| Panax notoginseng saponins?PNS?,as one of the main active components of Sanqi,which has anti-inflammatory,anti-aging,anti-tumor,relieving cardiovascular and cerebrovascular diseases and other pharmacological effects.The main monomer saponins in PNS are water-soluble macromoleculars,leading to a poor intestinal permeability and a low oral bioavailability.Chitosan,as a natural amino polysaccharide with positive charge,has a good bioadhesion and it can opens tight junction between intestinal epithelial cells,and promote the absorption of drugs through paracellular transport.To investigate the effect of low molecular weight chitosan nanoparticles?LMW-CTS-NPs?on the permeability of intestinal mucosa of PNS,the main works performed in this research is as follows:1.With a rotational viscometer,uv spectrophotometer and infrared spectrometer,the basic parameters of chitosan raw materials which with high molecular weight was determined.The results showed that the dynamic viscosity of chitosan was 541.25±2.56mPa·s and the degree of deacetylation was 95.1±0.2%.The infrared spectrum showed a characteristic absorption peak of chitosan.To improve the fluidity and solubility of chitosan,low molecular weight chitosan?LMW-CTS?was cleanly prepared by enzymatic degradation combined with ultrafiltration fractionation technology.The weight-average molecular weight of LMW-CTS was 5760 Da,and the polydispersity index was about 1.42,indicated a narrow molecular weight distribution.2.With LMW-CTS as the carrier and sodium tripolyphosphate as the cross-linking agent,the concentration ratio of both was adjusted to determine the conditions for the preparation of LMW-CTS-NPs by the ion gel method.The particle size,Zeta potential,morphology,and PNS loading capacity of LMW-CTS-NPs prepared under optimal conditions were characterized.The results showed that the average particle size was115.5 nm,PDI was 0.221,and the zeta potential was+37.1 mV,and the appearance is round and uniform in size.It showed different encapsulation capabilities for different components in PNS,the encapsulation efficiency ranges from 14.03%to 41.83%,and the drug loading ranges from 14.05?g/mg to 95.74?g/mg.In addition,infrared spectroscopy confirmed that the nanoparticles were crosslinked by chitosan and TPP via ion bonding.3.In order to verify the integrity,differentiation characteristics,compactness and permeability of Caco-2 cell model,its transepithelial electrical resistance,alkaline phosphatase activity and the permeability of the phenol red marker were detected.The results showed that,after being cultured for 21 days,the teer reached 682±66?·cm2,the alkaline phosphatase activity ratio of the intestinal cavity to basal side reached 3.3,and the apparent permeability coefficient of phenol red at each sampling time was ranged from?2.21±0.12?×10-7 cm/s to?3.28±0.23?×10-7 cm/s,indicating that the integrity,differentiation characteristics and permeability of the Caco-2 cell model meet the requirements which can be used for drug cross Membrane transport experiments.4.To screening the drug concentration,MTT toxicity test,LDH release test and transmembrane resistance test and transepithelial electrical resistance test were performed.The results showed that PNS had no significant effect on the viability of Caco-2 cells at a concentration of 200?g/mL;LMW-CTS and LMW-CTS-NPs had no significant effect on the viability and membrane integrity of Caco-2 cells at a concentration of 25?g/mL,but at this concentration,the teer was significantly reduced to below 20%of baseline.The PNS transmembrane transport experiments were performed at the selected concentration.The results showed that the apparent permeability coefficients of the PNS APIs were in the range of 110×10-7 cm/s,which indicated a poorly permeability of the mucosa;LMW-CTS had no obvious effect on Papp of Re,Rb1 and Rd,but increased Papp of R1 and Rg1 by 17.83%and 20.29%,respectively;Compared with the control group,LMW-CTS-NPs increased Papp of R1,Rg1,Re,Rb1 and Rd by 35.66%,23.28%,29.41%,37.99%and 36.00%respectively,which indicated that the permeability of intestinal of PNS can be improved by the transition from LMW-CTS molecular to nanoparticle. |
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