Surface-Layer Protein-Enhanced Immunotherapy Based on Cell

Subscriber access provided by LUNDS UNIV

Biological and Medical Applications of Materials and Interfaces

Surface-Layer Proteins Enhanced Immunotherapy Based on Cell Membrane Coated Nanoparticles for the Effective Inhibition of Tumor Growth and Metastasis Min Wu, Xingang Liu, Hongzhen Bai, Lihua Lai, Qi Chen, Guojun Huang, Bin Liu, and Guping Tang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b00294 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Surface-Layer Proteins Enhanced Immunotherapy Based on Cell Membrane Coated Nanoparticles for the Effective Inhibition of Tumor Growth and Metastasis

Min Wu,†,# Xingang Liu,†,# Hongzhen Bai,† Lihua Lai,‡ Qi Chen,† Guojun Huang,† Bin Liu,⊥,* and Guping Tang†,*

† Department

of Chemistry, Zhejiang University, Hangzhou 310028, China

⊥Department

of Chemical and Biomolecular Engineering, National University of

Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore ‡ Institute

of Immunology, Zhejiang University School of Medicine, Hangzhou,

Zhejiang 310058, China

ABSTRACT Chemo-immunotherapy is an important tool to overcome tumor immune suppression in cancer immunotherapy. Herein, we report surface-layer (S-layer) proteins enhanced immunotherapy strategy based on cell membrane-coated nanoparticles S-CM-HPAD for the effective malignant tumor therapy and metastasis inhibition. The S-CM-HPAD NPs could effectively delivery the tumor antigen, DOX and immunoadjuvant to the homotypic tumor by the homotypic targeting ability of the coated cell membrane. In addition to induce tumor cell death, the loaded DOX could enhance the immunotherapy response by inhibition of myeloid-derived suppressor cells (MDSCs). Due to the intrinsic adjuvant property and the surface epitopes and proteins displaying capability, the S-layers localized on the surface of S-CM-HPAD NPs potentiated the immune response to antigen. The results confirmed that the protective immunity against tumor occurrence was promoted effectively by prompting 1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 25

proliferation of lymphocytes and secretion of cytokine caused by tumor-associated antigen and adjuvant. The excellent combinational therapeutic effects on the inhibition of tumor growth and metastatsis in the melanoma tumor models demonstrated that the S-layer enhanced immunotherapeutic method is a promising strategy for tumor immunotherapy of malignant tumor growth and metastasis.

KEYWORDS:

cancer

cell

membrane,

biomimetic

nanoparticles,

cancer

immunotherapy, surface layer protein, myeloid derived suppressor cells INTRODUCTION Malignant tumor is a leading cause of human death in the world, and developing effective technology with high-specificity and low-damage to block tumor growth and metastasis is essential for cancer treatment.1 Along with the development of oncology, cancer immunotherapy has progressed rapidly and is considered as an alternative choice to traditional modalities.2 However, an obvious barrier of cancer immunotherapy is that tumors under immunotherapy often produce myeloid-derived suppressor cells (MDSCs) to mediate immune suppression,3 which significantly reduces its anti-tumor efficacy.4 Recent studies proved that MDSCs are sensitive to chemotherapeutic molecules including docetaxel, 5-fluorouracile and doxorubicin.5-7 In addition to elimination or inhibition of MDSCs, chemotherapy is capable of antigen shedding and presenting by inducing tumor cell death to enhance the immune response

of

host

against

cancer.8

Therefore,

the

combination

of

chemo-immunotherapy is a promising synergistic antitumor strategy. Traditional chemo-immunotherapies including chemotherapeutic molecules combined with checkpoint blockade, cytokine or vaccine, which showed exciting preclinical results.9-11 However, most of those strategies still have disadvantages such as high cost for preparation and preservation,12-13 poor tumor targeting and inaccurate activation of immune responses.14 Therefore, an ideal carrier for cancer chemo-immunotherapy should possess the following characteristics: 1) provide 2 ACS Paragon Plus Environment

Page 3 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


specific targeted delivery of drug and vaccine to tumors and alter tumor microenvironment to effectively activate the appropriate anti-tumor immune response; 2) protect antigen component from degradation; 3) allow for large-scale production at low cost. Biomimetic nanoparticles based on cancer cell membrane coating technology are promising carriers and have been successfully used in areas of cardio-vascular diseases, vaccination and cancer management.15 Due to the retained membrane structure and antigen, membrane coating endows NPs with additional and improved characteristics such as homotypic targeting, complex antigenic profile and low intrinsic immunogenicity, offering a promising delivery carrier for drug and vaccination.16 However, the cancer cell membrane vector which contains most of the surface antigens usually failed to induce maturation of the antigen-presenting cells (APCs).17 Crystalline Surface(S)-layers are cell surface structures that often observed in most prokaryotic organisms, such as bacteria. The natural S-layer are two-dimensional crystalline arrays of glycoprotein subunits with native self-assembly properties.18-21 Due to the inherent adjuvant property and the surface epitopes and proteins displaying capability, S-layers are usually used as carriers of antigen, either as self-assembly products, recrystallized on liposomes or displayed on bacteria.22,23 We speculate that the incorporation of S-layer protein in the membrane antigen would induce enhanced immune response and protect antigen components from degradation. As one of the most abundant biomolecules in nature, S-layer adjuvant from bacteria could greatly reduce the manufacturing cost due to their easy acquisition from the eukaryotic cell and microbial systems.24 In this study, we report new S-CM-HPAD NPs using supramolecular polymer DOX/polytheyleneimine- -modified (2-hydroxypropyl)-γ-cyclodextrin (HPAD) as the core.25 These NPs were coated with mouse melanoma B16F10 cancer cell membrane (CM) and self-assembled with S-layer protein collected from lactobacilli for combination chemo-immunotherapy. As showed in Scheme 1, the coated cancer cell membrane endowed S-CM-HPAD NPs with homologous targeting ability and 3 ACS Paragon Plus Environment


tumor-associated antigen properties. The DOX loaded inside the S-CM-HPAD NPs can not only kill the tumor cell but also eliminate the tumor-induced MDSCs to enhance the antitumor immunotherapy. As natural adjuvants (S), the S-layer proteins assembled on the S-CM-HPAD NPs surface to protect the antigen and activate antitumor immune response by increasing T cells proliferation and cytokines secretion. The cell membrane coated, surface-layer proteins enhanced immunotherapeutic nanoparticles provides a unique opportunity for effective chemo-immunotherapy at low cost.

