Constrution Of Self-assembled Nanoemulsions And Their Applications In Paclitaxel Delivery

According to statistical datas,hydrophobic drugs in the existing drug delivery systems account for 40%-60%.In the newly discovered active compounds,the proportion of hydrophobic compounds is about 70%.In addition,40% of the active compounds have been abandoned because of their poor water solubility.Improving the solubility and finding the proper dosages for hydrophobic drugs are the most urgent problems in pharmaceutics.Paclitaxel(trade name Taxol)is widely used as a first-line drug for the treatment of advanced ovarian cancer in clinic.Cremophor EL is used to improve the solubility of paclitaxel.However,Cremophor EL can cause the hypersensitivity reactions of human body,and therefore antianaphylactic treatment should be taken before injection of Taxol.Accordingly,the step of pretreatment is very tedious,and the consequence of the tedious steps is the low compliance of the patients.Therefore,the clinical value is very great to solve the problem through other delivery systems.A large number of studies show that the nanoemulsion drug delivery system can increase the solubility of hydrophobic drugs.Nanoemulsions can protect drugs against oxidation and hydrolysis,increase drug stability,increase drug retention in gastrointestinal sites and improve the bioavailability of hydrophobic drugs.As a result,nanoemulsions are the ideal dosage forms for insoluble drugs which have a low oral bioavailability.At present,the preparation of nanoemulsions needs a large amount of emulsifiers and co-emulsifiers which can cause some potential safety problems.Moreover,high energy dispersion methods must be used to obtain with small size.Based on the above problems,this work proposed a self-assembly method for the fabrication of nanoemulsions,which is used as a new delivery system for the hydrophobic drug paclitaxel to improve its solubility and bioavailability.To this end,we synthesized a hydrophilic block copolymer PEG-P[Asp(PPA)CD],with one block of polyethylene glycol(PEG)and another block containing ?-cyclodextrin(?-CD).We use the host-guest interaction between ?-CD and benzyl alcohol(BA)to fabricate oil in water(O/W)nanoemulsions through a self-assembly method.Also,we can use the paclitaxel containing BA solution as the oil phase to fabricate a paclitaxel loaded nanoemulsion.When we use the self-aseembly method to fabricate nanoemulsion,high concentration of emulsifier and co-emulsifier are avoided.Moreover,the preparation process can be very sample,and pretreatment before injection can be removed.Furthermore,the self-assembled drug-loaded nanoemulsion can effectively improve the oral bioavailability and the efficacy of paclitaxel,while reduce side effects.Methods1.Synthesis of a hydrophilic block copolymer PEG-P[Asp(PPA)CD]L-Aspartic acid benzyl ester was activated by triphosgene,and then copolymerized with 5 k Da amino terminated PEG to obtain polyethylene-glycol-block-poly-(?-benzyl L-aspartate)(PEG-PBLA),PEG-P[Asp(PPA)CD]was obtained by reaction of propargyl amine,and finally the hydrophilic copolymer PEG-P[Asp(PPA)CD] was synthesized by the 'click' chemistry reaction between mono-6-zaido-?-CD and PEG-P[Asp(PPA)],The hydrophilic copolymer was characterized by FTIR and NMR.2.Construction and characterization of the self-assembled nanoemulsionsThe water phase is PEG-P[Asp(PPA)CD] aqueous solution,the oil phase is benzyl alcohol.Fabrication of nanoemulsions is quite simple.Briefly,oil phase and water phase were mixed by vortexing to obtain self-assembled nanoemulsions through host-guest interaction between ?-CD and benzyl alcohol at the oil-water interface,the effects of different oil volumes on nanoemulsion formation was investigated.When the drug loaded nanoemulsion was prepared,the benzyl alcohol solution with pa clitaxel was dispersed in PEG-P[Asp(PPA)CD] aqueous solution.The morphology was characterized by confocal laser scanning microscopy,and the particle size,particle size distribution and polydispersity index(PDI)were measured by a laser particle size analyzer.3.Study on the stability of drug-loaded nanoemulsionsThe drug-loaded self-assembled nanoemulsion was stored at 4 ? or 37 ?.To evaluate the stability of self-assembly drug-loaded nanoemulsions,at different time point particle size and PDI were determined,changes in the drug content were detected by high performance liquid chromatography(HPLC).In addition,the change of nanoemulsion appearance was observed.4.The release of self-assembled drug-loaded nanoemulsions in vitroIn