A modular approach to enhancing cell membrane-coated nanoparticle functionality using genetic engineering

Abstract

Since their initial development, cell membrane-coated nanoparticles (CNPs) have become increasingly popular in the biomedical field. Despite their inherent versatility and ability to enable complex biological applications, there is considerable interest in augmenting the performance of CNPs through the introduction of additional functionalities. Here we demonstrate a genetic-engineering-based modular approach to CNP functionalization that can encompass a wide range of ligands onto the nanoparticle surface. The cell membrane coating is engineered to express a SpyCatcher membrane anchor that can readily form a covalent bond with any moiety modified with SpyTag. To demonstrate the broad utility of this technique, three unique targeted CNP formulations are generated using different classes of targeting ligands, including a designed ankyrin repeat protein, an affibody and a single-chain variable fragment. In vitro, the modified nanoparticles exhibit enhanced affinity towards cell lines overexpressing the cognate receptors for each ligand. When formulated with a chemotherapeutic payload, the modularly functionalized nanoparticles display strong targeting ability and growth suppression in a murine tumour xenograft model of ovarian cancer. Our data suggest genetic engineering offers a feasible approach for accelerating the development of multifunctional CNPs for a broad range of biomedical applications.

Fig. 1 |. Engineering and characterization of SpyCatcher-expressing cells and SpyTag-labeled ligands.

a, Wild-type cells are engineered to express surface-bound SpyCatcher, which forms an isopeptide bond with SpyTag. When the membrane from the SpyCatcher-expressing cells is coated onto nanoparticle cores, the resulting cell membrane-coated nanoparticles can be modularly functionalized with SpyTag-labeled ligands for enhanced functionality. Created with BioRender. b, Flow cytometric analysis of SpyCatcher expression on wild-type HEK293 and SpyCatcher-expressing HEK293 (HEK293-SC) cells. c, Fluorescent visualization of SpyCatcher expression on wild-type HEK293 and HEK293-SC cells. Blue: nuclei (DAPI), green: SpyCatcher (sfGFP); scale bar: 20 μm. d, Western blots probing for SpyTag-labeled ligands in control cell lysate, engineered cell lysate, and purified protein product.

Fig. 2 |. Functional characterization of SpyTag-labeled ligands.

a, Dose-dependent binding of ST-mKate2 with wild-type HEK293 or HEK293-SC cells (n = 3 biological replicates, mean ± SD). MFI: mean fluorescence intensity. b, Live cell fluorescent visualization of ST-mKate2 after binding with HEK293-SC cells. Blue: nuclei (DAPI), green: SpyCatcher (sfGFP), magenta: mKate2; scale bar: 10 μm. c-e, Dose-dependent binding of ST-αmCherry (c), ST-αEGFR (d), and ST-αHER2 (e) with wild-type HEK293 or HEK293-SC cells (n = 3 biological replicates, mean ± SD). f-h, Dose-dependent binding of ST-αmCherry to wild-type HEK293T or HEK293T-mCherry cells (f), ST-αEGFR to MDA-MB-453 (EGFR⁻) or SKOV3 (EGFR⁺) cells (g), and ST-αHER2 to MDA-MB-231 (HER2⁻) or SKOV3 (HER2⁺) cells (h) (n = 3 biological replicates, mean ± SD). i-k, Fluorescent visualization of ST-αmCherry (i), ST-αEGFR (j), and ST-αHER2 (k) binding with mixed cultures of HEK293T + HEK293T-mCherry cells (i), MDA-MB-453 + SKOV3 cells (j), and MDA-MB-231 + SKOV3 cells (k), respectively. Blue: nuclei (DAPI); scale bars: 20 μm.

Fig. 3 |. Nanoparticle synthesis and functionalization.