Scheme 1. Schematic illustration of cancer cell membrane based biomimetic nanoparticles for combination tumor chemotherapy and immunotherapy. A) Preparation process of S-CM-HPAD NPs. Cancer cell membrane (CM) extracted from B16F10 melanoma cell hybridized with cationic polymers (HPAD), then, surface layer protein (S) which collected from lactobacillus helveticus self-assembled on the the surface of CM-HPAD to form the biomimetic nanoparticle S-CM-HPAD NPs. B) Cartoon schematic of S-CM-HPAD NPs mediated chemotherapy and immunotherapy for solid or metastatic tumors. The S-CM-HPAD NPs nanoparticles deliver the drug, tumor-associated antigen and adjuvant to enhance systemic antitumor immunity by combinating chemotherapy and immunotherapy.

MATERIALS AND METHODS 4 ACS Paragon Plus Environment

Page 4 of 25

Page 5 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Materials. Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), BCA protein assay kit, 4',6-diamidino-2-phenylindole (DAPI), and Coomassie brilliant blue G250 were obtained from the Beyotime Institute of Biotechnology (Jiangsu, China). Doxorubicin hydrochloride (DOX • HCl, MW= 579.98, 99.5%) was acquired from Haida Pharmaceutical Co., Ltd. (Zhejiang, China). The fetal bovine serum (FBS), Dulbecco's Modified Eagle (DMEM) medium and trypsin-EDTA solution were acquired from Gibco (NY, USA). Mouse tumor necrosis factor α (TNF-α), interleukin 12 (IL 12) and interferon-γ (IFN-γ) ELISA Kits were supplied by from Boster (Pleasanton, USA). All antibodies were obtained from Abcam (Cambridge, MA). Cells and animals. Murine B16F10 Melanoma cell line, human A549 lung cancer cell line, rat C6 glioma cell line, human Gastric Cancer SGC 7901 and human Hep G2 liver cancer cell line were purchased from the American Type Culture Collection (ATCC, MD, USA). All cells were grown in DMEM or RPMI 1640 medium supplemented with 10 % FBS, 100 µg/mL streptomycin and 100 IU/mL penicillin (ThermoScientific) in a humidified 37 °C incubator with 5 % CO2. Female mice (C57BL/6 strain) with weight of 16-18 g (4-5 weeks old) were supplied by the Zhejiang Chinese Medical University and All animal experiments were presented according to the guidelines of the Animal Protection Law in China. Preparation of CM-HPAD nanoparticles. For cancer cell membrane (CM), we obtained from 5×107 B16F10 melanoma cells as beforehand reported with modification.32 Briefly, the cancer cells nurtured in serum-free DMEM including 20 μM cytochalasin B for 30 min at 37 °C. The cells and formed CMs were isolated after treated with trypsin-EDTA solution 37°C for 5 min. The collected CMs suspension was vigorously shaken for 1 minute, centrifuged at 500 g at 4 ℃ for 5 min to remove cells and large aggregated CMs. The supernatant was collected after centrifugation at

5 ACS Paragon Plus Environment


14000 rpm and 4 ℃ for 1 h to obtain the CMs. The protein concentration of CMs was detected via BCA protein assay kit for further use. CM-HPAD was fabricated by coating HPAD core with cancer cell membrane by a co-extrusion method. HPAD was prepared in our previous work. 25 In brief, the CMs was mixed with HPAD at different CM to HPAD weight ratio included 0.05, 0.1, 0.2 and 0.4, respectively. The mixtures were co-extruded with a 200 nm polycarbonate membrane 11 passes to prepare CM-HPAD. To test the adornment of the CMs, the particle sizes of CM-HPAD were measured at 25 °C by DLS using Brookhaven instruments corp. Extraction of S-layer protein. To fabricate S-CM-HPAD nanoparticles, the S layer proteins were prepared from Lactobacillus strains cultured under anaerobic environment in MRS-broth medium with 220 rpm at 37 ℃ . The method for separation and purification of S-layer protein involve decomposition of bacteria cells and differential centrifugation to isolate the cell wall fragments.33 In brief, S-layer protein from Lactobacillus cells was extracted with four different soultion includingM guanidinium hydrochloride buffer (5 M GHCl), 5M LiCl, PBS and 0.01M NaOH for 1 h at 25 ℃ . The cell wall fragments containing petidoglycan were separated from extracted S-protein via centrifugation at 40 000×g for 20 min. Subsequently, the supernatants were dialyzed in Milli-Q water overnight at 4 ℃ and freeze-dried. The BCA protein assay kit was used for detection of protein content of S layer protein. Preparation and Characterization of the S-CM-HPAD nanoparticles. To develop an optimal protocol for self-assembly and determine the maximum of S-layer protein on the CM-HPAD, different CM to S protein weight ratios ranging from 1:0.1, 1:0.2, 1:1, 1:2, 1:4, 1:8, 1:10 were incubated at 25℃ in a Test Tube Rotator with a rotation speed of 15 min-1 for 2 h before centrifugation at 40 000×g for 15 min to dislodge the excrescent S-layer protein. The particle size and zeta potential of the resulting biomimetic nanoparticles were measured via dynamic light scattering (DLS) using a Zetasizer equipment (Malvern). The Samples were suspended in deionized water at a concentration of 0.25 mg mL-1 and all tests were done in three times at 25℃. 6 ACS Paragon Plus Environment

