In Vitro and in Vivo Studies of Chlorin-based Carbon Nanocarrier with Photodynamic Therapy Features

Abstract

Carbonaceous materials have long been developed to utilize “nano-spaces” and numerous guest species could be encapsulated. A remarkable fluorescence difference has been observed after newly-designed pyropheophorbide-a-appended carbon nanohorns were incorporated into cellular medium and confocal microscopy was employed for the determination of the intracellular localization. Our study supported the role for carbon nanohorns as carriers of photodynamic therapy (PDT) agent and its heating behavior were discussed. We have developed the theranostic platform based on photosensitizer-conjugated carbon nanostructure and this system has been applied in an animal model. In addition, a negligible toxicity of CNH-Pyro was found in body weight experiments and histopathological examination of major organs.

Introduction

Organic fluorophores have been studied for several decades as a versatile source of functional objects and will be used in environmental sensing or biological transport processes [1–6]. However, most of the organic molecules are solvent-dependant and the stability would be suppressed. Hence, the incorporation of the organic structure in a solid host will be effective for reducing health and environmental hazards. In this regard, hybrid inorganic-organic materials with optical properties will be valuable for controlling the dispersion of the chromophores and preventing leaching problems. In view of nanotechnology, the application of nanoscale materials in therapy will offer opportunities for efficient treatments in health care [7–13]. Previously we have investigated the case of new chlorin-based carbon nanohorns (CNH-Pyro) (the detailed structure has been given in Scheme S1) and its photodynamic therapy effects were demonstrated [14]. However, the difficulty in monitoring the exact localization of nanosystems will prevent the identification of nanomedicine during tumor targeting process. Additionally, the real potentials of the anticancer activities require further exploration in vivo since it might be favorable for the fabrication of nano-formulation drugs.

In this study, it has been found that this hybrid nanomaterial could permeate into the cytoplasm of four cell lines and are sufficient to generate reasonably striking red luminescence, indicating its performance as an efficient cell-imaging stain will be satisfactory. To elucidate the in vivo uptake process, studies of bio-distribution on CNH-Pyro have been conducted by a tumor implantation mouse model. Near-infrared fluorescence (NIRF) and bioluminescence signals could be easily visualized near to the targeted tumors. The superimposed NIRF and bioluminescence images confirmed the exact localization of the tumor tissue. To the best of our knowledge, the optically followed bio-distribution of sp² carbon related hybrid has never been studied by way of dual imaging modalities.

Experimental section

Reagents and Materials.

N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide (EDC), N-Hydroxysuccinimide (NHS), Poly(ethylene glycol) diamine (H₂N-PEG-NH₂, Mn=2000) and pyropheophorbide-a were purchased from Sigma-Aldrich. Diamidino-2-phenylindole (DAPI), Fetal bovine serum (FBS), antibiotics penicillin (PS), Dulbecco’s modified medium (DMEM), RPMI1640, nutrient mixture F12 Ham Kaighn’s modification (F12K) medium were obtained from Life Technologies. D-Luciferin was purchased from Glod Biotechnology. Singlet oxygen sensor green (SOSG) was provided by ThermoFisher Scientific. The CellTiter 96®AQueous One Solution Reagent (MTS kit) was obtained from Promega. Single Layer Carbon Nanohorns (CNH) was purchased from Beijing Qingdajiguang technology Development Co., Ltd. The preparation of PEG functionalized carbon nanohorns (CNH-PEG), pyropheophorbide-a (Pyro) modified carbon nanohorns (CNH-Pyro) and the material structures have been reported in previous literature [14].

Live subject statement:

All animals were maintained at the University of Texas Southwestern Medical Center animal facility and animal experiments were performed with the approval of UT Southwestern Committee on Animal Care (APN#2007–0068).

Cell imaging.

