| Background and Purpose Glaucocalyxin A(GLA)is an active constitutent extracted from Rabdosia japonica,it is belong to diterpenoid,and has a wide range of pharmacological activity such as anti-tumor,antibacterial,antiviral,protective effect on cardiovascular system.However,the poor stability,poor water solubility and low bioavailability to some extent restrict the further clinical application of Glaucocalyxin A.In order to solve these problems,GLA nanoparticles(GLA NPs)were prepared using egg yolk lecithin and fetal calf serum as carrier materials,which have good biocompatibility and biodegradability.The resultant GLA nanoparticles were characterized and their in vitro drug release,in vitro and in vivo anti-tumor activity were investigated.Methods The Glaucocalyxin A concentration was determined by HPLC method with methodology examination.GLA nanoparticles were prepared by precipitation-combined ultrasonication method.Taking the particle size and short-term stability as the main index,The formulation and preparation conditions were optimized based on single factor such as type of carrier,drug-carrier ration,ultrasonic power,preparation temperature,etc.The particle size and Zeta potential were determined by Malvern laser particle size analyzer,the shape of GLA nanoparticles was observed by transmission electron microscopy,Ultracentrifugation method was used to separate the free drug from encapsulated drug in order to determine entrapment efficiency and drug loading capacity.The existence state of GLA in the nanoparticles was analyzed by differential scanning calorimetry.Drug release from GLA nanoparticles was evaluated by means of a dynamic dialysis technique.The in vitro cytotoxicity of HCPT injection,GLA nanoparticles and GLA solution were assessed by MTT against HepG-2 cells.In vivo antitumor efficacy was evaluated in H22 hepatoma-bearing ICR mice.Results The content of GLA was determined successfully by HPLC,which was proved to be an convenient,sensitive and specific method to accurately determine the GLA concentration in vitro with a good linear relationship between 0.1-1 ?g.mL-1(R2=1).After optimization,the resultant GLA nanoparticles were spherical in shape,no aggregation and no adhesion observed.The average size of GLA-NPs was 143.3±2.9 nm in diameter with PDI value of 0.188+0.01 and zeta potential of-9.86±1.59 mV.The average entrapment efficiency and drug loading content were 84.57±2.8%and 8.95±0.97%respectively.Glaucocalyxin A was proved to be in the amorphous state in nanoparticles.The in vitro drug release assay showd that GLA Solutions(GLA-Sol)rapidly released drug in 4 hours.GLA bulk podwer could only release 54%drug in 48 h,while GLA-NPs displayed an intial relatively rapid drug release within 10 hours followed by a slow drug release(the cumulative 89%release till the 48 hour).In vitro cytotoxicity tests demonstrate that the GLA can inhibit HepG-2 cells' proliferation over the range of 0.5-6 ?g.mL-l and GLA-NPs showed significant higher inhibition than GLA solution(IC50 1.793 ?g/mL vs 2.884 ?g/mL,P<0.05).The in vivo antitumor experiments showed that,in contrast to GLA solution,GLA-NPs exhibited improved anti-tumor activity(54.11%vs 36.02%,inhibition rate).Conclusion In conclusion,GLA nanoparticles were successfully prepared and characterized,the in vitro and in vivo experiments confirmed its improved antitumor activity.This research provides basis for GLA's further study and anti-tumor application.Background and Purpose Malignant tumor is one of the main diseases that have been threatening the health and life of human being.Till now,the conventional methods of tumor therapy are surgery,chemotherapy and radiationtherapy.However,chemotrerapy ofter exihibits serious side effect,due to the the non-specificity of chemotherapeutic drugs to tumor and the wide distribution of chemotherapeutic drugs to normal tissue and organs.So the enhanced turmor-targetability is very important for improved chemotherapeutic efficacy.Nanosuspensions are nearly pure drug nanoparticles stabilized by just a small amount of surfacants or other amphiphilic stabilizers.Nasosuspensions have very high drug-loading content,are easily to be prepared,thus very suitable for nano-preparation and passive tumor targeted delivery for hydrophobic drugs.The surface of nanosuspensions are also easily to be modified for special functions such as tumor-homing.Since the folate receptor(FR)has been detected at high concentration in ovarian and other gynecological cancers,while on the surface of the normal cells,they have little or virtually no expression.Hence,folate can be used as a ligangd to achieve the targeted drug delivery to the tumor.Stabilizers are the key factor for the preparation of nanosuspensions.In this study,we designed and custom-synthesizd cholesterol-mPEG1000 and used it as an effective stabilizer to prepare docetaxel(DTX)nanosuspensions of high drug payload,and then synthesizd cholesterol-PEG1000-FA for active tumor-targeting modification,so as to achieve targed DTX delivery to tumor and targeted therapy.Methods The docetaxel concentration was determined by HPLC method and its methodology was examined.DTX nanosuspensions were prepared by precipitation ultrasonication combined high pressure homogenization method with particle size as the evaluation index.The experimental preparation were optimized based on the pressure and cycle time of high pressure homogenzation.The particle size and Zeta potential were determined by Malvern laser particle size analyzer,the shape was observed by transmission electron microscopy,Drug release from DTX nanosuspensions was evaluated by means of a dynamic dialysis technique.The in vitro cytotoxicity of DTX-Sol,DTX-Nps,DTX-FA-Nps were assessed by MTT against 4T1 cells.In vivo antitumor efficacy was evaluated in 4T1 bearing BABL/C mice.Use DiR as fluorescent dye,investigating the distribution characteristics of drug in vivo by living imaging technology.Results The content of DTX was determined successfully by HPLC.The results showed that it was an convenient,sensitive method to accurate determine the DTX concentration in vitro,and there is a good linear relationship in the range of 1.5625-100 ?g.mL-'(R2=0.9999).Single factor optimization experiment resulted in drug-loaded nanoparticles of ideal size.These particles are spherical in shape,remain good dispersibility and no adhesion between each other.The average size of DTX-Nps and DTX-FA-Nps were 258.8±3.1 nm and 275.2 ±3.7 nm,the PDI were 0.118±0.018 and 0.124±0.021,the zeta potential was-17.0±2.21 mV and-14.6±1.57 mV respectively.The in vitro drug release assay of DTX showed that the release rate of DTX-Sol faster relatively and could release 60.21%of encapsulated drug.While the release rate of DTX-Nps and DTX-FA-Nps showed relative slower and only 36.3%and 31.47%encapsulated drug was released at 48h,no burst release observed.In vitro cytotoxicity tests demonstrated that,the inhibition against 4T1 cells were increased in order of DTX-Sol?DTX-Nps and DTX-FA-Nps with the IC60 being 0.5437?0.1926?0.1017 ?g/mL in suquence.The in vivo antitumor experiments showed that,at the dose of 10 mg/kg,DTX-Nps and DTX-FA-Nps showed significant higher anti-tumor inhibition rate than DTX-Nps(74.83%vsP<0.05),and meantime DTX-Nps showed better anti-tumor efficacy than DTX-Sol(66.19%vs 47.22%).The in vivo imaging technology further verified the tumor targeting of DTX-FA-Nps with significantly enhanced bio-distribution in tumor.Conclusion In this research a new folate targeting docetaxel nanosuspensions with high drug payload was developed successfully and it showed good anti-tumor effect and enhanced tumor accumulation. |
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