Objective To evaluate the effect of spicy foods on allergic contact dermatitis caused by cinnamic aldehyde.Methods Sixty mice were equally divided into 3 groups,i.e.,blank control group,cinnamic aldehyde group and spicy food+cinnamic aldehyde group.Spicy foods (including alcohol and bush redpepper fruit) were intragastrically given for 7 days to induce irritation in the mice of spicy food+cinnamic aldehyde group.Then,the mice of innamic aldehyde group and spicy food +cinnamic aldehyde group were challenged by cinnamic aldehyde on the skin.The difference in allergic reaction intensity was compared between the cinnamic aldehyde group and spicy food +cinnamic aldehyde group.Results After 7-day intragastric administration of spicy foods,increased scores of symptom and sign were observed in the spicy food+cinnamic aldehyde group compared with the blank control group receiving intragastric sodium chloride physiological solution (P ＜ 0.01 ).Intense allergic reactions were induced by cinnamic aldehyde,and the reaction intensity score was significantly higher in mice irritated with spicy foods than in those with sodium chloride physiological solution at 24 and 48 hours after the challenge,but was similar at 72 hours between the two groups regardless of a significant difference in pathological score (P ＜ 0.05).Conclusions The mixture of alcohol and bush redpepper fruit at the concentration used in this experiment could induce irritation in mice,and increase the intensity of allergic reaction caused by cinnamic aldehyde.

Objective To estimate the influence of pepper and alcohol on irritant contact dermatitis induced by cinnamaldehyde in rabbit ears.Methods Thirty-two Japanese large-ear white rabbits were used in this study,and equally divided into 4 groups to receive intragastric infusion of sodium chloride physiological solution twice daily (control group),sodium chloride physiological solution once and 50％ alcohol solution once daily (alcohol group),10％ pepper solution once and sodium chloride physiological solution once daily (pepper group),50％ alcohol solution once and 10％ pepper solution once daily (mixture group),for 7 days.The dose of these solutions for intragastric infusion was uniformly 8 ml/kg.After the appearance of symptoms of irritant contact dermatitis (including increase in water intake,dry stool,vasocongestion of auricle of ears),the back skin of 4 mice in each group was injured and served as the injured skin subgroup,and the back skin of the other 4 mice in each group remained uninjured and served as the intact skin subgroup.Then,skin irritation test was carried out according to the Hygienic Standard for Cosmetics on both sides of the back of these rabbits with 2％ cinnamaldehyde (irritant area) and 70％ alcohol (control area) respectively.Skin reaction at the irritated sites was observed and scored at 1,24,48 and 72 hours after the irritation.Analysis of variance was conducted to assess the differences in reaction intensity between these groups.Results After 7 days of intragastric infusion,the symptom score was 0.25 ± 0.46 in the control group,significantly lower than that in the pepper group (5.38 ±0.74,P＜ 0.01),alcohol group (7.25 ± 0.71,P＜ 0.01) and mixture group (12.75 ± 0.70,P＜ 0.01).In rabbits with intact skin,the intensity of irritant skin reaction was significantly stronger in the mixture group than in the pepper group at 24 and 48 hours (F =28.44,30.33,respectively,both P ＜ 0.05),while in rabbits with injured skin,the irritant skin reaction was more intense in the alcohol group and mixture group than in the pepper group at 24,48 and 72 hours (F =197.12,94.54,87.63,respectively,all P ＜ 0.01).Conclusions Pepper and alcohol alone or in combination at the tested concentration can induce irritation symptoms in rabbits,and both of them can enhance the response of skin to irratation by cinnamaldehyde.

