Oral Nanostructured Lipid Carriers Loaded with Near-Infrared Dye for

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Oral Nanostructured Lipid Carriers Loaded with Near-Infrared Dye for Image-Guided Photothermal Therapy Gang Chen,†,# Kaikai Wang,*,†,# Yiwen Zhou,† Ling Ding,† Aftab Ullah,† Qi Hu,† Minjie Sun,*,† and David Oupický*,†,‡ †

State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210028, China Center for Drug Delivery and Nanomedicine, Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States

‡

S Supporting Information *

ABSTRACT: Photothermal therapy exerts its anticancer effect by converting laser radiation energy into hyperthermia using a suitable photosensitizer. This study reports development of nanostructured lipid carriers (NLCs) suitable for noninvasive oral delivery of a near-infrared photosensitizer dye IR780. The carrier encapsulating the dye (IR780@NLCs) was stable in simulated gastric and intestinal conditions and showed greatly enhanced oral absorption of IR780 when compared with the free dye. As a result of increased oral bioavailability, enhanced accumulation of the dye in subcutaneous mouse colon tumors (CT-26 cells) was observed following oral gavage of IR780@NLCs. Photothermal antitumor activity of orally administered IR780@NLCs was evaluated following local laser irradiation of the CT-26 tumors. We observed significant effect of the photothermal IR780@NLCs treatment on the rate of the tumor growth and no toxicity associated with the oral administration of IR780@NLCs. Overall, orally administered IR780@NLCs represents a safe and noninvasive method to achieve systemic tumor delivery of a photosensitizing dye for applications in photothermal anticancer therapies. KEYWORDS: photothermal therapy, IR780, oral administration, nanostructured lipid carriers, tumor imaging

1. INTRODUCTION

Among the more recently developed photosensitizers, IR780 (Figure 1A) is a lipophilic organic dye that absorbs NIR light and exhibits excellent photothermal effects.17,18 Compared with the clinically used ICG, IR780 is more stable and has higher fluorescence intensity.19 IR780 can accumulate in a broad range of tumors and tumor cells without the need for any additional chemical conjugation to achieve tumor targeting (Figure S1).20,21 Despite the favorable properties, IR780 has poor aqueous solubility and limited photostability that limit its application in PTT.22 To the best of our knowledge, most currently published reports use IR780 formulations developed for parenteral (i.v., i.p.) administration. Oral administration is the most common route of drug administration in the world. When compared with intravenous route, for example, oral administration provides multiple benefits including noninvasive nature, high patient acceptance, and high treatment compliance, as well as availability of various pharmaceutical dosage forms at relatively low cost.23 Oral administration of NIR dyes has been mainly used for tracing drug delivery systems in the gastrointestinal (GI) tract24 and the use of oral administration to achieve systemic absorption

Photothermal therapy (PTT) is a minimally invasive anticancer method, which uses local hyperthermia generated by a photosensitizer exposed to a laser light.1,2 Compared with conventional chemotherapy, PTT promises to cause less adverse effects in tissues not exposed to the laser and potentially also a lower incidence of secondary cancers that are often associated with chemotherapy.3 Two main classes of photosensitizers include inorganic nanomaterials and organic small molecules. Gold nanorods,4−6 carbon nanotubes,7,8 and CuS nanostructures9,10 are examples of commonly used inorganic materials. The main advantage of the inorganic materials is their high photothermal conversion efficiency due to high absorbance in the near-infrared (NIR) part of the spectrum. Common examples of small molecule organic photosensitizers include porphyrins,11 phthalocyanins, and indocyanine green (ICG).12,13 Compared with the inorganic photosensitizers, the organic photosensitizers are often more easily secreted from the organism, which offers advantages regarding their potential long-term toxicity and potential for clinical translation. For example, ICG has already been approved by the FDA as a NIR imaging agent.14 However, many of these organic photosensitizers are limited by low aqueous stability, poor photostability, and off-target effects.15,16 © XXXX American Chemical Society

