Photochemistry and Photobiology, 2014, 90: 1144–1149

5-Aminolevulinic acid-Loaded Fullerene Nanoparticles for In Vitro and In Vivo Photodynamic Therapy Zhi Li, Li-Li Pan, Fei-Long Zhang, Xia-Li Zhu, Yang Liu and Zhen-Zhong Zhang* School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China Received 30 April 2014, accepted 2 June 2014, DOI: 10.1111/php.12299

ABSTRACT This report explores some properties of 80–200 nm nanoparticles containing 5-aminolevulinic acid (ALA) and fullerene (C60) for photodynamic therapy (PDT). Compared with ALA, the nanoparticles yielded more protoporphyrin IX (PpIX) formation in cells and tissues and to a significant improvement in antitumor efficacy in tumor-bearing mice. Maximum levels of PpIX were obtained 4 h after administration and selective PpIX formation in tumor was observed. These nanoparticles appear to be a useful vehicle for drug delivery purposes. In this study, a procedure for preparing fullerene nanoparticles containing ALA was developed. The product alone exhibited no detectable toxicity in the dark and was superior to ALA alone in promoting PpIX biosynthesis and PDT efficacy both in culture and in a murine tumor model. These results suggest that this procedure could be the basis for an improved PDT protocol for cancer control.

keep the drug incirculation for longer periods of time (by preventing renal clearance and non-specific uptake) and allow increased uptake within the tumors based on EPR (enhanced permeability and retention) effects (13–15). Fullerene(C60), the third allotrope of carbon after diamond and graphite, is a nanoscale carbon material with unique photo-, electro-chemical, and physical-properties, however, its inherent hydrophobicity limits use in biology and thus lead to its search in searching water-soluble fullerene derivatives (16–18). Recently, we reported some water-soluble fullerene derivatives which were modified with amino acid through an amide linker (19). 5-ALA was also with carboxyl and amino terminal, and several chemical modifications have been made both on the N- and C-termini of 5-ALA, which induced higher PpIX production and photosensitization (20–22). In this study, the conjugation of ALA to C60 nanoparticles has been prepared. C60-5-ALA nanoparticles were stable in water over multiple weeks. The photodynamic and inducing PpIX synthesis efficacy of these nanoparticles was examined using B16-F10 cells and tumor-bearing mice models.

INTRODUCTION Photodynamic therapy (PDT) has lower potential for peripheral toxicity than many conventional cancer therapies and has become an increasingly recognized alternative for traditional therapies in the clinic (1,2). PDT involves three components: a photosensitizer (PS) drug, a specific wavelength of drug-activating light and oxygen (3,4). A promising approach in PDT involves the exogenous administration of 5-aminolevulinic acid (5-ALA), a precursor of the heme biosynthesis pathway, which can be metabolized to protoporphyrin IX (PpIX) (5). PpIX is the endogenous photosensitizer needed for PDT. 5-ALA also has great potential as a photodynamic detection (PDD) in clinical practice (6–8). The use of 5-ALA-induced PpIX fluorescence is currently being exploited for diagnosis of bladder cancer, intraepithelial lesions of the cervix and lung cancer and fluorescence-guided surgery for resection of malignant gliomas. However, the efficacy of 5-ALA-PDT is limited by the hydrophilic nature of the molecule, 5-ALA has poor penetration through the natural barriers such as the intact skin, the intestinal walls, as well as through the cell membranes, leading to poor penetration through tumor (9–12). In recent years, the benefits of small molecule drugs have been significantly enhanced via the nanomedicine approach where the drug molecules are packaged within nanovehicles that *Corresponding author email: [email protected] (Zhen-Zhong Zhang) © 2014 The American Society of Photobiology

