Virus and cell culture
Mouse Cytomegalovirus (MCMV) provides a valuable model for viral pathology since it is closely related to human CMV. MCMV (MCMV-GFP, Smith strain) was kindly provided by Dr. Minhua Luo from the Wuhan Institute of Virology, China. NIH 3T3 and MLE12 cell lines were cultured in DMEM and DMEM/F12, supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 1% Pen-Strep (100 U/ml penicillin, 100 μg/ml streptomycin) and 1% glutamine, respectively.
MCMV was grown and purified on NIH 3T3 cells. Briefly, NIH 3T3 cells were seeded in 15-cm dishes in complete DMEM and were infected with MCMV at a multiplicity of infection (MOI) of 0.1 for 3–4 days. The culture supernatants were then passed through a 0.45 μm filter, and virus particles were pelleted by ultracentrifugation through a 30% sucrose cushion (25,000 rpm for 3 h at 4 °C, Beckman SW32Ti rotor). Then, aliquots of the virus were kept at − 80 °C, and viral titers were measured by plaque assay in NIH 3T3 cells. Ten-fold serial dilutions of MCMV were added to NIH 3T3 cells of overnight culture for 3–6 h. Cell medium was removed and replaced with overlay medium supplemented with agarose, and the cells were further cultured at 37 °C for 4–5 days. And plaque-forming unit (PFU) was counted, which were identified by GFP expression using a Leica DMi8 microscope (Leica, Wetzlar, Germany).
Isolation, culture, and identification of mBMSC
MSCs were isolated from the bone marrow of 4 ~ 6-week C57BL/6 mice under aseptic conditions. Briefly, bone marrow cells were separated and collected by flushing the bone marrow cavity from femurs and tibiae of mice. After red blood cells were lysed, the remaining cells were resuspended and cultured in L-DMEM complete medium containing 10% FBS at 37 °C and 5% CO2 incubator for 8–10 days. MSCs of passage 5 were detected by FACS for MSC surface antigens CD29 (eBioscience, 17-0291-80), CD44 (eBioscience, 12-0441-81), CD105 (eBioscience, 12-1051-81), SCA-1 (eBioscience, 45-5981-82), hematopoietic stem cells and endothelial cell markers CD45 (BD Pharmingen, 550994), CD31 (BD Pharmingen, 553372), CD34 (BD Pharmingen, 560230) and their isotypes. These cells were confirmed as MSCs by validating that they could differentiate into osteoblasts and adipocytes. All of the mBMSCs used in this study were collected in passages 3 to 8.
Preparation of antiviral-drug-loaded nanoparticles (PDGP)
PLGA/DOTAP nanoparticles loaded with antiviral drugs were synthesized by a double emulsification process, as previously reported . In short, 2 mg of GCV (Macklin, China) and 2 mg of PFA (Macklin, China) were dissolved in 500 μL of sterile normal saline at the weight ratio of 1:1, and the solution was added dropwise to 1 mL of the methylene chloride solution containing 20 mg of PLGA (Sigma-Aldrich, USA) and different mass of DOTAP (Alabama, USA) (1, 3, 6, 9, 12, and 15 mg) during a probe sonicator (60 W, 2 s on,1 s off). Through the second sonication of 60 s, the mixture was quickly added to 2 mL of 2% PVA solution. The obtained product was added to 10 mL of 2% PVA solution and stirred for 5 h at room temperature to evaporate the organic solvent, and then centrifuged at 12,000 rpm for 30 min. The pellet was washed thrice and suspended in sterile normal saline.
Fabrication of MPDGP
The mBMSCs were used to extract the cell membranes. In short, the mBMSCs were treated with 0.25% Trypsin–EDTA Solution (Life Technologies, USA) and collected. The cell membranes were extracted by the Membrane and Cytosol Protein Extraction Kit (Beyotime, China) according to the Manufacturer’s introduction. Specifically, the cells were scraped from the culture dish and centrifuged at 2000 rpm for 5 min to precipitate the cells, and then the supernatant was removed. The membrane protein extraction reagent A was added to resuspend the cells, and the cells were repeatedly freeze-thawed three times at liquid nitrogen and room temperature, followed by the centrifugation at 4 °C for 10 min at 700g to precipitate the nuclei and unbroken cells, the supernatant was collected into a new centrifuge tube, and the supernatant was centrifuged at 4 °C for 30 min at 14,000g to collect cell membranes.
