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Nanoparticle biointerfacing by platelet membrane cloaking

Nature volume 526, pages 118–121 (2015)Cite this article

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Abstract

Development of functional nanoparticles can be encumbered by unanticipated material properties and biological events, which can affect nanoparticle effectiveness in complex, physiologically relevant systems1,2,3. Despite the advances in bottom-up nanoengineering and surface chemistry, reductionist functionalization approaches remain inadequate in replicating the complex interfaces present in nature and cannot avoid exposure of foreign materials. Here we report on the preparation of polymeric nanoparticles enclosed in the plasma membrane of human platelets, which are a unique population of cellular fragments that adhere to a variety of disease-relevant substrates4,5,6,7. The resulting nanoparticles possess a right-side-out unilamellar membrane coating functionalized with immunomodulatory and adhesion antigens associated with platelets. Compared to uncoated particles, the platelet membrane-cloaked nanoparticles have reduced cellular uptake by macrophage-like cells and lack particle-induced complement activation in autologous human plasma. The cloaked nanoparticles also display platelet-mimicking properties such as selective adhesion to damaged human and rodent vasculatures as well as enhanced binding to platelet-adhering pathogens. In an experimental rat model of coronary restenosis and a mouse model of systemic bacterial infection, docetaxel and vancomycin, respectively, show enhanced therapeutic efficacy when delivered by the platelet-mimetic nanoparticles. The multifaceted biointerfacing enabled by the platelet membrane cloaking method provides a new approach in developing functional nanoparticles for disease-targeted delivery.

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Figure 1: Preparation and characterization of PNPs.
Figure 2: Collagen binding and immunocompatibility.
Figure 3: Adherence to damaged human and rodent vasculatures.
Figure 4: Binding to platelet-adhering pathogens.

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Acknowledgements

This work is supported by the National Institutes of Health under Award Numbers R01DK095168 (L.Z.), R01HL108735 (S.C.) and R01EY25090 (K.Z.), and partially by the Defense Threat Reduction Agency Joint Science and Technology Office for Chemical and Biological Defense under Grant Number HDTRA1-14-1-0064 (L.Z.). R.H.F. is supported by a National Institutes of Health R25CA153915 training grant from the National Cancer Institute.

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Author notes

  1. Che-Ming J. Hu, Ronnie H. Fang and Kuei-Chun Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of NanoEngineering, University of California, San Diego, La Jolla, 92093, California, USA

    Che-Ming J. Hu, Ronnie H. Fang, Soracha Thamphiwatana, Diana Dehaini, Pavimol Angsantikul, Ashley V. Kroll, Cody Carpenter, Manikantan Ramesh, Vivian Qu, Weiwei Gao, Kang Zhang & Liangfang Zhang

  2. Moores Cancer Center, University of California, San Diego, La Jolla, 92093, California, USA

    Che-Ming J. Hu, Ronnie H. Fang, Brian T. Luk, Soracha Thamphiwatana, Diana Dehaini, Pavimol Angsantikul, Ashley V. Kroll, Weiwei Gao & Liangfang Zhang

  3. Department of Bioengineering, University of California, San Diego, La Jolla, 92093, California, USA

    Kuei-Chun Wang, Brian T. Luk, Phu Nguyen & Shu Chien

  4. Institute of Engineering in Medicine, University of California, San Diego, La Jolla, 92093, California, USA

    Kuei-Chun Wang, Phu Nguyen, Kang Zhang, Shu Chien & Liangfang Zhang

  5. Shiley Eye Institute, University of California, San Diego, La Jolla, 92093, California, USA

    Cindy H. Wen, Sherrina H. Patel, Jie Zhu, William Shi & Kang Zhang

  6. Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, 90033, California, USA

    Florence M. Hofman & Thomas C. Chen

  7. Veterans Administration Healthcare System, San Diego, 92093, California, USA

    Kang Zhang

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  1. Che-Ming J. Hu
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Contributions

C.-M.J.H., R.H.F., K.-C.W., B.T.L., K.Z., S.C. and L.Z. conceived and designed the experiments; C.-M.J.H., R.H.F., K.-C.W., B.T.L., S.T., D.D., P.N., P.A., C.H.W., A.V.K., C.C., M.R., V.Q., S.H.P., J.Z., W.S., F.M.H., T.C.C. and W.G. performed all the experiments. The manuscript was written by C.-M.J.H., R.H.F., B.T.L., W.G. and L.Z. All authors discussed the results and reviewed the manuscript.

