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. 2018 Jun 19:13:3553-3570.
doi: 10.2147/IJN.S164967. eCollection 2018.

X-ray radiation-induced and targeted photodynamic therapy with folic acid-conjugated biodegradable nanoconstructs

Sandhya Clement #  1   2 , Wenjie Chen #  1 , Wei Deng  1   2 , Ewa M Goldys  1   2
Affiliations

X-ray radiation-induced and targeted photodynamic therapy with folic acid-conjugated biodegradable nanoconstructs

Sandhya Clement et al. Int J Nanomedicine. .

Abstract

Introduction: The depth limitation of conventional photodynamic therapy (PDT) with visible electromagnetic radiation represents a challenge for the treatment of deep-seated tumors.

Materials and methods: To overcome this issue, we developed an X-ray-induced PDT system where poly(lactide-co-glycolide) (PLGA) polymeric nanoparticles (NPs) incorporating a photosensitizer (PS), verteporfin (VP), were triggered by 6 MeV X-ray radiation to generate cytotoxic singlet oxygen. The X-ray radiation used in this study allows this system to breakthrough the PDT depth barrier due to excellent penetration of 6 MeV X-ray radiation through biological tissue. In addition, the conjugation of our NPs with folic acid moieties enables specific targeting of HCT116 cancer cells that overexpress the folate receptors. We carried out physiochemical characterization of PLGA NPs, such as size distribution, zeta potential, morphology and in vitro release of VP. Cellular uptake activity and cell-killing effect of these NPs were also evaluated.

Results and discussion: Our results indicate that our nanoconstructs triggered by 6 MeV X-ray radiation yield enhanced PDT efficacy compared with the radiation alone. We attributed the X-ray-induced singlet oxygen generation from the PS, VP, to photoexcitation by Cherenkov radiation and/or reactive oxygen species generation facilitated by energetic secondary electrons produced in the tissue.

Conclusion: The cytotoxic effect caused by VP offers the possibility of enhancing the radiation therapy commonly prescribed for the treatment of cancer by simultaneous PDT.

Keywords: PLGA nanoparticles; X-ray PDT; folic acid targeting; photodynamic therapy; singlet oxygen generation; verteporfin.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Characterization. Notes: (A) Photographs of the synthesized PLGA NPs (sample 1) and PLGA-VP NP matrix with different VP concentrations (samples 2–5 with VP concentrations of 4.5 µM, 15.9 µM, 26.4 µM and 39.6 µM, respectively) in water. (B) Absorption spectra of VP, PLGA and different PLGA-VP NP solutions. (C) Fluorescence spectra of VP in PLGA samples with 425 nm excitation. (D) TEM image of PLGA NPs (scale bar: 1 µm). Abbreviations: NPs, nanoparticles; PLGA, poly(D,L-lactide-co-glycolic acid); SOSG, singlet oxygen sensor green; TEM, transmission electron microscopy; VP, verteporfin.
Figure 2
Figure 2
Single oxygen detection. Notes: (A) Variation of SOSG intensity as a function of X-ray dose for different samples. (B) Comparison between the percentage increase in SOSG intensity of PLGA and PLGA-VP in the selected sample (sample 3). Inset shows the increase in fluorescence of SOSG for different radiation doses for PLGA-VP NPs (sample 3). Abbreviations: NPs, nanoparticles; PLGA, poly(D,L-lactide-co-glycolic acid); SOSG, singlet oxygen sensor green; VP, verteporfin.
Figure 3
Figure 3
Confirmation of conjugation of FA with the PLGA-VP nanoconstructs using (A) absorption spectra (insets highlight the FA and VP peaks in FA-PLGA-VP sample) and (B) FTIR spectra. Note: All graphs in the spectra are scaled for distinguishing the peaks. Abbreviations: FA, folic acid; FTIR, Fourier transform infrared; PLGA, poly(D,L-lactide-co-glycolide); VP, verteporfin.
Figure 4
Figure 4
Confocal fluorescence images of cellular uptake of HCT116 cells with FA-PLGA-VP under (A) normal condition (B) with FR blocking. Notes: Blue fluorescence, nuclei stained with Hoechst 33345; red fluorescence, emission of VP under 405 nm excitation. All images were taken with 20× magnification. Abbreviations: FA, folic acid; FR, folate receptor; PLGA, poly(D,L-lactide-co-glycolic acid); VP, verteporfin.
Figure 5
Figure 5
Confocal imaging of colocalization of FA-PLGA-VP in HCT116 cells. Notes: Green fluorescence, MitoTracker; red fluorescence, emission of VP under 405 nm excitation. All images were taken with 40× magnification. Abbreviations: FA, folic acid; PLGA, poly(D,L-lactide-co-glycolic acid); VP, verteporfin.
Figure 6
Figure 6
Image correlation analysis with ImageJ Costes map, scatter plot and cytofluorogram for the images as shown in Figure 5. Notes: M1 and M2 represent the Manders correlation coefficients. All images were taken with 40× magnification. Abbreviation: PCC, Pearson’s correlation coefficient.
Figure 7
Figure 7
Cell viability under X-ray radiation, Notes: (A) Viability of normal (CCD 841 CoN) and cancer cells (HCT116) toward different doses of 6 MeV X-ray radiation. (B) The viability of HCT116 cancer cells treated with different samples and different radiation doses. Abbreviations: FA, folic acid; PLGA, poly(D,L-lactide-co-glycolic acid); VP, verteporfin.
Scheme 1
Scheme 1
Illustration of conjugation and PDT mechanism. Notes: (A) Synthesis of FA-PLGA-VP conjugates. (B) FA-PLGA-VP targeting and interacting with cancer cells following X-ray radiation exposure. (i) Specific binding to FRs overexpressed in cancer cells and cellular internalization. (ii) Cellular uptake of the conjugates via the FR-mediated endocytosis pathway. (iii) Endosomal escape and accumulation around the mitochondria of NPs as well as 1O2-induced cell killing with X-ray-triggered PDT. Abbreviations: FA, folic acid; FR, folate receptor; NPs, nanoparticles; PDT, photodynamic therapy; PLGA, poly(D,L-lactide-co-glycolic acid); VP, verteporfin.
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