Method Research Of Encapsulation Efficiency And In Vitro Release Of Albumin Nanoparticles

Albumin is the most abundant protein in plasma,its good biocompatibility,biodegradability,no immunogenicity and other properties makes it to be a good carrier of nano-formulations.In addition to the traditional EPR effect,which is based on particle size and tumor vascular endothelial defects,albumin nanoparticles have active targeting to tumor cell.Albumin can be combined with gp60 which is highly expressed in tumor blood vessels,it penetrates into tumor cells intercellular space by concave,and then combines with SPARC which is highly expressed in tumor cells to transfer to intracellular.At present,PTX albumin nanoparticles(Abraxane(?))has been successfully developed by nabTM technology platform and sales good on market.Based on the development of the same technology platform,the DTX albumin nanoparticles and rapamycin albumin nanoparticles had been entered into clinical trials.Due to albumin nanoparticles' drug delivery advantages and antitumor efficacy,our laboratory adopted the patent technology "protein unfolding/refolding" to prepare DTX albumin nanoparticles?PTX albumin nanoparticles and carried on quality research.Encapsulation efficiency(E E)and in vitro release of nanoparticles is an important index to evaluate the quality of the nanoparticles,but there is no pharmacopoeia quality about encapsulation efficiency and in vitro release of nanoparticles exists,it seriously restrictes the progress of safety and product research and development of nanoparticles.This article mainly facus on method research of entrapment efficiency and in vitro release.In the first part,we established EE method of PTX albumin nanoparticles and carried out method validation.We used column chromatography to separate the encapsulated drug and free drug of PTX albumin nanoparticles,and the concentration of PTX was detected by high performance liquid chromatography(HPLC).In the method,sephadex G50 was used as filler,and the eluent was water,isopropanol:water:acetic acid=100:100:1,isopropyl alcohol:acetic acid=200:1.The encapsulated drug was eluted with 30ml pure water and then the free PTX in the gel column was eluted with the acidified organic phase.The average encapsulation efficiency of PTX albumin nanoparticles was 99.8%.In method validation,LOQ of PTX was 0.2070?g/ml.Linear range of PTX was 0.2070?31.0486?g/ml,the recovery rate of free drug was within 90%-110%,and the method was durable when the column length,eluent volume,detection of wavelength and other conditions had small changes,it indicated that this method was sensitive,accurate and feasible.In the second part,we established in vitro release method of PTX albumin nanoparticles and carried out method validation.Dialysis method was used to detect the in vitro release of PTX albumin nanoparticles.we chose intelligent dissolution tester,50rpm/min,100KD dialysis bag and 5%human serum albumin as the release medium,sample concentration was 200?g/ml,the volume of the medium was 100ml,we sampled at 0.5h,2h,4h,6h,8h,12h and then detected the concentration of PTX by HPLC.We verified the method from specificity,system applicability,linearity,precision,solution stability,durability and other aspects.In this method,release media?diluent had no interference with PTX detection,PTX in the range of 0.05881?1.76441?g/ml had a good linearity,small changes in release medium volume and concentration had no effect on PTX detection,it indicated that the method is good,sensitive and accurate,the method can be used for the detection of in vitro release of PTX albumin nanoparticles.In the third part,we have used the gel column chromatography,micro column centrifugation and ultrafiltration centrifugation to separate nanoparticles with free DTX to detect EE of DTX albumin nanoparticles.Using Gel column chromatography,we screened the packing from Sephadex G50?G25?G15?LH20 to Sephora G50HP?Sephora G75HP,screened the pH value of the eluent,screened the drug concentration and sample concentration,etc.but we have failed to separate the nanoparticles with free drug.Using Micro column centrifugation,we screened the micro column column length,sample volume and the centrifugal rotational speed,the EE of DTX nanoparticles was slightly lower,and there was a portion of free drug detected in entrapped drug using this method.In the subsequent ultrafiltration method of experiment,the ultrafiltration membrane material had strong adorption with DTX,nanoparticle samples would clogge the membrane material in the process of centrifugal and effected the result.To solve these two problems,I used ethanol to dissolve DTX and then used beta cyclodextrin to solubilize as a solution,and let nanoparticles centrifugal first and then took the supernatant to ultrafiltrate to test EE,I also deteced the sample recovery rate.Using this method,the EE of the DTX albumin nanoparticles was 90.73%,and the recovery rate of the free drug was 85.89%,and the sample recovery rate was 90.23%.Above all,we established method of encapsulation efficiency and in vitro release of PTX albumin nanoparticles and carried out method validation,we also developed encapsulation efficiency method of DTX albumin nanoparticles.It offered method support of nanoparticles quality research,and it was of great significance of the process optimization and quality control.