Vx3-Functionalized Alumina Nanoparticles Assisted Enrichment of

Samples were prepared in PBS at a concentration of 0.1 mg/mL; for TEM, .... cytotoxic T lymphocytes. DRiPs. defective ribosomal products. α-Al2O3–V...
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Article Cite This: Bioconjugate Chem. 2018, 29, 786−794

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Vx3-Functionalized Alumina Nanoparticles Assisted Enrichment of Ubiquitinated Proteins from Cancer Cells for Enhanced Cancer Immunotherapy Jinjin Zhao,† Ning Pan,† Fang Huang,† Mohanad Aldarouish,† Zhifa Wen,† Rong Gao,† Yuye Zhang,§ Hong-Ming Hu,†,⊥ Yanfei Shen,*,‡ and Li-xin Wang*,† †

Department of Microbiology and Immunology, Medicine School of Southeast University, Nanjing, Jiangsu 210009, P.R. China Department of Bioengineering, Medicine School of Southeast University, Nanjing, Jiangsu 210009, P.R. China § School of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu 210009, P.R. China ⊥ Laboratory of Cancer Immunobiology, Earle A. Chiles Research Institute, Providence Portland Medical Center, Portland, Oregon 97213 United States ‡

S Supporting Information *

ABSTRACT: A simple and effective strategy was developed to enrich ubiquitinated proteins (UPs) from cancer cell lysate using the α-Al2O3 nanoparticles covalently linked with ubiquitin binding protein (Vx3) (denoted as α-Al2O3−Vx3) via a chemical linker. The functionalized αAl2O3−Vx3 showed long-term stability and high efficiency for the enrichment of UPs from cancer cell lysates. Flow cytometry analysis results indicated dendritic cells (DCs) could more effectively phagocytize the covalently linked α-Al2O3−Vx3-UPs than the physical mixture of α-Al2O3 and Vx3-UPs (α-Al2O3/Vx3-UPs). Laser confocal microscopy images revealed that α-Al2O3−Vx3-UPs localized within the autophagosome of DCs, which then cross-presented α-Al2O3−Vx3-UPs to CD8+ T cells in an autophagosome-related cross-presentation pathway. Furthermore, α-Al2O3− Vx3-UPs enhanced more potent antitumor immune response and antitumor efficacy than αAl2O3/cell lysate or α-Al2O3/Vx3-UPs. This work highlights the potential of using the Vx3 covalently linked α-Al2O3 as a simple and effective platform to enrich UPs from cancer cells for the development of highly efficient therapeutic cancer vaccines.

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used directly as vaccines.28 Nevertheless, the immunotherapy efficiency by using inactivated tumor cells or tumor cell lysate is insufficient due to the inadequate amount of TAAs, which is thought to be one reason for inefficiency for immunotherapy of cancers.29 Therefore, it is highly desirable to develop a strategy to enrich TAAs from tumor cells for highly efficient cancer immunotherapy.30 We previously demonstrated that, with induction of autophagy and inhibition of lysosomal/proteosomal activity in tumor cells, a broad spectrum of defective ribosomal products (DRiPs) were sequestered into autophagosomes and secreted as DRiPs-containing blebs form known as DRibbles.21,22,31 We also documented that these DRibbles were efficient TAAs carriers for cross-presentation by dendritic cells (DCs) and could stimulate dramatic T-cell activation, leading to antitumor efficacy in several tumor models such as melanomas, lung carcinomas,22 breast carcinomas,27 and liver cancer.32,33 More importantly, we found that the antitumor efficacy induced by

INTRODUCTION During the 20th century, great effort has been devoted toward the development of cancer treatment1,2 by surgery,3 chemotherapy,4−6 radiation therapy,7,8 and targeted therapy.9 Although these approaches have great potential to improve the survival and life quality of patients,10,11 they are often aggravated by serious side effects.12−14 Moreover, these therapeutic approaches have been largely unsuccessful in providing a long-term response against cancer, which would contribute to tumor recurrence.15 In the past few years, there has been a growing interest in cancer immunotherapy based on its promising preliminary results in achieving meaningful and durable treatment responses with minimal manageable toxicity.16−18 Cancer immunotherapy has become an important modality for complementing and enhancing the current standard therapies to provide long-term management of cancer patients.19,20 During the immunotherapy process, crosspresentation of tumor-associated antigens (TAAs) from tumor cells, which induces cytotoxic T lymphocytes (CTLs), plays a pivotal role.21−27 However, the clinical successes of efficient vaccines have been underachieving. Because of the lack of skills for the effective extraction of TAAs from tumor cells, traditionally inactivated tumor cells or tumor cell lysate are © 2018 American Chemical Society