Four cancer cell lines, HeLa (human cervical cancer cell), LL2 (mouse lewis lung carcinoma cell), A549 (Human Caucasian lung carcinoma cell), and THP-1 (human leukemic monocytice cell), were used in this research. For cell imaging, the cells were plated in Lab-Tek chambered cover glass (8-well, Thermo Scientific, USA) with a density of 10⁴ cells/mL and grown to 70% confluency. The cells were washed with medium (Dulbecco’s modified medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotics penicillin) three times, and incubated in the fresh medium containing CNH-Pyro (10 μM for Pyro) for another 2 h in the dark. For the laser treatment group, the cells were cultured in the Lab-Tek chambered cover glass (8-well, Thermo Scientific, USA) with a density of 10⁴ cells / mL and grown to 70% confluency. Cell were exposed to light (using a 660 nm laser) irradiation for 3 min. Cells were washed three times with PBS and co-stained (one minute) by DAPI (4’,6-diamidino-2-phenylindole). The cells in well were then studied based on a Leica confocal laser scanning microscope (TCS SP2 AOBS)

Inhibition and cellular uptake experiments.

Inhibitors were diluted in DMEM medium. Hela cells were seeded in a 24-well plate and the medium in each well was replaced with the previous inhibitors containing solution (0.5 mL each well). One hour later, CNH-Pyro (10 μM) was added in the solution and incubated for another 2 h. The culture medium was replaced by DMEM medium and incubated for 24 h. Cells were washed three times and observed with a Leica confocal laser scanning microscope.

In Vitro toxicity.

Viabilities of HeLa, A549, LL2, and THP-1 cells after the incubation of CNH-PEG, Pyro, or CNH-Pyro were explored by MTS kit (with or without laser irradiation). Cells were incubated with CNH-PEG, Pyro, or CNH-Pyro (15 μM) for 24 h. For the phototoxicity groups, the cells were treated with a 660 nm laser for 3 min. 20 μL MTS solution was added to each well. After incubated for 2 h, the absorbance at 490 nm was recorded by using a Polarsarmicroplate reader.

Determination of singlet oxygen generation.

Singlet oxygen sensor green (SOSG) is a probe for sensing singlet oxygen (SO) in biological system. SO generated by the samples oxidizes SOSG and increase SOSG’s fluorescence. In this study, a suspension of CNH-Pyro (1 μM for Pyro) in PBS (pH 7.4) and SOSG (2.5 μM) in PBS containing 2% MeOH were stirred for 5 min in the dark. Then, the mixture subject to light irradiation from a 660 nm laser (100 mW/cm²). SOSG alone, free Pyro, and CNH-PEG in SOSG with light as the control groups. At each predetermined time interval, the SOSG’s fluorescence spectra (λ_ex = 494 nm, λ_em = 534) was recorded using a Hitachi F-7000 fluorescence spectrophotometer.

Photothermal effect.

For the photothermal effect studies, PBS solution containing CNH-Pyro (10 μM for Pyro) and pure PBS solution as control in quartz cells (4.5cm×1.2cm×1.2cm) were placed in an incubator (air, 37°C). A 660 nm laser (100 mW/cm²) was employed to irradiate the dispersion from 0 to 20 minutes. The temperature was monitored by a chromel-alumel thermocouple thermometer.

LL2 tumor implantationmodel and NIR and Bioluminescence imaging of CNH-Pyro in vivo.

The LL2 tumor implantation was performed essentially as we descried [15]. The LL2 cells modified with PLL3.7 expression luciferase-IRES-GFP were used in this study. 1×10⁶ of cells in 200 μl PBS were injected subcutaneously into the female C57BL/6 mice (4–6 weeks old). When the tumors reached a size of approximately 80 mm³, the mice were divided into several groups for subsequent experiments. For NIR imaging, the LL2 tumor implantation mice were intratumorally injected with 100 μL of CNH-Pyro (100 μM). After 10 hours, NIR images were collected on a Xenogen IVIS Lumina Imaging System. Then 150 μl D-luciferin (20 mg/mL) was subcutaneously injected. Five minutes later, the bioluminescence was collected via the same imaging system (Xenogen IVIS Lumina Imaging System, luminescence mode). After the irradiation treatment by a 660 nm laser (100 mW/cm²) for 20 min, the mouse was immediately studied by fluorescence imaging. In the next step, 150 μl D-luciferin (20 mg/mL) was subcutaneously injected again. Five minutes later, the bioluminescence after the irradiation was collected.

Photodynamic therapy in vivo and H&E staining.