Objective To estimate the effect of leptin antibody on transforming growth factor (TGF)-β1 mRNA expression in hypertrophic scar model in rabbit ears.Methods Fifteen New Zealand white rabbits were included in this study.Three circular incisions which measured 7 mm in diameter and reached the perichondrium,were made in each ear of these rabbits to establish 90 models of hypertrophic scar.After the operation,these models were randomly and equally divided into 3 groups to be treated with topical sodium chloride physiological solution for 40 days (saline group),topical leptin antibody of 2 ng/ml for 40 days (leptin antibody group),and topical leptin antibody of 2 ng/ml for 14 days followed by injection of leptin antibody of 2 ng/ml once a week for 3 weeks (combination group).Scar tissue was resected from these rabbit ears at 40 days after the operation,followed by the determination of scar elevation index,histopathological examination by using haematoxylin and eosin staining,and quantification of TGF-β31 mRNA expression by real-time fluorescence-based PCR.SPSS 13.0 software was used for data processing.Statistical analysis was carried out by one-way analysis of variance.Results A significant decrease was observed in scar elevation index (2.33 ± 0.33 and 2.35 ± 0.22 vs.3.33 ± 0.41,both P ＜0.05) and TGF-β1 mRNA expression in the leptin antibody group and combination group compared with the control group,whereas no significant difference was observed between the leptin antibody group and combination group in either of the two parameters.Pathologically,there was an apparent proliferation of capillaries in the saline group with numerous irregularly and densely arranged fibroblasts with large nuclei,while relatively few fibroblasts with small nuclei,which were arranged in a more regular way,were observed in the leptin antibody group and combination group.Conclusion Leptin antibody treatment can reduce the expression of TGF-β1 mRNA in hypertrophic scar tissue in rabbit ears.

Objective To evaluate effects of 4-hydroxyphenyl retinamide (4-HPR) in different vehicles on the proliferation and apoptosis of human keloid fibroblasts (HKFs).Methods A film-ultrasonic dispersion method was used to prepare 4-HPR liposome solution and 4-HPR microbubbles.Primary HKFs were in vitro treated with the 4-HPR liposome solution at different concentrations of 0-80 mg/L for 6-48 hours,and the proliferative activity of HKFs was evaluated by methyl thiazolyl tetrazolium (MTT) assay.Some other HKFs were divided into 3 experimental groups to be treated with 15 mg/L 4-HPR solution (4-HPR solution group),15 mg/L 4-HPR liposome solution (4-HPR liposome solution group) and 15 mg/L 4-HPR microbubbles (4-HPR microbubble group),respectively,and each group was divided into ultrasonic-treated and-untreated subgroups.HKFs without treatment served as control group.After 24-hour treatment,MTT assay was conducted to evaluate the proliferative activity of HKFs in the above groups,flow cytometry to detect apoptosis of HKFs in all groups except the 4-HPR solution group.Results The 4-HPR liposome solution and 4-HPR microbubbles were successfully prepared.MTT assay showed inhibitory effects of 4-HPR liposome solution at concentrations of 1-80 mg/L on the proliferation of HKFs,and the proliferation inhibition rate was positively associated with the drug concentrations (r =0.633,P ＜ 0.01).After the ultrasonic treatment,inhibitory effects on the proliferation of HKFs significantly differed among the 4-HPR microbubble group,4-HPR solution group and 4-HPR liposome solution group (P ＜ 0.01).The 4-HPR liposome solution group and the 4-HPR microbubble group both showed significantly increased apoptosis rates (21.81％ ± 3.73％,39.79％ ± 1.61％,respectively) compared with the control group (6.18％ ± 0.61％,both P ＜ 0.01).Conclusion The 4-HPR microbubbles are successfully prepared,and 4-HPR in different vehicles all can promote HKF apoptosis and suppress HKF proliferation,among which,4-HPR microbubbles in combination with ultrasonic treatment have stronger inhibitory effects than the 4-HPR liposome solution.