Special Issue: Bioconjugate Materials in Vaccines and Immunotherapies Received: September 27, 2017 Revised: January 12, 2018 Published: January 31, 2018 786

DOI: 10.1021/acs.bioconjchem.7b00578 Bioconjugate Chem. 2018, 29, 786−794

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Figure 1. Modification of α-Al2O3 nanoparticles. (a) Scheme for the synthesis of α-Al2O3−Vx3 nanoparticles. (b) FTIR spectra of α-Al2O3, α-Al2O3APTES, and α-Al2O3−CHO nanoparticles. (c) EDS of α-Al2O3, α-Al2O3-APTES, and α-Al2O3−CHO nanoparticles.

triethoxysilane (APTES).41−44 Then, Vx3 protein was covalently linked to α-Al2O3-APTES using glutaraldehyde45−47 as a linker (Figure 1a). Fourier transform infrared spectroscopy (FT-IR) was used to confirm the modification of α-Al2O3 nanoparticles by APTES and glutaraldehyde. We observed a broad absorption peak centered at ∼3448 cm−1 and a narrow absorption peak centered at ∼1632 cm−1, representing hydrogen-bonded OH stretching vibration and the scissoring vibration of adsorbed water, respectively48 (Figure 1b). Importantly, those two absorption peaks were significantly reduced after the reaction between α-Al2O3 and APTES. Moreover, a new absorption band was observed at 2932 cm−1 and could be ascribed to aliphatic γ (CH2) groups49 (Figure 1b). These data indicated that α-Al2O3 nanoparticles were successfully modified by APTES. α-Al2O3-APTES was then modified with glutaraldehyde, which was confirmed by a small absorption band centered at 1720 cm−1 and stronger bands centered at 2860−2960 cm−1, referred to as γ(CO) groups50 and aliphatic ν (CH2) groups,51 respectively. Energy-dispersive X-ray spectroscopy (EDS) was further used to confirm the modification of α-Al2O3−CHO nanoparticles.52,53 Si, C, and N elements were detected in α-Al2O3APTES but not in α-Al2O3. Many more C elements were detected on α-Al2O3−CHO after the reaction with glutaraldehyde (Figure 1c and Figure S1). After the successful functionalization and modification by APTES and glutaraldehyde, α-Al2O3 nanoparticles were ready to covalently link to Vx3 protein. Synthesis and Characterization of α-Al2O3−Vx3. Vx3 protein was then mixed with α-Al2O3−CHO at different ratios

DRibbles was mainly based on their content of ubiquitinated TAAs.23 Recent studies showed that ubiquitined proteins (UPs) can be enriched from tumor cells, after blocking their proteasomal degradation pathway, using Ni-NTA agarose beads conjugated with ubiquitin binding protein Vx3.34−36 We found that UPs that physically mixed with α-Al2O3 nanoparticles have the potential as a potent cancer vaccine.36,37 However, we found that those UPs were not highly immunogenic in addition to this approach being time-consuming. Moreover, Ni ions of Ni-NTA beads are not environmentally friendly.38 In this study, we applied a new design for efficient enrichment of UPs from tumor cell lysate using Vx3-covalently linked-α-Al2O3 (denoted as α-Al2O3−Vx3), where α-Al2O3 served as carrier and adjuvant simultaneously, and Vx3 functioned as a linker between α-Al2O3 and UPs. Compared with our previous work, the current design is expected to possess the following advantages: (1) By using Vx3-covalently linked α-Al2O3 nanoparticles, UPs could be simply enriched from tumor cell lysate by centrifugation. (2) Compared with Ni-NTA beads, α-Al2O3 is cheap, environmentally friendly, and sustainable. (3) Because the α-Al2O3 is the only licensed adjuvant in clinical applications,38−40 the α-Al2O3−Vx3-UPs could be used directly as a cancer vaccine without extracting UPs from α-Al2O3, and more importantly, the presence of αAl2O3 (as adjuvant) can enhance an efficient antitumor immune response.

