| Liposomes(LP)are double-layered or multi-layered lipid vesicles,which can be used as drug carriers to achieve transmembrane transport,can selectively and passively target diseased tissues,and can obtain active targeting ability when specific ligands are added.As an adjuvant,eucommia polysaccharide(EUPS)has an immune-enhancing effect.Dendritic cells are the most powerful antigen-presenting cells in the body.They have very strong antigen phagocytosis ability when they are immature.After ingesting antigen,they will differentiate into mature DC,which can express costimulatory factors and secrete cytokines.Dendritic cells are the key sentinels of the host’s immune response and play an important role in connecting innate immunity and adaptive immunity and maintaining tolerance.The DEC-205 receptor(also known as CD205)is one of the mannose receptors(MR)on DC cells,which mediates the uptake,processing and presentation of antigens.At present,there are few studies on EUPS and ovalbumin(OVA)being encapsulated in liposomes and targeted to dendritic cells.In this experiment,Eucommia ulmoides polysaccharide liposomes(EUPS-LP)were prepared by the thin film dispersion method,DEC-205 antibody was combined with liposomes by covalent modification method,and the quality of the prepared liposomes was evaluated,and finally the effect on dendritic cells was explored.The impact of immune activity lays the foundation for the development of new immune adjuvants and their clinical application.The main research results obtained from the experiment are as follows:First,the preparation and process optimization of EUPS-LP: The optimization process of the encapsulation efficiency of EUPS-LP was explored by response surface methodology.Firstly,EUPS-LP were prepared by membrane hydration-microporous membrane extrusion method,the liposomes encapsulating EUPS and free EUPS are separated by high-speed centrifugation,and the concentration of free polysaccharides was determined by phenol-Sulfuric acid method,the encapsulation efficiency of EUPS-LP was calculated.The encapsulation rate and drug loading of Eucommia ulmoides were used as indicators for single-factor tests.lipid-drug ratio,synthetic phospholipid to cholesterol ratio,hydration time and ultrasonic time were used as influencing factors.The three most significant factors affecting the encapsulation rate were selected,and then the three-factor three-level Box-Behnken design was used to optimize the preparation process of EUPS-LP,and finally the encapsulation efficiency under the optimal conditions was verified.The results showed that the optimal preparation process of EUPS-LP were:synthetic phospholipid to cholesterol ratio,4:1;lipid-drug ratio,4:1;ultrasound time,15 min,and the encapsulation rate of EUPS-LP prepared under optimal conditions was28.59% ±0.84%,which was only 0.3% away from the predicted value of the model group.Second,the preparation and quality evaluation of DEC-205 modified liposomes encapsulating Eucommia ulmoides polysaccharides and OVA(DEC-205-EUPS-OVALP): In this experiment,EUPS-OVA-LP was prepared by membrane hydration method,and the encapsulation efficiency of OVA was measured by BCA method;DEC-205-EUPS-OVA-LP was prepared by covalent modification method,and the connection efficiency of DEC-205-EUPS-OVA-LP was measured;the surface morphology of liposomes was observed by transmission electron microscope(TEM),and the internal particle size,PDI and Zeta potential of liposomes were measured by laser particle size analyzer within 28 days.At the same time,the in vitro release of liposomes was investigated.The results showed that the OVA encapsulation efficiency was85.14±2.51%,and the connection efficiency of DEC-205-EUPS-OVA-LP was75.07±3.31%.It was observed that the liposomes were round and nearly spherical in shape,uniform in size,without adhesion;Within 28 days,the particle size of various liposomes increased with the increase of storage time,but the amplitude was smaller,and the particle size was all below 220 nm,the PDI was all below 0.3,and they were all negatively charged,and the Zeta potential was between-11 to-15 m V,the absolute value of the zeta potential decreased with the increase of storage time,but the change was minimal and not significant(P>0.05);the release of Eucommia ulmoides polysaccharides coated with liposomes could achieve a certain sustained release effect,and the release mechanism of EUPS-LP was mainly Fick diffusion.Third,the investigation of DEC-205-EUPS-OVA-LP on the immune function of dendritic cells in vitro: In order to explore the effect of DEC-205-EUPS-OVA-LP on dendritic cells,ICR mouse bone marrow dendritic cells were used for in vitro tests.The CCK-8 method was used to detect the toxicity of DEC-205-EUPS-OVA-LP to mouse dendritic cells,and the best safe drug concentration was screened for later testing.The ability of dendritic cells to engulf the antigen of DEC-205-EUPS-OVA-LP was evaluated by laser confocal microscopy.The supernatant of dendritic cells was collected and tested for IL-2,IL-6,INF-γ,TNF-α,and cytokine levels were detected by ELISA.Dendritic cell phenotypic maturation markers MHC-Ⅱ,CD80,and CD86 were detected by flow cytometry;Finally,high-throughput sequencing was used to explore the effect of DEC-205-EUPS-OVA-LP on the gene expression of mouse dendritic cells.The results showed that the best safe drug concentration of DEC-205-EUPS-OVA-LP was 39.1μg/m L;it could significantly increase the secretion of IL-2,IL-6,INF-γ,TNF-α by dendritic cells.It could be seen from the laser confocal images that FITC-labeled OVA was phagocytosed by dendritic cells,and the fluorescence of the DEC-205-EUPS-OVA-LP group was significantly stronger than that of the other groups.At the same time,DEC-205-EUPSOVA-LP could promote the maturation of dendritic cells,which could promote the expression of dendritic cell phenotypes MHC-Ⅱ,CD80,and CD86.High-throughput sequencing of mouse dendritic cells exhibited that a total of 992 immune-relevant genes were differentially expressed,522 immune-related genes were significantly up-regulated,and 470 immune-related genes were significantly down-regulated,activating multiple immune-related pathways.In summary,the optimal preparation process of EUPS-OVA-LP was that the synthetic phospholipid to cholesterol ratio was 4:1,the lipid-drug ratio was 4:1,the ultrasound time was 15 min,and the maximum encapsulation efficiency was 28.59%.Based on the optimal preparation process,EUPS-OVA-LP was prepared by the thin film dispersion method and modified with DEC-205.DEC-205-EUPS-OVA-LP could promote the secretion of cytokines by dendritic cells in vitro,could promote the phagocytic efficiency of dendritic cells on antigens,and could significantly promote the maturation of dendritic cells,and also could activate multiple immune-related pathways.It was achieved by influencing the enrichment of differential genes to express immunerelated proteins. |
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