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An antifouling membrane-fusogenic liposome for effective intracellular delivery in vivo – Nature Communications

Ethical statement

The research presented here complies with all relevant ethical regulations. All experiments involving animals complied with the Guidelines for Care and Use of Laboratory Animals of Sun Yat-sen University and were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.

Materials

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) were obtained from Sunlipo Biotech Research Center for Nanomedicine (Shanghai, China). Cholesterol was obtained from Merck Ltd. (Beijing, China). Ammonium thiocyanate was bought from Macklin Inc. (Shanghai, China). DiD, DiR, and polymerase chain reaction (PCR) kit with Taq reagent were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). DiO, DiI, YOYO-1, LysoTracker Red (DND-99), and D-Luciferin luciferase substrate were bought from Yeasen Co., Ltd. (Shanghai, China). pCDNA3.1-Luc encoding luciferase (catalog: #185358) was purchased from Miaoling Biotechnology Co., Ltd. (Wuhan, China). CellMask plasma membrane stain and lipofectamine 2000 were bought from Thermo Fisher Scientific, Inc. (Pittsburgh, PA, USA). The commercial CRISPR/Cas9 plasmid (pX330, catalog: #42230) was purchased from Addgene (USA). The 1.2× HBV expression plasmid (pBB4.5–1.2×HBV) was kindly provided by Professor Ran Chen (Zhongshan School of Medicine, Sun Yat-sen University). The gRNA targeting HBV (5’-GACCGTGTGCACTTCGCTTC-3’) and PCR primers (5’-GGGTACCCAGCAGGTCTGGAGCAAA-3’ and 5’-GGAATTCGGAAAGAAGTCAGAAGGCAAA-3’) were purchased from Tsingke Biotechnology Co., Ltd (Beijing, China). The polyclonal anti-HBsAg antibody (catalog: #bs-1557G) was bought from Bioss Biotechlonogy Co., Ltd (Beijing, China). The Cy3-conjugated donkey anti-goat IgG H&L (catalog: #GB21404) was obtained from Servicebio Technology Co., Ltd (Wuhan, China). T7 Endonuclease I (T7E1) was bought from New England Biolabs, Inc. (Ipswich, MA, USA). The blood/cell/tissue DNA Isolation Kit was bought from Vazyme Biotech Co., Ltd. (Nanjing, China). SanPrep Column DNA Gel Extraction Kit was obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). The HBsAg, HBeAg, and HBV DNA ELISA kits were obtained from Shanghai Fusheng Industrial Co., Ltd. (Shanghai, China).

Cell line and animal experiments

The human cervical cancer cell line HeLa (catalog: #SCSP-504) was purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China) and cultured in DMEM with 10% FBS and 1% penicillin/streptomycin at 37 °C.

All animal experiments complied with Guidelines for Care and Use of Laboratory Animals of Sun Yat-sen University and approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (protocol number: SYSU-IACUC-2021-000714). Male C57/BL mice (3–5 weeks old) were obtained from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China). The mice were housed under SPF conditions and fed with standard food and water. The room was maintained at a controlled temperature of 25 °C and humidity of 30%-70% with a 12 h light/dark cycle.

Synthesis of membrane-fusogenic liposomes

The core of the plasmid/CaCO3 was constructed as follows. Solution 1 (50 μL) contained a mixture of CaCl2 solution (0.5 M, 16 μL), plasmid (1 μg μL−1, 1 μL), and sterile deionized water (33 μL). Solution 2 (50 μL) was obtained by mixing Na2CO3 solution (0.01 M, 16 μL) and sterile deionized water (34 μL). Next, solution 2 was pipetted gently to solution 1 to obtain a mixing solution (100 μL) with CaCO3 nanoparticles carrying plasmids.

