Formulation And Evaluation Of Astaxanthin-loaded Composite Micelles Via Coaxial Electrospray Technology

The development of water-insoluble drugs is a major challenge all over the worldwhile solubility is a key factor limiting bioavailability.Techniques commonly used to increase water-solubility include precursor drugs,cyclodextrins,surfactants,micronization,salt,crystalline screening,solid dispersions etc..Astaxanthin is the object of study in this paper.It was selected through preformulation study that the organic solvent system and pharmaceutical excipients applied to astaxanthin for solubilization and crystal suppression.And then coaxial electrostatic spray technique was used to prepare astaxanthin-loaded composite micelles.The technical process and formulation were optimized.Finally,the effect evaluation of astaxanthin-loaded composite micelles in vivo and antioxidant research in cell level were carried out.Chapter one:ReviewsIn this part,the property,pharmacological activities and safety of astaxanthin were reviewed.This part introduces the principle of electrospinning technology and the key parameters that must be controlled for the successful implementation of this technology.And then introduced the development of electrospinning technology and its application in pharmaceutical field,as well as its advantages in the formulation of insoluble drug amorphous solid dispersion.Moreover,the oral absorption of drugs was reviewed,including the absorption mechanism of gastrointestinal tract,the existing absorption obstacles and the physical and chemical factors influencing the absorption process.Finally,the construction idea of this topic was put forward.It mainly includes selecting pharmaceutical excipients applied to astaxanthin for solubilization and crystal suppression and the construction of the oral delivery system for astaxanthin by coaxial electrostatic spray technology and the study in vitro and in vivo.This topic was contributed to develop an oral delivery system that can be modeled for other insoluble drugs.Chapter two:Preformulation studiesIn this part,HPLC method for the determination of astaxanthin samples in vitro was established which had strong specificity,high precision,good repeatability and linearity in the concentration range from 0.5-10μg/m L,meeting methodological requirements.The solubility of astaxanthin in different solvents was determined and 50%-75%dichloromethane-methanol was selected as a mixed solvent for coaxial electrostatic spray.Lecithin was added into the inner layer of the prescription as absorption enhancer.The encapsulation efficiency as a major index,Soluplus and PVPVA64 were selected as the carriers of astaxanthin for enhancing the solubilization by using crystallization inhibition experiment.The critical micelle concentration（CMC）of polyethylene glycol grafted chitosan（PEG-g-CS）with different specifications was investigated to screen the outer layer materials of astaxanthin-loaded composite micelles.Chapter three:Preparation and characterization of astaxanthin-loaded composite micellesIn this part,astaxanthin-loaded composite micelles were prepared by coaxial electrostatic spray technology,and the process parameters and formulation were optimized.The optimized process parameter was that voltage of 18 KV,receiving distance of 15 cm,velocity of 0.3 m L/h in inner layer and outer layer solution,other factors such as temperature 25℃,35%relative humidity.The optimized formulation was that the inner layer:astaxanthin 15.0 mg,lecithin 27.5 mg,Soluplus 175.0 mg and PVPVA64 175.0 mg.In the outer layer:PEG-g-CS 25.0 mg,PEO 10.0 mg.The saturated concentration of astaxanthin in water is only 0.08735μg/m L but the significantly improved apparent concentration of astaxanthin in astaxanthin-loaded composite micelles prepared by coaxial electrostatic spraying technology could reach10942.13μg/m L in water.The astaxanthin-loaded composite micelles possess an average particle size 186.28±82 nm in water,with a good stability and a Zeta potential of-7.10±10 m V and the encapsulation efficiency of 93.96%and the astaxanthin loading rate of 3.38%.The results of scanning electron microscopy showed that the The astaxanthin-loaded composite micelles were regular round microspheres with relatively uniform distribution.The results of XRD,DSC and ATR-IR showed that astaxanthin changed from crystalline form to amorphous form and was highly dispersed in the polymer carrier.Finally,the results of in vitro dissolution study showed that the astaxanthin-loaded composite micelles could be rapidly dissolved in water,and the dissolution rate in water was as high as 94.08%within 2 hours.Chapter four:Pharmacokinetics and tissue distribution of astaxanthin-loaded composite micellesIn this part,a HPLC method was established for the analysis of astaxanthin in vivo.The plasma samples of rats showed a good linearity in the concentration of astaxanthin range from 0.04 to 1μg/m L and the different tissue samples of mice showed a good linearity in the concentration of astaxanthin range from 0.05 to 2μg/m L.The analytical results showed that the specificity and intra-day and inter-day precision of the method were good,and the recovery of the method were 95%with RSD less than 5%.All of the results meet methodological requirements.The results of pharmacokinetic in rats showed that the bioavailability of astaxanthin-loaded composite micelles in rats was significantly higher than that of astaxanthin API.The C_max of the formulation group could reach 311.75 ng/m L,which was more than 5 times of the concentration of API,and the relative bioavailability of API reached 308.33%.The results showed that the absorption degree of astaxanthin-loaded composite micelles in rats was much higher than that of astaxanthin API.Different tissue distribution in mice results show that astaxanthin-loaded composite micelles solves the problem of poor water solubility and low bioavailability.After astaxanthin API administrated in mice,the concentration of astaxanthin is low and even not measured but the concentration of astaxanthin after administrated with formulation in various tissues were much higher.The concentration of astaxanthin reached highest levels of 12.28 mg/kg.It is worth mentioning that astaxanthin-loaded composite micelles could improve the distribution of astaxanthin in mice brain.Chapter five:Cell uptake and antioxidant activity of astaxanthin-loaded composite micellesIn this part,the cytotoxicity of astaxanthin API and astaxanthin-loaded composite micelles,as well as the antioxidant effect at the cellular level,the uptake of astaxanthin API and astaxanthin-loaded composite micelles were evaluated with Caco-2 cells.Using H₂O₂ as oxidative damage substance,the relative cell viability and the scavenging effect of reactive oxygen species（ROS）pretreated with astaxanthin and astaxanthin-loaded composite micelles were evaluated.The results showed that astaxanthin API and astaxanthin-loaded composite micelles had no obvious cytotoxicity in the concentration of astaxanthin range of 0.5-10μg/m L.In addition,astaxanthin-loaded composite micelles could significantly improve the uptake of astaxanthin in Caco-2 cells.After administration of astaxanthin-loaded composite micelles,the oxidative damage of H₂O₂ in Caco-2 cells was significantly prevented,and the cell survival rate increased from 65.35%to 85.17%,while the cell survival rate of VC group was 77.86%,which proved that astaxanthin-loaded composite micelles had a better anti-hydrogen peroxide damage ability than Vc group.Finally,the ROS scavenging effect of astaxanthin-loaded composite micelles was comparable to that of Vc but significantly higher than that of free astaxanthin,which was consistent with the result of resistance to hydrogen peroxide damage study.