2.1 Materials
Polyvinyl alcohol (PVA) was purchased from J&K Science Co., Ltd. (Beijing, China) and dichloromethane (DCM) was purchased from Sinopretics Co., Ltd. (Shanghai, China). The pEGFP-n1 plasmid expressing enhanced green fluorescent protein was purchased from BD Biosciences Clontech (Palo Alto, CA, USA). Porcine kidney-15 (PK15) cells were purchased from the National Science and Technology Laboratory Cell Resource Platform (Beijing, China). 24-well plates, 96-well plates, Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from Gibco (Darmstadt, Germany).
2.2 Methods
2.2.1 Preparation of PLA microspheres
PLA microspheres were prepared by a combination of emulsion polymerization technology and an emulsion-solvent evaporation method, and the optimum shear rate and oil ratio of the PLA microspheres were investigated. To form the aqueous phase, 1 g PVA was added to 100 ml distilled water and stirred with a magnetic agitator at 500 rpm and 80 °C until completely dissolved. To prepare the oil phase, PLA (15 mg/ml) was completely dissolved in DCM using ultrasonication. Using a shear rate of 8000 or 10000 rpm, the oil phase was added into the water phase drop by drop at a ratio of oil phase: water phase = 1:3 or 1:5. After the addition of all the oil, the oil phase was continuously cut for 3 min. Then, the rotation speed of the shear machine was adjusted to 3000 rpm, and the oil was cut and mixed evenly to obtain a water-in-oil emulsion. The emulsion was ultrasonicated for 5 min and then the methylene chloride was volatilized overnight with a magnetic agitator at 500 rpm. The obtained PLA microspheres were dissolved in water and centrifuged at 10000 rpm for 10 min. The supernatant was discarded, and the PLA microspheres were obtained after freeze drying.
2.2.2 Synthesis of PLA/PEI microspheres
An appropriate amount of PEI polymers with molecular weights of 800, 10000, and 25000 were added into distilled water (1% solutions). The PEI polymers were added to the PLA microspheres at a PLA:PEI mass ratio = 1:3 and the mixture was stirred at 500 rpm for 4 h at room temperature, then, the solution was centrifuged at 8000 rpm for 10 min and the supernatant was discarded. Distilled water was added and the PLA/PEI microspheres were dissolved by ultrasonication, the solution was centrifuged at 8000 rpm for 10 min and after three or four repetitions of this washing process, the deposits were collected.
2.2.3 PLA/PEI microspheres loading DNA
Plasmid DNA containing green fluorescent protein (GFP) gene was dissolved in in DMEM solution and stirred evenly. Then, PLA/PEI microspheres were added at a mass ratio of PLA/PEI microspheres: plasmid DNA = 2:1 and the mixture was mixed well by oscillation. The mixtures were incubated at 37 °C for later use.
2.2.4 Particle size and Zeta potential measurements
The particle size and zeta potential were measured by the laser particle size analyzer (Malvern ZetasizerNano ZS, UK). At room temperature, the PLA microspheres and the PLA/PEI microspheres were diluted to 1 µg/mL in an aqueous solution. The average value of three measurements was used for the end result.
2.2.5 Morphology characterizations
The prepared PLA and PLA/PEI microspheres were dissolved with an appropriate amount of water, a drop of the solution was placed on a silicon wafer with a pipette. After completely dried at room temperature, they were observed under a scanning electron microscope (SEM, Hitachi SU8010 Japan). The PLA/PEI microspheres loaded with DNA were dissolved in water. Drop an appropriate amount of solution on the mica sheet. After air drying for 10 minutes, gently rinse the mica sheet with ultra-pure water, and then put it into the mica sheet until it is fully dried. Record the results at the scanning speed of 0.99 Hz under an atomic force microscope (AFM, Bruker Multimode Ⅷ, USA).
2.2.6 Agarose gel electrophoresis
Agarose was placed in a conical flask, then electrophoresis buffer was added to prepare an agarose gel with a mass fraction of 1%. The conical flask was rotated, and the solution was mixed well. After heating and dissolving, ethidium bromide, a nucleic acid stain, was added. When the temperature had dropped slightly, the gel was poured into the groove and allowed to set.
A series of PLA/PEI microspheres and plasmid DNA composite solutions were prepared with mass ratios of PLA/PEI microspheres: plasmid DNA = 2:1, 1:1, 1:2, 1:4, 1:6, and 1:8. 30 µl solution was taken into the sample tank. Electrophoresis was performed for 40 min at 100 V.
2.2.7 Cell transfection experiments
PK15 cells were cultured at 37 °C, 5% CO2, and a particular humidity in DMEM medium supplemented with 10% FBS and 1% antibiotics. The medium was changed after culturing for 48 h. An appropriate amount of DEME solution containing 10% FBS was added to a 24-well cell culture plate. Approximately 105 porcine kidney cells were inoculated into each well and cultured until the fusion rate reached 70%. The old culture medium was discarded, and the cells were rinsed once with PBS buffer. The medium was replaced with a medium that did not contain fetal bovine serum. Then, 200 µl of PLA/PEI/DNA complexes solution were added to each well. After a period of incubation at 37 ℃ and 5% CO2, the expression of GFP was observed under a fluorescence microscope (Olympus IX70, Japan).
2.2.8 Cytotoxicity test
The cytotoxicity of the microspheres was evaluated by the 3-(4, 5- dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. PK15 cells were seeded into a 96-well plate at a concentration of 104 cells/well and cultured at 37 °C and 5% CO2 for 24 h. Then, the PK15 cells were treated with PLA/PEI/DNA complexes and PLA/PEI microspheres. Untreated PK15 cells were used as a control. After incubation for 6 h, the medium was removed. The PK15 cells were cultured at 37 °C and 5% CO2 for 24 h, and were cultured for another 4 h in DMEM containing MTT (0.5%), then the medium was carefully removed. Dimethyl sulfoxide (150 µl/well) was added and the solution was oscillated gently to dissolve the crystals. The absorbance at 490 nm was measured using a microplate reader. The cell viability was expressed as [OD490(sample)/OD490(control)] × 100.
2.2.9 Animal experiments
The DNA vaccine against foot-and-mouth disease (FMD), experimental animals (guinea pigs) and test sites were provided by the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The guinea pigs were monitored to be pathogen-free and all animals were treated according to the regulations of Chinese law and the local ethical committee. Guinea pigs were given regular food and water to drink.
The study consisted of three groups with eight animals in each group: guinea pigs treated with PBS; guinea pigs treated with foot-and-mouth disease viral DNA; and guinea pigs treated with the PLA /PEI/DNA complexes. The weight of the guinea pigs was controlled to be approximately 300 g. Each guinea pig was given 200 µl of the vaccine, which contained 100 µg of DNA. Twenty-eight days after immunization, each guinea pig was injected with 200 µl of foot-and-mouth disease virus and the protection rate for each group was observed.