Preparation And Characterization Of Pyrene-loaded Polymeric Nanoparticles And Primary Study Of Its In Vitro And In Vivo Behaviour

Recently, nanomedicine, with advantages of improving the bioavailability of poorlysoluble drugs, sustained and controlled drug release as well as drug targeted delivery, hasbeen promoted by nanotechnology. To improve the therapeutic effects and reduce thesystem side effects, it plays a key role to alter the biopharmaceutics and pharmacokineticsbehaviors of nanomedicine. So it's very necessary and significant to study thepharmacokinetics of nanomedicine and to explore its rule.Overall speaking, some limitations of study idea and method still exist when studyingthe pharmacokinetics of nanomedicine. The details are as follows:(1) In the aspect of research idea, the data of pharmacokinetics is only describedapparently, and it is difficult to explore the pharmacokinetic mechanism and rule in depthby using conventional pharmacokinetic study pattern.(2) The pharmacokinetics model based on conventional mathematics theory can notreflect the complicated processes of nanomedicine in vivo, which is a "black box", and cannot describe the relationship between properties and effect of nanomedicine.(3) Encapsulated drugs, free drugs and protein-bound drugs are three possible drugforms existing in vivo when nanomedicine is administrated by intravenous injection.Presently, the pharmacokinetic study of different drug forms has not attracted interest.Besides the defect, effective methods for the discriminative determination of differentdrug forms in biological samples still lack.Among the three limitations mentioned above, the study of determination methods isthe basic of pharmacokinetic study of nanomedicine. Due to this background,MePEG-PLA polymeric nanoparticles which encapsulating model drug pyrene wasprepared and set as a representative of nanomedicine. Work including preparation andcharacterization of the polymeric nanoparticles, in virto drug release assessment of thepolymeric nanoparticles, establishment of method for determination of concentrations ofdifferent drug forms in biological samples and study of pharmacokinetics andbiodistribution of the polymeric nanoparticles were carried out. The major results are asbelow:(1)Two kinds of block copolymer i.e. MePEG-PLA with different molecular weight were used to prepare pyrene-loaded polymeric nanoparticles by nanoprecipitation method,the formulation and the preparation technique were also studied. On this basis, theobtained polymeric nanoparticles were characterized by several methods. The diameterand its PDI of the nanoparticles were determined by photon correlation spectroscopy(PCS), and theζpotential of nanoparticles was determined at the same time. Transmissionelectron microscope (TEM) and nuclear magnetic resonance (NMR) were used to observeand study the structure and shape of the nanoparticles. Differential scanning calorimetry(DSC) thermogram analysis and X-ray powder scattering (XRD) measurements werecarried out to investigate the presence state of pyrene in the nanoparticles. The resultsshowed that the mean diameters of both nanoparticles were smaller than 100 nm, and themean diameters were 76.5 and 80.1 nm, respectively. The nanoparticles were sphericalwith core-shell shape. The DSC and XRD results showed that pyrene was in theamorphous or molecular dispersion state in the nanoparticles. Encapsulation efficiencyand drug loading of the nanoparticles were determined by ultrafiltration method combinedwith HPLC. The results indicated that the nanoparticles had high encapsulation efficiency,which were 80.3% and 75.2%, while the drug loading were 1.42% and 1.35%, respectively.The lyophilization technique of the nanoparticle suspension was studied, and the resultsshowed that trehalose used as cryoprotectant had good effect. Stability of the nanoparticleswas also investigated by using diameter, encapsulation efficiency and drug loading asassessment index, the results showed that the nanoparticles had good stability and theindex had no significant changes within one month.(2) The apparent diffusion coefficients of pyrene in two different kinds of polymermembrane were determined, which were 3.84×10^-11 and 11.25×10^-11 cm²Â·s^-1, respectively.The results indicated that the apparent diffusion coefficient is relative bigger when pyrenediffused in the MePEG-PLA which has more MePEG fraction. Dialysis method wasapplied to study the in vitro drug release of pyrene-loaded MePEG-PLA nanoparticles.The release experiments were carried out under different conditions including in thepresence of phosphate buffer, plasma, base solution and lipase. The release data werefitted to various mathematical models including zero kinetic model, first kinetic model,Higuchi model and Power Law. The model fitting results indicated that between 0.5 and48 hours, the drug release behavior of pyrene-loaded nanoparticles in the presence ofphosphate buffer, plasma and base solution fitted first kinetic model, while between 0.5 and 12 hours, the drug release behavior of pyrene-loaded nanoparticles in the presence ofphosphate buffer fitted zero kinetic model and in the presence of plasma, the drug releasebehavior of pyrene-loaded nanoparticles fitted Higuchi model. The results suggested thatthe diffusion mechanism played a main role when the nanoparticles released in thepresence of phosphate buffer, plasma and base, while in the presence of lipase, thediffusion mechanism and degradation mechanism both controlled the drug release of thenanoparticles.(3) Methods for determination of total drug concentration and non-encapsulated drugconcentration in the biological samples including plasma, liver, spleen, lung and kidneywere studied and established, and the methods were comprehensively validated. Theresults showed that the method for determination of total pyrene concentration wassensitive, accurate and reproducible when methanol was used as protein precipitationagent to pretreat biological samples. When n-heptane was used as extractant to processbiological samples, the method has good specificity for it can separate the encapsulateddrug and non-encapsulated drug. The stability study results revealed that whendetermining the non-encapsulated drug concentration, the samples should be pretreatedafter thawing immediately, because the drug may release from the nanoparticles in theroom temperature.(4) Equilibrium dialysis method was applied to determine plasma protein binding rateof pyrene. When the concentration of pyrene was at 5, 25, 50 ng/mL, the correspondingaverage plasma protein binding rate were 25.1%, 40.0% and 62.7%, respectively. Theresults illustrated that the protein binding rate depends on the concentration of pyrene inplasma. The distribution of free pyrene and pyrene-loaded nanoparticles in blood wasstudied. The results showed that over 95% of the free pyrene was distributed into theerythrocyte, and the pyrene-loaded nanoparticles were less distributed in to the erythrocytethan free pyrene, but it was higher than 60%. The pharmacokinetics and biodistributionstudy were carried out in the Wistar rats and Kunming mice respectively, when pyrenesolution and pyrene-loaded nanoparticles were administrated by intravenous injection. Theresults revealed that when pyrene solution was administrated at a dose of 1 mg/kg, thevalues of the pharmacokinetic parameters V1, CL, K₁₀ and MRT were 17.555,10.87,0.619 and 1.493, but when pyrene-loaded nanoparticles were administrated at the same dose, the corresponding values of the pharmacokinetic parameters V1, CL, K₁₀ and MRTwere 23.999, 4.804, 0.205, 10.725 and 25.873, 5.452, 0.211, 8.018. The pyrene-loadednanoparticles had long circulating properties, and the non-encapsulated drug had adifferent pharmacokinetic behavior compared with that of the total drug. Thebiodistribution results also proved that pyrene-loaded nanoparticles showed a longcirculating property, and there existed difference of content change betweennon-encapsulated drug and total drug.In conclusion, the methods for determination of concentrations of different drugforms in biological samples were established, and the pharmacokinetics andbiodistribution of different forms drug were studied at the same time. The pharmacokineticcharacteristic of polymeric nanoparticles was disclosed to a certain degree by the studiespresented in this paper. The work described herein will provide a new strategy and methodfor the pharmacokinetic study of other nano drug delivery systems.