PDGP was mixed with the mBMSCs membrane at a weight ratio of 1:1, according to our previous work [17, 18]. The mixture was sonicated for 5 min and extruded back and forth 15 times with a LiposoFast-Basic extruder (Avestin Inc., Ottawa, Canada) to obtain MPDGP.
mBMSC membrane protein validation
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to characterize membrane proteins. In short, MPDGP was subjected to radioimmunoprecipitation analysis (RIPA) (Beyotime, China) with control of total mBMSC protein, mBMSC membrane, and MPDGP. The protein concentration was measured using the Pierce BCA protein assay kit (Life Technologies). All samples diluted with SDS-PAGE loading buffer (Invitrogen, USA) were boiled at 100 ℃ for 5 min. Then, samples of the same protein mass (30 μg/well) were resolved onto a 10% SDS-PAGE gel (Beyotime, China) by electrophoresis. The resulting gel was stained in Coomassie blue, washed, and then imaged with the Amersham Imager 600 system (GE Healthcare Life Sciences, USA).
Size, zeta potential, and morphology
Prepare PDGP and MPDGP suspension in ultrapure water, respectively. The average particle size and polydispersity index (PDI) of PDGP and MPDGP were measured by Zetasizer (Nano ZS, Malvern). PDGP and MPDGP were stained with 1% uranyl acetate (JEM-2000FX, Hitachi) and then observed under a transmission electron microscope (TEM).
In vitro release studies
MPDGP filled with DiD (Solarbio, China) and FITC (Beyotime, China) was used for this purpose. An aliquot of 2 mL of MPDGP was loaded into a dialysate tube (Molecular weight cutoff, 3500 Da) and immersed in 20 mL of dialysate with a given pH (pH 7.4 or 5.0). After incubation at room temperature for different time points (1, 2, 4, 6, 8, 10, 12, 24, 48, and 72 h), an aliquot of 10 mL dialysate was taken and added with 10 mL fresh dialysate. The ultraviolet intensity of the collected dialysate, which indicates the release of FITC and DiD from MPDGP was measured with an ultraviolet spectrophotometer.
To study the cellular uptake, the fluorescence dyes, FITC and DiD were loaded into the PLGA/DOTAP core. MLE-12 cells were seeded into confocal culture dishes at a density of 1 × 105 cells per well. When the cells grew to about 80% confluence, they were pretreated with TNF-α (Peprotech, USA, 10 ng/mL) and IFN-γ (Peprotech, USA, 10 ng/mL) for 24 h. The cells were washed and incubated in a freshly prepared Opti-mem medium containing MPD/DiD/FITC. The dosage of DiD or FITC was equivalent to 0.1% of the biomimetic nanoparticles, respectively. After a specified incubation time, the medium was discarded, and the cells were gently washed three times with PBS. The cell nuclei were stained with Hoechst and then washed with PBS repeatedly. Cell imaging was performed under a confocal laser scanning microscope (LSM 880, Zeiss). For FACS analysis, the adherent cells were separated from the culture plate with 0.25% trypsin–EDTA solution (Life Technologies, USA) and suspended in 50 μL PBS. The cell suspension was analyzed by FACS (ImageStreamX Imaging, Amnis Corporation, USA). In addition, cell uptake of MSCNP (80 μg/mL) overtime (4, 8, 12, and 16 h) was also performed as above.
FITC and DiD are loaded into the MPD to assess lysosomal escape. MLE-12 cells were pretreated with TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) for 24 h. The MLE-12 cells were incubated with MSCM/PLGA/DiD/FITC or MPD/DiD/FITC for 4 h and 12 h. Next, the cells were washed three times and stained with LysoBlue (KeyGEN BioTECH, China) for 60 min. The cells were washed with PBS and imaged under CLSM (LSM880, Zeiss, Germany).
In vitro cytotoxicity study
The vitality/cytotoxicity kit (Beyotime, China) was used to test the in vitro cytotoxicity of the nanoparticles. Briefly, MLE-12 cells were seeded into confocal dishes for a density of 1 × 105/well. To evaluate the cytotoxicity of MPDGP, when the cells grew to 80% confluence, the medium was replaced with a fresh medium containing MPDGP, which included an antiviral drug in indicated concentration. Cells treated with PBS or 75% EtOH were used as negative or positive controls, respectively. After incubation for 48 h, cells were stained with a Viability/ Cytotoxicity kit and imaged under CLSM (LSM880, Zeiss, Germany).
