Bioactive Polymeric Nanoparticles of Moringa oleifera Induced Phyto-Photothermal Sensitization for the Enhanced Therapy of Retinoblastoma

Abstract

Treatment of retinoblastoma is limited due to its delayed detection and inaccesbility of drugs to reach the retina crossing the blood-retinal barrier. With the advancements in nanotechnology, photothermal therapy (PTT) employing plasmonic nanomaterials and/or NIR dyes have emerged as an affordable alternative owing to the spatial control that is offered by the modality leading to localized and enhanced therapeutic efficacy with minimal invasiveness. However, the modality is limited in its clinical application owing to the increased heat shock resistance of the tumor cells in response to the heat that is generated via PTT. Hence, in this study, we explore the role of novel biomolecular fraction of Moringa oleifera (DFM) encapsulated within a polymeric nanosystem, for its anti-heat shock protein (HSP) activity. The MO extract was co-encapsulated with NIR sensitizing dye, IR820 into a biodegradable polycaprolactone (PCL) nano-delivery system (PMIR NPs). The photothermal transduction efficacy of PMIR NPs was validated in vitro against retinoblastoma cell lines. The inherent fluorescence of DFM was utilized to evaluate the cellular internalization of the PMIR NPs using fluorescence microscopy and flow cytometry. The overall oxidative protein damage and downregulation of HSP70 expression upon treatment with PMIR NPs and NIR laser irradiation was evaluated using densiometric protein analysis and Western blotting. Overall, the PMIR NPs exhibited excellent anti-cancer activity when combined with PTT with downregulated HSP70 expression against retinoblastoma cells.

Keywords: Moringa oleifera; heat shock proteins; phyto-photothermal therapy; polymeric nano system; retinoblastoma.

Figure 1

Characterization of the MI PNPs. (A) UV-Vis spectra of the blank, M, I, and MI PNPs. (B) Fluorescent spectra of the M and MI PNPs (ex/em: 415/680 nm). (C,D) Representative images of the various PNPs under bright and UV light. (E,F) DLS and zeta potential graphs for the MI PNPs. (G,H) SEM and TEM images of the MI PNPs.

Figure 2

Photothermal transduction efficacy studies of the MI PNPs during exposure to NIR 808 nm light. (A) Line graph indicating the temperature profiles of the various PNPs and controls. (B) Representative thermal images indicating the temperature increments. (C) Line graph indicating the temperature profiles of the various PNPs and controls for different power intensities of the NIR light. (D) Representative thermal images indicating the temperature increments for different power intensities of the NIR light.

Figure 3

PTT-mediated release/degradation studies of the MI PNPs during exposure to NIR 808 nm light. (A) Line graph indicating the UV-vis spectra before and post-NIR light irradiation. (B–D) UV-vis spectra of MI PNPs indicating the release/degradation using various power intensities of NIR light.

Figure 4

Fluorescent microscopy images of (A) untreated cells and (B) cells treated with MI PNPS indicating the cellular internalization efficacy of the MI PNPs.

Figure 5

Phantom PA imaging of the MI PNPs compared to ICG and Milli-Q water. (A) The ultrasound/ PA signal images of the samples at different wavelengths. (B) The line graph spectra indicate the PA signal in the scan window.

Figure 6

(A) Biocompatibility studies of the MI PNPs at various concentrations in HRMEC cell lines. (B) Photothermal-mediated cytotoxicity studies of the MI PNPs and reference controls in the presence or absence of the NIR light. (C) Fluorescent microscopy images of the FDA/PI-stained cells after treatment and exposure to NIR 808 nm light. (Scale bar represents 100 µm). A two-way ANOVA was used to perform the statistical analysis in (B). * p < 0.05 and *** p < 0.001.

Figure 7

Protein/DNA analysis for the samples that were treated with the MI PNPs in the presence or absence of the NIR light. (A) Plasmid denaturation assay indicating the formation of nicked, linear, or supercoiled plasmid content. (B) Protein denaturation assay. (C) bar graph indicating the relative intensities. (D) Western blot analysis for the cell lysates that were treated with the MI PNPs in the presence or absence of the NIR 808 nm light. A two-way ANOVA was used to perform the statistical analysis in (C,E) where ** corresponds to p < 0.01 and **** corresponds to p < 0.0001.