vitro release of drug-loaded self-assembled nanoemulsions was evaluated by a dialysis method.The PBS buffer of p H 7.4 or the simulated gastrointestinal tract p H conditions were used as the release medium.Samples were collected at different time points,and the drug content was measured by HPLC.5.The in vitro antitumor activity of drug-loaded self-assembled nanoemulsionHuman lung cancer cells A549 and murine melanoma cells B16F10 w ere incubated with different concentrations of drug-loaded nanoemulsions for 24 h.The cell viability was determined by MTT method to evaluate antitumor activity of self-assembled drug-loaded nanoemulsions.6.Study on in vivo pharmacokinetics of drug-loaded self–assembled nanoemulsionsSD rats were given with drug-loaded self –assembled nanoemulsions by intravenous injection or oral administration.The blood samples were collected at different time points,and the concentration of paclitaxel in blood was detected by liquid chromatography coupled with mass spectrometry(LC-MS).The pharmacokinetic parameters were analyzed by DAS software.7.Study on in vivo antitumor efficacy of drug-loaded self-assembled nanoemulsionsThe A549 transplanted tumor model in nude mouse was established.Drug treatment was performed when the tumor grew up to approximately 150 mm3.The changes of body weight and tumor volume were recorded.After 14 days,tumor tissues and typical organ were collected and weighted to calculate the organ index.Hematoxylin and eosin(H&E)staining was used to analyze the main pathological sections.Routine blood tests and the main biochemical levels were measured.8.Evaluation of acute toxicity of drug-loaded self-assembled nanoemulsionsA single dose of drug-loaded self-assembled nanoemulsions was injected in Kunming mice.The changes of body weight were recorded every 3 days.After 14 days,mice were killed and the main organs were collected to calculate organ index.H&E staining was used to analyze the main pathological sections.Typical hematological parameters and the biochemical makers were measured.Results1.A hydrophilic block copolymer PEG-P[Asp(PPA)CD] was synthesized.The NMR characteristic absorption peak and infrared absorption curve confirmed the expected product.2.Self-assembled o/w nanoemulsions mediated by the host-guest interaction were fabricated.The average particle size was 12 nm and the PDI was 0.26.Fluorescence microscope showed self-assembled nanoemulsions had a clear edge and homogeneous spherical appearance.3.The stability test for two months showed that there was no significant change in the particle size,PDI,and drug content of the drug-loaded self-assembled nanoemulsions.4.The cumulative release from the drug-loaded nanoemulsions in 24 h was 81.8% at p H 7.4,and 77.9% under the simulating gastrointestinal p H conditions.The cumulative release in the first 4 h was 74.3% at p H 7.4 and 62.3% under the p H conditions of simulating the gastrointestinal environment.5.The in vitro antitumor activity of drug-loaded self-assembled nanoemulsions is great.The cell viability of A549 was 22.6% after incubation with drug-loaded self-assembled nanoemulsions for 24 h,while cell viability of B16F10 cells was about 10% after incubation with self-assembled nanoemulsions for 24 h.6.The plasma concentration of paclitaxel in rats was quite high after oral administration or intravenous injection of drug-loaded nanoemulsions.The intravenous injection group had higher maximal plasma concentrations than the oral administration group.The highest blood concentration after oral administration was 4.22 ?g/m L,while it was16.22 ?g/ml after intravenous injection.7.The drug-loaded self-assembled nanoemulsions can inhibit the growth of A549 xenograft tumor either though oral administration or intravenous injection.Therefore,this paclitaxel-loaded nanaoemulsion exhibited good anti-tumor activity.8.The drug-loaded self-assembly nanoemulsion showed good safety profile in vivo with low side effects.ConclusionsA hydrophilic block copolymer PEG-P[Asp(PPA)CD] was synthesized.The self-assembled nanoemulsion was fabricated through a host-guest mediated self-assembly method to deliver a hydrophobic drug paclitaxel.The paclitaxel-loaded self-assembled nanoemulsion showed good stability and release rate in vitro.Furthermore,this self-assembled nanoemulsion can effectively improve the oral bioavailability of paclitaxel.Also,it can notably inhibit the growth of tumor in vivo.The treatment did not cause significant side effects.Preliminary safety evaluation showed that this nanoemulsion formulation had good safety in vivo.