| Objective: Nano drug delivery system of hyaluronic acid-modified panax notoginseng saponins targeting liposome(HA-PNS-LIP)and panax notoginsenoside liposome(PNS-LIP)were constructed,characterizing their basic properties and stability assay.HA-PNS-LIP targets the surface receptor CD44 of rabbit BMSCs,detecting the formation of calcium nodules,the expression of osteoblast-associated cytokines TGF-β1,ALP and OCN,preliminary study on the effect of HA-PNS-LIP targeting rabbit BMSCs deliver PNS on osteogenic differentiation.Methods: 1.Constructing HA-PNS-LIP and PNS-LIP drug-loaded nanoparticles,the drug concentration,encapsulation efficiency and drug loading were determined by HPLC,the particle size,PDI and Zeta potential were measured by particle size analyzer,the appearance was observed by transmission electron microscopy,and stability assay.2.Rabbit BMSCs were isolated,cultured and identified in vitro.3.Blank control group(BMSCs without intervention)and PNS group(BMSCs with 100 mg/L PNS medium)、PNS-LIP group and HA-PNS-LIP group(3 concentrations: 5,10,20 μg/ml BMSCs in culture medium)were established.The uptake efficiency of BMSCs at different concentrations of PNS-LIP and HAPNS-LIP was observed by inverted fluorescence microscope.4.The cytotoxicity of nanoparticles on BMSCs was detected by CCK-8 method.5.The formation of calcium nodules was detected by alizarin red staining on the 12 th day.6.On the 3rd,6th and 12 th day after the intervention,the mRNA expression of TGF-β1,ALP and OCN was detected by Real-time qPCR.7.On the 3rd,6th and 12 th day after the intervention,The protein expression of TGF-β1,ALP and OCN was detected by Western blot.8.On the 12 th day after the intervention of drug-loaded nanoparticles,The protein expression of TGF-β1,ALP and OCN was detected by immunofluorescence.Results: 1.The drug concentration of drug-loaded nanoparticles was 923 μg/ml;Encapsulation efficiency: HA-PNS-LIP: 86.65 ± 0.45%,PNS-LIP: 85.20 ± 0.90%;drug loading: HA-PNS-LIP: 7.89 ± 0.04%,PNS-LIP: 7.75 ± 0.08%;particle size: HA-PNS-LIP was 72.84±0.14 nm,PNS-LIP was 69.33±1.39nm;PDI:HA-PNS-LIP was 0.221±0.015,PNS-LIP is 0.237±0.011;Zeta potential: HA-PNS-LIP was-18.55±0.07 mV,PNS-LIP was-14.70±0.14mV;Transmission electron microscopy: the two drug-loaded nanoparticles showed circular doublelayer vesicle structure;Stability assay: the drug content of liposomes decreased significantly with the prolonging of time.2.Cellular uptake experimental results: 10,20μg / ml HA-PNS-LIP group cell uptake efficiency ≥ 50%;3.Cellular cytotoxicity results: 10μg/ml HA-PNS-LLP group and PNS-LIP group had the least cytotoxicity on BMSCs.4.Alizarin red staining results: The formation of calcium nodules in the 10μg/ml HA-PNS-LLP group was significantly higher than the other three groups.5.Real-time qPCR and Western blot results: analysis from mRNA and protein levels:(1)TGF-β1 expression changes: 10μg/ml HA-PNS-LLP group expression was higher than the other experimental groups at each time point,the difference was statistically significant,P<0.05;10μg/ml PNS-LLP group between 10μg/ml HA-PNS-LLP group and the 100 mg/L PNS group;the expression level of the experimental groups increased with time.(2)ALP expression changes: the expression of each experimental group showed significantly high expression on the 6th day,P <0.01,continued high expression on the 12 th day,P <0.05;The expression of 10μg/ml HA-PNS-LLP group higher than the other experimental groups at all time points,the difference was statistically significant,P<0.05;the expression of 10 μg/ml PNS-LLP group was between the 10 μg/ml HA-PNS-LLP group and the 100 mg/L PNS group.(3)OCN expression changes: The expression of each experimental group showed significantly high expression on the 12 th day,P<0.01;the expression of 10μg/ml HA-PNS-LLP was higher than the other experimental groups at each time point,the difference was statistically significant,P<0.01,the expression of 10μg/ml PNS-LLP group was between 10 μg/ml HA-PNS-LLP group and 100 mg/L PNS group.6.The results of immunofluorescence showed that on the 12 th day,the protein expression of TGF-β1,ALP and OCN in the 10 μg/ml HA-PNS-LLP group was strongly positive compared with the 10 μg/ml HA-PNS-LLP group.Conclusions: 1.The HA-PNS-LIP and PNS-LIP drug-loaded nanoparticles showed good characterization and stability within 2 weeks;2.10 g/ml HA-PNS-LIP targeting rabbit BMSCs to deliver PNS can significantly enhance the ability of calcium nodule formation,significantly promote the expression of mRNA and protein of TGF-1,ALP and OCN,which enhanced osteogenic differentiation. |
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