Non-Polymeric Nanogels as Versatile Nanocarriers: Intracellular Transport of the Photosensitizers Rose Bengal and Hypericin for Photodynamic Therapy
Torres-Martínez, Ana; Bedrina, Begoña; Falomir, Eva; Marín, María J.; Angulo-Pachón, César A.; Galindo, Francisco; Miravet, Juan
- Published
- 2021-04-01
- Publisher
- American Chemical Society
- Language
- en
Abstract
The use of nanocarriers for intracellular transport of actives has been extensively studied in recent years and represents a central area of nanomedicine. The main novelty of this paper lies on the use of nanogels formed by a low-molecular-weight gelator (1). Here, non-polymeric, molecular nanogels are successfully used for intracellular transport of two photodynamic therapy (PDT) agents, Rose Bengal (RB) and hypericin (HYP). The two photosensitizers (PSs) exhibit different drawbacks for their use in clinical applications. HYP is poorly water-soluble, while the cellular uptake of RB is hindered due to its dianionic character at physiological pH values. Additionally, both PSs tend to aggregate precluding an effective PDT. Despite the different nature of these PSs, nanogels from gelator 1 provide, in both cases, an efficient intracellular transport into human colon adenocarcinoma cells (HT-29) and a notably improved PDT efficiency, as assessed by confocal laser scanning microscopy and flow cytometry. Furthermore, no significant dark toxicity of the nanogels is observed, supporting the biocompatibility of the delivery system. The developed nanogels are highly reproducible due to their non-polymeric nature, and their synthesis is easily scaled up. The results presented here thus confirm the potential of molecular nanogels as valuable nanocarriers, capable of entrapping both hydrophobic and hydrophilic actives, for PDT of cancer.
Full text
S1 SUPPORTING INFORMATION Non-polymeric Nanogels as Versatile Nanocarriers. Intracellular Transport of the Photosensitizers Rose Bengal and Hypericin for Photodynamic Therapy Ana Torres-Martínez,a Begoña Bedrina,a Eva Falomir,a María J. Marín,b César A. AnguloPachón,a Francisco Galindoa,* and Juan F. Miraveta,* aDepartament de Química Inorgànica i Orgànica, Universitat Jaume I, Avda. Sos Baynat s/n, 12071 Castelló de la Plana, Spain. E-mail: [email protected] , [email protected] bSchool of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK
S2 460 480 500 520 540 560 580 600 620 640 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 Absorbance Wavelength / nm Original Baseline corrected Figure S1. Original and baseline corrected UV-Vis absorption spectrum of a sample of RB@1 in PBS. 500 520 540 560 580 600 620 640 660 680 700 0 0.02 0.04 0.06 0.08 0.1 Absorbance Wavelength / nm Original Baseline corrected Figure S2. Original and baseline corrected UV-Vis absorption spectrum of a sample of HYP@1 in PBS.
S3 Negative control 1:2 NPs 1:2 Negative control 1:3 NPs 1:3 0 20 40 60 80 100 % viable cells Figure S3. Trypan blue cell viability assays on HT-29 cells incubated with nanogel samples of 1 (NPs in the graph) diluted 1:2 and 1:3. 0 20 40 60 80 100 % Viable Negative control Free RB RB@1 Early apoptosis Figure S4. Cell viability and early apoptosis obtained for non-irradiated control experiments with HT-29 cells loaded with RB as a photosensitizer, using annexin VFITC/propidium iodide staining. Negative control corresponds to cells incubated with PBS. The results are the average of three different batches analyzed in duplicate (average [RB] in culture media was 2 M).
S4 Viable Early apoptosis 0 20 40 60 80 100 % cells Negative control NPs Figure S5. Cell viability and early apoptosis obtained for PDT experiments (2 min irradiation) of HT-29 cells incubated with unloaded nanogels samples, using Annexin VFITC/propidium iodide staining. Negative control corresponds to cells incubated with PBS. 0 10 20 30 40 50 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 y = -0,1171x + 0,0118 R² = 0,9927 y = -0,0079x - 0,0030 R² = 0,9968 RB@1 RB ln(C/C0) Irradiation time / min 0 50 100 150 200 250 300 -0.07 -0.06 -0.05 -0.04 -0.03 -0.02 -0.01 0.00 0.01 y = -0,000117x - 0,001500 R² = 0,935042 y = -0,000216x + 0,002036 R² = 0,983108 HYP@1 HYP (PBS) ln(C/C0) Irradiation time / s Figure S6. Representative examples of the linear fit of ABDA fluorescence emission (λex 380 nm, λem 407 nm) decay as a function of the irradiation time in the presence of PS@1 and PS in PBS (left: RB@1 and RB, right: HYP@1 and HYP).
S5 300 350 400 450 500 550 600 650 700 750 0.0 0.2 0.4 0.6 0.8 1.0 300 350 400 450 500 550 600 650 700 750 0.0 0.3 0.6 0.9 1.2 1.5 1.8 10 M HYP DMSO 10 M HYP PBS-10% DMSO Normalised absorbance Wavelength / nm Figure S7. Normalised UV-Vis absorption spectra of a 10 µM hypericin solution in DMSO (top) and in PBS, 10% DMSO (bottom). HYP (PBS) HYP (PBS-DMSO) HYP@1 Figure S8. Images from confocal laser scanning microscopy; λex 561 nm. The fluorescence intensity has been enhanced to show the localization of the dye. From left to right: merged, DIC and green channels. Scale bar = 27 m.
S6 0 20 40 60 80 100 % Negative control HYP in PBS Viable Apoptosis Non-viable Figure S9. Cell viability and apoptosis obtained for PDT experiments (2 min irradiation) with HT-29 cells and HYP as a photosensitizer (HYP in PBS), using YO-PRO®-1/propidium iodide staining. Negative control corresponds to cells incubated with PBS. The results are the average of three different batches analyzed in duplicate (average [HYP] in culture media was 0.02 M).
S7 0 20 40 60 80 100 % Negative control HYP PBS-DMSO HYP@1 Viable Early apoptosis Necrosis Figure S10. Results obtained by flow cytometry of cell viability and apoptosis in PDT experiments without irradiation with HT-29 cells and HYP as a photosensitizer. YO-PRO®- 1/propidium iodide was used for staining. Negative control corresponds to cells incubated with PBS. The results are the average of three different batches analyzed in duplicate. [HYP] = 0.2 M.
S8 Figure S11. Superposition of raw correlation data and cumulants fit from 6 measurements (11 scans each) by DLS of a sample of RB@1 Figure S12. Superposition of raw correlation data and cumulants fit from 6 measurements (11 scans each) by DLS of a sample of HYP@1.
S9 2 min irradiation No irradiation RB@1 RB control Negative control Figure S13. Representative dot plot obtained by flow cytometry in the analysis of HT-29 cells incubated with RB@1.