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RESULTS AND DISCUSSION Modification of α-Al2O3. To synthesize α-Al2O3−Vx3, we first modified α-Al 2O3 nanoparticles by 3-aminopropyl 787

DOI: 10.1021/acs.bioconjchem.7b00578 Bioconjugate Chem. 2018, 29, 786−794

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Bioconjugate Chemistry at 4 °C overnight and employed fluorescence microscopy to detect the covalent conjugation between Vx3 protein and αAl2O3−CHO nanoparticles. Figure 2a showed that, after

excitation at 488 nm54 by which the green color refers to the enhanced green fluorescent protein (eGFP) that fused with Vx3 protein. Moreover, ultraviolet light (UV) images revealed that the fluorescence intensity increased gradually with increasing ratios of Vx3 to α-Al2O3−CHO in the reaction (Figure S2). In contrast, the physical mixtures of Vx3 and αAl2O3 nanoparticles, which were prepared under the same conditions, showed almost no green fluorescence on the precipitation while the supernatant was still green (Figure S3a), and a fluorescence microscopy image of the precipitation displayed a faint green color under excitation at 488 nm (Figure S3b). These data demonstrated that Vx3 was successfully conjugated to α-Al2O3−CHO nanoparticles. Because the amount of conjugated Vx3 protein to α-Al2O3−CHO is critical for efficient enrichment of UPs from tumor cell lysate. Our results showed that the optimal amount of Vx3 linked to αAl2O3−CHO was observed at a ratio of 128 μg:1 mg (Figure 2b). Thus, a 128 μg:1 mg ratio of Vx3 protein to α-Al2O3− CHO was used as a preferable strategy to enrich UPs in the following experiments. The long-term stability of covalently linked α-Al2O3−Vx3 was determined after storage for 1, 2, and 4 weeks at 4 °C. Fluorescence microscopy analysis showed that α-Al2O3−Vx3 exhibited a bright fluorescence even after 4 weeks (Figure 2c). In contrast, the faint green fluorescence of α-Al2O3/Vx3-UPs completely disappeared after 1 week (Figure S3c). These data demonstrated that the covalently linked α-Al2O3−Vx3 was much more stable than the physical mixtures of Vx3 and αAl2O3. Enrichment of UPs by α-Al2O3−Vx3. We then prepared lysate from 4T1 tumor cells in which proteasome function was inhibited by bortezomib and ammonium chloride.36 Whole cell lysate was incubated overnight with α-Al2O3−Vx3 followed by centrifugation. Western blot analysis confirmed the enrichment of α-Al2O3−Vx3-UPs by which the amount of enriched UPs was directly proportional to the increasing ratio of 4T1 cell lysate to α-Al2O3−Vx3 until maximum UPs were captured (Figure 3a). The maximal level of enriched UPs (∼160 μg) was observed when the mass ratio of cell lysate to α-Al2O3−Vx3 was 300 μg:1 mg (Figure 3b). These results demonstrated that the covalently linked α-Al2O3−Vx3 was an efficient tool for the enrichment of UPs from tumor cells. Given the importance of the long-term stability of cancer vaccines in clinical applications, we stored α-Al2O3−Vx3-UPs for 1, 2, and 4 weeks at 4 °C, followed by suspension in PBS and centrifugation to detect their stability by Western blot

Figure 2. Vx3 was conjugated to α-Al2O3−CHO. (a) Fluorescence images of α-Al2O3−CHO nanoparticles conjugated with different amounts of Vx3. (b) Relationship between the amount of Vx3 conjugated onto α-Al2O3−CHO and that of Vx3 added for the reaction with 1 mg of α-Al2O3−CHO. (c) Fluorescence images of αAl2O3−Vx3 stored at 4 °C for 1, 2, 4 weeks after synthesis.

centrifugation, α-Al2O3−Vx3 nanoparticles were separated from the supernatant and exhibited bright green color under

Figure 3. UPs were enriched by α-Al2O3−Vx3. (a) Western blot analysis of UPs in precipitate after the reaction of different amounts of cell lysate with α-Al2O3−Vx3 followed by centrifugation. (b) The relationship between the amount of enriched protein and that of the cell lysate reacted with 1 mg of α-Al2O3−Vx3. (c) Western blot analyses of UPs in supernatant (S) and precipitate (P) after 1, 2, and 4 weeks of the first enrichment. 788

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Figure 4. TEM images of (a) α-Al2O3 and (b) α-Al2O3−Vx3-UPs.