For PDT in vivo studies, the mice were divided into eight groups (3 mice each group) for subsequent experiments, when the tumor volume reached approximately 80 mm³. Mice bearing LL2 tumors in different groups were intravenous injected with 100 μl of PBS, CNH-PEG (99.2 μg/mL), free Pyro (100 μM), and CNH-Pyro (152.3 μg/mL, 100 μM for Pyro), respectively. For the laser treated groups, the tumor region of mice was treated with a 660 nm laser (100 mW/cm²) for 20 min 10 hrs after injection. Tumor size was measured with a digital caliper every two day and tumor volume was calculated by (length of tumor)×(width of tumor)²/2 as we described [15]. The body weight of mice was also monitored. Fourteen days after treatment, major organs of those mice were collected, fixed in 4% formalin for at least 3 days. The tissues stained with hematoxylin and eosin (H&E), and observed under a BX50 bright field microscopy (Olympus).

Results and discussion

To evaluate the capability of CNH-Pyro to label tumor cells for the purpose of diagnosis and its promising therapy application, in vitro cell cultures were studied by confocal laser scanning microscopy (Fig. 1). Hela cells without CNH-Pyro encapsulation served as the control group. The red luminescence derived from chlorin indicated that CNH-Pyro was bound and internalized into the cells after incubation for 2 h, in contrast, no intracellular emission signals could be detected in the parallel control sample (Fig. 1, red channel). The striking red fluorescence was entirely distributed in the cell boundary without any visible signals in the extracellular medium, showing that a very high efficient cellular uptake has been achieved (Fig. S1). Since PDT effect is closely related to the quantities of photosensitizers in tumor cells, the intense red emission from CNH-Pyro predicts the possibility of PDT therapy.

Figure 1.

Hela cells were incubated with CNH-Pyro or free Pyro (10 μM) for 2 h at 37 ºC. The nuclei were stained by DAPI.

Visualization of a cell with a fluorescent probe and its localization will provide reliable information for the analysis of cell functions. Nucleus staining dye, diaminophenylindole (DAPI), was used to attach the DNA double helix of cell nucleus with blue emissions. The superimposed images demonstrated two independent colors (red and blue) simultaneously, suggesting that intracellular accumulation of CNH-Pyro mainly localized in cytoplasm (Fig. 1). Interestingly, under a laser (660 nm, 100 mW/cm²) irradiation for 3 min, the red fluorescent signals completely disappeared, whereas blue emission from the nuclei stained by DAPI still remained. The results corroborated that this hybrid inorganic-organic photosensitizer was very sensitive to the laser excitation and it has been decomposed after the singlet oxygen production. In Figure 2 to Figure 4, the staining patterns of CNH-Pyro in two adherent cell lines (A549 and LL2 cells) and one suspension cell line (human leukemic monocytice cells) were also observed. The fluorescent nanoprobe interacts with four living cells through the incorporation into the membrane and the in vitro performance under laser irradiation also displayed targeting efficiency (Fig. S2). The above results indicate that the combination of PDT therapeutic potentials with imaging features may permit the investigators to follow the distributions of anti-cancer agents and provides useful hints for the improvement of disease treatment.

Figure 2.

A549 cells were incubated with CNH-Pyro or free Pyro (10 μM) for 2 h at 37 ºC. The nuclei were stained by DAPI.

Figure 4.

THP-1 cells were incubated with CNH-Pyro or free Pyro (10 μM) for 2 h at 37 ºC. The nuclei were stained by DAPI.

With the aim of confirming the origin of the red luminescence, free pyropheophorbide-a was employed as another control group in Figures 1–4, the results also supported the cytoplasmic red fluorescence was caused by the accumulation of pyropheophorbide-a in cells. Compared with CNH-Pyro, the fluorescence intensity of free pyropheophorbide-a was slightly lower, indicating that the nano-carrier will lead to enhanced permeability and improve the uptake of photosensitizers by tumor cells.