Page 6 of 25

Page 7 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Transmission electron microscopy (TEM, JEM-1200EX, JEOL, Japan) was carried out by first fixing the S-CM-HPAD pellets with a solution containing 2.5 % glutaraldehyde and 2 % paraformaldehyde in 0.1 mol L-1 cacodylate buffer (pH 7.3) for 1 h. Then, the sample was immerged in 1 % Osmium tetroxide in the above buffer for 20 min and the ultrathin sections of the sample were observed by TEM at 100 kV. DOX release behaviors from S-CM-HPAD in different pH buffer was detected with dialysis method as described in previous research.34 Characterization of S-layer protein and membrane protein. S-layer protein and cancer cell membrane protein of S-CM-HPAD were identified with Sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ). The protein samples from B16F10 cancer cell lysate, CM, CM-HPAD, S-CM-HPAD and S-layer protein were prepared at a final protein concentration of 1.0 mg mL-1. The samples with loading buffer were denatured at 100 ℃ for 10 min. A same amount of protein was separated from the SDS-PAGE and stainned with Coomassie Blue and imaged after destaining. The protein gel was shifted and blocked over the nitrocellulose membrane, and then incubated at 4℃ overnight with anti-α-intergrin, anti-E-cadherin, anti-CD63, anti-β-tubulin and anti-histone H2B, following incubated with the secondary antibody for another 2-4 h at room temperature. The bands of different protein were visual after treated with the westzol enhanced chemiluminescence kit (Intron, Sungnam, Korea), and imaged via ChemiDoc MP gel imaging system (Bio-Rad). Cellular internalization and homotypic targeting in vitro. To investigate the colocalization of S-CM-HPAD, the CM of S-CM-HPAD nanoparticles was fluorescently labeled after stained with 10 μg/mL of CFDA-SE which was widely used for cell labeling. The CFDA-SE and DOX labeled S-CM-HPAD nanoparticles were incubated with B16F10 cells for 4 h, and the concerntration of DOX was 5 μg/mL. After incubated, the cells were washed for three times, and fixed with 4% paraformaldehyde and then nucleus stained with DAPI, cell visualization by CLSM



Page 8 of 25

(Bio-Rad). The green, red and blue fluorescence of digital images were obtained from CFDA-SE labeled CMs, DOX and DAPI. The cell uptake of DOX in B16F10 cells was observed by confocal scanning microscope. The B16F10 cells were cultured on 24-well for 20 h, the medium was then replaced and the cells were cultured with DMEM (serum free), DOX and S-CM-HPAD at a DOX concentration of 2 µg/mL. After incubating at different defined time points, the cells were washed and fixed. The cells

nucleus and

cytoskeleton were tained with DAPI (blue) and Alexa Fluor 488 phalloidin (green), respectively. Flow cytometry (Beckman-Coulter, USA) was employed to test the homotypic targeting effect of S-CM-HPAD NPs coated with B16F10 cell membranes. The procedure was described as follows. B16F10, A549, C6, Hep G2 and SGC 7901 cells were seeded in 6-well plates respectively and cultured for 18~20 h. After S-CM-HPAD nanoparticles ( the DOX concentration was fixed at 2.0 μg/mL) were co-incubated with these cells for 4 h, respectively, the cells were washed three times with PBS and collected by centrifugation at 500 g for 15 min. The collected cells were washed thrice and suspended with PBS and detection by flow cytometry. (BD FACSAria™ III, USA). The flow cytometry data obtained in B16F10, A549, C6, Hep G2 and SGC 7901 cells were used as the controls. Macrophage and Dendritic cell activation studies in vitro. To detection the reaction between S-CM-HPAD NPs and immune cells, macrophage Raw 264.7 and bone marrow-derived cells were plated at 6-well plates, respectively. S-CM-HPAD NPs, CM-HPAD NPs, the mixture of S, CM and DOX (termed as S+CM+DOX) samples were incubated in triplicate at a final concentration of 40 µg (HPAD) per mL culture media at a S : CM : HPAD core ratio of 0.8 : 0.2 : 1. Doxorubicin was incorporated at 5 wt % of HPAD polymer weight, and the final concentration of DOX was 2 µg per mL culture media. Samples were allowed to incubate for 48 h before the medium were collected from supermatant. Tumor necrosis factor (TNF-α), interferon


Page 9 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


gamma (IFN-γ) and alpha interleukin-12 (IL-12) concentration were tested by ELISA Kits ( eBioscience) accroding to the manufacture instructions. Cytotoxicity study in vitro.

B16F10 cells wered incubated with culture

medium in 96-well plates at a density of approximately 105/well overnight. The medium was renewed with 200 μL complete medium with different concentrations of DOX, HPAD, and S-CM-HPAD. After further incubation for 48h, the solutions were discarded and wash two times. 100 μL of the serum-free medium including 0.5 mg/mL of MTT was added and incubated for another 4 h. After removal of MTT medium, the formazan crystals were dissolved with 100 μL of DMSO and Absorbance at 490 nm was measured using a ELISA plate reader (Model 550, Bio-Rad). The analysis of MDSC, T cell and cytokine secretion in vivo. The C57BL/6 mice harboring B16F10 tumor model was used to evaluated for the influence of S-CM-HPAD on the frequency, proliferation of tumor-infiltrating lymphocytes (CD4+ and CD8+), macrophage and cytokine. Briefly, the mice were administered with PBS, SLP(S), DOX, HPAD, CM-HPAD or S-CM-HPAD 3 times within 2 weeks. Three mice per group were sacrificed and the lungs, spleens and blood were harvested for detection. The tissues were digested with DNase (30 U/mL), hyaluronidase (100 U/mL), and collagenase (175 U/mL) under 37 °C for 60 min.

The T cells were

separated through 75 μm filter sieves and collected for the following test. For T cell frequency detection, the TILs were labeled with anti-CD3-APC, anti-CD4-FITC, and anti-CD8-PE. For intracellular Ki67 detection, the T cells were fixed and permeabilized by a FoxP3 kit and labeled with anti-Ki67-FITC, anti-CD4-APC, and anti-CD8-PE. To detect MDSCs, the TILs were blocked with anti-CD45a-APC-Cy7, anti-F4/80+-PE, anti-CD11b-APC and Ly6G- FITC. The stained cells were then washed thrice and analyzed via flow cytometry. In order to quantify the intracellular transcription level of IL-12, IFN-γ, TNF-α, CD4 and CD8 genes (The primer sequences in table S1), total RNA was extracted from lung, spleen and blood using TRIzol ( Invitrogen,Carlsbad, CA, USA ). RNA 9 ACS Paragon Plus Environment