phagocytosis using flow cytometry. As shown in Figure 5a, the percentage of GFP+ DC2.4 cells incubated with α-Al2O3− Vx3-UPs was significantly higher than that of α-Al2O3/Vx3-UPs

analysis. There was an abundant level of UPs in precipitate samples even after 4 weeks of storage in contrast to supernatants that had no detectable level of UPs (Figure 3c). This result indicated that α-Al2O3−Vx3-UPs nanoparticles, which were prepared by specific binding, possessed long-term stability. Transmission electron microscopy (TEM) showed that the clean surface of the single-crystalline α-Al 2O 3 nanoparticles (Figure 4a) were coated with an amorphous layer after conjugation with UPs (Figure 4b), which further confirmed the successful conjugation of UPs to α-Al2O3−Vx3 nanoparticles. The Toxicity of α-Al2O3−Vx3-UPs. For evaluating whether α-Al2O3 was still safe after modification with APTES, BALB/c mice were subcutaneously vaccinated three times at 2 day intervals with PBS, α-Al2O3−Vx3 (containing 100 μg of Vx3), and α-Al2O3−Vx3-UPs (containing 100 μg of UPs), and were sacrificed 30 days later. H&E stain was used to identify features of liver and kidney sections. Results showed that there was no difference regarding the features of liver and kidney between three vaccinated groups (Figure S4), indicating that αAl2O3 had no cytotoxicity and was still safe after modification with APTES. Cross-Presentation of α-Al2O3−Vx3-UPs. Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that are critical for induction of cytotoxic lymphocyte (CTL) responses. Antigen cross-presentation, the process through which exogenous antigens are presented on MHC class I molecules by DCs, is crucial for the generation of tumor-specific CTLs. For testing whether the covalently linked α-Al2O3−Vx3-UPs could be more effectively acquired by DCs than the physical mixture of α-Al2O3 and Vx3-UPs (α-Al2O3/Vx3-UPs), αAl2O3−Vx3-UPs or α-Al2O3/Vx3-UPs were coincubated with DC2.4 cells for indicated times followed by evaluating

Figure 5. Cross-presentation of α-Al2O3−Vx3-UPs by DCs. (a) Flow cytometry analysis of α-Al2O3−Vx3-UPs or α-Al2O3/Vx3-UPs phagocytosis by DCs. (b) Confocal images of the localization of αAl2O3/Vx3-Ups (top row) or α-Al2O3−Vx3-UPs (bottom row) within DCs after staining with an antibody against LC3 (red). (c) Surface expression of major histocompatibility complex class I peptide complexes (H2Kb-SIINFEKL) on DCs after loading with α-Al2O3− Vx3-UPs or α-Al2O3/Vx3-Ups. (d) Flow cytometry analysis showing that the cross-presentation of α-Al2O3−Vx3-UPs by DCs, but not αAl2O3/Vx3-UPs, was blocked by 3-MA treatment. 789

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Figure 6. Antitumor immune response triggered by α-Al2O3−Vx3-UPs. (a) Levels of produced IFN-γ by splenocytes of vaccinated mice after restimulation with inactivated 4T1 tumor cells. (b) IFN-γ levels in the sera of tumor burden mice after vaccination. (c) Tumor growth and (d) median survival days of tumor burden mice with different treatments.

autophagy affects the cross-presentation of α-Al2O3−Vx3-UPs (OVA+), the phosphoinositide 3-kinase inhibitor 3-methyladenine (3-MA) was used to inhibit the autophagy of DCs. This chemical inhibitor nearly abolished the cross-presentation of αAl2O3−Vx3-UPs (OVA+) but not α-Al2O3/Vx3-UPs (OVA+) by loaded DCs (Figure. 5d). These findings suggested that, when loaded onto DCs, the covalently linked α-Al2O3−Vx3UPs were superior in activating CD8+ T cells as compared to that of the physical mixture of α-Al2O3 and Vx3-UPs (α-Al2O3/ Vx3-UPs), and the functional autophagy pathway is required for the efficient cross-presentation of α-Al2O3−Vx3-UPs (OVA+). Antitumor Immune Response. For investigating whether α-Al2O 3−Vx3-UPs could induce an antitumor immune response, BALB/c mice were subcutaneously vaccinated three times at 2 day intervals with different doses (0, 10, 30, 100 μg/ mouse) of α-Al2O3−Vx3-UPs. On day 12, mice were sacrificed, and their splenocytes were collected and stimulated with inactivated 4T1 tumor cells for 48 h. Splenocytes were also cultured without stimulation (culture medium, CM) as a negative control or with anti-CD3 antibody as a positive control. ELISA results of secreted interferon-γ (IFN-γ) by stimulated splenocytes revealed that vaccination with α-Al2O3− Vx3-UPs induced a potent immune response against 4T1 tumor cells in a dose-dependent manner. Because 30 μg of UPs induced the highest level of IFN-γ production (Figure S5a), it was used as a preferable dose in the next experiment. Moreover, we found that the ratio of inactivated 4T1 tumor cells to