a, Size of SC-NPs fabricated at varying membrane protein to PLGA core weight ratios in water or PBS (n = 3 technical replicates, mean + SD). b,c, Hydrodynamic diameter (b) and surface zeta potential (c) of bare PLGA cores, HEK293-SC membrane vesicles, and SC-NPs (n = 3 technical replicates, mean + SD). d, Western blots probing for SpyCatcher on cell lysate, cell membrane, and membrane-coated nanoparticles derived from wild-type HEK293 or HEK293-SC cells. e,f, Hydrodynamic diameter (e) and surface zeta potential (f) of SC-NPs and mKate2-NPs (n = 3 technical replicates, mean + SD). g, TEM image of mKate2-NPs negatively stained with uranyl acetate. Scale bar: 100 nm. h, Immunogold TEM image of mKate2-NPs probing for ST-mKate2. Scale bar: 100 nm. i, Size of SC-NPs and mKate2-NPs in an isotonic sucrose solution over 18 days (n = 3 technical replicates, mean ± SD). j, Fluorescence intensity of fractions collected from the size exclusion chromatography of ST-mKate2 (mKate2), SC-NPs (DiR), and mKate2-NPs (mKate2) prior to purification. k, Fluorescence intensity of fractions collected from the size exclusion chromatography of ST-mKate2 (mKate2), WT-NPs (DiR), and WT-NPs + ST-mKate2 (mKate2). l, Western blots probing for ST-mKate2 or SpyCatcher–ST-mKate2 complex in samples containing various combinations of SC-NPs and ST-mKate2.

Fig. 4 |. Functional characterization of modularly functionalized nanoparticles.

a-c, Dose-dependent binding of αmCherry-NPs to HEK293T-mCherry cells (a), αEGFR-NPs to SKOV3 cells (b), and αHER2-NPs to SKOV3 cells (c) (n = 3 biological replicates, mean ± SD); non-targeted SC-NPs were used as controls. MFI: mean fluorescence intensity. d-f, Fluorescent visualization of αmCherry-NP (d), αEGFR-NP (e), and αHER2-NP (f) binding with mixed cultures of HEK293T + HEK293T-mCherry cells (d), MDA-MB-453 + SKOV3 cells (e), and MDA-MB-231 + SKOV3 cells (f), respectively. Blue: nuclei (DAPI); scale bars: 20 μm. g-i, Binding of αmCherry-NPs to HEK293T-mCherry cells (g), αEGFR-NPs to SKOV3 cells (h), and αHER2-NPs to SKOV3 cells (i) in the presence of the corresponding free ligand as a blocking agent (n = 3 biological replicates, mean + SD). j-l, Dose-dependent cytotoxicity of αmCherry-[DTX]NPs against HEK293T-mCherry cells (j), αEGFR-[DTX]NPs against SKOV3 cells (k), and αHER2-[DTX]NPs against SKOV3 cells (l) measured at 72 h after 15 min of co-incubation (n = 3 biological replicates, mean ± SD); free DTX, non-targeted SC-[DTX]NPs, and the respective unloaded nanoparticles (αmCherry-NPs, αEGFR-NPs, and αHER2-NPs) were used as controls.

Fig. 5 |. In vivo tumor targeting, therapeutic efficacy, and safety.

a, Representative live fluorescent imaging of tumors at various timepoints after intravenous administration of SC-NPs, αEGFR-NPs, and αHER2-NPs. b, Representative ex vivo fluorescent imaging of tumors and major organs collected 24 h after intravenous administration of SC-NPs, αEGFR-NPs, and αHER2-NPs. c, Weight-normalized fluorescence of tumors and major organs collected 24 h after intravenous administration of SC-NPs, αEGFR-NPs, and αHER2-NPs (n = 4 biological replicates, mean + SEM). d, Growth kinetics of SKOV3 tumors treated intravenously with PBS, SC-[DTX]NPs, αEGFR-[DTX]NPs, and αHER2-[DTX]NPs (n = 6 biological replicates, mean ± SD). e, Survival of mice in (d) over time (n = 6 biological replicates). f,g, Serum biochemistry of immunocompetent mice on day 1 (f) and day 10 (g) after intravenous administration of PBS, SC-[DTX]NPs, αEGFR-[DTX]NPs, and αHER2-[DTX]NPs; injections were performed on days 0, 3, 6, and 9 (n = 3 biological replicates, mean + SD). ALB: albumin, ALP: alkaline phosphatase, ALT: alanine transaminase, AMY: amylase, BUN blood urea nitrogen, CA: calcium, CRE: creatinine, GLOB: globulin (calculated), GLU: glucose, K: potassium, NA: sodium, PHOS: phosphorus, TBIL: total bilirubin, TP: total protein. h-m, Red blood cell (RBC) (h,k), platelet (i,l), and total white blood cell (WBC) (j,m) counts of immunocompetent mice on day 1 (h-j) and day 10 (k-m) after intravenous administration with PBS, SC-[DTX]NPs, αEGFR-[DTX]NPs, and αHER2-[DTX]NPs; injections were performed on days 0, 3, 6, and 9 (n = 3 biological replicates, mean + SD).