Versatile RBC-derived vesicles as nanoparticle vector of photosensitizers for photodynamic therapy

Abstract

Various nanocarriers for photosensitizers have been developed to solve the problems of limiting the clinical utility of photodynamic therapy (PDT); however, to date, no carriers capable of supplying oxygen have been reported. We reported the development of a novel system composed of red blood cell (RBC)-derived vesicles (RDVs) generated by osmotic stress and demonstrated the capacity of RDVs for encapsulating and delivering external cargo into targeted cells due to the cellular uptake of RDVs. In this study, protoporphyrin IX (PpIX)-encapsulated RDVs (PpIX@RDVs) were prepared by the hypotonic incorporation of PpIX into RDVs in an aqueous environment, characterized, and utilized for PDT of cancer. PpIX@RDVs were rapidly uptaken by tumor cells via endocytosis in vitro, and the highly phototoxic effect of PpIX@RDVs was demonstrated upon irradiation. Superoxide anion (O₂˙) and singlet oxygen (¹O₂) were involved in PpIX@RDV-induced cell apoptosis and necrosis. Finally, we demonstrated that RDVs with an oxygen supply capacity have potential as versatile delivery vehicles for efficient PDT.

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Introduction

Photodynamic therapy (PDT) has emerged as a potential treatment for various cancers and other diseases.¹ The practical applications of PDT are based on the fact that the photosensitizers (PSs), after their systemic administration, can effectively and selectively accumulate in diseased targets, while decreasing nonspecific damage to healthy tissues.² Upon irradiation with appropriate light wavelengths, the excited PSs can transfer energy to surrounding oxygen, generating highly reactive oxygen species (ROS), such as singlet oxygen (¹O₂) or other free radicals, to induce cell death or tissue damage.^1–3 Therefore, PSs, light, and cellular or tissue oxygen are three crucial components for the clinical utility of PDT.

Most PSs, such as porphyrins, are hydrophobic and may easily aggregate in the physiological environment, which often limits their clinical utility.⁴ Even for hydrophilic PSs, the selective accumulation in the diseased targets is not high enough for clinical use. Therefore, various delivery carriers, such as liposomes,⁵ polymeric micelles,⁶ conjugated polymer nanoparticles,⁷ silica-based nanoparticles,^8,9 polyacrylamide-based nanoparticles,^10,11 and so on, have been developed for enhanced water solubility, improved stability, or higher accumulation selectivity of hydrophobic PSs. However, these methods show some but not comprehensive benefits to delivering PSs for clinical PDT. For instance, up to now, no carriers being capable of supplying oxygen have been reported. Therefore, well-rounded delivery carriers gathering multiple advantageous characteristics are still needed.

Previously, we developed a novel system composed of red blood cell (RBC)-derived vesicles (RDVs) generated by osmotic stress for efficient delivery of superparamagnetic iron oxide (SPIO) nanoparticles into human stem cells for in vitro and in vivo cellular magnetic resonance imaging (MRI),¹² demonstrating the capacity of RDVs for encapsulating external cargos and the efficient delivery of encapsulated cargos into targeted cells due to the cellular uptake of RDVs. The origination of osmotic stress-generated RDVs is based on the fact that in the aging process of RBC in the circulatory system, the hemoglobin and membrane components within erythrocytes can be diminished^13–15via the vesiculation facilitated by the spleen to generate endogenous RDVs.^16,17 Although endogenous RDVs and senescent erythrocytes may eventually be recognized and removed from circulation, the fact that endogenous RDVs can be harvested from peripheral blood¹⁶ suggests that RDVs could escape from spleen after their vesiculation into the circulation. In addition, the biocompatibility of autologous RDVs would improve the toxicology issue of the applications of systemic delivery systems in biomedicine.¹² Moreover, the hemoglobin inside RDVs holds the promise to be an oxygen source for PDT. On the basis of the above facts, we hypothesized that osmotic stress-generated RDVs would carry versatile properties such as effective delivery, biocompatibility, easy preparation, long in vivo circulation and, above all, oxygen supply for a highly efficient PDT drug delivery platform.