at different time points. These results indicated that α-Al2O3, covalently linked with Vx3-UPs proteins can be more effectively phagocytized by DCs. Subsequently, a confocal microscope was used to determine the subcellular localization of internalized αAl2O3−Vx3-UPs and α-Al2O3/Vx3-UPs. Results showed that αAl2O3−Vx3-UPs, but not α-Al2O3/Vx3-UPs, were colocalized with the autophagosome marker LC3 (Figure 5b). These results are consistent with those of a previous study that antigen conjugated to α-Al2O3 nanoparticles can be delivered into the autophagosome of DCs.48 Next, UPs were enriched from the cell lysate of EG7 tumor cells (EL-4 tumor cells transduced with OVA) and conjugated with α-Al2O3−Vx3, which we named α-Al2O3−Vx3-UPs (OVA+). The antibody specific for the peptide and MHC class I molecule (H2Kb-SIINFEKL) complexes was used to evaluate the efficiency of cross-presentation of α-Al2O3−Vx3UPs (OVA+) by DCs.48 Flow cytometry analysis showed that the DCs loaded with α-Al2O3−Vx3-UPs (OVA+) yielded a higher level of H2Kb-SIINFEKL complexes than the DCs loaded with α-Al2O3/Vx3-UPs (OVA+) (Figure 5c). We further examined the ability of DCs loaded with α-Al2O3−Vx3-UPs ̈ OVA(OVA+) or α-Al2O3/Vx3-UPs (OVA+) to stimulate naive + specific CD8 T cells in vitro. DCs loaded with α-Al2O3−Vx3UPs (OVA+) induced a stronger proliferation of OT-I T cells ̈ TCR transgenic mice, which recognize H2Kbfrom naive SIINFEKL complexes, than DCs loaded with α-Al2O3/Vx3-UPs (OVA+) (Figure 5d). For further examination of whether 790

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to improve cancer immunotherapy and support further studies to test the efficacy of this vaccine in clinical trials.

splenocytes plays an important role in inducing the production of IFN-γ by which a 1:1 ratio enhanced the secretion of the highest level of IFN-γ (Figure S5b). For investigating whether α-Al2O3−Vx3-UPs could induce a stronger antitumor immune response than α-Al2O3/cell lysate or α-Al2O3/Vx3-UPs, five groups (3 mice/group) of BALB/c mice were vaccinated with α-Al2O3−Vx3-UPs, PBS, α-Al2O3− Vx3, α-Al2O3/cell lysate, or α-Al2O3/Vx3-UPs. After restimulation with inactivated 4T1 tumor cells, splenocytes from αAl2O3−Vx3-UPs vaccinated mice produced the highest level of IFN-γ compared to those from the other four groups (Figure 6a). It is important to mention that splenocytes from α-Al2O3/ Vx3-UPs and α-Al2O3/cell lysate vaccinated mice also produced IFN-γ,36 but its level was significantly lower than that from splenocytes of α-Al2O3−Vx3-UPs vaccinated mice. Together, these findings demonstrated that α-Al2O3−Vx3-UPs have the potential to induce antitumor immune response, supporting it as a tumor vaccine candidate. The Antitumor Efficacy of α-Al2O3−Vx3-UPs Vaccination. The ability of α-Al2O3−Vx3-UPs to induce an efficient immune response prompted us to determine their therapeutic efficacy on an established murine tumor model. BALB/c mice were subcutaneously inoculated with 1 × 106 4T1 tumor cells. When tumors were palpable, tumor-bearing mice were divided randomly into 6 groups (6 mice/group); 5 groups were received triple subcutaneously vaccination of α-Al2O3−Vx3UPs (containing 30 μg of UPs), α-Al2O3−Vx3, α-Al2O3/cell lysate (containing 30 μg of total protein), α-Al2O3/Vx3-UPs (containing 30 μg of total protein), or PBS into both flanks on days 3, 5, and 7 after the first injection of tumor cells; one group received a caudal vein injection with Epirubicin (40 μg/ mouse) on days 3, 7, and 11. On day 14, blood sera were collected from the vaccinated mice, and the level of IFN-γ was monitored by ELISA. As shown in Figure 6b, the IFN-γ level in the serum of mice vaccinated with α-Al2O3−Vx3-UPs was higher than that in the serum of mice vaccinated with α-Al2O3/ cell lysate or α-Al2O3/Vx3-UPs. Moreover, the tumor growth curve showed that vaccination with α-Al2O3−Vx3-UPs significantly inhibited the tumor growth compared those with the other five groups (Figure 6c and Figure S6). Importantly, even the physical mixture of Vx3UPs and α-Al2O3 also had the ability to inhibit tumor growth, but their potential was obviously less than that of α-Al2O3− Vx3-UPs. Moreover, the inhibitory effect of Epirubicin, a chemotherapeutic agent commonly used in the treatment of breast cancer, on the tumor growth was remarkably less than that of α-Al2O3−Vx3-UPs. We also found that α-Al2O3−Vx3-UPs prolonged the median survival time of tumor bearing mice (49 days for α-Al2O3−Vx3UPs vs 40 days for α-Al2O3/Vx3-UPs, 32.5 days for α-Al2O3/ cell lysate, and 33.5 days for Epirubicin) (Figure 6d).