Objective To establish a simple and efficient method for developing a keloid model in nude mice with human keloid-derived fibroblasts.Methods Twenty-seven female BALB/c nude mice were randomly divided into five groups with 5,5,5,8 and 4 mice in group A,B,C,D and E respectively.The mice in group A,B and C were inoculated with 0.1 ml of suspension containing human keloid-derived fibroblasts at concentrations of 1.0 × 104,3.0 × 104 and 5.0 × 104 per microliter Matrigel,respectively,at the right axillary fossa.The tumors that formed in one mouse in group C were taken out,and cut into several parts measuring 5 mm × 5 mm × 5 mm in size,which were then subcutaneously transplanted into the right axillary fossa of mice in group D.The mice in group E were subcutaneously injected with 100 μl of Matrigel and served as the control group.The formation of tumor in mice was observed by naked eyes,and the size of tumors was measured until day 30 after tumor formation in group A,B and C as well as after tumor transplantation in group D.Mice were sacrificed on day 30 after tumor formation,and histopathologic examination was performed to analyze histological features of transplanted tumors and pathological changes in visceral organs such as heart,liver,spleen,lung and kidney.Results The tumor formation rate was consistently 100％ in group A,B and C,and the time required for tumor formation was (90.20 ± 3.96),(61.00 ± 2.92) and (39.60 ± 3.20) days in group A,B and C respectively.There was a significant difference in tumor volume on the 30th day after tumor formation between group A,B and C ((288.34 ± 25.29) vs.(1 370.63 ± 105.24) vs.(1 940.98 ± 184.37) mm3,F =138.74,P ＜ 0.05).The size of implanted tumor mass in group D firstly increased,then gradually decreased,but began to continuously increase since the 14~ day,and tumor finally formed in 7 out of 8 mice.There was no evidence of tumor formation in group E.Histopathologic examination showed uniform histological manifestations,which were similar to those of human scar,in tumor tissues from mice in group A,B,C and D.Neither pathological changes nor metastases were observed in visceral organs of these mice.Conclusion Keloid-bearing nude mouse model can be established by subcutaneous inoculation with human keloidderived fibroblasts,or by subcutaneous transplantation of tumor masses of a certain size that have formed in nude mice.

Objective: To explore the effects of N-(4-hydroxyphenyl) retinamide (4HPR), 4HPR liposome (4HPR-L), and 4HPR lipid microbubble (4HPR-LM) combined with ultrasound on proliferation, apoptosis, and cell cycle of human keloid fibroblasts (Fbs). Methods: (1) 4HPR-L and 4HPR-LM were prepared by hydration ultrasonic method. The appearance morphology, particle size distribution, Zeta potential, loading drug concentration, encapsulation efficiency, and drug loading rate of 4HPR-L were investigated by high performance liquid chromatography, dynamic light scattering, and transmission electron microscope. (2) Human keloid Fbs were cultured and divided into 13 groups by random number table (the same grouping method below), with 6 wells in each group. Cells in control group were given no treatment, while cells in 12 ultrasound groups including 0.5 W 30 s group, 0.5 W 60 s group, 0.5 W 120 s group, 0.7 W 30 s group, 0.7 W 60 s group, 0.7 W 120 s group, 1.0 W 30 s group, 1.0 W 60 s group, 1.0 W 120 s group, 1.5 W 30 s group, 1.5 W 60 s group, and 1.5 W 120 s group were treated by ultrasound with corresponding parameters. The cells viability was measured by a microplate reader after 24 hours of routine culture. Another batch of human keloid Fbs were divided into 5 groups, with 6 wells in each group. Cells in control group were given no treatment, while cells in 1, 10, 20, and 50 μg/mL blank lipid microbubble groups were treated with blank lipid microbubbles in corresponding mass concentration. The cells viability was measured as before after 24 hours of routine culture. Another