vitro. The B16.Mo5 cells were seeded at an initial density of 20,000 cells/well on 12-well plates. CAGE and CUGE containing 5 μM Ce6 were treated and incubated for 4 h in normoxia or hypoxia condition. After washing with cold DPBS, 10% neutral buffered formalin (NBF) solution was treated for 20 minutes. After several times of DPBS washing, the cells were stained with DAPI by following the manufacturer’s protocol and the images obtained by fluorescence microscopy. The cellular internalization was also determined by flow cytometry. The B16.Mo5 cells were seeded at an initial density of 200,000 cells/well on 6-well plates and incubated overnight. Same as fluorescence microscope imaging, CAGE and CUGE containing 5 μM Ce6 were treated and incubated for 4 h in normoxia or hypoxia condition. After incubation, each well was washed with DPBS several times and the cells were trypsinized, and resuspended in FACS buffer for flow cytometry analysis. Flow cytometry. To estimate the population of immune cells in the tumors, dissected tumors from mice were digested using tumor digesting buffer containing 4.4 mg/mL collagenase I, 10 μg/mL Dnase I in DMEM at 37 °C for 30 min. Single cell suspensions were pass through nylon mesh filters before PBS washing. The filtered cells were incubated for 30 min at 4 °C in dark with the following fluorophores conjugated Rat Anti-Mouse antibodies: BUV 395-CD45 (BD),


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BV 650-I-A/I-E(BD), PE-Cy7-CD11c(BD) FITC-CD80(BD),

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APC-CD86(BD), and APC-

H-2Kb bound to SIINFEKL(Biolegend). Stained cells analyzed using flow cytometry analysis. Monitoring tumor growth of mice. B16.Mo5 cells were subcutaneously (s.c.) inoculated at the right flank of six-to-eight week-old C57BL/6 female mice. When the size of tumor grown to ~100 mm3, the mice were divided into 6 groups (n=6) for intravenous injection with 200 μL of the each sample (PBS, Free Ce6/CpG, CAGE, CUGE/CpG and CAGE/CpG for two groups, 2 mg/kg Ce6 and 0.5 mg/kg CpG). Except for one CAGE/CpG treated group, the tumor site of mice were irradiated with 660-nm laser at a power density of 200 mW/cm2 for 15 min on a day after sample injection. The size of tumor as well as body weight of the mice were measured. Formula for a prolate ellipsoid was used to calculated the volume of tumors. (tumor volume = ab2/2, where a is the length and b is the width) Student’s t-test were carried out for all statistical analyses. After, H&E staining was performed to histologically analyze PDT-induced necrosis of the tumor tissue. In brief, after the mice had been given each treatment, the tumors were dissected and fixed in 10% NBF. The tumor masses underwent paraffin embedding and subsequently sectioning at 4 μm using a Finesse ME microtome (Thermo Fisher Scientific). Each tumor tissue sections were stained with H&E and observed using a microscope (Nikon eclipse 80i, USA). All animal experiments were approved by the POSTECH Biotech Center Ethics Committee.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed synthesis and analysis (quantification, UV/Vis, FT-IR, gel retardation, TGA) of CAGE; Scheme for hypoxia experiment in vitro and additional results; Hypoxia-responsive behavior of control samples; Flow cytometry of BMDC; Distribution of CAGE and CUGE ex vivo (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Sooseok Im: 0000-0003-4719-1513 Junseok Lee: 0000-0002-3709-1386 Dongsik Park: 0000-0003-0059-8973 Won Jong Kim: 0000-0002-4064-0999


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ACKNOWLEDGEMENT This research was supported by the National Research Foundation of Korea (NRF) grant (NRF2017R1E1A1A01074088), Bio & Medical Technology Development Program of the NRF funded by the Korea government (Ministry of Science and ICT) (NRF-2017M3A9F5030930), and Creative Materials Discovery Program through the NRF funded by Ministry of Science and ICT (NRF-2018M3D1A1058813).


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TOC FIGURE


Hypoxia-Triggered Transforming Immunomodulator for Cancer

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