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EXPERIMENTAL SECTION Materials. Alumina (α-Al2O3) nanoparticles (30 nm) were purchased from Aladdin (Cat number: A119402). (3-Aminopropyl) triethoxysilane, aldehyde, and deubiquitinating enzymes inhibitor (PR-619) were purchased from Sigma-Aldrich (St. Louis, MO). The antiubiquitin antibody was obtained from Cell Signaling Technology. Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from Gibco. Fetal bovine serum (FBS) was obtained from Hyclone. Penicillin-streptomycin solution and the BCA Protein Assay kit were purchased from Beyotime Institute of Biotechnology, China. Bortezomib was obtained from Millennium Pharmaceuticals, Cambridge, MA. RIPA lysis buffer was purchased from Millipore. Protease and phosphatase inhibitors were obtained from Roche. The mouse IFN gamma ELISA Ready SET Go! kit was purchased from eBioscience. Animals. BALB/c female mice (6−8 weeks old) were purchased from the Comparative Medicine Center, Yangzhou University (Yangzhou, China) and maintained in specific pathogen-free conditions. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Southeast University. Vx3 Protein Expression and Purification. Vx3 protein was purified according to our previous work.36 Briefly, His-Vx3eGFP-expressing plasmid-transferred E. coli was grown in LB medium at 37 °C. After that, at the logarithmic phase, 0.1 M isopropy-β-D-thiogalactoside (IPTG) was added for 16 h at 15 °C. Then, cells were harvested and centrifuged by high speed centrifugation, which was further treated with lysozyme on ice followed by sonication and centrifugation. The as-obtained supernatant containing Vx3 was poured into a column containing Ni-NTA resin and incubated at 4 °C overnight with stirring. After releasing the waste liquid, the column was washed with washing buffer (50 mmol/L NaH2PO4, 300 mmol/L NaCl, 10 mmol/L imidazole) 3 times, and the purified protein was collected by washing off the column with elution buffer (50 mmol/L NaH2PO4, 300 mmol/L NaCl, 250 mmol/ L imidazole). Preparation of Vx3-Functionalized α-Al2O3 Nanoparticles (α-Al2O3−Vx3). α-Al2O3 nanoparticles (20 mg) and (3-aminopropyl) triethoxysilane (APTES) (66.45 mg) were added to anhydrous ethanol (1.3 mL) and stirred at room temperature for 12 h. The APTES-functionalized α-Al2O3 (αAl2O3-APTES) was obtained after centrifugation and washing with anhydrous ethanol 3 times. Then, the α-Al2O3-APTES was further modified with glutaraldehyde. Glutaraldehyde (25%, 59 μL) and deionized water (530 μL) were mixed together and added drop by drop to the as-obtained α-Al2O3-APTES and stirred at room temperature for another 2 h. After washing with deionized water, the above product was further modified with purified Vx3 by stirring the mixture at 4 °C overnight. The Vx3functionalized α-Al2O3 (α-Al2O3−Vx3) was finally obtained by centrifugation, and the amount of Vx3 modified on α-Al2O3 was evaluated by collecting and calculating the amount of Vx3 in the supernatant. The successful conjugation of Vx3 to the modified α-Al2O3 was confirmed by images under UV light and a laser confocal microscope. For examining the stability of αAl2O3−Vx3, it was dispersed in PBS for 1, 2, and 4 weeks. Green fluorescence was detected by laser confocal microscope.