samples from different group (n = 3) were reverse-transcribed to cDNA with a first stand cDNA synthesis kit (TOYOBO, Japen). The relative expression of cytokine was measured via real-time PCR with a StepOne™ Real-Time PCR (Life technologies, USA) accroding to a standard protocol. In vivo tumor growth study. Briefly, B16F10 cells suspended in 200 μL were injected subcutaneously (2.0 × 105 cells) at the right abdominal of 4~5 weeks old female C57BL/6 mice. When the tumor grew to a volume of 100 mm3, the mice were randomly assigned to 6 groups (n = 8): control PBS, SLP(S), DOX, HPAD, CM-HPAD and S-CM-HPAD. Each group was intravenous injected with an equivalent dose at 0.5 mg/kg of DOX, 2.0 mg/kg of CM and 8 mg/kg of SLP. The treatment was performed every three days for consecutive 2 weeks. The tumor growth was monitored by measuring the tumor length and width with a calliper and the tumor volume was calculated as follows: tumor volume V (mm3) = π/6× length (mm) × width (mm)2. After 21 Days of administration, three mice from each group were sacrificed and the tumors were dissected, weighed, and then imaged. Immunohistochemical analysis of H&E, ki67 and TUNEL assay. The ex vivo tissues from the mice after treatments were fixed in 4 % fresh paraformaldehyde for 24-48 h. The tissues were dehydrated, embedded, and sliced with 4-5 mm thick sections. The fixed sections were deparaffinized and hydrated following the official protocol and stained with hematoxylin and eosin (HE) for microscopic detection. Apoptosis and proliferation of the cancer cells were detected with the TUNEL and ki 67 kits followed the standard instructions. In vivo metastasis study. To investigate the anti-metastasis effect of S-CM-HPAD, C57BL/6 mice was injected intravenously of 2×105 B16F10 cells in 200 μL oPBS (pH = 7.4) and randomly assigned to six different groups (n=6). The injected B16F10 melanoma cells spontaneously metastasized to the mouse lung and then i.v. injection of PBS, SLP(S), DOX, HPAD, CM-HPAD or S-CM-HPAD at DOX dose of 0.5 mg/kg DOX, 2.0 mg/kg CM and 8 mg/kg SLP. After 18 days, The mice were sacrificed and the lungs were harvested to be weighed and photographed. 10 ACS Paragon Plus Environment

Page 10 of 25

Page 11 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The lungs were fixed with fresh 4 % paraformaldehyde at room temperature for 24-48 h. The tissues were dehydrated in graded ethanol, embedded in pure paraffin and cut in 4-5 mm thick sections for further H&E staining and Ki 67 Immunohistochemical analysis. Micro-PET imaging. The melanoma lung metastasis model of C57BL/6 mice after treatment was monitored by micro-PET scans of lung. U-SPECT-II/CT scanner (Milabs, Utrecht, Netherlands) was used to perform the micro-PET imaging. CT scanning was exactly the same localization as PET scanning. All mice were kept limosis at least 8 h before undergoing 18F-FDG PET/CT scan. After anesthesia, the mice were positioned prone using a handmade holding device. 18F-FDG (37 MBq/kg) was administered via an ear vein, and PET/CT images were acquired 55–60 min afterwards on a U-SPECT-II/CT scanning apparatus. The scanning images were analyzed through commercially available software (Xeleris; GE Healthcare Bio-Sciences Corp). Statistical Analysis. The experiments were repeated at least 3 times. The data were performed as means ± standard deviations. The statistical significance (*p < 0.05 or **p < 0.01) was evaluated by the Student’ s t-test when twoor more groups were compared.

RESULTS AND DISCUSSION The cancer cell membrane coated HPAD NPs (CM-HPAD) were fabricated via a top-down method in which NP cores and cancer cell membranes were co-extruded through a porous polycarbonate membrane. To provide high membrane coating efficiency and optimal particle size, the membrane-to-HPAD weight ratio was optimized at 0.2:1 throughout the study (Figure 1A). Transmission electron microscopy (TEM), atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analysis (Figure S1 and S2) indicated that HPAD polymers were successfully coated by CMs with a diameter of ~200 nm. Furthermore, S-layer protein was extracted from lactobacilli (Figure S3 and 11 ACS Paragon Plus Environment


S4) and self-assembled onto CM-HPAD. To determine the maximum amount of S-layer protein on the CM-HPAD, different protein weight ratios of S to CM were incubated at room temperature for 2 h to yield S-CM-HPAD NPs. The protein concentration of S-CM-HPAD NPs reached the maximum at a CM to S weight ratio of 1:4 (Figure 1B). Based on the above results, the weight ratio of S: CM: HPAD was optimized at 0.8: 0.2: 1 throughout the study. The S-CM-HPAD NPs synthesized at 0.8: 0.2: 1 showed good stability in PBS, DMEM medium or fetal bovine serum solution (Figure 1C). From the ultrathin-section of S-CM-HPAD NPs observed by TEM, the S-layer proteins were observed to assemble on the surface of CM-HPAD with a monolayer thickness of 4~5 nm (Figure 1D).

Figure 1. A) DLS hydrodynamic diameters of CM-HPAD NPs in PBS with varying weight ratios of CM versus HPAD. The data were obtained immediately after synthesis and B) the protein absorption of S-CM-HPAD NPs with different weight ratio of S to CM-HPAD. C) Time-dependent particle stability of S-CM-HPAD NPs in PBS, 1640 medium and PBS containing 10% FBS at 37 C. D) Negative-staining TEM image of S-CM-HPAD NPs. E) SDS-PAGE protein analysis of S protein, S-CM-HPAD NPs, CM-HPAD, CM and cancer cell lysate, samples were run at the same protein concentration. F) Membrane protein characterization by western blotting analysis of cancer cell lysate, CM, CM-HPAD and S-CM-HPAD NPs.

The analysis of protein ingredients in S-CM-HPAD NPs was performed by comparing the functions of the biomimetic NPs with membrane antigens and S-layer 12 ACS Paragon Plus Environment

Page 12 of 25

Page 13 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


protein immune modulation. As showed in Figure 1E, the gel electrophoresis revealed that the S-layer and membrane proteins can be well retained in S-CM-HPAD NPs and the nuclear components were removed for the safety concerns. To verify specific antigens on S-CM-HPAD NPs, five typical proteins including α-integrin, E Cadherin, CD 63 (membrane protein), β-tubulin (cytosol marker) and Histone H2B (nucleus marker) were examined by western blotting analysis. It was reported that the transmembrane protein α-integrin and E Cadherin are associated with adherence and homotypic cancer cell-targeting capabilities26 and the tetraspanin (CD63) is essential for adaptive immune responses.27 As shown in Figure 1F, the amount of α-integrin, E Cadherin and CD63 protein on the S-CM-HPAD NPs was relatively high, while the β-tubulin and Histone H2B contents were quite low. The above results indicated that both the homotypic binding antigen and tumor associated antigen have been successfully transferred to the shell of S-CM-HPAD NPs.