In this study, to examine our hypothesis, we used RDVs for encapsulating protoporphyrin IX (PpIX), a hydrophobic PS, for PDT in cancer therapy. We encapsulated PpIX into RDVs by a simple hypotonic method that resulted in the synthesis of PpIX-encapsulated RDVs (PpIX@RDVs), which can, as PpIX@RDVs, be well-dispersed under aqueous conditions. To assay the advantages of RDVs as a nanoscale delivery carrier of PSs, we examined the characterization, cellular uptake and in vitro anti-tumor mechanism and efficacy of PpIX@RDVs. Moreover, we explored the potential of PpIX@RDVs for supplying oxygen for a superior PDT performance.

Materials and methods

Preparation of RDVs and PpIX-encapsulated RDVs (PpIX@RDVs)

Blood samples were collected from 3 healthy regular donors with informed consent approved according to the procedures of the institutional review board. The preparation of RDVs was performed as previously described.¹² Briefly, blood cell sediments collected by centrifugation were mixed with a gradient of CaCl₂ (1 M) and EDTA (390 mM) at 45 °C for 30 min to produce vesiculation. The reactant was in turn centrifuged at 1700 g at 4 °C for 10 min to remove large RBC ghosts. The ultrasmall RDVs in the supernatant was the harvested by centrifugation at 16 000 g at 4 °C for 10 min. After synthesis, the RDV pellet was resuspended in Dulbecco's phosphate buffered saline (DPBS, GIBCO) and then determined for the protein concentration by the Bio-Rad assay.

For encapsulation of PpIX into RDVs, RDVs (50 μg) were incubated in 90 μL hypoosmotic lysing buffer (100 mM Na₂HPO₄ in 20 μL, 100 mM NaH₂PO₄ in 20 μL, 50 μL ddH₂O, pH 8) containing 10 μL PpIX (10 μM) (Sigma-Aldrich) at 4 °C for 1 h. The mixture was then centrifuged at 16 000 g at 4 °C for 10 min. The pellet (PpIX@RDVs) was washed twice and resuspended in DPBS at 4 °C.

Characterization of RDVs and PpIX-encapsulated RDVs (PpIX@RDVs)

The particle size and zeta potential were measured by a particle size analyzer (90 plus, Brookhaeven, Instrument Corporation) and by ZetaPALS (Brookhaven, NY) in DPBS, respectively. For transmission electron microscopy (TEM) observations, RDVs or PpIX@RDVs were directly loaded onto the grid (Electron Microscopy Sciences) and extra DPBS was absorbed by nitrocellulose membrane, followed by incubation with 2% phosphotungstic acid (PTA) for 1 min. The loaded grid was stocked in a dry box at room temperature overnight for dehydration and then observed with TEM (Hitachi H-7650) at an accelerating voltage of 80 kV.

To determine the encapsulation efficiency, 50 μg PpIX@RDVs pellets were incubated with lysis buffer (0.15 M NH₄Cl, 10 mM NaHCO₃, 1 mM disodium EDTA, 0.3% triton X-100, pH 7.4) and sonicated at room temperature for 15 min in the dark. The solution was then examined spectrophotometrically at 632 nm using a microplate reader (Infinite M 200, TECAN). The encapsulated PpIX content was calculated from a standard plot of known concentrations of free PpIX vs. the corresponding absorbance density.

Cellular uptake of PpIX@RDVs

Huh7 cells were seeded in 35 mm dishes (2 × 10⁵ cells per dish) overnight. The cells were treated with free PpIX or PpIX@RDVs for 3 h, followed by washing with DPBS. Fresh media containing 100 nM LysoTracker Green DND-26 (Molecular Probes) were added into the dishes for 5 min, and the cells were washed three times with fresh media. The washed cells were observed with a confocal spectral microscope (Olympus FV10i) using a 60× oil immersion objective.

Phototoxicity and its mechanism of PpIX@RDVs

Huh7 cells (5 × 10³ cells) were seeded in a 96-well plate and allowed to attach for 24 h. To evaluate the roles of ROS in PpIX@RDVs-induced phototoxicity, in some experiments cells were pretreated with the indicated ROS scavengers such as tiron (1 mM) for superoxide anion (O₂˙), catalase (500 U) for hydrogen peroxide (H₂O₂), D-mannitol (20 mM) for hydroxyl radical (OH˙) and L-histidine (5 mM) for ¹O₂, for 1 h before the treatments of RDVs, free PpIX or PpIX@RDVs. After incubation with the indicated concentrations of RDVs, free PpIX or PpIX@RDVs in growth media for 3 h, the cells were illuminated using a diode-laser (175 ± 4 mW m⁻²) with the total energy at 1–20 J per well. Then, irradiated cells were incubated for 24 h and phototoxicity was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The data are expressed as the percentage variation of control cell viability as 100%.

