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In vitro or in vivo results of investigations into the development of photosensitizer drug delivery systems have demonstrated that the use of vehicles not only enhance the stability of photosensitizer,but also promote the accumulation of photosensitizer in tumor tissue,enhance the targetability of photodynamic therapy,the working depth,the yield of singlet oxygen and overcome many untoward effects.So the research of new photosensitizer drug delivery system has important application value in photodynamic therapy of tumor.
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Objective To increase the killing effect of aminolevulinic acid (ALA)-based photodynamic therapy (PDT) on a human skin squamous cell carcinoma cell line A431 by poly lactic-co-glycolic acid nanoparticles (PLGA NPs).Methods ALA-loaded PLGA NPs (ALA PLGA NPs) were prepared using a modified double emulsion solvent evaporation method,and characterized in terms of size,encapsulation efficiency,loading capacity and morphology.Transmission electron microscopy was carried out to observe the morphology of A431 cells after uptake of ALA PLGA NPs.To optimize incubation time,multi-mode microplate reader was used to describe the fluorescence kinetics of protoporphyrin Ⅸ generated by A431 cells during 24 hours of incubation with 0.1 mmol/L ALA,1 mmol/L ALA,2.7 g/L ALA PLGA NPs containing about 0.1 mmol/L ALA,and PLGA NPs without ALA separately.Some A431 cells were divided into 10 groups:control group receiving neither treatment nor irradiation,0.1 and 1 mmol/L ALA dark/PDT group incubated 0.1 and 1 mmol/L ALA respectively,ALA PLGA NP dark/PDT group incubated with ALA PLGA NPs of 2.7 g/L,PLGA NP dark/PDT group incubated with PLGA NPs of 2.7 g/L,simple irradiation group irradiated with a He-Ne laser (wavelength:635 nm,power density:8.6 mW/cm2,energy density:8 J/cm2) only.The dark groups were kept in darkness strictly by wrapping in aluminum foil,and PDT groups were irradiated using a He-Ne laser.After another 24 hours of culture following irradiation,methyl thiazolyl tetrazolium (MTF) assay was conducted to estimate the survival rate of cells.To study the effect of ALA PLGA NPs on cell apoptosis,some A431 cells were divided into three groups:control group receiving neither treatment nor irradiation,ALA-PDT group and ALA PLGA NP PDT group receiving PDT after incubation with ALA and ALA PLGA NPs respectively.Flow cytometry was performed to detect the apoptosis of A431 cells at 12 and 24 hours,separately,after the photodynamic therapy.Data were statistically analyzed using SPSS 13.0 software package by means of a t test.Results The prepared ALA PLGA NPs were spherical with a mean particle size of (65.6 ± 26) nm,encapsulation efficiency of (65.8 ± 7.2) ％,and drug loading capacity of (0.62 ± 0.27)％.ALA PLGA NPs could be uptaken by A431 cells and gathered in the cytoplasm.The PpIX fluorescence kinetic study showed that the fluorescence intensity increased with time within 24 hours in A431 cells incubated with ALA or ALA PLGA NPs.After 6 and 24 hours of incubation,the A431 cells incubated with 2.7 g/L ALA PLGA NPs showed a significant increase in the protoporphyrin Ⅸ fluorescence intensity compared with those incubated with 0.1 mmol/L ALA (both P ＜ 0.01).Further more,the survival rate of A431 cells was statistically lower in the ALA PLGA NP PDT group than in the 0.1 mmol/L ALA PDT group at 6 and 24 hours (t =35.685,5.262,respectively,both P ＜ 0.01).Elevated apoptosis rate was observed in the ALA PLGA NP PDT group compared with the ALA PDT group at 12 ((13.10 ± 0.50)％ vs.(4.90 ± 0.13)％,t =9.074,P＜ 0.01) and 24 ((30.17 ± 1.02)％ vs.(11.6 ± 0.59)％,t =9.095,P ＜ 0.01) hours.Conclusions ALA PLGA NPs can promote the formation of protoporphyrin Ⅸ,strengthen the killing effect of ALA-PDT on A431 cells in vitro,and enhance the apoptosis induced by ALA-PDT in tumor cells.
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Objective To investigate the protective effect of butyl flufenamate ointment against ultraviolet (UV)-induced skin damage,skin aging,and cutaneous squamous cell carcinoma (CSCC) in SKH-1 hairless mice.Methods A total of 128 mice were randomly and equally divided into four groups:UV group receiving UV irradiation only,butyl flufenamate ointment group and matrix cream group receiving UV irradiation after 30-minute pretreatment with topical butyl flufenamate ointment and matrix cream respectively,and blank control group receiving neither pretreatment nor irradiation.In the sunburn experiment (n =24),mice were exposed to single session of UV irradiation (1.5 minimal erythema doses (MEDs)),and 24 hours later,erythema and swelling response was observed,and skin tissue was obtained from the irradiated area on the back of mice followed by the determination of COX-2 expression using the streptavidin biotin peroxidase complex (SABC) method.To establish a photoaging (n =24) and CSCC (n =80) model,mice were exposed to four sessions of UV irradiation every week for 12 and 28 successive weeks respectively,with the irradiation dose starting at 0.9 MED and increasing gradually.After 12-week irradiation,skin tissue was resected from the back of photoaged mice and subjected to Masson staining for the evaluation of collagen changes as well as immunohistochemical analysis for the quantification of Bax,Bcl-2 and Caspase 3 expression.The initiation and progression of CSCC were observed in mice on a once-a-week basis from 12 to 28 weeks.SPSS 21.0 software was used for statistical analysis.One way analysis of variance was carried out for multiple-group comparisons of numerical data,Ridit analysis for the comparison of immunohistochemical staining intensity.Kaplan-Meier method and log-rank test were utilized for the comparison of tumor-free survival time.Results Both the degree of erythema and swelling response and expression level of COX-2 were significantly lower in the butyl flufenamate ointment group than in the other two UV-irradiated groups (all P ＜ 0.05).After 12-week irradiation,the butyl flufenamate ointment group showed milder degree of skin aging,together with higher density of collagen in dermis,weaker expression of Bcl-2 but stronger expression of Bax and Caspase 3,by comparison with the other two UV-irradiated groups (all P ＜ 0.05).During the 28 weeks of irradiation,the median tumor-free survival time was statistically longer in the butyl flufenamate ointment group than in the matrix cream group and UV group((25.0 ± 0.4) months vs.(24.0 ± 0.3) months and (23.0 ± 0.4) months,P ＜ 0.05 and 0.01 respectively).Conclusion Butyl flufenamate ointment has a certain photoprotective effect.