batch of human keloid Fbs were divided into 6 groups, with 12 wells in each group. Cells in control group were given no treatment, while cells in 1, 10, 20, 50, and 100 μg/mL 4HPR-L groups were added with 4HPR-L carrying corresponding mass concentration of 4HPR. The cells viability in 6 wells of each group was detected after 24 and 48 hours of routine culture, respectively. Another batch of human keloid Fbs were divided into 4 groups, with 6 wells in each group. Cells in control group were given no treatment, while cells in 4HPR, 4HPR-L, and 4HPR-LM+ ultrasound groups were treated with 4HPR, 4HPR-L, and 4HPR-LM (all the mass concentration of 4HPR was 20 μg/mL), respectively, and cells in 4HPR-LM+ ultrasound group were given 0.5 W 60 s ultrasound treatment immediately after drug administration. The cells viability was measured as before after 24 hours of routine culture. (3) Another batch of human keloid Fbs were divided into control group, 4HPR group, 4HPR-L group and 4HPR-LM+ ultrasound group, with 3 wells in each group, and the cells in each group were treated as before. Apoptosis of the cells was detected by flow cytometer after 24 hours of routine culture. (4) Another batch of human keloid Fbs were grouped and treated as in (3), and then the cell cycle distribution was detected by flow cytometer after 24 hours of routine culture. Data were processed with one-way analysis of variance and t test. Results: (1) 4HPR-L particles had a spherical or spheroidal structure and were uniform in size, with particle size of (100.1±1.3) nm and Zeta potential of (-34.3±2.3) mV. The mass concentration of 4HPR in 4HPR-L solution was about 1 400 μg/mL, with the encapsulation efficiency of (95.8±1.2)% and drug loading rate of (8.3±0.4)%. (2) The viability of cells in the 12 ultrasound groups was higher than 93.0%, and the viability of cells in 1, 10, 20, and 50 μg/mL blank lipid microbubble groups was higher than 95.0%. The viability of cells in 1 μg/mL 4HPR-L group at administration hour 24 was similar to that at 48 (t=0.393, P>0.05). The viability of cells in 10, 20, 50, and 100 μg/mL 4HPR-L groups at administration hour 24 was significantly higher than that at administration hour 48 (t=44.593, 22.961, 32.224, 35.337, P<0.01). The viability of cells in 4HPR group, 4HPR-L group, and 4HPR-LM+ ultrasound group was (47.3±0.7)%, (42.3±1.7)%, and (38.6±0.8)%, respectively. The viability of cells in 4HPR group was significantly higher than that in 4HPR-L group and 4HPR-LM+ ultrasound group (t=4.551, 15.895, P<0.05 or P<0.01). The viability of cells in 4HPR-L group was significantly higher than that in 4HPR-LM+ ultrasound group (t=-3.360, P<0.05). (3) The percentages of total apoptotic cells in 4HPR group, 4HPR-L group, and 4HPR-LM+ ultrasound group were (32.8±2.4)%, (42.5±2.4)%, and (58.5±6.3)%, respectively, which were significantly higher than the percentage of control group [(14.9±1.6)%, t=8.748, 13.637, 9.500, P<0.01]. The percentages of total apoptotic cells in 4HPR-L group and 4HPR-LM+ ultrasound group were significantly higher than the percentage in 4HPR group (t=4.049, 5.393, P<0.05 or P<0.01), and the percentage of total apoptotic cells in 4HPR-LM+ ultrasound group was significantly higher than that in 4HPR-L group (t=3.371, P<0.01). (4) The percentage of G2/M phase cells in 4HPR group was higher than that in control group, but there was no statistically significant difference (t=2.107, P>0.05). The percentage of G2/M phase cells in 4HPR-L group was significantly higher than that in 4HPR group or control group (t=18.169, 30.026, P<0.01). The percentage of G2/M phase cells in 4HPR-LM+ ultrasound group was significantly higher than that in 4HPR-L group, 4HPR group, and control group (t=4.932, 25.854, 66.231, P<0.01). Conclusions 4HPR can inhibit proliferation, induce apoptosis, and arrest G2/M phase of human keloid Fbs, and the effects of 4HPR-LM combined with ultrasound are better than those of 4HPR-L and free 4HPR.