Alternatively, Huh7 cells (2 × 10⁵ cells) were seeded in a 6-well plate and allowed to attach for 24 h. After incubation with the indicated concentrations of RDVs, free PpIX or PpIX@RDVs in growth media for 3 h, the cells were illuminated with the total energy at 5 J per well. Then, irradiated cells were incubated for 24 or 1 h and then directly imaged using a microscope (Olympus CKX41) for cell morphological observation or processed for being stained with Annexin V-FITC and propidium iodide (PI) (Annexin V-FITC assay kit; AbD, Serotec), respectively, according to the manufacturer's protocol, for flow cytometry.

Preparation and spectral absorbance of oxyhemoglobin (HbO₂)- and carboxyhemoglobin (HbCO)-containing PpIX@RDVs

As modified from a previous study,¹⁸ HbO₂-containing PpIX@RDVs and HbCO-containing PpIX@RDVs were prepared from as-synthesized PpIX@RDVs supplied with gaseous mixtures of O₂/CO₂ (94.4 : 5.6 v/v) and N₂/CO (95 : 5 v/v), respectively, for 15 min at a pressure of 1.5 kg cm⁻² and their absorbance spectra were immediately determined by a Varian Cary 50 UV/Vis spectrophotometer.

Phototoxicity of PpIX@RDVs, HbO₂-containing PpIX@RDVs and HbCO-containing PpIX@RDVs

Huh7 cells (5 × 10³ cells) were seeded in a 96-well plate and allowed to attach for 24 h. After incubation with the indicated concentrations of PpIX@RDVs, HbO₂-containing PpIX@RDVs, and HbCO-containing PpIX@RDVs in growth media for 3 h, the cells were illuminated with the total energy at 5 J per well. To further demonstrate the important role of RDVs on oxygen supply, an iron chelator, desferrioxamine (DFO; 30 μM), was used following the incubation of RDVs to mimic the environment of hypoxic conditions (Fig. S2†). Then, irradiated cells were incubated for 24 h and phototoxicity was assessed using the MTT reduction assay.

Statistical analysis

Data are presented as the mean ± standard error of mean (SEM) for the indicated numbers of separate experiments. The results were compared using Student's t-test. Statistical significance was assigned if the p-value was less than 0.05.

Results and discussion

Synthesis and characterization of PpIX@RDVs

The particle size and size distribution of RDVs slightly varied from donor to donor. In one of the representative donors, these RDVs had an average diameter of 258.9 nm (Fig. 1a). After encapsulation of PpIX into RDVs, PpIX@RDVs were highly monodispersed as native RDVs in the aqueous solutions, such as phosphate buffered saline and cultured medium, and had an average diameter of 255.2 nm (Fig. 1a). No significant difference of particle size between RDVs and PpIX@RDVs was observed. Also, surface charge was not significantly different between RDVs and PpIX@RDVs. The zeta potentials of RDVs and PpIX@RDVs were about −15.99 ± 0.53 and −16.32 ± 1.70 mV, respectively.

Fig. 1 The preparation and characterization of PpIX-encapsulated RDVs (PpIX@RDVs). RDVs were generated by osmotic stress from human RBC. PpIX@RDVs were synthesized with the incorporation of PpIX into RDVs in a hypoosmotic solution. (a) The particle sizes of RDVs and PpIX@RDVs were analyzed by a particle size analyzer. The y axis plots intensity, and the x axis plots particle size. The polydispersity index (PDI) values of RDVs and PpIX@RDVs measured by Nano ZS, ZEN 3600 (Malvern Instruments) are 0.31 and 0.32, respectively. (b) RDVs and PpIX@RDVs were observed under TEM. Scale bar, 200 nm.

Fig. 1b shows the TEM images of RDVs and PpIX@RDVs. As can be seen, RDVs and PpIX@RDVs are quasi-spherical and display no morphological differences. In the samples of Fig. 1b, 38.89 ± 2.15% of loaded PpIX (∼56 μg), about 21.9 μg of PpIX, was encapsulated into RDVs (50 μg).