In order to further clarify the endocytic process, the inhibition effects on the uptake of the nanomaterial have been investigated. The cellular entry mechanism would provide useful information for the localization of CNH-Pyro. Four inhibitors were used to explore the internalization pathways and they included glucose, NH₄Cl, DMA (Na⁺/H⁺ exchange inhibition) and colchicines (microtubule inhibition) [16]. The results showed DMA and colchicines had negligible influence on the cellular uptake (Fig. S3). However, a significant reduction in emission intensity was detected in cells treated with glucose or NH₄Cl (Fig. S3). Glucose is closely related to inhibition of clathrin-depedent endocytosis. The luminescence change indicated a clathrin-mediated endocytosis pathway would be involved in the internalization process. As for NH₄Cl, it has been used to neutralize the pH value of endosomal and lysosomal effectively to inhibit a low-pH-dependent endosomal escape [17]. The striking change induced by NH₄Cl suggested the involvement of endolysosomal trafficking in the uptake of CNH-Pyro. But the whole cellular uptake process may involve multiple routes and the exact information of intracellular trafficking still needs further exploration.

In addition, the in vitro toxicity experiments were performed (Fig. S4). Without laser irradiation, almost no cytotoxicity were observed based on either CNH-PEG, Pyro or CNH-Pyro after incubation as determined by MTT assay. Under laser irradiations, cells treated with CNH-PEG, Pyro and CNH-Pyro all showed apparent cell death rates. Especially CNH-Pyro exhibited more toxicity to cells at the same concentrations under irradiation by the same laser power density.

The efficiency of CNH-Pyro for reactive oxygen species is of great importance. The photophysical features of singlet oxygen sensor green (SOSG), a commercially available optical probe for singlet oxygen, were examined. It has been incorporated into solution at a concentration of 2.5 μM. The ¹O₂ formation has been expressed by the emission of SOSG at 530 nm depending on irradiation time. The significant increase in the emission intensity has provided reliable information that the interaction of CNH-Pyro with the light produced powerful oxidizing agents (Fig. 5, left). The generation of ¹O₂ in three different chemical species was assessed in the presence of irradiation. Pure SOSG or CNH-PEG alone did not result in any fluorescence output, showing that direct detection of singlet oxygen-related luminescence requires efficient photosensitizers. Under 660 nm light exposure, CNH-Pyro displayed remarkable and time-dependent fluorescence improvement, which was relatively stronger than that of free pyropheophorbide-a at the same optical dose (Fig. 5, right). This enhanced PDT potential of CNH-Pyro over pyropheophorbide-a suggested that the nano-carrier, which is able to conjugate with the pigment could improve the efficiency of singlet oxygen production.

Figure 5.

Fluorescence emission spectra of CNH-Pyro in SOSG solution with the increase ofthe laser irradiation time (left). Emission peak intensity changes for the characteristic band of SOSG (530 nm) over different periods of time under irradiation time (each point is the average of triplicate determinations and error bars represent one standard deviation) (right).

It is well known that photothermal therapy is a noninvasive laser-based technique for tumor therapy [18,19]. Light energy would be converted to heat by specific substrate. In order to figure out the carbon nanohorns-mediated photothermal ablation effects, the temperature of PBS buffer and its solution containing CNH-Pyro was measured by the 660 nm laser irradiation (Fig. S5). It was found that the temperature of CNH-Pyro dispersion dramatically increased according to the laser irradiation duration and reached 45°C within 20 min. Additionally, the results showed CNH-PEG had a comparable PTT efficiency with that of CNH-Pyro. Previous literature described that the cell destruction by the hyperthermic treatment would occur between 40 and 43°C [20]. In this way, the study confirmed the feasibility of guiding photothermal therapy by using chlorin-loaded carbon nanohorns. Therefore, this multifunctional nanoprobe offers a synergistic therapeutic effect in contrast to photodynamic or photothermal therapy alone, providing the possibility for potential multi-mode clinic therapy.