Objective To evaluate the effect of fenretinide (4-HPR)-loaded liposomes (4-HPR-L) on the proliferation,apoptosis and migration of A375 and B16F10 melanoma cells.Methods A film-ultrasonic dispersion method was used to prepare 4-HPR-L.In vitro cultured A375 cells,as well as B16F10 melanoma cells,were divided into the following groups:blank control group treated with fresh culture medium alone,4-HPR groups treated with 4-HPR at concentrations of 0.1,1,15,30,50 and 70 mg/L separately,and 4-HPR-L groups treated with 4-HPR-L at concentrations of 0.1,1,15,30,50 and 70 mg/L separately.Cell counting kit-8 (CCK8) assay was conducted to detect cell proliferation,Hoechst33258 staining and flow cytometry to detect cell apoptosis,wound healing assay to evaluate cell migration ability,and laser scanning confocal microscopy to observe endocytic uptake of the liposomes.Statistical analysis was done by one-way analysis of variance (ANOVA) for intergroup comparison,and a two-sample t-test for comparisons between 2 groups with SPSS 20.0 software.Results Both 4-HPR and 4-HPR-L showed inhibitory effects on the proliferation of A375 and B16F10 melanoma cells.After 48-hour treatment,the survival rates of A375 cells in the 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR groups were (94.3 ± 1.4)％,(91.7 ± 2.5)％,(84.4 ± 2.5％),(78.8 ± 2.1)％,(59.0 ± 1.1)％ and (42.8 ± 2.0)％ respectively,and those in the 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR-L groups were (86.0 ± 0.2)％,(76.5 ± 0.6)％,(60.9 ± 1.5)％,(49.0 ± 0.5)％,(32.9 ± 0.2)％ and (18.9 ± 0.5)％ respectively.After 48-hour treatment,the survival rates of B16F10 cells in 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR groups were (95.4 ± 1.9)％,(90.5 ± 2.6)％,(77.0 ± 0.8％),(64.4 ± 3.5)％,(59.1 ± 2.9)％ and (49.9 ± 1.9)％ respectively,and those in the 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR-L groups were (88.4 ± 2.0)％,(80.9 ± 3.4)％,(60.9 ± 2.2)％,(51.5 ± 2.9)％,(41.1 ± 1.2)％ and (33.5 ± 2.4)％ respectively.The survival rates of A375 and B16F10 cells were significantly higher in the 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR groups than in the 4-HPR-L groups at the same concentrations (A375 cells:t =8.019,8.298,11.455,19.978,33.672,16.314 respectively,all P ＜ 0.01;B16F10 cells:t =3.573,3.153,9.953,4.019,8.097,7.53 respectively,all P ＜ 0.05).Hoechst33258 staining showed that the morphology of the melanoma cells in the control group and 4-HPR groups did not change obviously,but the cells.in the 4-HPR-L groups became smaller,with the cytoplasm concentrated,nuclei dissociated into fragments,and apoptotic bodies formed.Flow cytometry showed that the apoptosis rates of A375 and B16F10 cells were significantly higher in the 4-HPR-L groups than in the 4-HPR groups (all P ＜ 0.01).Cell wound healing assay showed that the inhibitory effect of 4-HPR-L on the migration of cells was stronger than that of 4-HPR,and 4-HPR-L could markedly decrease the degree of wound healing.Laser scanning confocal microscopy showed that C6 liposomes could be rapidly and successfully internalized into both A375 and B16F10 cells.Conclusion The 4-HPR-L can be internalized into A375 and B16F10 cells better than 4-HPR,effectively inhibit the proliferation and migration of A375 and B16F10 cells,and induce the apoptosis of these cells.