Figure 2. A) Flow cytometric profiles of the five cell lines SGC7901, A549, C6, HepG2 and B16F10 incubated with S-CM-HPAD NPs for 2 h. B) CLSM images of murine melanoma B16F10 cells incubated with S-CM-HPAD NPs for 4 h (CM: green, DOX: red, Nucleus: blue), the scale bar is 10 μm. C) Cell uptake of S-CM-HPAD NPs in murine melanoma B16F10 cells detected under CLSM at 0.5 h, 2 h, 4 h, 12 h and 24 h, respectively. The cell nucleus and cytoskeleton were stained with DAPI (blue) and Alexa Fluor 488 phalloidin (green),respectively. The scale bar is 40 μm. D) Cytotoxicity of S-CM-HPAD NPs, HPAD and DOX at various DOX concentrations on B16F10 cells after 48 h incubation. Data are given as the mean ± SD (n = 4).

To investigate the homologous targeting ability, S-CM-HPAD NPs were incubated with different cell lines including B16F10, SGC7901, A549, C6 and HepG2 cells. The results were assessed by flow cytometry. B16F10 group exhibited the 14 ACS Paragon Plus Environment

Page 14 of 25

Page 15 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


highest fluorescence intensity, which confirmed the specific targeting ability of S-CM-HPAD NPs to homologous B16F10 cells (Figure 2A). The in vivo biodistribution of 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR) labeled S-CM-HPAD NPs (S-CM-HPAD NPs@DiR) was studied in nude mice bearing B16F10 murine melanoma model. The fluorescent signals of S-CM-HPAD NPs@DiR in tumor tissue were detected at 8 h in vivo imaging, which showed S-CM-HPAD NPs can target to the tumor successfully (Figure S5 and S6).28 Next, we evaluated the intracellular delivery of S-CM-HPAD NPs. First, DOX release behaviors of S-CM-HPAD NPs were tested in pH 6.5 and pH 7.4 phosphate buffer solutions (Figure S7). At pH 7.4, only 11.4 ± 3 % of entrapped DOX were released from the S-CM-HPAD NPs. However, 48.1 ± 3 % of DOX were released in simulated acidic tumor microenvironment (pH 6.5) with a sustained way. These results indicated that the drug release of S-CM-HPAD NPs was pH-responsive. Then, S-CM-HPAD NPs were incubated with B16F10 cell lines and subsequent observed by CLSM at different time interval. CLSM images in Figure 2B show good co-localization between CFDA-SE labeled CM (green fluorescence) and DOX (red fluorescence) after incubation with the NPs for 4 h. With the extension of incubation time, the red fluorescence emitted from DOX increased gradually (Figure 2C). After 12 h incubation, the red fluorescence was higher than that of 4 h. Furthermore, it can be observed that the red fluorescence from S-CM-HPAD apparent increased in nuclei after 24 h incubation. As showed in figure S8, the red fluorescence from DOX and green fluorescence from NPs also increased with time. Notably, the green fluorescence from NPs was always observed in the cytoplasm even after 24 h incubation. With large particle size, the S-CM-HPAD NPs were difficult to directly enter into nuclei. The red fluorescence in nuclei should be attributed to the sustained release of DOX molecule from S-CM-HPAD NPs. The above results demonstrated that S-CM-HPAD NPs could be internalized with a time-dependent behavior by the B16F10 cells and release the DOX to the nucleus successfully.



The in vitro cytotoxicity of S-CM-HPAD NPs against B16F10 cells was examined by MTT assay. Enhanced cytotoxicity of S-CM-HPAD NPs was identified compared with HPAD and free DOX after incubation with B16F10 cells for 48 h (Figure 2D). To explore the compatibility of S-CM-HPAD NPs, we measured the hemolytic potential of S-CM-HPAD NPs. The RBC solutions treated with S-CM-HPAD NPs and PBS were clear in the supernatant, while those treated with water turned red. Hemolysis rate represents the percentage of the destroyed red blood cells (RBCs). S-CM-HPAD NPs only lead to a hemolysis rate of 4% when the concentration was 100 µg/mL, indicating its good hemocompatibility (Figure S9).

Figure 3. Induction immune response in C57BL/6 mice immunized with S-CM-HPAD NPs nanoparticles. A) Schematic illustration of the experimental design followed by the evaluation of the effects of S-CM-HPAD NPs on MDSC, T cell and cytokines in the B16F10 melanoma mode. B) Flow cytometric examination of proportion of MDSC (CD11b+ and ly6G+) and proliferation of the C) CD8+ and D) CD4+ tumor-infiltrating lymphocytes in the tumor of B16F10


Page 16 of 25

Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tumor-bearing mice after treatment. E) Frequency of tumor-infiltrating CD8+ T cells within tumor, spleen and blood. F) Semiquantitative analysis of CD4, CD8, IL-12, IFN-γ and TNF-α content within tumor, spleen and blood, respectively.

The ability of S-CM-HPAD NPs to deliver tumor-associated antigens to activate immune response was tested both in vitro and in vivo. To study the interaction between S-CM-HPAD NPs and immune cells, murine macrophage (RAW264.7) and bone marrow-derived dendritic cells were employed as typical immune cells in protecting the tumor microenvironment by producing cytokines to activate anti-tumour immune responses.29 Enzyme-linked immunosorbent assay (ELISA) was used to quantify pro-inflammatory cytokines level of factor-α (TNF-α), interferon-γ (IFN-γ) and Interleukin-12 (IL-12), which were secreted by RAW264.7 and bone marrow-derived dendritic cells after incubated with different formats for 48 h. The final concentration of DOX in S-CM-HPAD was 2 µg per mL culture media, which was a relatively safe concentration to immune cells (Figure S10).

