Nanoparticle biointerfacing by platelet membrane cloaking

Abstract

Development of functional nanoparticles can be encumbered by unanticipated material properties and biological events, which can affect nanoparticle effectiveness in complex, physiologically relevant systems. 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 substrates. 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.

Figures

Extended Data Fig. 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 Fig. 2. PNP preparation and storage

(a) Isolation of platelet rich plasma (PRP) was achieved via centrifugation at 100 × g. 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 following a freeze-thaw cycle and re-suspension upon lyophilization (n=3). Bars represent means ± SD. (g) Transmission electron micrograph shows retentions of PNPs’ core-shell structure following a freeze-thaw cycle in 10% sucrose. Scale bar = 100 nm. (h) Transmission electron micrograph shows retentions of PNPs’ core-shell structure upon resuspension following lyophilization in 10% sucrose. Scale bar = 100 nm.

Extended Data Fig. 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 prior to 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 Fig. 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 are 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 following DiD-loaded PNP incubation shows 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 ± SEM.

Extended Data Fig. 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 sec. 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 in order 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 ± SD (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 following PNP incubation. Left: Scale bar = 1 µm; Right: Scale bar = 500 nm.

Extended Data Fig. 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 following intravenous nanoparticle administration through the tail vein (n=6). (c) Comprehensive metabolic panel of rats following 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 following the PNP administration. All bars and markers represent means ± SD.

Extended Data Fig. 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 Fig. 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 as compared to 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 ± SD.

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

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

Extended Data Fig. 10. PNP adherence to MRSA252 bacteria

(a) Flow cytometric analysis of MRSA252 bacteria following incubation with different DiD-loaded nanoformulations. (b) Pellets of MRSA252 following 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 pseudocolored SEM image of PNPs binding to MRSA252 under high magnification (MRSA colored in gold, PNP colored in orange). Scale bar = 400 nm.

Figure 1. Preparation and characterization of PNPs

(a) Physicochemical characterization of platelets, platelet vesicles, bare NPs, and PNPs (n=3). (b) TEM images of bare NPs (left) and PNPs (right) negatively stained with uranyl acetate. Scale bar = 100 nm. (c) Particle diameter of bare NPs and PNPs in water and in 1X PBS (n=3). (d) Representative protein bands resolved using western blotting. (e) TEM image of PNPs primary stained with extracellular-domain-specific anti-CD47, and secondary stained by an immunogold conjugate. Scale bar = 40 nm. (f–h) Platelet-activating contents including (f) thrombin, (g) ADP, and (h) thromboxane in platelets, platelet vesicles, and PNPs were quantified (n=3). (i) Platelet aggregation assay in which citrate-stabilized platelet rich plasma (PRP) was mixed with PBS, PNPs, or thrombin followed by spectroscopic examination of solution turbidity. All bars represent means ± SD.

Figure 2. Collagen binding and immunocompatibility

(a) Fluorescence quantification of nanoparticle retention on collagen-coated and non-coated plates (n=6). (b) Localization of PNPs (stained in red) on collagen-coated tissue culture slides seeded with HUVECs (nuclei stained in blue). Cellular periphery is outlined based on cytosolic staining. Scale bar = 10 µm. (c) A pseudocolored SEM image of the extracellular matrix of a decellularized human umbilical cord artery following PNP incubation (PNPs colored in orange). Scale bar = 500 nm. (d) Flow cytometric analysis of nanoparticle uptake by human THP-1 macrophage-like cells (n=3). (e) Classical complement activation measured by C4d split products and (f) alternative complement activation measured by Bb split products for bare NPs, platelet vesicles, and PNPs in autologous human plasma (n=4). Zymosan and untreated plasma are used as positive and negative controls respectively. All bars represent means ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 3. Adherence to damaged human and rodent vasculatures

(a) H&E-stained cross-sections of undamaged (top) and damaged (bottom) human carotid arteries. Scale bar = 200 µm. (b) Fluorescence images of the cross-section (scale bar = 200 µm) and (c) the luminal side (scale bar = 500 µm) of undamaged (top) and damaged (bottom) arteries following PNP incubation (tissue in green and PNPs in red). (d,e) 3D reconstructed images of intact (top) and balloon-denuded (bottom) arterial walls from multisectional images following intravenous administration of PNPs in rats (cell nuclei in blue and PNPs in red). Dimensions: 152.5 µm × 116 µm × 41 µm. (f) Retention of PNPs at the denuded and the intact arteries over 120 h following PNP administration (n=6). (g) Representative H&E-stained arterial cross-sections from different treatment groups in a rat model of coronary restenosis. Scale bar = 200 µm (h) Zoomed-in H&E-stained arterial cross-sections highlight the different vascular remodeling from the different treatment groups. I, intima; M, media. Scale bar = 100 µm. (i,j) Quantitative analysis of intima-to-media area ratio and luminal obliteration from the different treatment groups (n=6). All bars represent means ± SD. NS = no statistical significance.

Figure 4. Binding to platelet-adhering pathogens

(a) SEM images of MRSA252 bacteria following incubation with PBS (top left), bare NPs (top right), RBCNPs (bottom left), and PNPs (bottom right). Scale bar = 1 µm. (b) Normalized fluorescence intensity of DiD-loaded nanoformulations retained on MRSA252 based on flow cytometric analysis. Bars represent means ± SD (n=3). (c)In vitro antimicrobial efficacy of free vancomycin, vancomycin-loaded RBCNPs (RBCNP-Vanc), and vancomycin-loaded PNPs (PNP-Vanc). Bars represent means ± SD (n=3). (d–i)In vivo antimicrobial efficacy of free vancomycin at 10 mg kg−1 (Vanc-10), RBCNP-Vanc-10, and PNP-Vanc-10, and free vancomycin at 6 times the dosing (Vanc-60, 60 mg kg−1) was examined in a mouse model of systemic infection with MRSA252. Following 3 days of treatments, bacterial loads in different organs including (d) blood, (e) heart, (f) lung, (g) liver, (h) spleen, and (i) kidney were quantified. Bars represent means ± SEM (n=14). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.