The emergence of new imaging instrumentation has provided near-infrared fluorescence as an efficient way to explore key biological processes and the interventions in living organisms. But the fluorescence technique has inherent disadvantages. At first, the optical probe incorporated into living matters would be pumped by the external light source. Tissue or organ scattering and absorption could be main obstacles for the sensitivity and light transmission should circumvent twice (in and out) the problem of tissue scatter. Consequently, the signal intensity would be low. In addition, the red fluorescence (although very near to near infrared spectral region) arising from CNH-Pyro still has some difficulties in completely avoiding the autofluorescence generated from endogenous entities. As a straightforward modality to monitor the physiological changes as an effective supplement, bioluminescence does not contain the external light source for the illumination. The mutual interaction between luciferase and substrate will induce visible light through a chemiluminescence reaction. To realize this molecular imaging hybridnano-probe in the tumor detection including anti-cancer therapy and determine its potential in vivo, we developed a C57BL/6 mice implantation model created subcutaneous inoculation with LL2 cancer cells as we described [15]. Notably, after injecting CNH-Pyro for 10h, the near infrared fluorescence activity within the mouse can be observed (Fig. 6, top). The tumor tissue has been clearly identified and guided by the red luminescence spot. With the aim of precise identification of the disease progression, imaging acquisition has been performed via bioluminescence and signals from the two different modalities were very closely overlapped in tumor growth (Fig. 6, bottom). The above analysis of whole-body medical imaging results again confirmed the localization of the tumors. Subsequently, in vivo PDT study was conducted in the presence of a laser (660 nm, 100 mW/cm²) irradiation for 20 min. The administration of CNH-Pyro gave ablation results of the subcutaneous tumor. Moreover, the superimposed bioluminescent signal also substantiated the tumor regression behavior (Fig. 6).

Figure 6.

In vivo images of LL2 tumor implantation mice that expressed PLL3.7 luciferase-IRES-GFP construct by both NIR-fluorescence (top) and bioluminescence (bottom) imaging with or without laser irradiation.

In parallel experiments, mice were administered intratumorally with 100 μl of PBS, CNH-PEG (99.2 μg/mL), free Pyro (100 μM), or CNH-Pyro (152.3 μg/mL, 100 μM for Pyro) in each group. For the mice treated in the absence of laser irradiations, continuous expansion volumes based on the time elapse was observed (Fig. S6, left). Tumor volumes increased dramatically regardless of the injected species (including CNH-PEG, Pyro or CNH-Pyro). Partial tumor volume shrinkage after injection of CNH-PEG (or pyropheophorbide-a) was illustrated in Fig. S6 in the presence of irradiation, showing that each of the two independent curing agents (carbon nanohorns and pyropheophorbide-a) could demonstrate individual photothermal or photodynamic therapy efficiency. However, in mice treated with CNH-Pyro, the most striking reductions in tumor volumes were achieved (Fig. S6, right), suggesting that the synergistic effects between photothermal therapy and photodynamic therapy were realized in the hybrid nano-network (CNH-Pyro). This nano-carrier could possess the therapy potentials of organic photosensitizer.

It has been discussed treatments with nano-materials or photo-irradiations would induce undesirable side effects [21], toxicity was evaluated by measurement of body weights of mice treated under various conditions (Fig. 7). The mice that were administered CNH-Pyro started to gain weight within the first 4 days and maintained body weight almost equal to the beginning stage of the treatment. Essentially no apparent weight loss was observed in the whole administrations, implying that the toxicity of the treatments were negligible. Major organs, including lung, heart, liver, kidney, and spleen, were also examined for signs of abnormalities. As shown in Figure 8, hematoxylin and eosin (H&E) staining revealed no distinguished signs of organ damage with any of the treatments.

Figure 7.

Body weight changes of mice after different treatments.

Figure 8.

H&E stained images of major organs. No noticeable abnormality was observed in major organs including liver, spleen, kidney, heart, and lung.

Conclusions

In order to explore the chlorin photodynamic therapy behavior, pyropheophorbide-a has been anchored onto carbon nanomaterials and the promising theranostic features have been discussed. In particular, the exact position of action for the carbonaceous-based PDT agents has been identified via dual optical modalities. The ¹O₂ formation has been indicated by the presence of SOSG and CNH-Pyro demonstrated the most effective production of singlet oxygen. This new therapeutic treatment exhibited minimum side effects in terms of the systematical toxicity investigation. More importantly, it is appealing that the therapy principle derived from chlorin-appended carbon nanohorns have been realized both in vitro and in vivo.

Figure 3.

LL2 cells were incubated with CNH-Pyro or free Pyro (10 μM) for 2 h at 37 ºC. The nuclei were stained by DAPI.