As showed in

Figure S11 and S12, the secretion of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and Interleukin-12 (IL-12) were significantly enhanced by S-CM-HPAD NPs, which suggested that S-CM-HPAD NPs were more easily to elicit an antigen-specific response compared with the CM-HPAD NPs without S-layer self-assembled. To detect whether S-CM-HPAD NPs could induce immune response in vivo, PBS, S, DOX, HPAD, CM-HPAD or S-CM-HPAD NPs were intravenously injected (i.v.) into B16F10 melanoma tumor-bearing C57BL/6 mice, respectively. After treatment, tumors, spleens and blood from tumor-bearing mice were collected and dissociated, which were further used for detection of MDSCs, T cell and cytokines level (Figure 3A).8 As shown in Figure 3B, the MDSCs percentage in tumor of the control group (19.2 %) was much higher than the DOX contained HPAD (8.78 %), CM-HPAD (7.48 %) and S-CM-HPAD NPs (6.82%). A similar trend was observed in blood and spleen (Figure S13). These results demonstrated the MDSCs recruitment in tumor was significantly resisted by S-CM-HPAD NPs, since the doxorubicin released from S-CM-HPAD NPs could eliminate MDSCs. 17 ACS Paragon Plus Environment


CD8+ cytotoxic T cells and CD4+ helper T cells were reported to play an important role in the protective immunity against tumors, which promoted the secretion of tumor-specific T cells to enhance antitumor immunity. Here we tested the influence of S-CM-HPAD NPs on the proliferation and frequency of CD8+ and CD4+ tumor-infiltrating lymphocytes. As shown in Figure 3C, the proliferation of CD8+ T cells in the tumor administered with PBS as control is only 12.4 %, while the promoted CD8+ T cells proliferation level was found in S-CM-HPAD NPs which seemed to be the most effective in activating CD8+ T cells proliferation (33.2 %). In case of CD4+ T cells (Figure 3D), the CD4+ T cells proliferation level was significantly promoted to 36.2% when the tumor-bearing mice were administered with S-CM-HPAD NPs. The proportions of CD8+ (Figure 3E) and CD4+ T (Figure S14) cells in tumor, blood and spleen were increased when S-CM-HPAD NPs were administered, which indicated that the biomimetic NPs promoted CD8+ and CD4+ T cells infiltration in tumor.

Figure 4. A) Inhibition of tumor growth in C57BL/6-derived B16F10 melanoma tumor-bearing models treated with different formulations. Survival curves B) and average weight C) tumor-bearing mice treated with the different formulations. D) H&E staining, Ki 67 and TUNEL immunohistochemical images of tumor tissues after mice were sacrificed at the last treatment post intravenous injection of various formulas. All scale bars represent 100 μm. E) Dissected tumor tissues after successive 21 days of vaccination of different formulations in tumor-bearing mice. The size of each grid is approximately 2 cm × 2 cm. Two-tailed unpaired t test was used to calculate P values, *p < 0.05, **p < 0.01.


Page 18 of 25

Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Besides T cells related antitumor immunity, lots of cytokines which were secreted in response to immunity can exert detraction effect against the development and progression of tumor.30 An increase of tumor, spleen and serum interleukin-12 (IL-12), interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), CD4 and CD8 levels were observed after S-CM-HPAD NPs treatment (Figure 3F and S15). Collectively, the results validate that S-CM-HPAD NPs were capable of not only modulating tumor immune escape by inhibiting MDSCs recruitment in tumor but also altering tumor microenvironment via improving T cell proliferation, stimulating the secretion of cytokines. Therefore, the co-delivery of DOX, antigen (CM) and adjuvant (S-layer protein) exerted positive effect in activating antitumor immunity, which was expected to promote tumor suppression.

Figure 5. A) Typical photographs of the metastatic foci in lung. B) H&E staining and Ki 67 analysis, all scale bars represent 100 μm. C) The weight of lungs dissected from mice 25 days after first treatment. D) In vivo MicroPET/CT imaging study of S-CM-HPAD NPs nanoparticles in a lung metastasis of murine melanoma model. The images of B16F10 melanoma lung metastasis bearing mice were acquired after treatment with different formulations. In each picture, the left is the segmented image, the right is the whole body image of C57BL/6 mouse.Two-tailed unpaired t test was used to calculate P values, *p < 0.05, **p < 0.01.

To investigate the synergistic effects of chemo-immunotherapy, mice with subcutaneous

B16F10 melanoma model were administrated with PBS, S and

various DOX formulation including free DOX, HPAD, CM-HPAD and S-CM-HPAD NPs. In the group treated with PBS (Figures 4A and 4E), the tumor volume increased 19 ACS Paragon Plus Environment


rapidly during the therapy process. The tumor growth of the mice administrated with S-layer protein or free DOX were similar to that of PBS group and all three groups showed high proliferation and low cell apoptosis (Figure 4D). Due to the homotypic targeting ability to tumor, the cell membrane coated S-CM-HPAD NPs group displayed stronger antitumor efficacy when compared with DOX and HPAD. Furthermore, S-CM-HPAD NPs group exhibited higher anti-tumor growth efficacy compared with CM-HPAD, which indicated that the S-layer protein as an adjuvant could boost the immune response successfully. The survival studies showed that 100 % mice treated with S-CM-HPAD NPs survived over 21 days (Figure 4B) and no obvious body weight loss was observed (Figure 4C). Meanwhile, there were no significant pathological changes in heart, liver, spleen, lung and kidney after S-CM-HPAD NPs treatment (Figure S16). These results indicated that the S-CM-HPAD NPs have high synergistic chemo-immunotherapy antitumor efficacy with excellent biocompatibility. Next, mice with B16F10 melanoma metastasis model were used to evaluate the synergistic therapy efficiency of metastatic tumors. The C57BL/6 mice treated with the S-CM-HPAD NPs showed inhibited tumor growth compared with other treated groups. Evident black nodules were observed in the lung of the control group, which represented the localization of metastasized B16F10 cells in the lung tissues. While there were little nodules found in the S-CM-HPAD NPs treated group (Figure 5A). The results of hematoxylin and eosin (H&E) staining and Ki 67 assay indicate that the tumor cell proliferation in S-CM-HPAD NPs treated group was significantly inhibited (Figure 5B). The weight of the lungs was also found to be lower in S-CM-HPAD NPs than other treated groups (Figure 5C). To further confirm the increased anti-metastasis efficacy of S-CM-HPAD NPs, micro-PET/CT scans were performed after all the treatment was completed.31 As shown in Figure 5D, significant yellow signals were observed in the control group which represented the existing of much metastatic nodules on the lung. There were dramatically decreased yellow signals in the


Page 20 of 25

Page 21 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


S-CM-HPAD NPs group, which suggested the effective treatment against tumor metastasis.