Objective To evaluate the inhibitory effect of fenretinide-loaded liposomes(4-HPR-L) on subcutaneous transplanted malignant melanomas in nude mice.Methods A film-ultrasonic dispersion method was used to prepare 4-HPR-L.BALB/c nude mice were subcutaneously inoculated with A375 melanoma cells in the right axillary fossae to establish malignant melanoma-bearing nude mouse models.Ten nude mouse models were randomly and equally divided into 2 groups to be injected with near-infrared fluorescent cell membrane label (DiR) solution or DiR liposomes (DiR-L)at the same concentration in the caudal vein,and a live imaging system was used to observe the distribution of DiR or DiR-L in nude mice at 6,12,24 hours after the injection.Another 30 nude mice were randomly and equally divided into 3 groups to be injected with 5％ (mass fraction) glucose solution at a single-dose of 0.2 ml (control group),25 mg/kg 4-HPR solution (4-HPR group)and 25 mg/kg 4-HPR-L solution (4-HPR-L group) respectively on days 8,10,12,14,16,18,20 and 22 after the inoculation with A375 cells.The mouse body weight and tumor volume were dynamically monitored in the above groups after the injection,and the survival situation was observed.The nude mice were sacrificed on day 2 after the final injection,and the heart,liver,spleen,lung,kidney and tumor tissues were resected.These tissues were subjected to hematoxylin-eosin staining and immunohistochemical staining to observe the metastasis of melanoma in mice,and terminal deoxyribonucleotidyl transferase-mediated nick end labelling was performed to detect the apoptosis in tumor cells.One-way analysis of variance and independent-sample t test were used to analyze measurement data.Results The live imaging system showed that DiR-L could be retained in melanoma for a long time,and strong fluorescence of DiR-L could be still observed in the tumors at 24 hours after injections.Quantitative fluorescence analysis revealed that the fluorescence intensity of DiR-L (22.85 ± 1.66) was significantly higher than that of DiR in tumor tissues (8.45 ± 0.97,t =12.957,P ＜ 0.01).Compared with the control group and 4-HPR group,the resected tumor weight on day 2 after the final injection was significantly decreased in the 4-HPR-L group (F =27.055,t =4.768,6.640,respectively,both P ＜ 0.05).Hematoxylineosin staining showed that liver metastasis occurred in 2 nude mice in the 4-HPR-L group,but in all the nude mice in the control group and 4-HPR group.All the nude mice in the 4-HPR-L group died within 76 days after inoculation,and the mice in the control group and 4-HPR group all died within 56 and 59 days respectively after inoculation.There were significant differences in the apoptotic index among the control group (12.14‰ ± 1.33‰),4-HPR group (67.17‰± 15.18‰) and 4-HPR-L group (152.73‰ ± 11.27‰;F =167.588,P ＜ 0.05),and the apoptotic index was significantly higher in the 4-HPR-L group than in the control group and 4-HPR group (t =18.162,11.075 respectively,both P ＜ 0.05).Conclusion 4-HPR-L can effectively inhibit the growth of subcutaneous melanoma in nude mice and metastasis of melanoma cells,and prolong the survival duration of nude mice.

Objective:To investigate the effect of ultrasound combined with 4-hydroxyphenyl-retinamide (4-HPR) lipid microbubbles on type Ⅰ collagen α1 chain (COL1A1) protein expression in keloid-derived fibroblasts.Methods:In vitro cultured keloid-derived fibroblasts were divided into 3 groups: control group receiving conventional culture with incomplete Dulbecco′s modified Eagle′s medium (DMEM) , 4-HPR lipid microbubble group cultured with incomplete DMEM containing 15 mg/L 4-HPR lipid microbubbles, and ultrasound + 4-HPR lipid microbubble group cultured with incomplete DMEM containing 15 mg/L 4-HPR lipid microbubbles under ultrasound treatment. After 24-hour treatment, reverse transcription (RT) -PCR and Western blot analysis were performed to determine the mRNA and protein expression of COL1A1 in keloid-derived fibroblasts in each group. Intergroup comparison was carried out by using t test. Results:The mRNA relative expression level of COL1A1 was 1.00 ± 0.18, 0.69 ± 0.15 and 0.35 ± 0.18 in the control group, 4-HPR lipid microbubble group and ultrasound + 4-HPR lipid microbubble group respectively, and the protein relative expression level of COL1A1 was 0.93 ± 0.03, 0.74 ± 0.07 and 0.44 ± 0.06 in the above 3 groups respectively. Moreover, the mRNA and protein expression of COL1A1 was significantly lower in the 4-HPR lipid microbubble group and ultrasound + 4-HPR lipid microbubble group than in the control group ( P < 0.05 or 0.001) , and lower in the ultrasound + 4-HPR lipid microbubble group than in the 4-HPR lipid microbubble group ( P < 0.05) . Conclusion:Ultrasound combined with 4-HPR lipid microbubbles could markedly inhibit the mRNA and protein expression of COL1A1 in keloid-derived fibroblasts.

