Microwave assisted drug delivery of titanium dioxide/rose Bengal conjugated chitosan nanoparticles for micro-photodynamic skin cancer treatment in vitro and in vivo

Abstract

Background: Micro-photodynamic therapy (MWPDT) combines photo-dynamic (PDT) and microwave-dynamic (MWDT) therapies with sensitizers, offers new avenues for cancer treatment. Despite the fact that novel sensitizers for MWPDT have been successfully synthesized, only a few are being employed effectively. The low tumor-targeting specificity, inability to transport sensitizer's deeper intratumorally, and deteriorating tumor microenvironment all restrict their anti-tumor efficacy. The current work was done aiming at microwave assisted drug delivery of titanium dioxide / rose Bengal conjugated chitosan nanoparticles (TiO₂/RB@CSNP) for micro- photo-dynamic skin cancer (SKCA) treatment in vitro and in vivo as activated cancer treatment up-to-date modality.

Materials and methods: The study was conducted in vitro on human SKCA cells (A-375) and the study protocol application groups in vivo on Swiss albino mice treated with 7,12-dimethylbenz[a]anthracene (DMBA)/croton oil only and were not received any treatment for inducing SKCA, and only after SKCA induction the study treatment protocol began, treatment was daily with TiO₂/RB@CSNP as MWPDT sensitizer with or without exposure to laser (IRL) or microwave (MW) or a combination of them for 3 min for two weeks.

Results: Revealed that CSNP can be employed as effective TiO₂/RB delivery system that directly targets SKCA cells. Additionally TiO₂/RB@CSNP is a promising MWPS for and when combined with MWPDT can be very effective in treatment of SKCA-A-375 in vitro (cell viability decreased in a dose-dependent basis, the cell cycle progression in G0/G1 was slowed down, and cell death was induced as evidenced by an increase in the population of Pre-G cells, an increase in early and late apoptosis and necrosis, and an increase in autophagic cell death) and DMBA/croton oil SKCA-induce mice in vivo (induced antiproliferative genes (caspase 3,9, p53, Bax, TNFalpha), suppressed antiapoptotic and antiangiogenic genes (Bcl2,VEGF respectively) effectively reducing the tumors growth and leading to cancer cell death as well as decreased oxidative stress (MDA), and ameliorated enzymatic and non-enzymatic antioxidants (SOD, GR, GPx, GST, CAT, GSH, TAC) as well as renal (urea, creatinine) and hepatic (ALT, AST) functions. This process could be attributed to MWPDT; microwave and/or photo-chemical TiO₂/RB activation mechanism and antioxidant potential of non activated TiO₂/RB as well.

Conclusion: The results indicate that TiO₂/RB@CSNP has great promise as an innovative, effective delivery system for selective localized treatment of skin cancer that is activated by MWPDT.

Keywords: Chitosan nanoparticle; Micro-photodynamic; Rose Bengal; Skin cancer; Titanium dioxide.

Fig. 1

Schematic representation of TiO₂/RB-CSNP synthesis and MWPDT

Fig. 2

Characterization of TiO₂/RB@CSNP; a. TEM, b. SEM, c. particle size, d. zeta potential of TiO₂/RB@CSNP, e. UV–Vis spectra, f. PL, g. FTIR transmittance and absorbance, h. XRD of (1. TiO₂/RB@CSNP, 2. RB, 3. TiO₂), i. EDX of TiO₂/RB@CSNP

Fig. 3

The effect of different treatment modalities on skin cancer (A-375) cell viability; Cells were exposed to different treatment modalities with serial dilution of TiO₂/RB@CSNP for 24 h, a. microscopic investigations, b. A375 containing TiO₂/RB@CSNP (arrow), Ne; nuclear envelop, Pm; plasma membrane, m; mitochondria, rer; rough endoplasmic reticulum and c. dose response curve in all in vitro study groups; cell viability was determined using SRB assay. cell viability (%): F = 5.822 p < 0.001*. Mean ± SD is used to illustrate the results (n = 3). F stands for the ANOVA test value. ^a,b,c,d,e Significance to (skin cancer untreated group, TiO₂/RB@CSNP treated non activated group, laser-, microwave-, laser + microwave- exposed groups)

Fig. 4

The effect of different treatment modalities on skin cancer (A-375) cell cycle distribution in all in vitro study groups; 1. Cells were exposed to different treatment modalities for 24 h, 2. Cell cycle distribution was determined using DNA cytometry analysis and different cell phases were plotted as percentage of total events; SubG1(%): F = 329.212 p < 0.001*, G0/G1(%): F = 88.084 p < 0.001*, S(%): F = 92.935 p < 0.001*, G2/M(%): F = 173.137 p < 0.001*. Mean ± SD is used to illustrate the results (n = 3). F stands for the ANOVA test value..^a,b,c,d,e Significance to (skin cancer untreated group, TiO₂/RB@CSNP treated non activated group, laser-, microwave-, laser + microwave- exposed groups)

Fig. 5

The effect of different treatment modalities on skin cancer (A-375) apoptosis and necrosis in all in vitro study groups; 1. Cells were exposed to different treatment modalities for 24 h, 2. Cells were stained with annexin V-FITC/PI and different cell populations were plotted as percentage of total events. early apoptosis (%): F = 16.884 p < 0.001*, late apoptosis (%): F = 26.304 p < 0.001*, early and late apoptosis (%): F = 23.439 p < 0.001*, necrosis (%): F = 18.719 p < 0.001*, total cell death (%): F = 23.288 p < 0.001*. Mean ± SD is used to illustrate the results (n = 3). F stands for the ANOVA test value..^a,b,c,d,e Significance to (skin cancer untreated group, TiO₂/RB@CSNP treated non activated group, laser-, microwave-, laser + microwave- exposed groups)

