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Optimum Preparation Method for Self-Assembled PEGylation Nano-Adjuvant Based on Rehmannia glutinosa Polysaccharide
Yee Huang1, Li Nan1,2, Chenwen Xiao1
1Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Science, Hangzhou 310021, People's Republic of China.
Background:
Rehmannia glutinosa polysaccharide is the main reason that contributes to the immunological function of R. glutinosa. Due to its disadvantages in clinical use, here we designed the PEGylation nano-RGP (pRL) to improve the drug-targeting effect and the immunological function. Our present work aims to establish the optimum condition of preparing the pRL and to investigate its immunological function on macrophages.
Methods:
pRL was prepared by thin film hydration method combined with ultra-sonication technique. And its preparation conditions were optimized with response surface methodology. Also, the lyophilization method was optimized. The characteristics of the pRL were evaluated, including particle size, drug loading, encapsulation efficiency and morphology. The immunological function of pRL on macrophage was investigated through CCK-8 test, ELISA and flow cytometry.
Results:
The lipid-to-cholesterol molar ratio of 8:1, the addition of DSPE-PEG2000 of 9% and the lipid-to-drug ratio of 5.4:1 were the optimum preparation technology for pRL. The encapsulation efficiency (EE) of pRL under this preparation technology was 95.81±1.58%, with a particle size of 31.98 ± 2.6 nm. The lactose-to-lipid ratio (2:1) was the optimal lyophilization method. pRL promoted macrophage proliferation, which is significantly better than that of nano-RGP without PEGylation (RL). pRL-stimulated RAW264.7 cells showed a high secretion of pro-inflammatory cytokines, which is the characteristic indicator of M1 polarization. Enhanced cellular uptake through macropinocytosis-dependent and caveolae-mediated endocytosis was observed in pRL-treated RAW264.7 cells.
Conclusion:
Our study concluded that PEGylation effectively overcame the poor targeting effect of Rehmannia glutinosa polysaccharide (RGP) and significantly improved the immunological profile of its nano-formulation, which suggested that pRL could serve as an immune adjuvant in clinical application.
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