Cellular uptake of PpIX@RDVs

To determine the intracellular uptake and distribution of free PpIX and PpIX@RDVs, fluorescence imaging using confocal microscopy was performed on Huh 7 cells. Huh7 cells were incubated with free PpIX (0.42 μM in Fig. 2a–c; 10 μM in Fig. 2g–i) or PpIX@RDVs with 0.42 μM PpIX (Fig. 2d–f) for 3 h, followed by LysoTracker Green staining. We tracked PpIX and lysosomes individually using different fluorescence filters. The fluorescence images of LysoTracker Green in Huh7 cells indicated lysosomes (Fig. 2a, d, and g). Cells treated with free PpIX at 0.42 μM (Fig. 2b) or even at 10 μM (Fig. 2h) showed no significant red fluorescence of PpIX; however, a very strong fluorescence signal of PpIX was observed in cells treated with PpIX@RDVs with 0.42 μM PpIX (Fig. 2e), indicating a highly efficient uptake of PpIX@RDVs. Moreover, a colocalization of PpIX and LysoTracker signals (yellow fluorescence) was exhibited (Fig. 2f), suggesting an endocytosis of PpIX@RDVs.

Fig. 2 Cellular endocytosis of PpIX@RDVs. Huh7 cells were incubated with free PpIX at 0.42 μM (a–c), at 10 μM (g–i), or with PpIX@RDVs (0.42 μM of PpIX; d–f) for 3 h, followed by LysoTracker Green staining. (a), (d) and (g): the images of lysosomes with LysoTracker Green (excitation at 405 nm, emission at 524 nm); (b), (e) and (h): the observation of PpIX (excitation at 405 nm, emission at 620 nm); (c), (f) and (i): colocalization of PpIX with lysosomes (yellow fluorescence), particularly in (f). Scale bar 20 μm.

Phototoxic effect and mechanism of PpIX@RDVs in vitro

We incubated Huh7 cells with RDVs (10, 50, and 100 μg), free PpIX (0.076, 0.38, and 0.76 μM), or PpIX@RDVs (0.076 μM PpIX@10 μg RDVs, 0.38 μM PpIX@50 μg RDVs, and 0.76 μM PpIX@100 μg RDVs) in a 96-well cell plate for 3 h, and then irradiated them with a diode-laser (635 nm, 175 mW cm⁻²) with the total energy at 5 J per well (Fig. 3a). After irradiation, we determined cell viability using the MTT assay. Although we observed minor but significant cytotoxicity in RDV-treated or free PpIX-treated cells with irradiation, when PpIX@RDVs-treated cells were irradiated, cell viability dramatically decreased (Fig. 3a), which was dependent on the doses of PpIX@RDVs. Moreover, the phototoxicity of PpIX@RDVs was higher than that of free PpIX at the same PpIX concentrations, suggesting that the enhanced phototoxicity of PpIX@RDVs is due to the enhanced cellular uptake of PpIX by RDVs, as demonstrated in Fig. 2. In addition, we incubated Huh7 cells with 50 μg RDVs, free PpIX at 0.38 μM and PpIX@RDVs (0.38 μM PpIX@50 μg RDVs) in a 96-well cell plate for 3 h, and then irradiated treated cells with the various energies at 1, 5 or 20 J per well (Fig. 3b). In RDV-treated or free PpIX-treated cells, minor but significant cytotoxicity was only observed when the irradiation energy was more than 5 J. In PpIX@RDVs-treated cells, compared with free PpIX-treated cells, the enhanced phototoxicity of PpIX@RDVs could be observed at different irradiation energies (Fig. 3b). Moreover, the decrease of cell viability in PpIX@RDV-treated cells was highly irradiation-energy dependent. The observation of cell morphology also demonstrated the enhanced phototoxicity of PpIX@RDVs (Fig. 3c). After a 5 J irradiation, the morphology of the Huh7 cells did not change compared with that of the Huh7 cells before irradiation (Fig. 3c, control), and there was no morphological change observed in RDVs-treated cells. Although free PpIX-treated Huh7 cells showed some morphological changes, no more obvious changes were observed as the concentration of free PpIX increased (0.49 μM PpIX vs. 2 μM PpIX), suggesting that a low solubility of hydrophobic PpIX in culture medium limited the cellular utility even at a high concentration of PpIX. Importantly, great changes could be observed in PpIX@RDV-treated cells, and the cell structures were rounded and destroyed at a low encapsulated-concentration of PpIX (0.49 μM). These results demonstrate the capacity of RDVs for stabilizing and intracellularly delivering PpIX for efficient PDT for cancer therapy.

