| Ginsenoside Rg3 is one of the important active ingredients of the traditional Chinese herbal medicine ginseng,and"Shenyi Capsule"is the only ginsenoside Rg3monomer that is currently marketed to improve the efficacy against lung and liver cancer.The inhibitory effect of ginsenoside Rg3 on glioma has been confirmed,but its water solubility is poor and there is a hepatic first-pass effect when administered orally.How to cross the blood brain barrier（BBB）and achieve safe and efficient targeted delivery to the brain is the key to the anti-brain tumor efficacy of ginsenoside Rg3.Nasal drug delivery is non-invasive and convenient,and can bypass the blood-brain barrier to achieve brain targeting,which has become a hot research topic in the central nervous system（CNS）drug delivery pathway.However,the retention time of common drug formulations in the nasal cavity is short,and they are easily degraded by the nasal mucosal environment and they may irritate the nasal mucosa and damage the nasal cilia,and the physicochemical properties and formulation factors of drugs seriously affect the efficiency of nasal drug delivery into the brain.Nanolipid carriers can enhance the control and targeting of drug delivery,and have the advantages of high encapsulation rate,high drug loading capacity and low toxicity.To enhance the targeting of nanolipid carriers,lactoferrin（Lf）,which can bind to surface receptors on epithelial cells and brain endothelial cells,was used to modify the negatively charged nanolipid carriers to a positive charge,allowing better contact with the negatively charged nasal mucosa.Objective:In this project,we propose to prepare Lactoferrin Modified Ginsenoside Rg3 nano lipid carrier（Lf-Rg3-NLC）to enhance the targeted delivery of ginsenoside Rg3 to the brain.Method:1.An in vitro method for the analysis of ginsenoside Rg3 was established using high performance liquid chromatography,and its precision,recovery and stability were investigated.2.The methods for breaking the emulsion of nanolipid carriers were screened,and four methods for determining the encapsulation rate of nanolipid carriers were compared,namely,low-speed centrifugation,ultracentrifugation,fisetin precipitation and dialysis.The optimal preparation prescription and process of ginsenoside Rg3nanolipid carrier were screened by single-factor and response surface tests using a combination of high shear and wet stirred media milling（WSMM）with particle size,encapsulation rate and drug loading capacity as indicators.3.The optimum incubation time and concentration of lactoferrin and nanolipid carriers were screened by using the particle size,zeta potential,PDI,encapsulation rate and drug loading capacity of the modified nanolipid carriers as indicators.The appearance,type,p H,encapsulation rate,drug loading capacity,particle size,potential,stability and in vitro release of lactoferrin-modified ginsenoside Rg3 nanolipid carriers were also investigated.4.An in vivo method for the analysis of ginsenoside Rg3 was established using high performance liquid chromatography.Pharmacokinetic experiments were performed on rats,which were divided into four groups.The experimental groups were given lactoferrin-modified ginsenoside Rg3 nanolipid carrier（Lf-Rg3-NLC）and ginsenoside Rg3 nanolipid carrier（Rg3-NLC）intranasally,and the control groups were given ginsenoside Rg3 mixed with blank nanolipid carrier（Rg3-blank NLC）intranasally and Shenyi Capsule solution by gavage,respectively.The concentration of ginsenoside Rg3 in serum and brain tissue at each time point after administration was determined,and the pharmacokinetic parameters in serum and brain tissue were calculated for each group,and the data were analyzed by ANOVA to evaluate the brain targeting of ginsenoside Rg3 in each group by targeting index（DTI）.Result:1.An in vitro high performance liquid chromatographic method was established for the analysis of ginsenoside Rg3 with acetonitrile-water（45:55,v/v）as the mobile phase and the standard curve equation was Y=3817.3x+3639.4,R2=0.9996,and the RSD values of precision,stability and spiked recoveries were less than 3%.2.Ethanol was selected