Development Of Thermosensitive Injectable Hydrogel And Its Application As NDV DNA Vaccine Delivery System

Thermosensitive hydrogel is liquid at room temperature,forming gel when temperature increases.This feature makes it injectable at room temperature and be able to control the drug release in vivo,extending the half-life and improving the bioavailability of drugs.There are lots of advantages of hydrogel,such as simple preparation process,convenient storage,high drug loading,controllable gelation condition,which make it a promising,stable and efficient vaccine delivery system to realize the long-time release,improve the stability,enhance the immune effects,reduce the vaccination times and prolong the immune protection of vaccines.In this study,PEO-PPO-PEO and PLGA-PEG-PLGA polymers were selected as the candidate materials,while the optimal prescriptions were screened out and characterized.Then the gel dissolution and drug release of the drug-loaded gels with the drug model(OVA)in vitro were investigated and the pharmacodynamics was evaluated.Based on the above research,in order to evaluate the feasibility of the thermosensitive hydrogels as DNA vaccine delivery system,NDV gel vaccine was prepared with Newcastle disease as a disease model and its characters,release behavior in vitro and security were investigated.Finally,the immune effects of NDV gel vaccine and the molecular mechanism of the immune enhancement effects were preliminarily explored.The main contents and results are as follows:1.Preparation and characterization of thermosensitive injectable hydrogelsThe thermosensitive hydrogels of different prescriptions were prepared with PEO-PPOPEO and PLGA-PEG-PLGA block copolymer,respectively.The optimal prescription of each copolymer was screened out with Tm as the main evaluation index and the gelation temperature(Tm),gelation time(Gt),pH were characterized.Meanwhile,the appearance,viscosity,microstructure,micelle size,Zeta potential and stability were evaluated.The results were as follows.The optimal prescription of PEO-PPO-PEO thermosensitive hydrogel is made up of P407 of 17%,P188 of 5%and HPMC of 0.4%,with Tm of 35.6?,Gt of 47.72 s,pH of 7.18,viscosity of 54 mPa s,micellar particle size of 70.66 nm and zeta potential of 1.30 mV.The polymer solution is colorless transparent liquid with good liquidity at 25?,while it presents a semi-solid gel state when T>Tm.PLGA-PEG-PLGA block copolymer was synthesized by open-loop polymerization and the purified polymer was prepared with distilled water at different concentrations(10%?30%,w/w).The optimal PLGA-PEGPLGA thennosensitive hydrogel is at the concentration of 20%with Tm of 35.3?,Gt of 64.66 s,pH of 6.47,viscosity of 50 mPa·s,micellar particle size of 26.02 nn and zeta potential of-0.16 mV.The polymer solution is a light blue transparent liquid at room temperature,while it presents a white semi-solid gel state when the temperature is higher than 35.3?.The two thermosensitive hydrogels have simple preparation methods,good liquidity at low temperature,rapid gelation at phase-transition temperature and high stability,which are expected to become promising injectable vaccine delivery system to control the release and improve the release kinetics of biological macromolecular(e.g.,proteins,peptides)and other treatment components(e.g.,vaccine,antibody).2.Pharmacokinetic and pharmacodynamics of drug-loaded thermosensitive gelsIn this section,ovalbumin(OVA)was used as a model drug and enveloped into PEOPPO-PEO and PLGA-PEG-PLGA,respectively.The PEO-PPO-PEO gel with OVA of 2.0 mg·mL-1 has Tm of 33.8?.Gt of 40.17 s,pH of 7.21,particle size of 89.09 nm,multiple dispersion index(PDI)of 0.372 and zeta potential of 4.11 mV;The PLGA-PEG-PLGA drugloaded gel has Tm of 35.3?,Gt of 64.04 s,pH of 6.50,particle size of 36.84 nm PDI of 0.472 and zeta potential of 5.73 mV.It was observed by scanning electron microscopy(SEM)that a three-dimensional network structure with honeycombed appearance in the two drugloaded gels(pore size 100?200 ?m),in which OVA was evenly distributed.After that,the release behavior in vitro of the drug-loaded gels was investigated by membrane-free dissolution method.The results showed that OVA was released from PEO-PPO-PEO for 6 days with a cumulative release rate of 94.06%.The release process followed the HixsonCrowell kinetic model,indicating gel dissolution was the main factor controlling the release of OVA.It taked 28 days to release OVA of 94.42%from PLGA-PEG-PLGA.The release followed the Ritger-Peppas kinetic equation,showing that the release mechanism was the synergistic effect of drug diffusion and gel dissolution.The structure of OVA remained stable during the preparation and in vitro release processe of the two drug-loaded gels detected by SDS-PAGE.In order to assess whether the drug can be truly continuously released from these two hydrogels and maintain its biological activity in animals,the Balb/c mice aged 6 weeks were administered by subcutaneous injection with the two drug-loaded gels,respectively.The pharmacodynamics of OVA as antigen model in mice was evaluated.The results revealed that the levels of IgG antibody titer,lymphocyte proliferation and immune cytokines in serum were significantly higher in the two drug-loaded gels than those of mice immunized with OVA solution alone(P<0.05).In summary,PEO-PPO-PEO and PLGA-PEG-PLGA both have good drug-loading capacity and sustained release performance,maintaining the structure of drugs to play effective biological activities in vivo,which are expected to be developed into long-term injections.It should be emphasized that the PLGA-PEG-PLGA drug-loaded gel had longer antibody duration and stronger immune response,which has more obvious advantages as a vaccine carrier.Therefore,PLGA-PEG-PLGA was elected as the carrier for our subsequent studies.3.Preparation and characterization of thermosensitive gel DNA vaccine for Newcastle diseaseIn order to evaluate the feasibility of the thermosensitive