MATERIALS AND METHODS Materials. Fullerene (C60, purity > 95%) were purchased from Henan Fengyuan Chemicals Co. Ltd. 5-aminolevulinic acid (5-ALA) and protoporphyrin IX (PpIX) were received from Aladdin Chemistry Co. Ltd. Sulforhodamine B (SRB), DMEM cell culture medium, penicillin, streptomycin, fetal bovine serum (FBS) were bought from Gibco Invitrogen. Synthesis of C60-5-ALA and characterization. C60-5-ALA was synthesized according to the procedure of our previous study (12). Briefly, 5-ALA (70 mg) and NaOH (120 mg) were added to ethanol–water mixture (ethanol:water 5:1, 2.4 mL), and then added dropwise to a toluene solution (6 mL), containing C60 (25 mg). Afterward, the solution was stirred with magnetic agitation at room temperature until the organic layer was nearly colorless. Using a rotary evaporation machine to remove organic solvents, the lower layer black solution was mixed with ethanol to extract the water-soluble fraction of the C60 derivative precipitate. The precipitate was purified by rinsing with deionized water, and filtrated to remove the unreacted reagents to obtain C60-5-ALA complex. C60-5-ALA was characterized using an UV–VIS spectrometer (Lambda35, PerkineElmer). FT-IR spectra were recorded on an IR spectrophotometer (Nicolet iS10, Thermo). The relative amount of 5-ALA linked to C60 was tested using a thermal gravimetric analysis (TGA, PerkinElmer) with the experimental conditions of scanning from 25 to 800°C under nitrogen at a heating rate of 20°C min 1. Dynamic lights scattering (DLS) measurements of particle size were carried out using a Zetasizer Nano-ZS90 light scattering instrument (Malvern Instruments, Enigma Business Park, UK). Cell culture and viability measurements. B16-F10 mice melanoma cell line was obtained from Chinese Academy of Sciences Cell Bank (Catalog No. TCM36). Cells were cultured in normal DMEM culture medium with 10% FBS and 1% penicillin/streptomycin in 5% CO2 at 37°C in an incubator.


Photochemistry and Photobiology, 2014, 90 On cell phototoxicity measurements, B16-F10 cells were plated in 96well plates and then incubated for 24 h. After incubating B16-F10 cells with various concentrations of free 5-ALA or C60-5-ALA for 2 h, replace them with fresh culture medium, then further cultured for 4 h. After that, B16-F10 cells were exposed to 630 nm lasers (100 mW cm 2) for 0.5 min. The samples were further incubated at 37°C for 24 h. At last, cell phototoxicity was evaluated using Sulforhodamine B (SRB, Sigma) assay. Cell survival rate(% of control) = (ODtre)/ (ODcon) 9 100%, where ODtre was the absorbance value of treated cells cultured with C60 derivatives or ALA, ODcon was that of control cells cultured with culture medium only. Protoporphyrin IX (PpIX) extraction from cells. B16-F10 cells were seeded at 5 9 105 cells per well in six-well plates. When cells reached 70% confluence, they were treated with C60-5-ALA or 5-ALA (5-ALA dose:90 lg mL 1) for 2 h, then replace them with fresh culture medium after cultured for 2, 4,5, 8 and 12 h, respectively. After cell disruption through repetitive freeze-thawing, the cells were centrifuged and the supernatant was collected, 0.3 mL supernatant added with 0.7 mL methanol/ethanol absolute/acetonitrile (50:40:10), vortex mixed for 5 min, then centrifuged and the supernatant was employed for PpIX determination by high-performance liquid chromatography (HPLC, Waters) with the following conditions: an Eclipse XDB-C18 column (150 mm 9 4.6 mm, 5.0 mm); mobile phase methanol/ethanol absolute/acetonitrile 50:40:10; column temperature 25°C; the excitation and emission wavelengths of light used producing the highest fluorescence were 403 nm and 628 nm, respectively; and flowrate 1.0 mL min 1. PpIX was used as a reference standard. PpIX-specific fluorescence in B16-F10 cells was harvested by fluorescence microscopy (Nikon, 80i). Cells were treated with 5-ALA or