CONCLUSION In conclusion, we demonstrated the biomimetic nanoparticles S-CM-HPAD with homotypic targeting, multiantigenic immunity activation and drug delivery capability to inhibit the melanoma tumor growth and metastasis. The cancer cell membranes derived from B16F10 melanoma cells were coated onto the surface of doxorubicin (DOX) loaded polymer HPAD to obtain CM-HPAD, which brought the NPs superior targeting efficiency to the homotypic tumor. Natural adjuvant S-layer protein (S) collected from lactobacilli was then self-assembled on the surface to form S-CM-HPAD NPs. Owing to the intrinsic adjuvant ability as well as the capability to surface display proteins and epitopes, the S-layers localized on the surface of S-CM-HPAD NPs potentiated the immune response to antigen. The S-CM-HPAD NPs could not only modulate tumor immune escape by inhibiting MDSCs recruitment in tumor but also alter tumor microenvironment via improving T cell proliferation and stimulating the secretion of cytokines. The results of inhibiting tumor growth and tumor metastasis demonstrated that the S-layer enhanced chemo-immunotherapeutic method provided a promising strategy for effective cell-membrane immunotherapy.

ASSOCIATED CONTENT Supporting Information Additional details on the physicochemical and biological characterizations of S-CM-HPAD are provided (PDF)

AUTHOR INFORMATION Corresponding Author 21 ACS Paragon Plus Environment


* Email: [email protected] * Email: [email protected] ORCID Guping Tang: 0000-0003-3256-740X Bin Liu: 0000-0002-0956-2777 Min Wu: 0000-0002-3956-0107 Xingang Liu: 0000-0001-7071-0209 Hongzhen Bai: 0000-0002-0886-3906 Author Contributions # M.

W. and X.-G. L. contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors greatly acknowledge for the financial support of the following programmes: the National Natural Science Foundation of China (Grant No. 51573161), the National Science Foundation for Young Scientists of China (Grant No. 21807092), and the National University of Singapore (R-279-000-482-133).

REFERENCES (1) Steeg, P. S.; Theodorescu, D. Metastasis: a Therapeutic Target for Cancer. Nat. Clin. Pract. Oncol. 2008, 5, 206. (2) Mellman, I.; Coukos, G.; Dranoff, G. Cancer Immunotherapy Comes of Age. Nature 2011, 480, 480-489. (3) Marigo, I.; Dolcetti, L.; Serafini, P.; Zanovello, P.; Bronte, V. Tumor ‐ Induced Tolerance and Immune Suppression by Myeloid Derived Suppressor Cells. Immunol. Rev. 2008, 222, 162-179. (4) Talmadge, J. E.; Gabrilovich, D. I. History of Myeloid-Derived Suppressor Cells. Nat. Rev. Cancer 2013, 13, 739-752. (5) Bracci, L.; Schiavoni, G.; Sistigu, A.; Belardelli, F. Immune-Based Mechanisms of Cytotoxic Chemotherapy: Implications for the Design of Novel and Rationale-Based Combined Treatments Against Cancer. Cell Death And Differ. 2013, 21, 15-25.


Page 22 of 25

Page 23 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(6) Alizadeh, D.; Trad, M.; Hanke, N. T.; Larmonier, C. B.; Janikashvili, N.; Bonnotte, B.; Katsanis, E.; Larmonier, N. Doxorubicin Eliminates Myeloid-Derived Suppressor Cells and Enhances the Efficacy of Adoptive T-Cell Transfer in Breast Cancer. Cancer Res. 2014, 74, 104-118. (7) Vincent, J.; Mignot, G.; Chalmin, F.; Ladoire, S.; Bruchard, M.; Chevriaux, A.; Martin, F.; Apetoh, L.; Rébé, C.; Ghiringhelli, F. 5-Fluorouracil Selectively Kills Tumor-Associated Myeloid-Derived Suppressor Cells Resulting in Enhanced T Cell–Dependent Antitumor Immunity. Cancer Res. 2010, 70, 3052-3061. (8) Veglia, F.; Perego, M.; Gabrilovich, D. Myeloid-Derived Suppressor Cells Coming of Age. Nat. Immunol. 2018, 19, 108-119. (9) Sierro, S. R.; Donda, A.; Perret, R.; Guillaume, P.; Yagita, H.; Levy, F.; Romero, P. Combination of Lentivector Immunization and Low‐Dose Chemotherapy or PD‐1/PD‐L1 Blocking Primes Self ‐ Reactive T Cells and Induces Anti ‐ Tumor Immunity. Eur. J. Immunol. 2011, 41, 2217 -2228. (10) Song, Q.; Yin, Y.; Shang, L.; Wu, T.; Zhang, D.; Kong, M.; Zhao, Y.; He, Y.; Tan, S.; Guo, Y.; Zhang, Z. Tumor Microenvironment Responsive Nanogel for the Combinatorial Antitumor Effect of Chemotherapy and Immunotherapy. Nano Lett. 2017, 17, 6366-6375. (11) Mahoney, K. M.; Rennert, P. D.; Freeman, G. J. Combination Cancer Immunotherapy and New Immunomodulatory Targets. Nat. Rev. Drug Discov. 2015, 14, 561-584. (12) Ledford, H. Ledford H. Immunotherapy’s Cancer Remit Widens. Nature 2013, 497, 544. (13) Palucka, K.; Banchereau, J. Cancer Immunotherapy via Dendritic Cells. Nat. Rev. Cancer 2012, 12, 265-277. (14) Gotwals, P.; Cameron, S.; Cipolletta, D.; Cremasco, V.; Crystal, A.; Hewes, B.; Mueller, B.; Quaratino, S.; Sabatos-Peyton, C.; Petruzzelli, L.; Engelman, J. A.; Dranoff, G. Prospects for Combining Targeted and Conventional Cancer Therapy with Immunotherapy. Nat. Rev. Cancer 2017, 17, 286-301. (15) Zhai, Y.; Su, J.; Ran, W.; Zhang, P.; Yin, Q.; Zhang, Z.; Yu, H.; Li, Y. Preparation and Application of Cell Membrane-Camouflaged Nanoparticles for Cancer Therapy. Theranostics 2017, 7, 2575-2592. (16) Fang, R. H.; Hu, C.-M. J.; Luk, B. T.; Gao, W.; Copp, J. A.; Tai, Y.; O’Connor, D. E.; Zhang, L. Cancer Cell Membrane-Coated Nanoparticles for Anticancer Vaccination and Drug Delivery. Nano Lett. 2014, 14, 2181-2188. (17) Kroll, A. V.; Fang, R. H.; Jiang, Y.; Zhou, J. R.; Wei, X. L.; Yu, C. L.; Gao, J.; Luk, B. T.; Dehaini, D.; Gao, W. W.; Zhang, L. F. Nanoparticulate Delivery of Cancer Cell Membrane Elicits Multiantigenic Antitumor Immunity. Adv. Mat. 2017, 29, 1703969. (18) Pum, D.; Toca-Herrera, J.; Sleytr, U. S-Layer Protein Self-Assembly. Int. J Mol. Sci. 2013, 14, 2484-2501. (19) Zhu, J.-Y.; Zheng, D.-W.; Zhang, M.-K.; Yu, W.-Y.; Qiu, W.-X.; Hu, J.-J.; Feng, J.; Zhang, X.-Z. Preferential Cancer Cell Self-Recognition and Tumor Self-Targeting by Coating Nanoparticles with Homotypic Cancer Cell Membranes. Nano Lett. 2016, 16, 5895-5901. (20) Chen, Z.; Zhao, P.; Luo, Z.; Zheng, M.; Tian, H.; Gong, P.; Gao, G.; Pan, H.; Liu, L.; Ma, A.; Cui, H.; Ma, Y.; Cai, L. Cancer Cell Membrane–Biomimetic Nanoparticles for Homologous-Targeting Dual-Modal Imaging and Photothermal Therapy. ACS Nano 2016, 10, 10049-10057.