hydrogel as vaccine delivery system,the NDV gel vaccine was prepared with PLGA-PEG-PLGA as the carrier and the NDV DNA vaccine as the target vaccine.Firstly,the gene fragment of NDV HN gene with good immunogenicity was amplified and recombined into the efficient eukaryotic expression vector pVAX1.The recombinant plasmid pVAXl-HN was obtained and transfected into the chicken macrophages(HD11)using LipoFiter liposome.The HN protein was widely expressed in HD 11 cytoplasm detected by the immunofluorescence.Then the pVAXl-HN was loaded into PLGA-PEG-PLGA to obtain NDV gel vaccine which gelation characteristics,plasmid encapsulation degree,degradation resistance to DNase I and other properties were characterized.The results showed that the Tm and Gt of the gel vaccine rised to 37.1? and 78.57 s respectively,with 6.46 of its pH and 30 mPa·s of the viscosity at room temperature.When the plasmid concentration was 0.5 mg·mL-1,it could be completely wrapped by the gel and effectively resisted to be degraded by DNase I.In vitro release kinetics of the plasmid was analyzed by membrane-free dissolution method.The results showed that NDV gel vaccine could continuously release plasmid for 22 d,with the cumulative release rate of 95.07%and the effective drug loading of 0.47 mg·g-1.The release media collected at different time point was detected by electrophoresis and transfected into HD 11,showing that the plasmid structure was not significantly affected.The plasmids released from the vaccine could express the antigenic proteins in HD 11 and be targeted by NDV positive serum determined by immunofluorescence and western blot.Finally,the NDV gel vaccine was inoculated into commercial chickens aged 14 days by subcutaneous injection and intramuscular injection,respectively.The acute toxicity,degradability and tissue irritation of the vaccine in vivo were investigated.Meanwhile,the blood compatibility and cytotoxicity in vitro were evaluated.The results showed that the NDV gel vaccine did not cause obvious acute toxic reaction,hemolysis and blood cell coagulation,with no obvious cytotoxicity to CEF.It could be eliminated by body fluid without obvious damage to surrounding tissues.In summary,NDV gel vaccine with good biological safety can sustain the release of the plasmids and maintain their biological activity.These results provide a theoretical basis for the widespread application of thermosensitive hydrogel as vaccine delivery system in clinical practice.4.The immune effects and action mechanism of NDV gel vaccineIn order to evaluate the immune effects of NDV gel vaccine,some chickens aged 14 days were inoculated with the vaccine by intramuscular injection and the serum samples were collected at each predetermined time point after immunization.The HI titers and the concentrations of NDV antibody(NDV-Ab)and IL-2,IL-4,IFN-? were determined.Meanwhile,the peripheral blood lymphocytes were isolated and cultured to determine the proliferation activity.At the end of the monitoring period,the chickens were challenged with the virulent strain of NDV to detect the protection rates of the gel vaccine.The results were as follows.The serum HI titers increased at the first week after immunization and steadily rised to the maximum of 7.0log2,while maintained at a high level(greater than 6.0log2)at the sixth week.The concentrations of NDV-Ab peaked(1686±54 ng·mL-1)at the third week after immunization,and remained at high levels until the end of monitoring,which was significantly higher than the levels of the naked plasmid group(P<0.05).At the same time,the secretion of IL-2,IL-4 and IFN-y was significantly promoted and maintained at high levels for a long time.The proliferation activity of T lymphocytes was significantly higher than that of the naked plasmid group as well as the commercial vaccine group(P<0.05).The protection rate against the virulent strain of NDV was 100%.In order to explore the molecular mechanism of the enhancement effects on immune levels induced by NDV gel vaccine,the mRNA expression of the transcription factors from the peripheral blood lymphocyte was detected by qRT-PCR.Then the ratios of T lymphocyte subsets were determined by flow cytometry.Further more,the effects of PI3K/Akt signaling pathway on the lymphocyte proliferation,apoptosis,and the cytokines secretion were examined.The results showed that the mRNA expression of TBX21 and GATA3(specific transcription factors of Thl and Th2 lymphocytes)in the peripheral blood lymphocyte was increased after the vaccination with NDV gel vaccine,promoting the secretion of the Th1 cytokines(IL-2,IFN-y)and Th2 cytokines(IL-4).Besides,the ratios of CD4+ and CD8+T lymphocytes increased significantly,indicating the vaccination of NDV gel vaccine could promote the differentiation from Th0 lymphocyte to Th1,Th2 cells.After immunization,the PI3K/Akt pathway in peripheral blood lymphocytes was significantly activated.But the enhancement effects on the lymphocyte proliferation,anti-lymphocyte apoptosis and cytokine production induced by NDV gel vaccine were significantly eliminated when using the PI3K inhibitor.These results proved that the immune enhancement effects of NDV gel vaccine were depended on the activation of PI3K/Akt pathway in the lymphocytes.In summary,this study developed a kind of thermosensitive hydrogel which has good biocompatibility,self-degradability and sustained release capability as the vaccine delivery system.Then the NDV gel vaccine was prepared by loading the NDV recombinant plasmid within the delivery system.This vaccine was found to be able to enhance humoral immunity,cellular immunity and the antiviral ability of the body with longer immunity protection.It is proved that the thermosensitive hydrogel can be used as an effective delivery system for the DNA vaccine,and it is possible to be used as carrier with safety and efficiency for other vaccines,which has important theoretical significance,practical significance and broad application prospect.