The core-shell structured AFMFlip was prepared using the following film hydration/extrusion method. Briefly, DSPE-PEG, DOPC, and DOTAP were dissolved in chloroform and stored at 4 °C. DiD or DiR (5 mM) was mixed in the organic solvent for lipophilic dye incorporation. Then, lipid films of DSPE-PEG, DOPC, and DOTAP with specific molar ratios were prepared by evaporating the organic solvent and then drying in a vacuum desiccator overnight. Next, the lipid films were rehydrated with the solutions of plasmid-loaded CaCO3 nanoparticles. The resultant mixture solution was repetitively extruded under polycarbonate nanoporous membranes with a decreasing pore size (800, 400, and 200 nm). Similarly, traditional membrane-fusogenic liposomes (MFlips) are synthesized with the same processes but using different lipid components at different molar ratios according to the previous reports (Supplementary Fig. 11a). Finally, the solution containing the plasmid-loaded nanoparticles underwent purification through centrifugation at 21,000 × g for 50 min at 4 °C. Subsequently, the precipitates were collected. To determine the encapsulation efficiency of plasmids within nanoparticles, the concentration of unencapsulated free plasmid in the supernatant of the solution after centrifugation was quantified using a NanoDrop 2000 spectrophotometer (Wilmington, DE, USA). The encapsulation efficiency was calculated using the following formula: EE (%) = (CTCF)/CT × 100%, where EE represents the encapsulation efficiency, CT is the total concentration of plasmids, and CF denotes the concentration of unencapsulated, free plasmids in the supernatant.

Physicochemical characterization

Nanoparticle size and zeta potential were measured by a dynamic light scattering (DLS) system with a Litesizer 500 particle analyzer (Anton Paar, Austria) at room temperature. The images of morphology were acquired by transmission electron microscopy (FEI Tecnai G2 F30, Philips-FEI) at a 120 kV accelerating voltage. Freshly prepared samples were dropped onto the 200-mesh carbon-coated copper grid. The excess solvent was dried off after 10 min, and then 5 μL of phosphotungstic acid was applied for negative staining.

Membrane fusion effect observation

For the optimization of stable membrane fusion in the FBS-containing medium, the various DiD-labeled MFlips consisting of DOPC, DSPE-PEG, and DOTAP were prepared with different molar ratios (DSPE-PEG/DOPC/DOTAP = 5/95/0, 5/90/5, 5/85/10, 5/75/20, 5/65/30). HeLa cells in 2 mL of DMEM culture medium were seeded in a 6-well plate at a density of 2 × 105 per well and incubated at 37 °C for 24 h. The medium was then replaced with 1 mL of DMEM solution containing various nanoparticles. After 1-h co-incubation, cells were washed carefully with PBS, and the plasma membranes were labeled with CellMask. Then, DAPI was used to stain the cell nuclei for 10 min, followed by another careful PBS washing. After that, cells were observed by CLSM imaging (Leica STELLARIS STED).

The DiD-labeled AFMFlip (DSPE-PEG/DOPC/DOTAP/DiD = 5/90/5/5), DiD-labeled MFlip-a (DSPE-PEG/DMPC/DOTAP/DiD = 3.8/76.2/20.0/5.0), and DiD-labeled MFlip-b (DSPE-PEG/DOPE/CHEMS/DiD = 12.3/69.5/18.2/5.0) were then constructed. The plasmid cargos were labeled with YOYO-1 following the manufacturer’s instructions. HeLa cells in 2 mL of DMEM culture medium were seeded in a six-well plate at a density of 2 × 105 cells per well and incubated at 37 °C for 24 h. The medium was then replaced with 1 mL of DMEM solution containing various formulations (2000 ng mL−1 plasmid). After 1-h co-incubation, cells were washed carefully with PBS, and the plasma membranes were labeled with CellMask. Then, DAPI was used to stain the cell nuclei for 10 min, followed by another careful PBS washing. After that, cells were observed by CLSM imaging.

FRET-based membrane-fusogenic quantitative assay

The liposomes were labeled with both DiO and DiI, which can exhibit the FRET effect. When these liposomes fused with cell membranes, the increased distance significantly reduced FRET efficiency35,36. The prepared liposomes were added to HeLa cells at a density of 2 × 106 cells per mL without or with 10% FBS for 1 h at 37 °C. The initial FRET effect of the liposome (excitation using Ex (DiO) = 483 nm, but emission at Em (DiI) = 565 nm) was measured as 0% fusion using a Tecan Spark spectrofluorometer. The spectrum of 100% fusion was determined after adding Triton X-100 at 0.1% (v/v). After incubation with cells in the presence/absence of 10% FBS for 1 h at 37 °C, the fluorescence spectra of liposome/cell mixtures were recorded to determine the membrane-fusion performance using the formula: Fusion (%) = (FDiOF0)/(F100F0).

