Enhanced Tumor Retention Effect by Click Chemistry for Improved

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Biological and Medical Applications of Materials and Interfaces

Enhanced Tumor Retention Effect by Click Chemistry for Improved Cancer Immunochemotherapy Ling Mei, Yayuan Liu, Jingdong Rao, Xian Tang, Man Li, Zhirong Zhang, and Qin He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02954 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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ACS Applied Materials & Interfaces

Enhanced Tumor Retention Effect by Click Chemistry for Improved Cancer Immunochemotherapy Ling Mei, Yayuan Liu, Jingdong Rao, Xian Tang, Man Li, Zhirong Zhang and Qin He*

Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University. No. 17, Block 3, Southern Renmin Road, Chengdu 610041, China

*Corresponding author E-mail: [email protected]

Abstract Due to the limited drug concentration in tumor tissues and inappropriate treatment strategies, tumor recurrence and metastasis are critical challenges for effectively treating malignancies. A key challenge for effective delivery of nanoparticles is to reduce uptake by reticuloendothelial system and to enhance the permeability and retention effect. Herein, we demonstrated Cu(I)-catalyzed click chemistry triggered the aggregation of azide/alkyne-modified micelles, enhancing micelles accumulation in tumor tissues. In addition, combined doxorubicin with the adjuvant monophosphoryl lipid A, an agonist of toll-like receptor4, generated immunogenic cell death, which further promoted maturity of dendritic cells, antigen presentation and induced strong effector T cells in vivo. Following combined with anti-PD-L1 therapy, substantial anti-tumor and metastasis inhibitory effects were achieved because of the reduced PD-L1 expression and regulatory T cells. In addition, effective long-term immunity from memory T cell responses protected mice from tumor recurrence. Keywords: drug delivery; click chemistry; immunochemotherapy; immune checkpoint inhibitor; immunogenic cell death (ICD) 1. Introduction Chemotherapy is the common choice in the treatment of most cancers, especially solid tumors. However, the anti-tumor effect is usually limited. Chemotherapeutic agents are nonspecifically distributed, killing both tumor and normal cells. Thus, interest in researching tumor-targeted nanoparticles has been 1

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great1. In addition, since the drug concentration in tumor site is restricted; more doses are required, which inevitably leads to higher toxicity, and increased possibilities of multidrug resistance and metastasis. Recently, based on the development of a range of tools that scrutinize the progress of any anti-tumor responses, novel therapies have been developed including immunotherapy2. The ability of nanoparticles for enhanced accumulation in tumors mainly due to the leaky nature of tumor vasculature3; for this process, particle size is a key factor. Nanoparticles with smaller sizes uuaually showed greater penetration within limits. In contrast, the retention capacity is significantly better with larger nanoparticles than smaller ones4-6. Currently, conventional nanoparticles with fixed sizes cannot solve the contradiction. To address this problem, a new nanoparticle system with changeable particle sizes, based on click chemistry reactions, was established. DSPE-PEG micelles modified with azide or alkyne group had small sizes of approximately 25 nm. After the micelles (~25 nm) penetrated tumor tissues, cycloaddition occurred between the groups, leading to an obvious increase in micelle size (~120 nm), which was beneficial for retention and accumulation. Currently combination therapies with chemotherapy and immunotherapy have been progressing rapidly and have shown potential for eliminating tumor cells, and preventing metastasis and tumor recurrence. Though chemotherapy and immunotherapy are generally considered unrelated or even antagonistic in tumor treatment, a good deal of academic and clinical studies showed that appropriate immunochemotherapy can achieve a stronger anti-tumor effect than monotherapy7. Apoptotic cell death was assumed poorly immunogenic (or even tolerant)for a long time, whereas necrotic cell death is truly immunogenic8. Recently, immunogenic cell death (ICD), which could induce immune responses, was widely accepted 9-10. Anthracycline-treated tumor cells usually cause immune responses mainly through the exposure of calreticulin and other factors11. This response facilitates the phagocytosis of tumor antigens the antigen presentation by dendritic cells (DCs) , leading to a potent immune response, including increased levels of related cytokines and cytotoxic T lymphocytes (CTLs)12. Furthermore, monophosphoryl lipid A (MPLA), approved by FDA can generate faster, stronger, and longer lasting immune responses when combined with vaccine antigens13. Though doxorubicin (Dox) induced ICD ultimately lead to an immune response with the aid of adjuvant MPLA, strong CTLs responses are lacking. Programmed death receptor-1 (PD-1) and ligand 1 (PD-L1) are a pair of important immunosuppressive molecules that prevent excessively strong immune response from damaging normal tissues and are thus called an immunological check point14. PD-L1 can bind to PD-1 expressed in T cells, which is a key pathway for suppressing T cell responses15. In addition, MPLA-induced interferon gamma (IFN-γ) expression by T cells can further promote PD-L1 expression, forming a feedback loop that maintains immunosuppression in a dominant manner16 and promotes the proliferation of infiltrating regulatory T cells. Therefore, inhibiting of PD-1/PD-L1 may restore the activation of CTLs. Several immune checkpoint inhibitors approved by the FDA showed impressive clinical anti-tumor effects. 2


