Preparation,in Vitro And In Vivo Study Of Resveratrol Nanoparticles And Honokiol Prodrug Nanoparticles

Resveratrol(RES)is an edible polyphenolic phytoalexin with extensive biological and pharmacological activities,including antitumor,antioxidant,anti-inflammatory,antiviral,cardioprotection,hepatoprotective and neuroprotective et al.However,the application of RES is limited for it's instability,poor solubility,rapid metabolization,and low bioavailability.To overcome these problems,drug delivery systems have been developed to protect and stabilize RES and to enhance its bioavailability.Nanosuspensions are nanoscale colloidal dispersions of pure drug particles stabilized by small amount of surfactants or polymers.Over the past few years,nanosuspensions have been widely used to tackle problems associated with poor solubility and low bioavailability.A reduction of particle size would lead to a dramatic increase in the dissolution rate of a drug,and hence the bio-availability of poorly water-soluble drugs was increased.In this study,we prepare Resveratrol nanosuspensions(RES-Nps)using nanoprecipitation and high pressure homogenization method to improve the drug loading capacity and increase the drug dissolution rate.The main contents include RES-Nps formulation,synthesis of mPEG1000-RES,preparation and characterization of RES-Nps.In addition,their in vitro release,in vivo pharmacokinetics and biodistribution were also evaluated.1.RES-Nps were prepared by nanoprecipitation and high pressure homogenization method.PEG2000-PCL2000?PEG2000-PLA2000?PEG2000-PLGA2000?cholesterol-PEG1000(chol-PEG1000)?TPGS?SDS and SPC were used for formulation study,and the particle size and PDI value of the resultant RES nanoparticles were measured.It was found that the RES-Nps stabilized by PEG2000-PCL2000 was optimal with particle size(235.5 ± 4.13)nm and PDI(0.14±0.02).2.In order to obtain RES-Nps with smaller particle size(<200 nm),a novel stabilizer,mPEG1000-RES,was designed and synthetized.It was hypothesized that an amphiphilic stabilizer with the hydrophobic moiety highly similar to the encapsulated drugs,in polarity and/or structure,may be the efficient stabilizer for the preparation of the nanosuspensions of that drug.The 1HMR and MALDI-TOF analysis showed that the mPEG1000-RES was synthetized successfully.3.RES-Nps stabilized by mPEG1000-RES was easily obtained even at high drug-stabilizer ratio of 10:1,with the resultant particle size being(187.7 ±4.87)nm,the PDI value being(0.13 ±0.04)and the zeta potential being(-15.94±2.37)mV.The drug payload was determined to be(86.10±1.74)%and the encapsulation efficiency was(95.6810.81)%.The long term stability of RES-Nps under 4? was good and no hemolysis was observed even at concentration of 6 mg/mL.TEM analysis revealed the nanoparticles were almost sphere,and both the DSC and XRD analysis confirmed that drug was in crystalline form in the nanosuspensions.RES-Nps could exhibit a sustained release in vitro in contrast with RES solution while an increased dissolution rate in comparison with RES corse suspensions.4.The Pharmacokinetic studies in rats revealed that the Cmax and AUC of RES-Nps were 8.5-fold and 15.6-fold greater than that of RES solution after iv adminstration.Also the T1/2 value was also much longer and the CL was much lower for RES nanosuspensions.This may be because the hydrophobic segment of the stalizer RES-PEG1000 is RES itself and the interaction between drug molecular and stablizer is relatively strong.In addition the long hydrophilic chains of PEG on the surface of the nanoparticles provide a steric stabilization which would protect the nanoparticles from enzymolysis of the plasma enzymes to some extent.5.Biodistribution of RES-Nps and RES solution were investigated following a single i.v.administration to mices.The results indicated that RES distributed widely in vivo and mainly accumulated in organs with abundant blood flow like liver and spleen.The amount of RES disributed in the heart was also high which is beneficial to heart diseases therapy.Honokiol(HK),a small molecule with phenolic hydroxyl groups extracted from Magnolia officinalis,has been reported to possess multiple biological activities including anti-cancer,antioxidant,anti-inflammatory,cardioprotection,neuroprotective effect and hepatoprotection.However,its poor water solubility and instability greatly hampers its clinical application.Therefore,improved solubility and stability become the key to solve this problem.In this study,honokiol was modified to form its ester prodrugs,and the resultant prodrugs were further made into nanoparticles using proper stabilizers.1.HK caproate and HK decyl decanoate were synthesised and purified successfully.The 1HMR analysis indicated that characteristic protons signal from the phenolic hydroxyl groups of HK disappeared and the signals of hexanoyl and decanoyl appeared.The purities of HK caproate and HK decyl decanoate determined by HPLC were 98.13%and 98.05%respectively.The hydrolysis tests demonstrated that both these two prodrugs were stable in pH 2.0 and pH 7.4 solutions and HK decyl decanoate showed slower hydrolysis rate because of the longer carbon chain.2.The feasibility of preparation nanoparticles for HK caproate and HK decyl decanoate were invesgated and different stabilizers,such as PEG2000-PCL2000,TPGS,SPC,chol-PEG1000,a-CD and HP-?-CD,were tried as the stabilizers to prepared HK prodrugs nanoparticles.The results displayed that the two prodrugs could be easily made into nanoparticles and PEG2000-PCL2000 was an optimal stabilizer.At the high drug-stabilizer ratio of 5:1,the resultant HK caproate nanoparticles was(164.2±3.15)nm in mean diameter with PDI value of(0.17±0.03)and zeta potential of(-27.2±2.14)mV.While the HK decyl decanoate nanoparticlesdemonstrate were(131.9 ± 1.26)nm in particle size,with PDI of(0.17±0.06)and zeta potential of(-27.4±2.45)mV.However,it was found only when drug-stabilizer ratio was reduced to of 1:10,the hemolysis of nanoparticles made from the two prodrugs could be decreased to be proper for iv administration.3.At low drug-stabilizer ratio of 1:10,HK decyl decanoate nanoparticles were(23.6±2.24)nm in the average diameter with the PDI and zeta potential being(0.23±0.02)and(-15.3 ±0.82)mV.TEM analysis showed that the nanoparticles were sphere and the DSC indicated HK decyl decanoate was in the same form of crystalline in nanoparticles as in the bulk HK decyl decanoate.The hemolysis rate was only 2.1%even at high drug concentration of 4mg/mL.HK decyl decanoate nanoparticles could release the encapsulated drug in a sustained release manner and the accumulative release rate was 80%within 192 h.4.The Pharmacokinetic studies of HK decyl decanoate nanoparticles in rats revealed that the Cmax,AUC and T1/2 of HK decyl decanoate nanoparticles were 2.97-fold,2.05-fold and 1.36-fold greater than that of HK nanoparticles respectively after iv administration.The results suggested that HK decyl decanoate may be used as a depot of HK and it could transform into HK sustainedly after administration.5.Biodistribution of HK decyl decanoate nanoparticles and HK nanoparticles were investigated in comparison following a single i.v.administration to mices.The results demonstrated that the MRT of HK in HK decyl decanoate nanoparticles group was prolonged in different degrees,and also the amount of HK distributed in the heart and liver were significant higher than that of HK nanoparticles group.These results indicated that HK prodrug could prolong the residence time of HK in different organs,what's more,it can alter the in vivo distribution of free HK.