(21) Stel, B.; Cometto, F.; Rad, B.; De Yoreo, J. J.; Lingenfelder, M. Dynamically Resolved Self-Assembly of S-Layer Proteins on Solid Surfaces. Chem. Commun. 2018, 54, 10264-10267. (22) Taverniti, V.; Stuknyte, M.; Minuzzo, M.; Arioli, S.; De Noni, I.; Scabiosi, C.; Cordova, Z. M.; Junttila, I.; Hämäläinen, S.; Turpeinen, H.; Mora, D.; Karp, M.; Pesu, M.; Guglielmetti, S. S-Layer Protein Mediates the Stimulatory Effect of Lactobacillus Helveticus Mimlh5 on Innate Immunity. Appl. Environ. Microbiol. 2013, 79, 1221-1231. (23) Ilk, N.; Egelseer, E. M.; Sleytr, U. B. S-Layer Fusion Proteins-Construction Principles and Applications. Curr. Opin. Biotechnol. 2011, 22, 824-831. (24) Middelberg, A. P. J.; Rivera-Hernandez, T.; Wibowo, N.; Lua, L. H. L.; Fan, Y.; Magor, G.; Chang, C.; Chuan, Y. P.; Good, M. F.; Batzloff, M. R. A Microbial Platform for Rapid and Low-Cost Virus-Like Particle and Capsomere Vaccines. Vaccine 2011, 29, 7154-7162. (25) Wu, M.; Liu, X.; Jin, W.; Li, Y.; Li, Y.; Hu, Q.; Chu, P. K.; Tang, G.; Ping, Y. Targeting ETS1 with RNAi-Based Supramolecular Nanoassemblies for Multidrug-Resistant Breast Cancer Therapy. J. Control. Release 2017, 253, 110-121. (26) Cano, A.; Gamallo, C.; Kemp, C. J.; Benito, N.; Palacios, J.; Quintanilla, M.; Balmain, A. Expression Pattern of the Cell Adhesion Molecules E ‐ Cadherin, P ‐ Cadherin And α6β4 Integrin is Altered in Pre‐Malignant Skin Tumors of P53‐Deficient Mice. Int. J. Cancer 1996, 65, 254-262. (27) Engering, A.; Kuhn, L.; Fluitsma, D.; Hoefsmit, E.; Pieters, Differential Post‐Translational Modification of CD63 Molecules During Maturation of Human Dendritic Cells. J. Eur. J. Biochem. 2003, 270, 2412-2420. (28) Cho, H.; Indig, G. L.; Weichert, J.; Shin, H.-C.; Kwon, G. S. In vivo Cancer Imaging by Poly (Ethylene Glycol)-B-Poly (Ɛ-Caprolactone) Micelles Containing a Near-Infrared Probe. Nanomed-nanotechnol. 2012, 8, 228-236. (29) Zhang, J. Q.; Zeng, S.; Vitiello, G. A.; Seifert, A. M.; Medina, B. D.; Beckman, M. J.; Loo, J. K.; Santamaria-Barria, J.; Maltbaek, J. H.; Param, N. J.; Moral, J. A.; Zhao, J. N.; Balachandran, V.; Rossi, F.; Antonescu, C. R.; DeMatteo, R. P. Macrophages and CD8+ T Cells Mediate the Antitumor Efficacy of Combined CD40 Ligation and Imatinib Therapy in Gastrointestinal Stromal Tumors. Cancer Immunol. Res. 2018, 6, 434-447. (30) Hu, Q.; Wu, M.; Fang, C.; Cheng, C.; Zhao, M.; Fang, W.; Chu, P. K.; Ping, Y.; Tang, G. Engineering Nanoparticle-Coated Bacteria as Oral DNA Vaccines for Cancer Immunotherapy. Nano Lett. 2015, 15, 2732-2739. (31) Gambhir, S. S. Molecular Imaging of Cancer with Positron Emission Tomography. Nat. Rev. Cancer 2002, 2, 683-693. (32) Peng, L.-H.; Zhang, Y.-H.; Han, L.-J.; Zhang, C.-Z.; Wu, J.-H.; Wang, X.-R.; Gao, J.-Q.; Mao, Z.-W. Cell Membrane Capsules for Encapsulation of Chemotherapeutic and Cancer Cell Targeting in Vivo. ACS Appl. Mater. Interfaces 2015, 7, 18628-18637. (33) Kontro, I.; Wiedmer, S. K.; Hynönen, U.; Penttilä, P. A.; Palva, A.; Serimaa, R. The Structure of Lactobacillus Brevis Surface Layer Reassembled on Liposomes Differs from Native Structure as Revealed by SAXS. Bba-biomembranes 2014, 1838, 2099-2104. (34) Shen, J.; Wang, Q.; Hu, Q.; Li, Y.; Tang, G.; Chu, P. K. Restoration of Chemosensitivity by Multifunctional Micelles Mediated by P-gp siRNA to Reverse MDR. Biomaterials 2014, 35, 8621-8634.


Page 24 of 25

Page 25 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ToC figure:


Surface-Layer Protein-Enhanced Immunotherapy Based on Cell

Recommend Documents