| Objective:The main active ingredient of Panax notoginseng（Burk）F.H.Chen is the Panax notoginseng saponins（PNS）,notoginsenoside R1（R1）is one of the active ingredient of the PNS.Preliminary study found that R1 can significantly treatmented dextran sulfate sodium-induced ulcerative colitis in mice by improving colon length,alleviating the symptoms of diarrhea and bloody stools,so it is expected to develop into a new medication for the treatment of ulcerative colitis.R1 is one kind of dammarane tetracyclic triterpenoid saponin with instability under acidic conditions,poor lipid solubility,short half-life in vivo,which made its oral bioavailability low and limited its clinical application.In order to improving the druggability of R1,based on transferrin receptor（Tf R）exhibited low expression on normal cells,but high expressed on colonic epithelial cells under the condition of inflammation,we prepared transferrin modified notoginsenoside R1-loaded PEG-PLGA nanoparticles（R1@Tf-PEG-PLGA NPs）and evaluated physical characterization,in vitro release rate,cell targeting,absorption mechanism,pharmacodynamics and intestinal tissue distribution.Methods:Preparation of notoginsenoside R1-loaded PEG-PLGA nanoparticles（R1@PEG-PLGA NPs）:R1@PEG-PLGA NPs was prepared by nanoprecipitation method and took encapsulation rate and drug loading as indicators.Based on sigle factor test,the prescription factors influencing the preparation were also studied,such as surfactant concentration,PEG-PLGA concentration,the ratio of oil and water phase,reaction medium,the ratiol of drug and carrier material.Then,the best prescription was determined by orthogonal experiment.Preparation of transferrin modified notoginsenoside R1-loaded PEG-PLGA nanoparticles（R1@Tf-PEG-PLGA NPs）:With the participation of catalyst,the R1@PEG-PLGA NPs was modified by Tf.Taking the grafting rate as indexes,the molar ratio of Tf and PEG-PLGA,the amount of catalyst and incubation time were investigated,and the preparation process were established.Physical characterization,in vitro release,cell targeting and absorption mechanism:The particle size,polydisperse index and Zeta potential were determined by malvern particle size analyzer,two nanoparticles were characterized by TEM.In vitro release was determined by dialysis bag method for 24 h.5-aminofluorescein was modified onto the surface of nanoparticles,using Caco-2 cells as model cells,intracellular fluorescence intensity were observed by fluorescence microscopy.The effects of incubation time,incubation temperature and endocytosis inhibitors were compared for the uptake of nanoparticles,and the absorption mechanism were discussed.Pharmacodynamics,safety and intestinal tissue distribution:Rats with ulcerative colitis induced by oxazolone as a model.The effects of the preparation on disease activity index（DAI）,colon length,spleen coefficient,colon morphology and inflammatory factors were investigated.By detecting serum biochemical indexes and observing the pathological sections of heart,liver,spleen,lung and kidney,the in vivo toxicity were evaluated preliminarily.The model rats were treated with drug after 4,8,12 and 24 h,intestinal tissue and its contents were collected.The content of R1 was determined,and the distribution of R1 and its preparation in different intestinal segments was investigated.Results:The preparation process of R1@PEG-PLGA NPs was confirmed as following:PEG-PLGA and R1 are both dissolved in acetone-ethanol mixed solution as the organic phase,the concentration of PEG-PLGA was 10 mg·m L-1,the mass ratio of PEG-PLGA to R1 was 1:1,the volume ratio of acetone to ethanol was 1:9.And 1%poloxamer188 solutions as the aqueous phase,the volume ratio of the organic phase to the aqueous phase was 1:20.Then,the organic phase was extracted with a syringe and