Objective To assess the effect of fenretinide (4-HPR) on proliferation,apoptosis and migration of B16F10 and A375 melanoma cells,and to evaluate the effect of liposomes and RGD peptidemodified liposomes on its uptake and therapeutic effects.Methods A film-hydration method was used to prepare 4-HPR liposomes (4-HPRL),which were modified with RGD peptide to prepare RGD-4-HPRL,and the concentration,particle size,electric potential,drug loading capacity and encapsulation efficiency were measured for 4-HPRL and RGD-4-HPRL.In vitro cultured B16F10 and A375 cells were divided into several groups:4-HPR group,4-HPRL group and RGD-4-HPRL group treated with Dulbecco's minimum essential medium (DMEM) containing 4-HPR bulk drug,4-HPRL and RGD-4-HPRL respectively at the same concentration of 4-HPR,and control group treated with culture solution at the same volume.After different durations of treatment,cell counting kit-8 (CCK8) assay was performed to evaluate cellular proliferative activity,annexin V/propidium iodide staining to detect apoptosis,and scratch wound healing assay to evaluate the effect of drug treatment on cell migration ability.Then,4-HPR was replaced by coumarin 6 (C6) to prepare C6 liposomes (C6L) and RGD-C6L,and flow cytometry was conducted to evaluate C6 uptake by B16F10 cells.Statistical analysis was carried out with SPSS22.0 software by one-way analysis of variance (ANOVA) for the comparison among several groups and t test for the comparison between two groups.Results The concentration of 4-HPR in the prepared 4-HPRL solution was over 1 300 mg/L.The encapsulation efficiency and drug loading capacity of 4-HPRL were (95.51 ± 1.22)％ and (7.27 ± 0.11)％ respectively,and those of RGD-4-HPRL were (95.82 ± 0.81)％ and (7.14 ± 0.13)％ respectively.The particle size distribution of 4-HPRL and RGD-4-HPRL was uniform,and their average particle size was below 100 nm.CCK8 assay showed that 4-HPR could markedly inhibit the proliferative activities of B16F10 and A375 cells.The cell proliferation inhibition rate of 4-HPRL was higher than that of 4-HPR at the same concentration of 4-HPR (P ＜ 0.01),and the inhibition rate of RGD-4-HPRL was higher than that of 4-HPRL (P ＜ 0.01 or P ＜ 0.05) and 4-HPR (P ＜ 0.01).As annexin V/propidium iodide apoptosis assay showed,when the concentration of 4-HPR was 10 mg/L,the total apoptosis rates of B16F10 cells in the control group,4-HPR group,4-HPRL group and RGD-HPRL group were (4.44 ± 0.35)％,(28.33 ± 0.66)％,(46.43 ± 0.77)％ and (51.33 ± 0.37)％ respectively.When the concentration of 4-HPR was 20 mg/L,the total apoptosis rates of A375 cells in the above 4 groups were (4.97 ± 0.62)％,(16.68 ± 3.81)％,(32.62 ± 1.24)％ and (44.85 ± 4.92)％ respectively.The apoptosis rates of B16F10 and A375 cells were significantly higher in the 4-HPRL group than in the 4-HPR group (both P ＜ 0.01),and higher in the RGD-4-HPRL group than in the 4-HPRL group (both P ＜ 0.01) and 4-HPR group (both P ＜0.01).Scratch wound healing assay showed that 4-HPR could inhibit scratch healing and migration of B16F10 and A375 cells,and the inhibitory effects of 4-HPRL and RGD-4-HPRL were distinctly superior to those of 4-HPR bulk drug.C6 uptake assay revealed that the fluorescence intensity of C6 in B16F10 cells in the control group,C6 group,C6L group and RGD-C6L group were 2.15 ± 0.28,8.56 ± 0.36,20.48 ± 0.13 and 22.55 ± 0.07 respectively,and there were significant differences between the 4 groups (F =67 194.186,P ＜ 0.01).Additionally,the fluorescence intensity of C6 was significantly higher in the C6L group and RGD-C6L group than in the C6 group (both P ＜ 0.01),and higher in the RGD-C6L group than in the C6L Group (P ＜ 0.01).Conclusions 4-HPR can inhibit the proliferation and migration of A375 and B16F10 cells,and induce their apoptosis.Liposomes and RGD-targeted liposomes can markedly enhance the effect of 4-HPR on melanoma cells.