Fig. 3 Photocytotoxicity of free PpIX, RDVs and PpIX@RDVs against Huh 7 cells. (a) Dose dependent cytotoxicity. Huh 7 cells were treated with RDVs (10, 50, and 100 μg), free PpIX (0.076, 0.38, and 0.76 μM), or PpIX@RDVs (0.076 μM PpIX@10 μg RDVs, 0.38 μM PpIX@50 μg RDVs, and 0.76 μM PpIX@100 μg RDVs) for 3 h, irradiated at 5 J, and then grown for 24 h before MTT assay. (b) Irradiation energy-dependent cytotoxicity. Huh 7 cells were treated with RDVs (50 μg), free PpIX (0.38 μM), or PpIX@RDVs (0.38 μM PpIX@50 μg RDVs) for 3 h, irradiated at different energies, and then grown for 24 h before MTT assay. For (a) and (b) all data are expressed as mean ± standard error of three determinations (each in quadruplicate). (**, P < 0.01; ***, P < 0.001 as compared with control). (c) Transmission images depict the cell morphology. Scale bar 3 μm.

It has been deduced that PDT for cancer cells is due to the generation of ROS or other radicals. Following the absorption of light, the activated PSs can undergo two types of reaction. In type I reaction, the activated PSs can react directly with the substrate and transfer a hydrogen atom to form radicals. These radicals then interact with oxygen to produce oxygenated products. Alternatively, the activated PSs can transfer their energy directly to oxygen to form ¹O₂ in the type II reaction.¹ Therefore, we examined the effects of various scavengers such as tiron (1 mM) for superoxide anion (O₂˙), catalase (500 U) for hydrogen peroxide (H₂O₂), D-mannitol (20 mM) for hydroxyl radical (OH˙) and L-histidine (5 mM) for ¹O₂ on PpIX@RDV-mediated phototoxicity (Fig. 4a). When cells were treated with PpIX@RDVs (0.42 μM PpIX@50 μg RDVs), tiron and histidine but not catalase or mannitol partly protected PpIX@RDV-treated cells from phototoxicity, suggesting that O₂˙ and ¹O₂ are crucially involved in PpIX@RDV-mediated phototoxicity in Huh7 cells in the present study. We also examined the cell death pattern by annexin V and PI staining (Fig. 4b). Annexin V positive and PI negative (annexin V+/PI−) indicated that cells were in early apoptosis; on the contrary, annexin V negative and PI positive (annexin V−/PI+) suggested that cells underwent a necrosis spurt. When cells are in late apoptosis or early necrosis, annexin V and PI are both positive (annexin V+/PI+). Fig. 4b shows that significant increases in annexin V+/PI+ cells and annexin V−/PI+ cells were observed only in the PpIX@RDV treatment group, suggesting a concurrent occurrence of apoptosis and necrosis. Although cell death following PDT can occur by apoptosis or necrosis, or a combination of the two depending on cell type, concentration, the intracellular localization of various PSs as well as the light doses,^19–21 in many cases apoptosis has been shown to be a rapid and dominant form of cell death.^22,23 Since most PSs for PDT are efficient in producing ¹O₂, type II photochemistry is assumed to be the dominant mechanism for PDT;²³ however, both ¹O₂ and O₂˙ can induce apoptosis. As shown in Fig. 4b, apoptosis was more dominant than necrosis, suggesting that PpIX@RDVs could induce efficient apoptosis and subsequent necrosis via type I and type II reactions.

Fig. 4 Mechanism study of PpIX@RDV-induced cell death. (a) The antagonizing effects of various antioxidants on PpIX@RDV-induced cytotoxicity. Data are expressed as mean ± standard error of three determinations (each in quadruplicate) (***, P < 0.001 as compared with Control; #, P < 0.05; ##, P < 0.01 as compared with PpIX@RDVs). (b) The percentage of apoptotic and necrotic cells was quantified by the Annexin V-propidium iodide (PI) flow cytometry method. Huh7 cells were treated with RDVs (50 μg), free PpIX (0.38 μM), or PpIX@RDVs (0.38 μM PpIX@50 μg RDVs) for 3 h, irradiated at 5 J, and then incubated for 1 h. These cells were stained using the Annexin V-FITC assay kit and then subjected to FACS analysis. Cells were gated into four quadrants: annexin V−/PI−, viable cells; annexin V+/PI−, early apoptosis; annexin V+/PI+, late apoptosis/early necrosis; annexin V−/PI+, necrosis.

