| Objective To prepare N-(4-hydroxyphenyl)retinamide-loaded lipid microbubbles(4HPR-LM)and evaluate the performance of 4HPR-LM combined with ultrasound(US)on keloid.Methods 4HPR-loaded liposomes(4HPR-L)were first prepared by film-ultrasound dispersion method,and then drug loaded lipid microbubbles were manufactured from mixtures of 4HPR-L and perfluoropentane(PFP)by ultrasonic cavitation method.Single-factor test and Orthogonal Test were employed to optimize the preparation conditions and prescription of 4HPR-L.The pharmacy characters,such as morphology,sizes,Zeta potentials,entrapment efficiency,4HPR loading,Temperature and ultrasonic sensitivity,were evaluated by transmission electron microscopy(TEM),dynamic light scattering and high-performance liquid chromatograph(HPLC),respectively.MTT essay were used to conduct a preliminary screening for ultrasound parameters in vitro;cellular uptake of Coumarin 6-lipid microbubbles were observed by Confocal Laser Scanning Microscopy;Cell Proliferation Assay were employed to evaluate the biocompatibility of Blank-loaded lipid microbubbles(Blank-LM)and the inhibotional effect of free 4HPR,4HPR-LM and 4HPR-LM combined with ultrasound to Human Keloid Fibroblasts(HKFs);Quantitative analysis and Morphological evaluation of apoptosis induced by free 4HPR and 4HPR-LM combined with ultrasound were examined by AnnexinV-FITC/PI dual staining and Hoechst33258 staining.In vivo study was assessed in BALB/c nude mice bearing subcutaneous xenograft keloid model through intralesional injections.The safety of free 4HPR and 4HPR-LM combined with ultrasound were observed at the same time.Results The optimum preparation conditions were obtained as follow:chloroform was 2 ml;the time,temperature and speed of rotary evaporation were 30 min,50 and 120 rpm/min,respectively;5%glucose solution was 8 ml;rotating time of hydration was 20 min;ultrasound time was 5 min;times of passing through 0.22?m filtration membrane was 7.The optimum prescription were EPC:Chol(w:w)(11:1),mPEG-DSPE2000(3 mg),4HPR(3 mg).The TEM image showed 4HPR-L were spherical or approximately spherical shape visually and the mean particle sizes and Zeta potentials were 122.87±5.06 nm and-43.80±1.00 mV respectively.Afterwards,the mean particle size and Zeta potential of 4HPR-L,which were stored at 4? for 30 days,had no obvious changes and were stable in short term.The leakage rates were 1.30±0.89%,3.49±0.19%,5.93±1.31%at 7,15 and 30 d,respectively.The mean particle sizes,Zeta potentials,entrapment efficiency and 4HPR loading of 4HPR-LM were 113.80±3.27 nm,-35.50±1.10 mV,94.48±1.05%and 8.31±0.33%respectively.4HPR-LM were sensitive to temperature and ultrasound which could help drug release in vitro and in vivo under ultrasound irradiation.As a result of MTT essay,ultrasound parameters were set as 3 MHz,0.7 w/cm2,60 s in vitro study;The reuslts of CLSM indicated that lipid microbubbles could lead a well cellular internalization ability in 10 minutes,which could be obviously enhenced by ultrasound irradiation.Cell Proliferation Assay showed that the inhibition of HKFs incubated with Blank-LM were all less than 9.67%in our experimental concentration range,which indicated that materials of lipid microbubbles had good biocompatibility as a drug-delivery material.Both 4HPR and 4HPR-LM could meaningfully inhibited the growth of HKFs in a concentration and time dependent manner.Compared with free 4HPR,4HPR-LM had higher cell proliferation inhibition rate(P<0.05).Furthermore,4HPR-LM+US group have significantly stronger inhibitition than free 4HPR and 4HPR-LM group at all experimental concentration and time points,the difference between two groups are significant(P<0.01).Cell apoptosis could be induced more efficacious by 4HPR-LM combined with ultrasound than free 4HPR(P<0.01).4HPR-LM combined with ultrasound also led higher anti-keloid rates than free 4HPR(P<0.01).The HE staining of heart,liver,spllen and kidney did not show obvious pathological change.During our study,these mice's general physical status were well and the body weight had no obvious difference and no obvious adverse reaction occurred.Conclusions 4HPR-L had high encapsulation efficiency and good stability and significantly enhanced 4HPR solubility in aqueous media,which laid a good foundation for the further study of 4HPR.The further preparation 4HPR-LM combined with ultrasound could effectively inhibit cell proliferation,induce apoptosis and inhibit the growth of keloid in vitro and in vivo,which could be considered as a promising drug targeting delivery system for clinic and supplied the possibility of a novel therapeutic technology for keloid treatment.It is worth of further study. |
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