To determine the quantitative membrane-fusion efficiency of AFMFlip, MFlip-a, and MFlip-b at different time intervals, liposomes labeled with DiO and DiI were added into the cells in a 96-well plate in the presence/absence of 10% FBS with different incubation times at 37 °C. The fluorescence intensity of DiO was measured at an excitation of 483 nm and an emission of 501 nm using a microplate reader (BioTek Synergy).

Analysis of protein corona on MFlips

The solution containing 20% (v/v) FBS or mouse serum was added to the equivalent solution containing AFMFlip, MFlip-a, or MFlip-b. Then, the mixture was incubated with shaking at 100 × g for 1 h at 37 °C. To remove unbound serum proteins, the liposomes were pelleted by centrifugation at 21,000 × g for 50 min at 4 °C. After that, the collected precipitates were washed carefully with sterile deionized water, followed by measurements of particle sizes and zeta potentials at room temperature.

Following the manufacturer’s instructions, these precipitates dispersed in PBS were detected by the BCA to calculate protein concentrations. Generally, the standard curve was performed by a gradient concentration dilution to a protein standard sample, starting from 1 μg/mL. The reagents A and B at a 50:1 mixed ratio were added with 200 μL per well in the 96-well plate. The standard or unknown samples were dispensed with 20 μL of each well, followed by a 30-min incubation at 37 °C. After that, the microplate reader (BioTek Synergy) was used to measure the absorbance at 574 nm.

For the SDS-PAGE analysis, the samples were resuspended in the SDS-PAGE loading buffer, followed by 10-min sonication. After that, the solutions were boiled for five min, and then they were loaded in 4–20% Tris-Gly Plus Precast PAGE Gel (Beyotime). The gel was started by running at 80 mV for 5 min, then at 120 mV until the loading buffer band reached the end of the gel. The dying process with EZ Blue Gel was carried out overnight at room temperature; subsequently, samples were washed with distilled water for 2 h.

For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the proteins were separated with 12.5% SDS-PAGE (constant current 14 mA, 90 min) and then digested in-gel with trypsin. The peptides were analyzed by a Q Exactive mass spectrometer (Thermo Scientific) that was coupled to Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific) for 60/120/240 min. The spectra were under analysis via the MaxQuant engine. The mass tolerance was twenty ppm. In the meantime, MS/MS tolerance was 0.1 Da. The UniProt database was searched for protein identification.

Membrane-fusion mechanism investigation

In 500 μL of DMEM culture medium, HeLa cells were seeded in a 24-well plate at a density of 5 × 104 cells per well and incubated at 37 °C for 24 h. Z-Phe-Phe-Phe-OH (a membrane-fusion transportation mediator), chlorpromazine (an inhibitor of clathrin-mediated endocytosis), amiloride (an inhibitor of macropinocytosis), and nystatin (an inhibitor of caveolae-mediated endocytosis) were added separately to the medium at their recommended concentrations (100 μg mL−1, 10 μg mL−1, 100 μg mL−1, and 15 μg mL−1, respectively). After the pretreatments of different inhibitors for 30 min, these solutions were replaced with DMEM solutions containing various MFlip formulations with the plasmid concentration of 2000 ng mL−1 in different FBS concentrations. After incubation for two hours, the cells were removed with the medium followed by careful PBS washing, collected, and then analyzed by flow cytometry using a BD FACS Calibur flow cytometer (BD Biosciences) equipped with a 488 nm laser and a 525/40 nm filter to obtain the mean fluorescence intensity (MFI) from YOYO-1-labeled plasmids.