Oxygen supply for PDT

Each of the three crucial components, light, PSs and oxygen, for PDT is multifactorial and these factors are interdependent.¹ To date, various carriers, in particular, nanoscale carriers have been extensively used to improve the issues associated with PSs; however, no nanoscale particles capable of improving oxygen availability have been reported. To explore the capacity of RDVs for supply oxygen for efficient PDT, HbCO-containing PpIX@RDVs were generated and their phototoxic effect was examined. Fig. 5a illustrates spectral absorption curves of PpIX@RDVs, HbO₂-containing PpIX@RDVs, and HbCO-containing PpIX@RDVs between 500 and 600 nm and suggests that PpIX@RDVs had been mainly occupied by oxygen and that HbCO-containing PpIX@RDVs were indeed derived. When cells were treated with PpIX@RDVs (0.36 μM PpIX@50 μg RDVs), HbO₂-containing PpIX@RDVs, and HbCO-containing PpIX@RDVs for irradiation, PpIX@RDVs and HbO₂-containing PpIX@RDVs induced a similar degree of cell death, but HbCO-containing PpIX@RDVs showed less cytotoxicity (Fig. 5b). Moreover, we used an iron chelator, desferrioxamine (DFO), to mimic the environment of hypoxic conditions (Fig. S2†) to demonstrate the important role of RDV hemoglobin in oxygen supply. The cell viability data showed that with the treatment of DFO, HbO₂-containing PpIX@RDVs indeed induced much more cell death than PpIX@RDVs or HbCO-containing PpIX@RDVs (Fig. 5c). Because PpIX@RDVs have efficiently killed cancer cells already (Fig. 3), the increased cell death induced by HbO₂-containing PpIX@RDVs under hypoxia compared to PpIX@RDVs or HbCO-containing PpIX@RDV suggests that the oxygen of HbO₂-containing PpIX@RDVs could be used to kill the remaining cells difficult to handle. All the data suggest that RDVs play a crucial role as an oxygen carrier for efficient PDT.

Fig. 5 (a) Absorption spectra of PpIX@RDVs, HbO₂-containing PpIX@RDVs, and HbCO-containing PpIX@RDVs. No difference of absorptivity between PpIX@RDVs and HbO₂-containing PpIX@RDVs suggests the oxygen occupation of as-synthesized PpIX@RDVs. A unique absorption curve of HbCO indicates the availability of HbCO-containing PpIX@RDVs; (b) and (c), photocytotoxicity of PpIX@RDVs, HbO₂-containing PpIX@RDVs, and HbCO-containing PpIX@RDVs. Huh 7 cells were treated with PpIX@RDVs (0.36 μM PpIX@50 μg RDVs), HbO₂-containing PpIX@RDVs (0.36 μM PpIX@50 μg RDVs pre-saturated with O₂), or HbCO-containing PpIX@RDVs (0.36 μM PpIX@50 μg RDVs pre-saturated with CO) for 3 h, treated with (c) or without (b) 30 μM DFO, irradiated at 5 J, and then grown for 24 h before MTT assay. Without the treatment of DFO, HbCO-containing PpIX@RDVs induced less cell cytotoxicity. With the treatment of DFO, HbO₂-containing PpIX@RDVs indeed induced much more cell death than PpIX@RDVs or HbCO-containing PpIX@RDVs. The data are expressed as the percentage variation of PpIX@RDV-induced cell death as 100% (***, P < 0.001 as compared with PpIX@RDVs).

Conclusions

In summary, we showed that RDVs efficiently encapsulated hydrophobic PpIX. The as-synthesized PpIX-encapsulated RDVs (PpIX@RDVs) were of nanosize, easy to prepare, highly dispersed, and stable in aqueous conditions. The drug carrier was rapidly internalized into Huh7 cells via endocytosis in vitro. The irradiation of cells resulted in the generation of the cytotoxic superoxide anion and singlet oxygen, and thus resulted in apoptotic and necrotic cell death. Moreover, the current study first verified that RDVs could offer significant promise as novel photosensitizers for efficient PDT due to the oxygen supply capacity.

Acknowledgements

This work was supported by grants from the National Health Research Institutes (NHRI) (NM-099-PP02, NM-099-PP-09, NM-100-PP-02 and NM-100-PP-09) and the National Science Council (99-2320-B-400-002-MY3), both of Taiwan.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr32506c

‡ These authors contributed equally to this work.

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