Corresponding authors

Correspondence to Kang Zhang, Shu Chien or Liangfang Zhang.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Schematic preparation of PNPs.

a, Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are enclosed entirely in plasma membrane derived from human platelets. The resulting particles possess platelet-mimicking properties for immunocompatibility, subendothelium binding, and pathogen adhesion. b, Schematic depicting the process of preparing PNPs.

Extended Data Figure 2 PNP preparation and storage.

a, Isolation of platelet rich plasma (PRP) was achieved by centrifugation at 100g. PRP was collected from the top layer (yellow) separated from the red blood cells (red, bottom layer). b, Collected human platelets under light microscopy, which possess a distinctive morphology from c, red blood cells. Scale bars, 10 μm. d, Transmission electron micrographs of platelet membrane vesicles and e, PNPs, both of which were negatively stained with 1% uranyl acetate. Scale bars, 200 nm. f, Dynamic light scattering measurements of PNPs in 10% sucrose show that the particles retain their size and stability after a freeze-thaw cycle and re-suspension upon lyophilization (n = 3). Bars represent means ± s.d. g, Transmission electron micrograph shows retentions of the core-shell structure of PNPs after a freeze-thaw cycle in 10% sucrose. Scale bar, 100 nm. h, Transmission electron micrograph shows retentions of the core-shell structure of PNPs upon resuspension after lyophilization in 10% sucrose. Scale bar, 100 nm.

Extended Data Figure 3 Overall protein content on PNPs resolved by western blotting.

Primary platelet membrane protein/protein subunits including CD47, CD55, CD59, αIIb, α2, α5, α6, β1, β3, GPIbα, GPIV, GPV, GPVI, GPIX, and CLEC-2 were monitored in platelet rich plasma, platelets, platelet vesicles, and PNPs. Platelets derived from four different protocols, including commercial blood anti-coagulated in EDTA, freshly drawn blood anti-coagulated in EDTA, freshly drawn blood anti-coagulated in heparin, and transfusion-grade platelet rich plasma anti-coagulated in acid-citrate-dextrose (ACD), were examined to compare the membrane protein expression. Each sample was normalized to equivalent overall protein content before western blotting. It was observed that the PNP preparation resulted in membrane protein retention and enrichment very similar across the different platelet sources.

Extended Data Figure 4 Platelet membrane sidedness on PNPs.

a, Transmission electron micrograph of PNPs primary-stained with anti-CD47 (intracellular), secondary-stained with immunogold, and negatively stained with 2% vanadium. The immunogold staining revealed presence of intracellular CD47 domains on collapsed platelet membrane vesicles, but not on PNPs. b, Transmission electron micrograph of PNPs primary-stained with anti-CD47 (extracellular), secondary-stained with immunogold, and negatively stained with 2% vanadium. PNPs were shown to display extracellular CD47 domains. All scale bars, 100 nm. c, 2 μm polystyrene beads were functionalized with anti-CD47 against the protein’s extracellular domain, anti-CD47 against the protein’s intracellular domain, or a sham antibody. Flow cytometric analysis of the different beads after DiD-loaded PNP incubation showed the highest particle retention to beads functionalized with anti-CD47 against the protein’s extracellular domain. d, Normalized fluorescence intensity of PNP retention to the different antibody-functionalized beads. Bars represent means ± s.e.m.

Extended Data Figure 5 PNP binding to collagen and extracellular matrix.

a–f, Collagen-coated tissue culture slides seeded with human umbilical vein endothelial cells (HUVECs) were incubated with PNP solution for 30 s. Fluorescence microscopy samples demonstrate differential PNP adherence to exposed collagen versus covered endothelial surfaces. a–c, Representative fluorescence images visualizing DiD-loaded PNPs (red), cellular cytosol (green), and cellular nuclei (blue). d–f, Images showing only the red and blue channels to highlight the differential localization of PNPs. Scale bar, 10 μm. g, Fluorescence quantification of PNP per unit area on collagen and endothelial surfaces. Bars represent means ± s.d. (n = 10). h, i, PNP adherence to arterial extracellular matrix (ECM) as visualized by SEM. h, SEM images of the ECM of a decellularized human umbilical cord artery. Left, scale bar, 1 μm; right, scale bar, 500 nm. i, SEM images of the ECM of a decellularized human umbilical cord artery after PNP incubation. Left, scale bar, 1 μm; right, scale bar, 500 nm.