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Herein, to optimize anti-tumor efficacy, we established DSPE-PEG-based micelles with variable particle sizes for the co-delivery of Dox and adjuvant MPLA (Figure 1a). Due to the ability to manipulate penetration capacity and retention by controlling the particle size, the delivery platform achieved maximal accumulation in the tumor. Due to the endoplasmic reticulum stress in response to Dox-induced ICD, tumor cells exposed calreticulin on the outer cell membrane. This exposure and other released signaling proteins facilitated the tumor antigen engulfment by DCs and antigen presentation. The processes led to cytokines mediated immune response involving increased CTLs, which was further enhanced by an immune checkpoint inhibitor, an anti-PD-L1 monoclonal antibody. In this manuscript, we have carried out a series of in vivo investigations, and our combined immunochemotherapy strategy notebly suppresed tumor growth and prevented tumor metastasis and recurrence. 2. Materials and methods 2.1 Materials Monophosphoryl lipid A (MPLA) and DSPE-PEG2000-OH were bought from Avanti Polar Lipids (Alabaster, AL, USA). NaN3, propargyl bromide, copper sulfate, sodium ascorbate and all other chemical reagents obtained from Aladdin Bio-chem Technology Co., Ltd (Chengdu, China) were analytical grade or better. Doxorubicin hydrochloride (Dox) was purchased from Beijing Huafeng United Technology Co., Ltd (Beijing, China). Anti-PD-L1 used in vivo was purchased from Bioxcell (West lebanon, NH, USA). Antibodies against cell surface markers for flow cytometry assays and Elisa kits were obtained from eBioscience. Anti-mouse calreticulin, anti-mouse CD34, FITC-labeled goat anti-rabbit secondary antibody and Rhodamine labeled goat anti-rabbit secondary antibody were obtained from Zen Bioscience (Chengdu, China). 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT),1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD), 4,6-diamidino-2-phenylindole (DAPI) were purchased from Biotium (Hayward, California, USA).

2.2 Biodistribution Study and Fluorescent Imaging Mixed DiD-loaded micelles were injected into tumor-bearing C57 mice via tail vein.Catalysts (copper sulfate and sodium ascorbate, 50 μL) were intratumorally injected 4 h later in M-DiD (+) group. Then mice were dealed with Bio-Real in vivo imaging system (Caliper, Hopkington, MA, USA) at 1, 4, 8, 12 and 24 h. Then, main organs from sacrificed mice were also imaged. The semi-quantitative evaluation of the fluorescence intensity at the tumor site from the in vivo images was also carried out. For the micelle tumor accumulation and distribution studies, the tumors from mice at were deal with 4% paraformaldehyde, 10% and 30% sucrose solution. Tumor sections were abtained at different depths with a 10 μm thickness. Then, the tumor sections incubated with anti-CD34 antibody overnighwere incubated with FITC-labeled anti-rabbit IgG antibody for 1 h. Finally, the sections were incubated with DAPI and imaged by a laser confocal (FV1000, Olympus, Japan). 3