C60-5-ALA (5-ALA dose:90 lg mL 1) for 2 h, then replace them with fresh culture medium, further cultured for 4 h. Cells were stained with Hoechst 33342 for 30 min, washed three times with PBS, the cells were imaged by a Fluorescence Microscope. In vivo PDT effect. All animal experiments were performed under a protocol approved by Henan laboratory animal center. For the in vivo antitumor experiments, the tumor-bearing mice were divided into four groups (five mice per group), minimizing the differences in weights and tumor sizes in each group. The mice were administered with [1] saline (0.1 mL), [2] 630 nm laser alone, [3] C60-5-ALA/630 nm laser (5-ALA dose: 30 mg kg 1) and [4] 5-ALA/630 nm laser (5-ALA dose: 30 mg kg 1); C60-5-ALA and 5-ALA in saline were intravenous injected into mice via the tail vein every 2 days, and then the tumor regions were irradiated with 630 nm laser (100 mW cm 2, 0.5 min) at 4 h post injection. The mice were observed daily for clinical symptoms and the tumor sizes were measured by a caliper every other day and calculated as the volume = (tumor length) (tumor width)2/2. Tumor and normal tissue PpIX extraction. The tumor-bearing mice were given C60-5-ALA or 5-ALA (5-ALA dose: 30 mg kg 1). After treatment for 2, 3, 4 and 4.5 h, the mice were killed and tissues/organs were collected and weighed, 0.3 mL tissue homogenate added with 0.7 mL methanol, vortex mixed for 5 min, then centrifuged, 0.3 mL supernatant added with 0.7 mL mobile phase, vortex mixed for 5 min, after that centrifugation separated. The supernatant was employed for PpIX in tissues/organs by high-performance liquid chromatography (HPLC, Waters) with the following conditions: an Eclipse XDB-C18 column (150 mm 9 4.6 mm, 5.0 lm); mobile phase methanol/ethanol absolute /acetonitrile 50:40:10; column temperature 25°C; excitation and emission wavelengths of light used producing the highest fluorescence

Figure 1. Characterization of fullerenes (A): FT-IR spectrum of (a) pristine C60, (b) C60-5-ALA and (c) 5-ALA; (B): UV spectrum of (a) C60-5-ALA, (b) 5-ALA; (C): Photos of C60-5-ALA in (1) cell culture, (2) water medium for 4 weeks; (D): Size of C60-5-ALA; (E): Thermal gravimetric analysis curves of pristine C60, C60-5-ALA, 5-ALA.


Zhi Li et al.

were 403 nm and 628 nm, respectively; and flow rate 1.0 mL min 1. PpIX was used as a reference standard. For fluorimetric determination, C60-5-ALA in saline was intravenous injected into mice via the tail vein, after 4 h, frozen sections of tumor tissue were collected and imaged by a Fluorescence Microscope. Statistical analysis. All data are expressed as the mean  SD. The statistical significance of differences was calculated by Student’s t-test. P < 0.05 was considered as a significant difference.

RESULTS AND DISCUSSIONS Synthesis and characterization of C60-5-ALA The inherent hydrophobicity limits the use of C60 in drug delivery. 5-ALA complexed with fullerene will provide greater interaction between the fullerene and a biological environment leading to novel medical applications. Figure 1A shows the FT-IR spectra of C60, C60-5-ALA and 5-ALA. It can be observed that three bands of (~1181 cm 1), (~576 cm 1) and (~515 cm 1) correspond to C60 core (23). Conjugation of 5-ALA to C60 was confirmed by the strong C-N (~1108 cm 1) vibrations, N-H (~1641 cm 1, ~3406 cm 1) vibrations and C-H (2924 cm 1) vibrations of