Extended Data Figure 6 Pharmacokinetics, biodistribution and safety of PNPs.

a, DiD-loaded PNPs were injected intravenously through the tail vein of Sprague–Dawley rats. At various time points, blood was withdrawn via tail vein blood sampling for fluorescence quantification to evaluate the systemic circulation lifetime of the nanoparticles (n = 6). b, Biodistribution of the PNP nanoparticles in balloon-denuded Sprague–Dawley rats at 2 h and 48 h after intravenous nanoparticle administration through the tail vein (n = 6). c, Comprehensive metabolic panel of rats after injections with human-derived PNPs and PBS (n = 6). The rats received intravenous injections of PNPs and PBS on day 0 and day 5, and the blood test conducted on day 10 did not reveal significant changes between the two groups, indicating normal liver and kidney functions after the PNP administration. All bars and markers represent means ± s.d.

Extended Data Figure 7 PNP targeting of damaged vasculatures upon intravenous injection to rats with angioplasty-induced arterial denudation.

a, Fluorescence microscopy of the aortic branch revealed selective PNP binding to the denuded artery (right) as opposed to the undamaged artery (left) (PNP fluorescence in red). b, Fluorescence images acquired from the control artery, which did not reveal PNP fluorescence upon focusing on either the endothelium (top) or the smooth muscle layer (bottom) (nuclei in blue). c, Fluorescence images acquired from the denuded artery, which revealed significant PNP retention as fluorescent punctates (PNP fluorescence in red) above the smooth muscle layer. d, Fluorescence image of arterial cross-section acquired from the control artery, which showed nuclei of endothelial cells above the collagen layer (autofluorescence in green) and an absence of PNP fluorescence. e, Fluorescence image of arterial cross-section acquired from the denuded artery, which showed PNP retention as fluorescent punctates on the collagen layer (PNP fluorescence in red; collagen autofluorescence in green) and an absence of endothelial cell nuclei. All scale bars, 100 μm.

Extended Data Figure 8 Characterizations of drug-loaded cell membrane cloaked nanoparticles.

a, Physicochemical properties of drug-loaded cell membrane cloaked nanoparticles. b, TEM visualization of docetaxel-loaded PNPs (PNP-Dtxl). Scale bar, 200 nm. c, Drug release profile of PNP-Dtxl compared to polyethylene glycol (PEG)-PLGA diblock nanoparticles of equivalent size and docetaxel loading (n = 3). d, TEM visualization of vancomycin-loaded PNPs (PNP-Vanc). Scale bar, 200 nm. e, Drug release profiles of PNP-Vanc and RBCNP-Vanc (n = 3). Bars represent means ± s.d.

Extended Data Figure 9 Treatment of an experimental rat model of coronary restenosis.

a–e, H&E-stained arterial cross-sections reveal the vascular structure of non-damaged arteries (serving as baseline, a) and denuded arteries after treatment with PNP-Dtxl (b), PBS (c), PNP with no docetaxel content (d), or free docetaxel (e). Scale bar, 200 μm.

Extended Data Figure 10 PNP adherence to MRSA252 bacteria.

a, Flow cytometric analysis of MRSA252 bacteria after incubation with different DiD-loaded nanoformulations. b, Pellets of MRSA252 after incubation with DiD-loaded RBCNPs (left) and DiD-loaded PNPs (right) show differential retention of nanoformulation with MRSA252 upon pelleting of the bacteria. c, A pseudocoloured SEM image of PNPs binding to MRSA252 under high magnification (MRSA coloured in gold, PNP coloured in orange). Scale bar, 400 nm.

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Hu, CM., Fang, R., Wang, KC. et al. Nanoparticle biointerfacing by platelet membrane cloaking. Nature 526, 118–121 (2015). https://doi.org/10.1038/nature15373

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