added to the aqueous phase at the stirring rate of 1 000 rpm,R1@PEG-PLGA NPs was obtained with 1 000 rpm at atmospheric temperature after 4 h.The encapsulation efficiency,drug loading and particle size of R1@PEG-PLGA NPs were 61.04%,30.39%and 129.83 nm,respectively.The R1@PEG-PLGA NPs was prepared with uniform size,good dispersibility and suitable Zeta potential.The preparation process of R1@Tf-PEG-PLGA NPs was determined as fllowing:EDC/NHS catalytic solution was added to R1@PEG-PLGA NPs suspension with 230rpm for 4 h at atmospheric temperature,the mole ratio of EDC to PEG-PLGA was 10:1,the mole ratio of EDC to NHS was 4:1.Tf solution was dripped into the above suspension and contimuously mixed for 2 h,the mole ratio of PEG-PLGA to Tf was750:1.The encapsulation efficiency,drug loading and particle size of R1@Tf-PEG-PLGA NPs were 50.32%,24.26%and 153.50 nm,respectively.The R1@Tf-PEG-PLGA NPs was prepared with uniform size,good dispersibility and suitable Zeta potential.Physical characterization,in vitro release,cell targeting and absorption mechanism:The morphology of R1@PEG-PLGA NPs and R1@Tf-PEG-PLGA NPs were spherical or quasi-spherical under TEM,and they had good dispersion.Within 24 h,the cumulative release of R1@PEG-PLGA NPs in p H5.8 and p H7.4 dissolve medium was91.22±4.39%and 91.11±3.56%,respectively.The cumulative release of R1@Tf-PEG-PLGA NPs in p H5.8 and PH7.4 dissolve medium was 84.27±2.69%and80.48±3.25%,respectively.Under the same conditions,the cumulative release of R1@PEG-PLGA NPs was higher than R1@Tf-PEG-PLGA NPs.The release of R1 was compatible with the first-order model.The release of the two nanoparticles was compatible with the Riger-Peppas model,and showed that the release mechanism of R1@PEG-PLGA NPs and R1@Tf-PEG-PLGA NPs was the combined effect of diffusion and matrix erosion.Caco-2 cells got more Tf-PEG-PLGA NPs than PEG-PLGA NPs under the same condition.PEG-PLGA NPs was mainly entered into Caco-2 cells by fossae-mediated endocytosis,while Tf-PEG-PLGA NPs was mainly entered into Caco-2 cells by fossae-mediated and clathrin mediated endocytosis.Pharmacodynamics,safety and intestinal tissue distribution:From the 5th day of administration,R1,R1@PEG-PLGA NPs and R1@Tf-PEG-PLGA NPs could significantly relieved colonic injury（p<0.05,p<0.05,p<0.01）,and relieved the symptoms of alleviated diarrhea,diarrhea,hematochezia of UC rats;R1,R1@PEG-PLGA NPs and R1@Tf-PEG-PLGA NPs could improved colon length（p<0.05,p<0.01,p<0.001）;R1@Tf-PEG-PLGA NPs significantly inhibited the increase of spleen weight（p<0.05）;R1,R1@PEG-PLGA NPs and R1@Tf-PEG-PLGA NPs obviously mitigated the colon tissue lesions（p<0.001）,reduced the degree of inflammatory cell infiltration;R1,R1@PEG-PLGA NPs and R1@Tf-PEG-PLGA NPs significantly inhibited the expression of myeloperoxidase（p<0.05,p<0.01,p<0.01）and tumor necrosis factor-α（p<0.01）,and increased the expression of interleukin-4（p<0.01）and interleukin-13（p<0.05,p<0.01,p<0.001）.The preliminary safety evaluation results showed that R1 and its preparation had good safety in vivo.The results of tissue distribution showed that R1@PEG-PLGA NPs and R1@Tf-PEG-PLGA NPs both can prolonged the retention time of R1 in vivo.The main absorption sites of R1@PEG-PLGA NPs were in the duodenum and cecum,the main absorption sites of R1@Tf-PEG-PLGA NPs were absorbed in the cecum and colon.It suggested that R1@Tf-PEG-PLGA NPs could be highly enriched in the colon.Conclusion:In this study,R1@Tf-PEG-PLGA NPs was successfully prepared,which had a certain sustained-release effect and could be specifically absorbed by colonic epithelial cells,and showed significant therapeutic effect on UC model rats,and had good safety in vivo.Thus R1@Tf-PEG-PLGA NPs had high clinical application value. |
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