2.3 Therapeutic Effect on B16F10 Xenograft Tumor Models C57 mice were divided into seven groups including the Saline, M-MPLA (+), M-Dox (+), Free-Dox/MPLA, M-Dox/MPLA, M-Dox/MPLA (+), and M-Dox/MPLA (+) plus anti-PD-L1. B16F10 cells (5 × 105) in PBS were subcutaneously injected inmice. Beginning on the 8th day after tumor implantation, the different drug formulations were intravenously injected every three days for three cycles. The MPLA and Dox doses were 5 μg/ per mice and 2 mg/kg. For catalyst (+) groups, copper sulfate and sodium ascorbate were intratumorally injected (50 μL）4 h after the micelles were injected, and for the M-Dox/MPLA (+) plus anti-PD-L1 group, the anti-mouse PD-L1 (100 μg per mouse) was administered intraperitoneally on days 9, 12 and 15. The tumor volumes of mice were monitored and recorded after the implantation (tumor volume = 0.52 × length × width2). 2.4 Therapeutic Effect on Lung Metastatic Tumor Models B16F10 cells (5 × 105) in PBS were subcutaneously injected in mice; 7 days later, B16F10 cells were also intravenous injection to establish the metastatic lung tumor model. The mouse groups and dosage regimens were identical to those in the B16F10 xenograft tumor model therapeutic study. On the 21st day after implantation, lungs from sacrificed mice in all groups were carefully collected and photographed. The metastatic nodules were counted and recorded. The average number of metastatic lung nodules in the treatment groups was compared with that of the Saline group in order to evaluate lung metastasis inhibition. Moreover, the lung tissues of all the groups were assessed by histological examinationto detect the metastatic lesions. 2.5 Long-term Immune Memory Effects and In Vivo Survival Monitoring B16F10 cells (5 × 105) in PBS were subcutaneously injected int mice, and the mouse groups and dosage regimens were identical to those in the B16F10 xenograft tumor model therapeutic study. On the 16th day after implantation, primary and uncured xenograft tumors were surgically removed. At 30 days after the surgery, 5 × 105 B16F10 cells were subcutaneously injected in mice again, in order to evaluate the long-term immune-memory effects. For the M-Dox/MPLA (+) plus anti-PD-L1 group, the anti-mouse PD-L1 (100 μg per mouse) was administered intraperitoneally on days 31, 34 and 37 after the surgery. In addition, beginning on the 38th day after the surgery, the volumes of the secondary tumors were measured and the survival time of each group was recorded. 2.6 Evaluation of the In Vivo Immune Status and Cytokine Level in the Serum B16F10 cells (5 × 105) in PBS were subcutaneously injected in mice, and the mouse groups and dosage regimens were identical to those in the B16F10 xenograft tumor model therapeutic study. On the 16th day after implantation, the lymph nodes were isolated, filtered via 70 mm cell strainers in complete DMEM media to form a 4


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single cell suspension. To detect immune cells, the cells were incubated with different antibodies and were measured. Dual staining with FITC-anti-CD11c and four kinds of PE labeled maturation marker antibodies (anti-CD86, anti-CD80, anti-CD40 and anti-MHC-II) were used to detect DCs maturation. T cells were also analyzed. On the 16th day after implantation, the tumors were collected and digested. The tumor cells were filtered via cell strainers and washed. Then, the suspension was incubated with different antibodies to detect the corresponding T cells. FITC-anti-CD8α/PE-anti-CD3 for CD8α+ T cells, FITC-anti-CD4/PE-anti-CD3 for CD4+ T cells, and FITC-anti-CD4/PE-anti-Foxp3 for Tregs. FITC-anti-CD14 was used for macrophages. All cells were measured by flow cytometer. For measuring cytokine levels, serum samples were collected and diluted to be measured on the 16th day. Tumor necrosis factor (TNF), interferon γ (IFN-γ) and interleukin 2 (IL-2) were analyzed by ELISA kits in accordance with vendor protocols. 3. Results and discussion 3.1 Characterization of M-Dox/MPLA Because of the efficiency and high catalytic activity of the ‘click’-type reactions, Cu(I)-catalyzed azide/alkyne cycloaddition (CuAAC) enables the surface modification of peptides, nucleic acids, supramoleculars and a large variety of other ligands. Another advantage of “click chemistry” reactions has also been reported: the crosslinking reaction of nanoparticles via CuAAC leads to particle aggregation and permanent stabilization17. To implement the CuAAC-based strategy to enhance tumor accumulation of drug-loaded nanoparticles, DSPE-PEG-N3 and DSPE-PEG-alkynyl (DSPE-PEG-Alk) were first synthesized by conjugating N3 and alkynyl to DSPE-PEG-OH, respectively (Figure S2 and S3). These modified amphiphilic conjugates were self-assembled into micelles and the initial size was about 25 nm (Figure 1b). Then, MPLA and Dox were loaded easily due to the hydrophobic properties and electrostatic interactions of these drugs. The M-Dox/MPLA was obtained by mixing equal volumes of the DSPE-PEG-N3 (Dox/MPLA) micelles and DSPE-PEG-Alk (Dox/MPLA) micelles. The particle size of the drug loaded micelles was similar to blank micelles and the related physical characteristics are listed in Table S1. We also examined micelle stability in serum-supplemented PBS, and the particle size of all micelles exhibited little change over 24 h (Figure S4). To characterize this aggregation process, we utilized transmission electron microscopy (TEM) to image the M-Dox/MPLA (+) micelles before and after the Cu(I)-triggered cycloaddition reaction. Indeed, the TEM images revealed the interaction between micelles (M-Dox/MPLA) and the corresponding aggregation after Cu(I) exposure for 4 h. The micelles appeared “glued” to each other and even fused. Dynamic light scattering further verified the substantial increase in hydrodynamic size (Figure 1b). This size increase exhibited a time-dependent manner in the presence of catalysts, and even to be hundreds of nanometers in vitro (Figure S1a and S1b). The in release profile was evaluated by dialysis in vitro. The fluorescence was assessed by HPLC. Dox released from the M-Dox/MPLA was slower at pH 7.4 than that at pH 5.0 (Figure S5). Approximately 50% of the total Dox was released 5