as the emulsion breaker of the nanolipid carrier,and the encapsulation rate of the nanolipid carrier was determined by low-speed centrifugation method.The optimal preparation process of nano-lipid carrier was obtained by single-factor screening and response surface experimental design:the mass ratio of stearic acid to monooleate was 0.21,the input mass of ginsenoside Rg3 accounted for 0.0625 of the mixed lipid,the drug and the mixed lipid were melted at 76℃,injected into Tween-80solution with a concentration of 1%at the same temperature,and emulsified for 2 min using a high shear dispersion emulsifier.The primary milk was added with the same volume of zirconia beads and ground at 1500 rpm for 4 h at 76℃and placed in a refrigerator at 4℃for low temperature curing.The particle size of ginsenoside Rg3nanolipid carrier made by this prescription was（162.60±3.72）nm,the encapsulation rate was（91.56±0.56）%,and the drug loading was（6.67±0.04）%.3.The optimum incubation time for the electrostatic binding of lactoferrin and nanolipid carrier was 6 h,the optimum concentration was 160 mg-m L-1,and the particle size of the lactoferrin-modified ginsenoside Rg3 nanolipid carrier was（169.90±6.07）nm,the PDI was 0.21±0.01,the zeta potential was（5.68±0.33）m V,the encapsulation rate was（83.39±1.33）%,and the drug loading capacity was（3.41±0.17）%.4.An in vivo method for the analysis of ginsenoside Rg3 was established using high performance liquid chromatography with good specificity,precision,stability and recovery.The pharmacokinetic parameters of transnasal Lf-Rg3-NLC,transnasal Rg3-NLC,transnasal Rg3-blank NLC and gavage of Shenyi Capsule solution were calculated using PKSolver software.The results of the pharmacokinetic experiments showed that the transnasal Rg3-NLC and transnasal Lf-Rg3-NLC reached the body circulation and brain tissue with greater AUC0-t and more drug amount reached the brain tissue compared with the transnasal Rg3-blank NLC and gavaged Ginseng I capsule solutions.The brain targeting index DTI of nanolipid carriers before and after lactoferrin modification was calculated to be 2.11 for transnasal Lf-Rg3-NLC and 1.19for transnasal Rg3-NLC with lactoferrin modification as the control group,and 2.77 for transnasal Lf-Rg3-NLC and 1.56 for transnasal Rg3-NLC with gavage of Sen I capsule solution as the control group.the DTI was 1.56,both of which were greater than 1.Conclusion:1.The specificity,linearity,precision and stability of the established ginsenoside Rg3 in vitro methodology are good for the in vitro analysis of ginsenoside Rg3.2.For the first time,lactoferrin modified ginsenoside Rg3 nano lipid carrier delivery system was prepared by high shear and wet stirring medium grinding process,which is easy to operate and does not cause toxic residues and metal contamination.The preparation of lactoferrin modified ginsenoside Rg3 nano lipid carrier is simple,has high encapsulation rate and drug loading,and has stable physical properties.3.The in vivo pharmacokinetic experiments showed that the intranasal administration of ginsenoside Rg3 nanolipid carriers before and after lactoferrin modification could enhance the brain targeting compared with the intranasal ginsenoside Rg3 blank nanolipid carrier mixture and the gavaged Shenyi Capsule solution,and the lactoferrin-modified nanolipid carriers were more effective in enhancing the targeting.4.The results of intranasal delivery of ginsenoside Rg3 nanolipid carriers provide new ideas for the development of new nano-delivery systems and new drug delivery routes for ginsenoside Rg3.Innovation point:1.Ginsenoside Rg3 nanolipid carriers were prepared by using wet media stirring and grinding method innovatively,which is economical and easy to operate without causing toxic residues and metal contamination.2.For the first time,in vivo pharmacokinetic and targeting study of lactoferrin-modified ginsenoside Rg3 nanolipid carriers by intranasal administration to rats demonstrated that the lactoferrin-modified ginsenoside Rg3 nanolipid carriers prepared in this experiment were brain-targeting. |
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