CONCLUSIONS

These findings provide clear evidence that the covalently linked α-Al2O3−Vx3 can be used to enrich UPs from tumor cell lysate with high efficiency and long-term stability. Furthermore, the covalently linked α-Al2O3−Vx3-UPs could be effectively phagocytized by DCs and induce efficient autophagy-dependent cross-presentation. More importantly, results showed that α-Al2O3−Vx3-UPs have the potential to induce potent antitumor immune responses and antitumor efficacy in the established tumor model. This work presents a novel strategy 791

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Bioconjugate Chemistry Characterization of Modified α-Al2O3. Fourier transform infrared spectroscopy (FT-IR) was used to confirm the modification of α-Al 2O 3 nanoparticles by APTES and glutaraldehyde. After the reaction, the α-Al2O3, α-Al2O3APTES, and α-Al2O3−CHO were detected by Fourier transform infrared spectrometer (Thermo Fisher IS10). Enrichment of UPs from Cancer Cells by α-Al2O3−Vx3. 4T1 tumor cells were cultured in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL of streptomycin. For enriching UPs from tumor cells, the tumor cells were treated with bortezomib (velcade, 200 nmol/L) and ammonium chloride (20 mmol/L) for 9 h. The treated cells were collected and washed with PBS 3 times; then, the precipitated cells were resuspended in RAPI buffer supplemented with protease inhibitors, phosphatase inhibitors, and PR-619. After being incubated on ice for 20 min, the cell lysate was centrifuged at 15000g for 30 min to remove cell debris; the supernatant was collected, and the total protein concentration was quantified by the bicinchoninic acid kit according to the manufacturer’s protocol. α-Al2O3−Vx3 was added to the supernatant, and the mixture was stirred overnight at 4 °C. The covalently linked α-Al2O3−Vx3-UPs was obtained by centrifugation; the amount of UPs conjugated to α-Al2O3− Vx3 was calculated by protein reduction in the cell lysate. Western Blotting. α-Al2O3−Vx3-UPs were mixed with SDS-PAGE loading buffer, boiled for 5 min, and centrifuged at 15000g for 10 min. The supernatant was resolved by 4−8% SDS-PAGE. Proteins were transferred to a PVDF membrane, blocked by 5% dry milk for 2 h, and incubated with antiubiquitin antibodies overnight. Horseradish peroxidase (HRP)-conjugated secondary antibody was added for 1 h. Membrane was exposed using chemiluminescent reagents (Thermo Fisher). For confirming the stability of α-Al2O3− Vx3-UPs, Western blot analysis was used; α-Al2O3−Vx3-UPs were first resuspended in PBS for 1, 2, and 4 weeks. After centrifugation, the supernatant and precipitation were resuspended in SDS-PAGE loading buffer, and Western blotting was performed as described above. Transmission Electron Microscopy (TEM). Samples were prepared in PBS at a concentration of 0.1 mg/mL; for TEM, a droplet of the sample was deposited on a carbon-coated 400 mesh Cu grids and dried at 40 °C. Samples were detected using a HITACHI JEM-2100 operating at 120 kV, and photos were captured using a Gatan imaging system. Evaluation of the Toxicity of α-Al2O3−Vx3-UPs. BALB/ c mice were subcutaneously vaccinated three times at 2 day intervals with PBS, α-Al2O3−Vx3 (containing 100 μg of Vx3), and α-Al2O3−Vx3-UPs (containing 100 μg of UPs); 30 days later, the mice were sacrificed. Liver and kidney sections were collected for H&E staining. Detection the Cross-Presentation of α-Al2O3−Vx3-UPs and α-Al2O3/Vx3-UPs. α-Al2O3−Vx3-UPs or α-Al2O3/Vx3UPs were incubated with DC2.4 cells for 6 h. After washing, cells were fixed with 4% formaldehyde and permeabilized with 0.2% TritonX-100 in PBS for 15 min. Cells were stained with rabbit anti-LC3 antibody and Alexa fluor 568-labeled donkey antirabbit secondary antibody; images were captured by confocal microscope. The percentage of GFP+ DCs was evaluated by flow cytometry after the incubation of α-Al2O3− Vx3-UPs or α-Al2O3/Vx3-UPs with DC2.4 cells for 6 h. UPs were enriched from OVA-expressing tumor cells (EG7). The antibody specific for the peptide and major histocompatibility complex class I molecule (H-2Kb-SIINFEKL) complexes was

