Biomimetic nanoparticles (NPs) combine the flexibility and reproducibility of synthetic materials with the functionality of biological materials. Here, we developed and characterized biomimetic poly­(lactic-co-glycolic acid) (PLGA) NPs coated with human cancer cell membrane fractions (CCMFs) to form CCMF-coated PLGA (CCMF-PLGA) NPs. We evaluated the ability of these CCMF-PLGA NPs to disrupt cancer cell–stromal cell interactions and to induce an immune response. Western blot analysis verified the plasma membrane purity of CCMFs. Confocal fluorescence microscopy and flow cytometry confirmed the presence of intact membrane-associated proteins including CXCR4 and CD44 following membrane derivation and coating. CCMFs and CCMF-PLGA NPs were capable of inhibiting cancer cell migration toward human mammary fibroblasts. Intravenous injection of CCMF-PLGA NPs significantly reduced experimental metastasis in vivo. Following immunization of Balb/c mice, near-infrared fluorescence imaging confirmed the migration of NPs to proximal draining lymph nodes (LNs). A higher percentage of CD8+ and CD4+ cytotoxic T-lymphocyte populations was observed in spleens and LNs of CCMF-PLGA NP-immunized mice. Splenocytes isolated from CCMF-PLGA NP-immunized mice had the highest number of interferon gamma-producing T-cells as detected by the ELISpot assay. CCMF-PLGA NPs hold promise for disrupting cancer cell–stromal cell interactions and for priming the immune system in cancer immunotherapy.

Synechocystis sp. strain PCC6803 (Synechocystis) is a model microorganism and its mechanosensitive (MS) channels play important roles in its osmoadaptation mechanism. When the osmotic concentration of the culture environment changes, the inner pressure of the cell also changes due to the transportation of water through ion channels. Because the tension in the cell membrane relates to the inner pressure, we expect that the response of the MS channels to an osmotic concentration change could be evaluated by measuring their mechanical properties. Here, we propose a system for the measurement of the mechanical properties of a single Synechocystis cell. We developed a robot-integrated microfluidic chip combined with optical tweezers. The chip has an external actuated pushing probe and a force sensor probe. A single cell was located between the tip of both probes using the optical tweezers and was then deformed using the probes. As a result, we could measure the force and deformation and compare the Young's moduli of two groups: a group of wild type cells and a group of mutant (genetically modified) cells with a defect in the MS channels, at three different osmotic concentrations. The results showed that the Young's modulus of each group changed according to the osmotic concentration, while changes in cell size were too small to be detected. These results confirmed that the proposed evaluation method provides an understanding of the physiological function of MS channels for keeping the cell integrity of microorganisms when the cells are exposed to different external osmotic changes.

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Abstract

Introduction: Biomimetic nanoparticles (NPs) combining synthetic and biological materials have flexibility and functionality for drug delivery and immunomodulation.1 Red blood cell (RBC) membranes coated poly(lactic-co-glycolic acid) (PLGA) NPs that mimic RBCs and act as nanosponges for toxins were recently described.2-3 Here, we coated cancer cell membranes onto PLGA NPs in a “right-side” out manner, to translocate membrane anchored proteins onto NPs. Cancer cell membrane coated PLGA NPs hold promise for disrupting cancer cell-stromal cell interactions, and for priming the immune system in cancer immunotherapy. Method: Plasma membrane fractions (MFs) of U87 (low CXCR4) and U87-CXCR4 (high CXCR4) cells were isolated upon homogenization, and sucrose density gradient centrifugation. MFs and PLGA NPs were mixed and physically extruded through a porous membrane to obtain MF-coated PLGA NPs. MFs were probed with antibodies against cell fraction markers, and CXCR4. MFs-coated NPs, MFs and PLGA NPs were characterized on size, morphology, and zeta-potential. The orientation of MFs and MF-coated NPs was investigated by confocal microscopy and flow cytometry. Transwell migration assays were performed to investigate the migration of cancer cells towards human mammary fibroblasts (HMFs). Immune-competent Balb/c mice were immunized with IR700-labeled MFs or MF-coated NPs via subcutaneous injection through hock and imaged in vivo and ex vivo by near-infrared (NIR) fluorescence. Immune responses to MFs or MF-coated NPs were examined on activating CD4+ and CD8+ T lymphocytes in lymph nodes and spleens by flow cytometry. Results: Plasma membrane purity was confirmed from western blot analysis that showed the significant enrichment of Na+/K+-ATPase, negligible amount of GPR78 or GAPDH in MFs. PLGA NPs, U87-CXCR4 MFs and U87-CXCR4 MFs-coated NPs had average diameters of 50 nm, 200 nm, and 70 nm, respectively. Z-average diameters and zeta-potential of PLGA NPs, U87-CXCR4 MFs, and U87-CXCR4 MFs-coated NPs were 79.8 nm, 336 nm and 168 nm, and -34.3 mV, -24.9 mV and -25.0 mV, respectively. Confocal fluorescence microscopy and flow cytometry detected intense PE fluorescence and higher CXCR4 in U87-CXCR4 MFs and U87-CXCR4 MFs-coated NPs than U87 counterparts, confirming a “right-side” out orientation. When U87 or U87-CXCR4 MFs were added to HMFs in the transwell assay, fewer cancer cells migrated towards HMFs, identifying the unique ability of MFs in disrupting HMF-cancer cell interactions. U87-CXCR4 MFs and MF-coated NPs were observed in the popliteal lymph nodes, and triggered the induction of CD8+ T lymphocytes, identifying a role for MF-coated NPs in providing cancer cell membranes to antigen presenting cells to induce a tumor-specific immune response. References: 1. Fang, R. et al. Small 2015; 2. Hu, C. et al. PNAS 2011; 3. Hu, C. et al. Nat. Nanotechnol. 2013. Supported by NIH R21 CA198243 and P50 CA103175. Citation Format: Jiefu Jin, Di Chang, Samit Chatterjee, Balaji Krishnamachary, Yelena Mironchik, Sridhar Nimmagadda, Zaver M. Bhujwalla. Cancer cell membrane coated biomimetic nanoparticles: Synthesis, characterization, and functionality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2198. doi:10.1158/1538-7445.AM2017-2198