incubating at pH 5.0 after 48 h, while approximately 33% Dox was released at pH 7.4. Drug release patterns can be affected by the interaction between the drugs and nanoparticles. Dox contains an amino group, which is protonated under acidic conditions (pH 5.0), enhancing the electrostatic repulsion between Dox and DSPE-PEG, and DSPE-PEG is also positively charged under acidic conditions. Thus, the release of Dox in acidic medium increases rapidly. A hemolytic exxperiment was carried out to validate the safety of modified micelles. Mixed micelles (M-Dox/MPLA) varying from 0.25 to 2.5 mg/mL showed little hemolytic toxicity, and the hemolysis rate was lower than 0.5%. After exposure to micelles, the microscopic images revealed that the blood erythrocyte membrane structure presented with little change or aggregation (Figure S6).

Figure 1. (a) Design of enhanced accumulation of micelles for cancer immunochemotherapy. M-Dox/MPLA(+) composed of DSPE-PEG-N3 and DSPE-PEG-Alk, is engineered for Cu(I)-catalyzed azide/alkyne cycloaddition with size increase to improve tumor accumulation of drugs. As a result of preapoptotic exposure of calreticulin induced by sufficient Dox, tumor cells undergoing ICD release 6


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ATP and other proteins. This facilitates the maturity of DCs, resulting in elicitation of CTLs responses with the help of immunologic adjuvant MPLA. Activated CTLs recognize tumor cells and exert strong anti-tumour efficacy. Combined immunochemotherapy and immune regulatory checkpoint inhibitor anti-PD-L1 further amplify the potency to eliminate tumors. (b) In vitro study of Cu(I)-catalyzed size increase detected by DSL and TEM. Scale bar means 50 nm.

3.2 In Vitro Cellular Uptake, Cytotoxic Effect and ICD Assessment The M-Dox/MPLA (+) herein was designed to be effective immuno-stimulants. Prior to evaluating the capacity of Dox contained formulations to induce ICD, we investigated the cellular uptake and cytotoxic effect in tumor cells. Confocal laser microscopy images of B16F10 cells revealed that the fluorescence intensity was stronger in cells incubated with M-Dox/MPLA (+) than in the cells without Cu(I) (Figure S7), which might be because the increased size reduced the exocytosis of nanoparticles. The results were further comfirmed by flow cytometric (Figure 2a). Catalysts were added after 2 h of cellular uptake. At 8 h, the fluorescence intensity was much stronger in M-Dox/MPLA (+)-treated mice than in mice without catalysts. In addition, the in vitro cytotoxicity of M-Dox/MPLA (+) exhibited an enhanced anti-tumor effect (Figure 2b). Next, we analyzed the surface exposure of calreticulin, non-histone chromatin protein high-mobility group box 1 (HMGB1) and ATP. Surface calreticulin exposure on B16F10 cells was detected by immunofluorescence staining. Compared with the control group, B16F10 cells in the presence of Free-Dox, M-Dox/MPLA or M-Dox/MPLA (+) exhibited enhanced substantial increase in calreticulin exposure (Figure 2f), which was further confirmed by western blotting analyses of the isolated plasma membrane proteins (Figure 2e). In contrast to the Dox groups, paclitaxel (PTX), cyclophosphamide (CTX) and mitomycin C did not induce calreticulin exposure. ATP is a kind of important signaling molecule, which cound be released by tumor cells undergoing chemical or physical stress18. Released ATP by tumor cells is also an essential mechanism for anti-tumor immune responses19. To assess the extracellular ATP released from B16F10 cells, an ATP assay kit was used. As shown in Figure 2c, the corresponding extracellular ATP content was considerably increased after incubating with Free-Dox, M-Dox/MPLA or M-Dox/MPLA (+), which also rapidly acted as an identification signal for attracting antigen-presenting cells to apoptotic tumor cells20. Moreover, the better antigens presentation of dying cell by DCs was also related to the phagocytosis of TLR4 triggered by HMGB121. As determined by flow cytometry, HMGB1 was substantially increased by stimulation with Dox-containing groups (Figure 2d). Overall, the ICD induced by M-Dox/MPLA (+) was varified by a series of changes in the plasma membrane elements (including calreticulin and HMGB1 exposure) and microenvironment (such as ATP release) of dying cells. These immunogenic signals with the help of adjuvant MPLA may induce the immune responses against tumors in vivo, which affects the long-term success of anti-tumor effects.