used to evaluate the efficiency of cross-presentation of OVA by DCs. The ability of DCs loaded with α-Al2O3−Vx3-UPs ̈ OVA(OVA+) or α-Al2O3/Vx3-UPs (OVA+) to stimulate naive ̈ OVAspecific CD8+ T cells in vitro was examined. Naive specific CD8+ T cells were collected from OT-1 transgenic mice and labeled with CFSE. α-Al2O3−Vx3-UPs (OVA+) or αAl2O3/Vx3-UPs (OVA+) were incubated with DCs without or with 3-MA treatment for 12 h. After washing, CFSE-labeled OT-1 CD8+ T cells were pulsed and cocultured for 60 h, and the percentage of divided OT-I CD8+ T cells was determined by flow cytometry analysis. Detection of the Antitumor Immune Responses. Mice were subcutaneously vaccinated with α-Al2O3−Vx3-UPs (containing 30 μg of UPs), α-Al2O3−Vx3, α-Al2O3/cell lysate (containing 30 μg of total protein), α-Al2O3/Vx3-UPs (containing 30 μg of UPs), and PBS on day 1. The same vaccination schedule was performed on days 3 and 5 after the first injection. On day 12, mice were sacrificed and their splenocytes were cultured with inactivated 4T1 tumor cells for 48 h. The immune response was evaluated by IFN-γ release using the Mouse IFN gamma ELISA Ready SET Go! kit according to the manufacturer’s instructions. Detection of the Antitumor Efficacy. For tumor-bearing mice to be established, 1 × 106 4T1 cells were subcutaneously injected into the right flank of BALB/c female mice. Subsequently, when tumors were palpable, mice were randomly divided into 6 groups and vaccinated with α-Al2O3−Vx3-UPs (containing 30 μg of UPs), α-Al2O3−Vx3, α-Al2O3/cell lysate (containing 30 μg of total protein), α-Al2O3/Vx3-UPs (containing 30 μg of UPs), and PBS, respectively, into both flanks of the tumor-bearing mice on days 3, 5, and 7 after the first injection of tumor cells; one group received triple caudal vein injection with epirubicin (40 μg/mouse) on days 3, 7, and 11. On day 14, blood sera were collected from the vaccinated mice to detect the level of IFN-γ by ELISA. Tumor growth was assessed by measuring the perpendicular diameters three times per week. Mice were sacrificed when the tumor diameter or tumor area reached a size larger than 15 mm and 200 mm2, respectively.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00578. Elemental analyses, fluorescence images, H&E assays, immune responses, and tumor growth curves (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yanfei Shen: 0000-0003-0369-5920 Li-xin Wang: 0000-0002-3480-4491 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the National Natural Science Foundation of China Nos. 31670918 and 31370895 (to 792

DOI: 10.1021/acs.bioconjchem.7b00578 Bioconjugate Chem. 2018, 29, 786−794

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Bioconjugate Chemistry

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L.X.W.) and 21675022 and 21305065 (to Y.F.S.), the Fundamental Research Funds for the Central Universities, the Natural Science Foundation of Jiangsu Province (BK20170084), and the Research and Innovation Program for Doctoral Graduate Students in Jiangsu (KYLX15_0178).

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ABBREVIATION UPs, ubiquitinated proteins; Vx3, ubiquitin binding protein; CM, culture medium; TAAs, tumor-associated antigens; CTLs, cytotoxic T lymphocytes; DRiPs, defective ribosomal products; α-Al2O3−Vx3, Vx3 covalently linked α-Al2O3; APTES, aminopropyl triethoxysilane; FT-IR, Fourier transform infrared spectroscopy; EDS, energy-dispersive X-ray spectroscopy; eGFP, enhanced green fluorescent protein; UV, ultraviolet light; TEM, transmission electron microscopy; α-Al2O3/Vx3UPs, mixture of α-Al2O3 and Vx3-UPs; α-Al2O3/cell lysate, mixture of α-Al2O3 and cell lysate; α-Al2O3−Vx3-UPs, UPs conjugated with α-Al2O3−Vx3; α-Al2O3−Vx3, Vx3 conjugated to α-Al2O3

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DOI: 10.1021/acs.bioconjchem.7b00578 Bioconjugate Chem. 2018, 29, 786−794