Cell uptake and intracellular distribution analysis

Before the experiments, HeLa cells in 2 mL of DMEM culture medium were seeded in a 6-well plate at a density of 2 × 105 cells per well and incubated at 37 °C for 24 h. The medium was then replaced with 1 mL of DMEM solution containing various formulations (2 μg mL−1 plasmid) in different concentrations of FBS, in which the plasmid cargos were labeled with YOYO-1 following the manufacturer’s instructions. After staining for two hours, the cells were removed with the medium, followed by careful PBS washing, collected, and then analyzed by flow cytometry to obtain the MFI.

For intracellular distribution analysis, after co-incubation for 1 h, the cells were washed three times with PBS. Then, the cells were stained with LysoTracker Red for 30 min at 37 °C. Then the nuclei were stained with DAPI for 10 min. After three PBS washes, the cells were imaged by CLSM.

Effects of FBS concentration on gene transfection of AFMFlips

For EGFP gene transfection, HeLa cells were seeded in a 24-well plate at a density of 5 × 104 cells per well and incubated at 37 °C for 24 h. The medium was then replaced with 1 mL of DMEM solution containing AFMFlippEGFP (2 μg mL−1 plasmid) in a variety of FBS concentrations (0%, 10%, 20%, 30%, 40%, and 50%). After 48-h transfection, the cells were removed with the medium followed by careful PBS washing, then either collected for EGFP-positive cell analysis by the flow cytometer equipped with a 488 nm laser and a 525/40 nm filter, or the cells were applied to a 10-min DAPI staining for imaging by an inverted fluorescence microscope (Nikon Ti2-U).

In vivo investigation of the anti-protein-adsorption capacity of MFlips

DiR-AFMFlipYOYO-1, DiR-MFlip-aYOYO-1, and DiR-MFlip-bYOYO-1 were intravenously administrated into the C57BL/6 mice (male, 3–5 weeks). After injection for 1 h and 3 h, the nanoparticles were recovered by collecting nearly 500 μL blood samples in tubes through cardiac puncture. After striation at 4 °C overnight, the samples were centrifugated for ten min at 1500 × g at 4 °C, followed by the supernatant collection of plasma.

Subsequently, size exclusion chromatography was performed to separate excess plasma proteins from the nanoparticles of DiR-AFMFlipYOYO-1, DiR-MFlip-aYOYO-1, and DiR-MFlip-bYOYO-1. Generally, each of the above-collected plasma was loaded onto a Sepharose CL-4B column with PBS equilibration. Then, 30 chromatographic fractions (1 mL per fraction) were collected. Each chromatographic fraction was analyzed by DLS and Stewart assay. To quantify lipid concentration using the Stewart assay, each chromatographic fraction that might contain nanoparticles was under a mixture with both chloroform solution and ammonium ferrothiocyanate for 1 min. Then, after centrifugation at 20,000 × g for 1 min, the partitions from the chloroform phase were collected to measure their absorbance values at 485 nm on a UV-2600 UV-Vis Spectrophotometer (Shimadzu, MD, USA).

Accordingly, membrane ultrafiltration was performed for nanoparticle purification and collection. The above fractions containing nanoparticles were collected together and then concentrated to nearly 500 μL by centrifugation at 9600 × g using protein concentrator spin columns (10000 MWCO). The collected samples above were detected by BCA and SDS-PAGE analysis as described above in “Analysis of protein corona on MFlips”. Besides, nanoparticle-corona was further observed by TEM using the aforementioned protocol.

In vivo intracellular transportation pathway observation

DiR-AFMFlipYOYO-1, DiR-MFlip-aYOYO-1, and DiR-MFlip-bYOYO-1 were intravenously administrated into the C57BL/6 mice (male, 3–5 weeks). After injection for 1 h and 3 h, the mice were sacrificed. The liver sections were harvested for freezing sectioning and CLSM observation. Phalloidin was used to label the cell membranes for 40 min10. After that, DAPI was used to stain the nuclei for ten min. After three times of careful PBS washing, the sections were imaged by CLSM.

In vivo biodistribution study

AFMFlip, MFlip-a, and MFlip-b were labeled with DiR dye, followed by intravenous administration into the C57BL/6 mice (male, 3–5 weeks). In vivo imaging was performed using the VISQUE Invivo Smart-LF system at 1, 2, 3, 5, and 8 h post injection. Furthermore, 3 and 8 h after injection, the mice were sacrificed to collect major organs (heart, liver, spleen, lung, and kidney) and blood samples for ex vivo imaging analysis.

