The Effect Of 4-HPR-loaded Lipid Microbubbles Combined Ultrasonic Therapy On Keloid And NF-κB Signaling Pathway

Objective To prepare N-(4-hydroxyphenyl) retinamide-loaded lipid microbubbles (4HPR-LM) and investigate the effect of 4-HPR-LM combined with ultrasound (US) on keloid, and elucidate the role of NF-kB signaling pathway.Methods 4-HPR-loaded liposomes (4HPR-L) were first prepared by film-ultrasound dispersion method, and then drug loaded lipid microbubbles were manufactured from mixtures of 4-HPR-L and perfluoropentane (PFP) by ultrasonic cavitation method. Single-factor test and Orthogonal Test were employed to optimize the preparation conditions and prescription of 4-HPR-L. The pharmacy characters, such as morphology, sizes, Zeta potentials, entrapment efficiency,4-HPR loading, temperature and ultrasonic sensitivity, were evaluated by dynamic light scattering (DLS), transmission electron microscopy (TEM), and high-performance liquid chromatograph (HPLC), respectively. MTT essay were used to conduct a preliminary screening for concentrations of the drug; Cell Proliferation Assay were employed to evaluate the inhibitional effect of free 4-HPR,4-HPR-L,4-HPR-LM and 4-HPR-LM combined with ultrasound on Human Keloid Fibroblasts (HKFs); Quantitative analysis and morphological evaluation of apoptosis induced by 4-HPR and 4-HPR-LM combined with ultrasound were examined by AnnexinV-FITC/PI dual staining and DAPI staining. For further study the effect of ultrasound mediated 4-HPR-LM in the treatment of keloid, RT-PCR and Western Blotting were used to detect the impact of 4-HPR on the expression of type I collagen in HKFs. The abnormal expression of NF-kB signaling pathway related molecules in keloid tissue was also observed, and the mechanism of therapeutic effect of 4-HPR in the treatment of keloid was also discussed. HKFs were inoculated and tumor masses were transplanted subcutaneously into BALB/c nude mice to establish xenograft keloid model, then the growth, formation rate, histopathological and immunohistochemical of the tumors were analyzed.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 μm filtration membrane was 6. The optimum prescription was EPC (28 mg), Chol (2.5 mg), mPEG-DSPE2000 (3 mg),4HPR (3 mg). The TEM image showed 4-HPR-LM was spherical or approximately spherical shape visually and the mean particle sizes. The mean particle sizes, Zeta potentials, entrapment efficiency and 4-HPR loading of 4-HPR-LM were 110.80±3.27 nm,-45.50±1.10 mV,96.48±1.05% and 8.01±0.33%, respectively.4-HPR-LM was sensitive to temperature and ultrasound which could help drug release in vitro and in vivo under ultrasound irradiation. MTT essay showed that 4-HPR-L could meaningfully inhibit the growth of HKFs in a concentration and time dependent manner. Compared with 4-HPR-L, free 4-HPR had higher cell proliferation inhibition rate (P<0.05). Furthermore,4-HPR-LM combined with ultrasound have significantly stronger inhibition than free 4-HPR and 4-HPR-L group at half maximal inhibitory concentration, the difference between two groups are significant (P<0.01). Cell apoptosis could be induced more efficacious by 4-HPR-LM combined with ultrasound than 4-HPR (P<0.01).4-HPR can inhibit the expression of collagen type I alpha 1 (COL1A1), and the mRNA and protein expression levels of COL1A1 were significantly decreased in 4-HPR-LM combined with ultrasound group. We also observed that 4-HPR-LM combined with ultrasound suppress the abnormal expression of NF-kB signaling pathway related molecules in HKFs. All mice were in good state, no significant difference in body weight, no significant adverse reactions. When HKFs were inoculated with the concentration of 1.0×106/100 μL and 3.0 × 106/100 μL, the tumors formation time was up to 90 days and 60 days.4 weeks after the tumor formation, the life status of nude mice has gradually decreased and tumor growth was slow. When HKFs were inoculated with the concentration of 5.0 × 106/100 μL and 7.0 × 106/100 μL, the average time of tumors formation was 50 days and 40 days, the tumor growth rate of roughly the same, no uncontrolled growth and tumor necrosis was found. They are more appropriate inoculation concentrations which were conducive for use in animal models of keloid.Conclusions 4-HPR-L had high encapsulation efficiency and good stability and significantly enhanced 4-HPR solubility in aqueous media, which laid a good foundation for the further study of 4-HPR. The further preparation 4-HPR-LM combined with ultrasound could effectively inhibit cell proliferation and induce apoptosis.4-HPR-LM combined with ultrasound could improve the imbalanced biosynthesis of various types of collagen in HKFs, reduce the generation and accumulation of type I collagen.4-HPR-LM combined with ultrasound may play a role in treatment of keloid by regulating of NF-κB signaling pathway. In this study, we have successfully established keloid fibroblasts inoculation model and tumor masses xenograft model, and provide evidence for a further study of the effect of 4-HPR-loaded lipid microbubbles combined ultrasonic therapy on keloid in vivo.4-HPR-LM combined with ultrasound 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.