Fig. 6

The effect of different treatment modalities on skin cancer (A-375) autophagy in all in vitro study groups; 1. Cells were exposed to different treatment modalities for 24 h; and were stained with Cyto-ID autophagosome tracker. 2. Net fluorescent intensity (NFI; red color) were plotted and compared to basal fluorescence of control group (green color). autophagy (%): F = 24.876 p < 0.001*. Mean ± SD is used to illustrate the results (n = 3). F stands for the ANOVA test value..^a,b,c,d,e Significance to (skin cancer untreated group, TiO₂/RB@CSNP treated non activated group, laser-, microwave-, laser + microwave- exposed groups)

Fig. 7

The effect of different treatment modalities on skin cancer (A-375) migration; 1. A-375 untreated, 2. A-375 treated with laser + microwave only, 3. A-375 treated with TiO₂/RB@CSNP + laser + microwave. wound clousure-24 h (%): F = 2.416E3 p < 0.001*, wound clousure-48 h (%): F = 694.712 p < 0.001*, wound clousure-72 h (%): F = 2.855E3 p < 0.001*. Mean ± SD is used to illustrate the results (n = 3). F stands for the ANOVA test value..^a,b,c Significance to (skin cancer untreated group, laser + microwave only group, laser + microwave + TiO₂/RB@CSNP groups)

Fig. 8

The effect of different treatment modalities on MDA, antioxidants activities, and capacities in all study groups; F: value for ANOVA test 1. GSH(mg/dl): F = 871.530 p < 0.001*, 2. GR (mU/ml): F = 161.065 p < 0.001*, 3. GST (U/ml): F = 1.221E3 p < 0.001*, 4. GPx (mU/ml): F = 4.155E4 p < 0.001*, 5. SOD (U/ml): F = 3.819E6 p < 0.001*, 6. CAT(mU/ml): F = 1.676E5 p < 0.001*, 7. TAC(mM/L): F = 453.839 p < 0.001*, 8. MDA(nmol/ml): F = 9.736E4 p < 0.001*. Mean ± SD is used to illustrate the results (n = 10). F stands for the ANOVA test value..^a,b,c,d,e Significance to (skin cancer untreated group, TiO₂/RB@CSNP treated non activated group, laser-, microwave-, laser + microwave- exposed groups)

Fig. 9

The effect of different treatment modalities on hepatic, renal biomarkers biomarkers and tissues histopathology in all study groups; F: value for ANOVA test 1. ALT (U/l): F = 7.865E3 p < 0.001*, 2. AST (U/l): F = 1.651E5 p < 0.001*, 3. Urea (mg/dl): F = 4.230E3 p < 0.001*, 4. Creatinine (mg/dl): F = 401.771 p < 0.001*. 5. H&E stained liver and kidney tissues; (1. Normal untreated group, 2. DMBA/croton oil induced SKCA group without any treatment, 3. DMBA/croton oil induced SKCA group treated with TiO₂/RB@CSNP without activation, 4. DMBA/croton oil SKCA induced group subjected to laser only, 5. DMBA/croton oil SKCA induced group subjected to laser in TiO₂/RB@CSNP presence, 6. DMBA/croton oil induced SKCA group subjected to microwave only, 7. DMBA/croton oil SKCA induced group subjected to microwave in TiO₂/RB@CSNP presence, 8. DMBA/croton oil SKCA induced group subjected to combined modalities laser/microwave only, 9. DMBA/croton oil SKCA induced group subjected to combined modalities laser/microwave in TiO₂/RB@CSNP presence. Mean ± SD is used to illustrate the results (n = 10). F stands for the ANOVA test value..^a,b,c,d,e Significance to (skin cancer untreated group, TiO₂/RB@CSNP treated non activated group, laser-, microwave-, laser + microwave- exposed groups)

Fig. 10

The effect of different treatment modalities on VEGF, TNF alpha, Bax, Caspase (9,3), Bcl-2 and p53, qRT-PCR relative genes expressions in all study groups; F: value for ANOVA test. p53: F = 406.566 p < 0.001*, Bax: F = 985.443 p < 0.001*, Caspase 9: F = 1.053E3 p < 0.001*, Caspase 3: F = 761.080 p < 0.001*, TNFalpha: F = 435.687 p < 0.001*, VEGF: F = 147.444 p < 0.001*, Bcl-2: F = 191.970 p < 0.001*. Mean ± SD is used to illustrate the results (n = 10). F stands for the ANOVA test value..^a,b,c,d,e Significance to (skin cancer untreated group, TiO₂/RB@CSNP treated non activated group, laser-, microwave-, laser + microwave- exposed groups)

Fig. 11

The in vivo study groups images and H&E skin tissue stained section demonstrating the effect of different treatment modalities on cellular level in all study groups; 1. Normal untreated group, 2. DMBA/croton oil induced SKCA group without any treatment, 3. TiO₂/RB@CSNP treated group without activation, 4. DMBA/croton oil SKCA induced group subjected to laser only, 5. DMBA/croton oil SKCA induced group subjected to laser in presence of TiO₂/RB@CSNP, 6. DMBA/croton oil induced SKCA group subjected to microwave only, 7. DMBA/croton oil SKCA induced group subjected to microwave in presence of TiO₂/RB@CSNP, 8. DMBA/croton oil SKCA induced group subjected to combined modalities laser/microwave only, 9. DMBA/croton oil SKCA induced group subjected to combined modalities laser/microwave in presence of TiO₂/RB@CSNP