Triazine-Carbosilane Dendrimersomes Enhance Cellular Uptake and Phototoxic Activity of Rose Bengal in Basal Cell Skin Carcinoma Cells

Abstract

Background: The search for new formulations for photodynamic therapy is intended to improve the outcome of skin cancer treatment using significantly reduced doses of photosensitizer, thereby avoiding side effects. The incorporation of photosensitizers into nanoassemblies is a versatile way to increase the efficiency and specificity of drug delivery into target cells. Herein, we report the loading of rose bengal into vesicle-like constructs of amphiphilic triazine-carbosilane dendrons (dendrimersomes) as well as biophysical and in vitro characterization of this novel nanosystem.

Methods: Using established protocol and analytical and spectroscopy techniques we were able to synthesized dendrons with strictly designed properties. Engaging biophysical methods (hydrodynamic diameter and zeta potential measurements, analysis of spectral properties, transmission electron microscopy) we confirmed assembling of our nanosystem. A set of in vitro techniques was used for determination ROS generation, (ABDA and H₂DCFDA probes), cell viability (MTT assay) and cellular uptake (flow cytometry and confocal microscopy).

Results: Encapsulation of rose bengal inside dendrimersomes enhances cellular uptake, intracellular ROS production and concequently, the phototoxicity of this photosensitizer.

Conclusion: Triazine-carbosilane dendrimersomes show high capacity as drug carriers for anticancer photodynamic therapy.

Keywords: amphiphiles; carbosilane; dendrimersomes; dendrons; photodynamic therapy; rose bengal.

Graphical abstract

Figure 1

Synthesis of the amphiphilic dendron: (i) n-C₁₂H₂₅NH₂, CHCl₃, NaOH (aq.); (ii) piperazine, CHCl₃; (iii) BrG_nV_m, K₂CO₃, 18-crown-6, KI, acetone; (iv) HS(CH₂)₂N(CH₃)₂·HCl, DMPA, 365 nm UV, THF:CH₃OH.

Figure 2

Structures of amphiphilic triazine–carbosilane dendrons of 2nd and 3rd generation.

Figure 3

DLS profiles for supramolecular associates of the amphiphilic dendron G3 (100 μM) exposed to different pH in 10 mM Na-phosphate buffer.

Figure 4

Changes in fluorescence and absorbance spectra of RB after encapsulation within dendrimersomes. The measurements of absorbance and fluorescence intensity were carried out for the tested compounds at RB concentrations of 5 and 1 μM, respectively.

Figure 5

Release of RB from G2-RB and G3-RB dendrimersomes at pH 5.5. Percentage of release was determined relative to the first sample (0 h, 100% of initial absorbance). *Statistically significant difference at p < 0.05 between the two generations of dendrimersomes. Data are presented as means ± SD, n=3.

Figure 6

Singlet oxygen generation by free RB and dendrimersomes loaded with RB (G2-RB and G3-RB), determined using an ABDA probe. *Statistically significant difference at p<0.05 between G3-RB or G2-RB and RB. ^ Statistically significant difference at p<0.05 between G2-RB and G3-RB. Data are presented as means ± SD, n=3.

Figure 7

(A) Uptake of free RB and dendrimersomes loaded with RB (G2-RB and G3-RB) by AsZ cells. Data are presented as means ± SD, n=3. *Statistically significant difference at p<0.05 between evaluated compounds and RB. (B) Confocal micrographs: (1) Phalloidin–Atto 633; (2) Hoechst 33342; (3) rose bengal; (4) bright field image; (5) merge of channels 1, 2, and 3. Scale bar represents 20 µm.

Figure 8

(A) ROS generation and (B) phototoxic activity of free RB and RB-loaded dendrimersomes (G2-RB and G3-RB) on AsZ cells. Data are presented as means ± SD, n=3. *Statistically significant difference at p<0.05 between RB and G2-RB or G3-RB. ^ Statistically significant difference at p<0.05 between G2-RB and G3-RB.