5-ALA (Fig. 2A, c)(21). As seen in the UV–VIS spectrum of 5-ALA and C60-5-ALA (Fig. 1B), an absorption peak at 267 nm was observed both in 5-ALA and C60-5-ALA, also indicating that 5-ALA was linked to C60. C60-5-ALA was stable in water and cell culture medium over multiple weeks without significant aggregation (Fig. 1C). We found that C60-5-ALA tend to form monodisperse aggregates in the size range 100–200 nm as confirmed by DLS. The particle size of C60-5-ALA was about 80–200 nm (Fig. 1D). The relative amount of 5-ALA transferred onto the surface of C60 was tested by TGA. 5-ALA degraded completely at about 617°C, C60 and C60-5-ALA showed about 18.67% and 63.79% weight losses at 617°C, respectively, thus the relative amount of 5-ALA grafted onto C60 was 45.12% (Fig. 1E). PpIX extraction in vitro The cumulative results of PpIX extraction in B16-F10 cells are graphically represented in Fig. 2A. This showed that PpIX synthesis was much higher at 2 h for 5-ALA, while PpIX synthesis



Figure 2. Increased cytoplasmic PpIX generation in B16-F10 cells exposed to C60-5-ALA or 5-ALA. (A) PpIX accumulation in B16-F10 cells, cells were exposed to 5-ALA or C60-5-ALA for 2 h, then replace them with fresh culture medium, further cultured for 1–5 h. Each data point is the average of three determinations. (B) Fluorescence microscopic images of B16-F10 cells with a incubation of C60-5-ALA. (a: bright field; b: fluorescence; c: stained with Hoechst 33342).

Photochemistry and Photobiology, 2014, 90 from C60-5-ALA was much higher at 4 h. When exposure of the cells to C60-5-ALA for some times, PpIX generation was sustained over a much longer period, up to 5 h, compared to 5ALA which exhibited peak PpIX production after 2 h, thereafter declining to basal levels. These results indicated that C60-5-ALA significantly increased themselves cancer cells retention time. We also observed that B16-F10 cells displayed an intense homogeneous cytoplasmic red fluorescence, cells were treated with C60-5-ALA (5-ALA dose: 90 lg mL 1) for 2 h, replace them

Figure 3. Phototoxicity of PDT against B16-F10 cells. Data are presented as mean  SD (n = 6).


with fresh culture medium, then further cultured for 4 h. As shown in Fig. 2B, cells displayed an intense homogeneous cytoplasmic red fluorescence with C60-5-ALA nanoparticles, indicating that a lot of PpIX was formed based on the effective entry of nanoparticles into cells. The strong fluorescence intensity of intracellular PpIX predicts that PDT of C60-5-ALA will be effective. Phototoxicity of PDT against cancer cells All the cytotoxic effects studies against cultured B16-F10 cells were using the SRB assay. The dark cytotoxicity study of C605-ALA on B16-F10 cells was carried out at different concentrations of C60-5-ALA to determine the systemic toxicity of the nanoparticles. As shown in Fig. 3, cell survival rate of all groups remained above 95%. This result indicated that C60-5-ALA was noncytotoxic to B16-F10 cells without irradiation. B16-F10 cells were also exposed to 630 nm laser (100 mW cm 2) for 0.5 min, cell survival rate remained above 95%, indicating that only irradiation would not affect the cancer cells growth. The C60-5ALA group has an equivalent 5-ALA dosage to free 5-ALA group. Both the C60-5-ALA and 5-ALA groups were irradiated by 630 nm laser. As seen from Fig. 5, a dose-dependent cytotoxicity of all C60-5-ALA groups is shown. At a 5-ALA

Figure 4. PpIX synthesis from 5-ALA or C60-5-ALA in tissues as a function of time. The tumor-bearing mice were intravenous injected with C60-5ALA or 5-ALA (5-ALA dose: 30 mg kg 1). After treatment for 2, 3, 4 and 4.5 h, PpIX generation was determined by HPLC. Each data point is the average of three determinations.