In vitro inflammation targeting
MLE-12 cells in confocal culture dishes at about 80% confluence were washed and incubated in DEME/F12 medium containing 10 ng/mL TNF-α and INF-γ for 24 h to manufacture an in vitro inflammation model. Then, after incubating with MPDGP and loaded with DiD and FITC, respectively, for a specified time. Samples were analyzed under CLSM and by FACS, as mentioned above.
Viral inhibition in vitro
MLE-12 cells were cultured in complete DMEM/F12, which contained 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Add the virus to MLE-12 cells (MOI = 0.1) to simulate cell infection, then add different concentrations of MPDGP and incubate for a certain time. Observe the fluorescence intensity of GFP carried by the virus and detect the expression of MIE1 (MCMV immediate-early gene 1) mRNA by quantitative real-time polymerase chain reaction (qPCR) to determine the inhibitory virus effect.
RNA isolation, cDNA preparation, and qPCR
Total RNA was extracted using TRIzol reagent (Invitrogen), and complementary DNA (cDNA) synthesis was performed using PrimeScript RT Master Mix (Perfect Real Time) (Takara, Japan). The target gene was amplified by SYBR Green qPCR (Takara RR820L). Normalized analysis of mRNA expression in vivo and in vitro using the 2 –∆ΔCt method against GAPDH. The primers sequence used are:
Sense: 5’- TGCACCACCAACTGCTTAGC-3’.
MCMV pneumonia model
All animals were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). We raised the animals in a specific pathogen-free (SPF) environment. All in vivo studies were performed in accordance with the Institutional Authority for Laboratory Animal Care of Guangzhou Medical University. MCMV pneumonia model was created in 4 ~ 6-weeks-old BALB/c-nu female mice. Briefly, after general anesthesia, Mice were infected with 2 × 105 PFU of MCMV in 40 μL saline through intratracheal administration. Then the mice were randomized grouped to receive one of the following treatments: (1) Saline group: MCMV pneumonia + tail vein injection of 200 μL saline to MCMV pneumonia mice; (2) MPDG group: MCMV pneumonia + tail vein injection of 200 μL MPDG; (3) MPDP group: MCMV pneumonia + tail vein injection of 200 μL MPDP; (4) GP group: MCMV Pneumonia + tail vein injection 200 μL GP; (5) PDGP group: MCMV pneumonia + tail vein injection 200 μL PDGP; (6) MPDGP Group: MCMV pneumonia + tail vein injection of 200 μL of MPDGP. Mice of all groups also received GCV or PFA (5 mg/kg), respectively.
Viral inhibition in vivo and cytokine determination
After treatment, the lung tissue of each mouse was taken for determination of viral MIE1 mRNA via qPCR and expression of viral GFP protein via Western Blotting. The expression of cytokines TNF-α and IL-6 were also accessed via qPCR and ELISA (Neobioscience, China). The primers sequence used are:
Biodistribution in vivo
To detect the distribution in vivo, MPD/DiR was injected into the tail vein (DiR is equivalent to 0.1% weight of the MPD) and was tracked by the IVIS Lumina XRMS Series III (PerkinElmer, USA) at time points of 1, 6, 12, 24 and 48 h. The mice were sacrificed, and organs were imaged. The fluorescence was analyzed with Living Image V4.5.5 software.
Histological analysis and immunofluorescence staining
Tissues were fixed in 10% buffered formalin and embedded in paraffin. The tissue sections were obtained from lung tissue and stained with hematoxylin–eosin (H&E). For immunofluorescence staining, the sections were rehydrated and washed in PBS, pretreated for 1 h at room temperature with protein block solution (Dako, Carpinteria, CA). After incubation with CD68 (Servicebio, GB14043, 1:200) and LY-6G (Servicebio, GB11229, 1:200) overnight at 4 °C, sections were washed and incubated with fluorescence-labeled secondary antibodies. After the nucleus was stained with Hoechst (Invitrogen, USA), samples were examined under the microscope.