In vitro luciferase gene delivery by MFlips

For luciferase gene transfection, pCDNA3.1-Luc encoding luciferase was used as a reporter plasmid. HeLa cells were seeded in a 24-well plate at a density of 5 × 104 cells per well and incubated at 37 °C for 24 h. The medium was then replaced with 1 mL DMEM solution containing AFMFlippLuc, and MFlip-bpLuc (2 μg mL−1 plasmid) with or without 10% FBS. After transfection for 24 h, 48 h, and 72 h, the cells were under careful PBS washing, followed by cell lysis to collect proteins to measure the luciferase gene expression level. The total protein contents from the lysis solution were measured by BCA. After adding D-Luciferin luciferase substrate for 10 min, the luciferase expression with emitted light was detected using BioTek (SYNERGY H1MF) plate reader as the relative light unit per mg protein.

In vivo luciferase delivery using MFlips

AFMFlippLuc, MFlip-apLuc, and MFlip-bpLuc were intravenously administrated into the C57BL/6 mice (male, 3–5 weeks). After injection for 48 h, the mice were under intraperitoneal injection with luciferase substrate for in vivo bioluminescence imaging. Immediately following that, the mice were sacrificed, and the heart, liver, spleen, lung, and kidney were resected from each mouse for ex vivo bioluminescence imaging.

AFMFlip-based nanomedicine for HBV infection treatment in mice

To build HBV-replication models, C57BL/6 mice (male, 3–5 weeks) received a hydrodynamic tail vein injection with 6 μg of pBB4.5–1.2× HBV in PBS solution per mouse48,49. After 24 h, PBS solution, AFMFlippCas9-gHBV solution (containing 6 μg pCas9-gHBV), MFlip-apCas9-gHBV solution (containing 6 μg pCas9-gHBV), and MFlip-bpCas9-gHBV solution (containing 6 μg pCas9-gHBV) were injected into different mouse groups via the tail vein according to the scheme (Fig. 6a). The mouse models were then under sacrification at the endpoint of treatments. The blood samples and major organs of mice were collected and harvested for further biochemistry analysis, immunofluorescent staining, and viral load quantification. The serum samples were diluted in PBS to measure serum HBsAg, HBeAg, and HBV DNA using ELISA kits. For immunofluorescent staining of liver sections, the goat anti-HBsAg antibody (bs-1557G) was used at a dilution of 1:200, followed by the incubation and staining of Cy3-conjugated donkey anti-goat IgG H&L (catalog: #GB21404) at a dilution of 1:200. The results were visualized using an inverted fluorescence microscope (Nikon Ti2-U). The collected mouse liver tissues were lysed on ice, and the supernatant was diluted with PBS to measure HBsAg, HBeAg, and HBV DNA using ELISA kits.

DNA extraction kits were applied to purify DNA from the liver tissues. DNA fragment with CRISPR/Cas9 target location was PCR amplified using target-specific primers and purified by Tiangen gel extraction kit (Tiangen). Direct sequencing by Thermo Scientific 3730xl DNA Analyzer as well as T7E1 analysis were conducted after acquiring the amplified PCR products.

In vivo biosafety evaluation

In vivo biosafety evaluation was performed by weight monitoring, blood biochemical test, and the H&E staining of tissues. After the HBV mouse models were sacrificed, serum levels of ALB, ALT, AST, ALP, BUN, CK, CREA, and LDH were determined using their respective activity assay kits with provided protocols. The major organs, including the heart, liver, spleen, lung, and kidney, were also collected for H&E staining to assess the biosafety of the nanoparticles.

Statistical analysis

All the data displayed represent the results from multiple independent experiments, presented as mean ± SD unless otherwise mentioned. GraphPad Prism (GraphPad Software) was used for all of the calculations. Data comparisons were performed with two-tailed t test and one-way analysis of variance. A difference of p < 0.05 was considered statistically significant; ns: not significant (p > 0.05).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.