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Figure 2. (a) Uptake assays of B16F10 cells incubated with different formulations for 1, 4 and 8 h. Error bars indicate SD (n = 3), *p < 0.05. (b) Cytotoxicity assay using MTT. Error bars indicate SD (n = 3), *p < 0.05 and **p < 0.01. (c) Extracellular release of ATP of B16F10 cells measured by luminometer. Error bars indicate SD (n = 3), *p < 0.05 and **p < 0.01 vs M-Dox/MPLA (+) treated micelles. (d) The release of nuclear HMGB1 detected by flow cytometric. Error bars indicate SD (n = 3), *p < 0.05 and **p < 0.01 vs M-Dox/MPLA (+) treated micelles. (e) The exposure of calreticulin on the surface of immunogenic cell death detected by western blot. (f) Immunofluorescence detection of calreticulin. The concentration of DOX was 5 µg/mL. 3.3 In Vivo Distribution After being intravenously injected of DiD-loaded micelles (M-DiD) into B16F10 melanoma-bearing mice, the biodistribution was detected by the IVIS imaging system. At 1 h after the injection, the fluorescence could be observed at the tumor site (Figure 3a). Then, the fluorescence intensity of M-DiD was increased over time, suggesting that M-DiD targeted tumor via the EPR effect. To achieve efficient extravascular accumulation in tumor tissue, effective NP extravasation from blood vessels and retention in tumor tissues are necessary22. Nanoparticles with small sizes present with a stronger penetration ability, varying within a certain range23, and easily pass through vessel pores, such as the DSPE-PEG micelles in this manuscript24. Second, nanoparticles that diffuse through tumor tissues should be efficiently retained for maintaining a sufficient drug concentration. However, large nanoparticles (~100 nm) are required for the retention effect25. Thus, the next step of our strategy was to increase NP accumulation at the tumor site by adjusting the particle size. After 4 h, soluble copper sulfate (Cu(II)SO4) and sodium ascorbate were intratumorally injected in the M-DiD (+) group. After 4 h, the fluorescence intensity was gradually stronger at the tumor site of the M-DiD (+) group than at that of the M-DiD (-) group, which was further determined by semi-quantitative data (Figure 3b). The average M-DiD (+) signal increased approximately 1.25-, 1.49-, and 1.31-fold compared with that of the control group at 8, 12, 24 h. Moreover, ex vivo fluerescence showed that the fluorescent signal was reduced in the other main organs of M-DiD (+) group than that of groups without the copper catalyst (Figure 3c, 8


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Figure S8). Furthermore, the frozen tumor sections were imaged at different depths using confocal laser scanning microscopy. As showed in Figure 3d, the distribution of DiD was throughout the tumor sections at 50 μm, with or without catalyst, and the red fluorescence of the M-DiD (+) was much stronger than in M-DiD (-) group, which was in accordance with in vivo results. For a deeper site, at 1000 μm, the fluorescence intensity decreased significantly. At 24 h post M-DiD (+) injection, DiD fluorescence was maintained, but minimal fluorescence was observed in the absence of catalyst. Therefore, the strategy for increasing particle size indeed improved drug accumulation in the tumor.

Figure 3. In vivo distribution of M-Dox/MPLA (+) in B16F10 melanoma-bearing mice. (a) C57BL/6 mice were administered by tail intravenous injection with M-DiD. In vivo near-infrared fluorescent images were abtained at different time after intravenous injection. (b) Semi-quantitative data of fluorescence in tumors at different time intervals, error bars indicate SD (n = 3), *p < 0.05 and ***p

Enhanced Tumor Retention Effect by Click Chemistry for Improved

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