Zhi Li et al. concentration of 90 lg mL 1, the survival rate of C60-5-ALA/ 630 nm laser was 51.4  15.07%, indicating a higher cytotoxicity than 5-ALA/630 nm laser (68.8  10.41%). These results indicated C60-5-ALA/630 nm laser enhanced cell-killing effect, thus, a synergistic therapeutic effect of PDT induced by C60-5ALA was observed in B16-F10 cells. PpIX extraction in vivo

Figure 5. Fluorescence microscopic images of tumor tissue-frozen sections through intravenous injection of C60-5-ALA. (a: bright field; b: fluorescence).

Following intravenous injection of C60-5-ALA or 5-ALA to tumor-bearing mice, PpIX production was measured in tissues as a function of time (Fig. 4). 5-ALA and C60-5-ALA were compared at equal drug equivalent doses for PpIX synthesis. It was observed that significant differences in the PpIX generation in the two compounds existed. The PpIX generation induced by 5-ALA shows a peak between 2 and 3 h in most tissues. In contrast, maximal PpIX levels induced by the C60-5-ALA were found in the tumor with a peak at around 4 h, which was more pro-

Figure 6. Tumor volume and changes of body weight after PDT with 5-ALA or C60-5-ALA (5-ALA dose: 30 mg kg 1). Inset, a photo of representative tumors of mice with different treatments, 1–4: saline, saline/630 nm laser, C60-5-ALA/630 nm laser, 5-ALA/630 nm laser. Data are presented as mean  SD (n = 5).

Photochemistry and Photobiology, 2014, 90 nounced than in other tissues. This is probably due to that 5-ALA can be transferred into cells faster, and as a result, its effects of increasing PpIX extraction are earlier than C60-5-ALA. However, when exposing the cells to C60-5-ALA for some times, PpIX generation was sustained over a much longer period, up to 4.5 h. These results confirmed the EPR effects. These results also indicate that C60-5-ALA significantly increased retention time of 5ALA in cancer cells. Frozen sections of tumor tissue displayed an intense red fluorescence, indicating that a lot of PpIX was formed based on the effective entry of nanocomposites into tumor tissue (Fig. 5). The PpIX content induced by these nanoparticles was not detected in the heart and the spleen (Fig. 4). PDT studies in vivo PDT of C60-5-ALA nanoparticles in vitro indicated that these nanoparticles were noncytotoxic to cancer cells without irradiation. The tumor growth following PDT in vivo is shown in Fig. 6. The tumor-bearing mice were divided into four groups. All groups, except saline groups, were irradiated with a 630 nm laser. There was no statistically difference in the tumor volume between the saline groups (5494.6  856.2 mm3) and the 630 nm laser groups (6077.8  381.4 mm3) after 11-day treatment (P > 0.05), suggesting that 630 nm laser alone would not affect the tumor growth. C60-5-ALA (5-ALA dose: 30 mg kg 1) resulted in a significant delay of tumor growth compared with 630 nm laser groups (P < 0.05), and the final therapeutic outcome of PDT with C60-5-ALA resulted a higher tumor tissue suppression than 5-ALA (P > 0.05), suggesting that the PDT efficacy of C60-5-ALA was higher than that of 5-ALA, the growth of tumor tissue was successfully suppressed by PDT of C60-5-ALA. Body weight was observed in all groups, and no weight loss was observed (Fig. 6), implying that the toxicity of treatments was not obvious.

CONCLUSION In summary, a method for preparation of fullerene particles containing 5-ALA was developed. The nanoscale C60-5-ALA showed no observable toxicity without irradiation, and enhancing PpIX synthesis in vitro and in vivo in a murine tumor model using an intravenous injection. C60-5-ALA can significantly increase the accumulation of photosensitizer in tumor and showed excellent PDT efficacy, indicating that there is a great potential of C60-5-ALA for cancer therapeutic applications.

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5-Aminolevulinic acid-loaded fullerene nanoparticles for in vitro and in vivo photodynamic therapy.

This report explores some properties of 80-200 nm nanoparticles containing 5-aminolevulinic acid (ALA) and fullerene (C60) for photodynamic therapy (P...
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