Received: June 19, 2016 Accepted: August 31, 2016

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DOI: 10.1021/acsami.6b07425 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

prepared and used as a control. CMC-Na was dissolved in distilled water at the concentration of 0.5% (w/v). Fine powder of IR780 was added to the CMC-Na solution and dispersed in an ultrasonic bath for 5 min to achieve dye concentration of 0.65 mg/mL. 2.3. Physicochemical Characterization of Nanostructured Lipid Carriers. Particle size was determined by Zeta Plus (Brookhaven, USA). Absorption spectra were recorded by UV−vis spectro-photometer. Particle morphology was assessed by transmission electron microscopy (TEM, H-600, Hitachi, Japan). 2.4. Stability of Nanostructured Lipid Carriers. To test photostability of IR780@NLCs, the particles were prepared in buffers with different pH values (1.0, 2.0, and 7.0), and changes in UV−vis absorption of IR780 were followed. The stability of NLCs in simulated gastric and intestinal conditions was tested at 37 °C by incubating the particles with pepsin at pH 2.0 for 2 h, followed by incubation with a 1:1 mixture of pepsin and trypsin (pH 3) for additional 2 h, and final incubation with trypsin (pH 7.4) for another 2 h.33,34 Particle size and UV−vis absorption of IR780 were monitored throughout the experiment. 2.5. In Vitro Toxicity and Cell Uptake. The cytotoxicity of IR780@NLCs in Caco-2 cells and CT-26 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the cells were seeded in 96-well plates (1 × 104 cells per well) and cultured overnight. The culture medium was replaced with a fresh medium containing increasing concentrations of IR780@NLCs. For Caco-2 cells, after incubation for 2 or 24 h, the medium was removed, cells were washed with phosphate-buffered saline (PBS), and cell viability was measured by the MTT assay. To assess phototoxicity, the CT-26 cells were incubated with formulations for 2 or 4 h and then irradiated by an 808 nm laser (1 W/cm2) for 3 min. After that the cells were cultured for 24 h, and cell viability was measured by MTT assay. Cells were incubated with MTT for 4 h, dimethyl sulfoxide (DMSO) was added, and the absorbance was measured at 450 nm by a microplate reader.35 CT-26 cells were seeded at a density of 5 × 105 cells/well in a six-well plate containing a PDL-coated glass coverslip per well, were treated with IR780@NLCs (2, 4, 6, 8, and 10 μg/mL) for 2 h, following irradiation by an 808 nm laser (1 W/cm2) for 3 min per well. Afterward, the cells with fragmented and condensed nuclei were determined using DAPI staining as described.36 2.6. In Vivo Pharmacokinetics, Biodistribution, and Therapeutic Activity of IR780@NLCs in Subcutaneous Tumor Model. All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee of China Pharmaceutical University. Pharmacokinetics study following single-dose intravenous injection and oral gavage was conducted in tumor-free male ICR mice. The mice were randomly divided into IR780@NLCs group and free IR780 (ethanol and Cremophor EL, 1:1, v/v) group or IR780@CMC-Na group (five mice per group). Both formulations were administered via intravenous injection and oral gavage with the IR780 dose of 1.4 mg/kg and 6.5 mg/kg, respectively. Blood samples were collected by retro-orbital bleeding at different time points (5 min to 120 h) after administration. The content of IR780 in the serum samples was measured using a Varioskan Flash Spectral Scanning multimode plate reader (Thermo Fisher Scientific, Waltham, MA, USA). For tumor studies, pathogen-free eight-week-old Balb/c male mice were allowed to acclimate for one week before the experiment. The mice were used to establish xenograft tumor model as follows. Briefly, 1 × 107 CT-26 cells in 0.2 mL of PBS were first subcutaneously injected into the right flank of mice. Two weeks later, the developed tumor was excised and cut into ∼1 mm3 pieces. The pieces of tumors (one per mouse) were implanted into the right flank to establish subcutaneous tumors for the biodistribution and therapeutic studies. The mice were used in subsequent experiments when the tumors reached ∼200 mm3.37 To investigate biodistribution of IR780, the mice were given a single dose of IR780@NLCs (1 mg/kg IR780) by oral gavage (three mice per group). Whole-body fluorescence imaging was performed using IVIS Lumina imaging system (Xenogen Co., USA) with excitation/emission

Figure 1. Oral PTT by IR780@NLCs. (A) Structure of IR780. (B) Preparation and mechanism of action of IR780@NLCs.

for imaging, and PTT has not been extensively investigated. Multiple nanotechnology approaches have been developed for improving oral delivery of poorly soluble drugs, including liposomes, nanoemulsions, micelles, and solid lipid nanoparticles.25,26 Nanostructured lipid carriers (NLCs) are composed of a solid lipid matrix with a certain amount of a liquid lipid and represent an improved generation of lipid nanoparticles.27 NLCs offer a number of advantageous features, including physical stability, biocompatibility, and ease of manufacturing on a large scale.28 NLCs have been widely used for oral drug delivery due to their remarkable biocompatibility and biodegradability.29,30 In this study, we report the first example of a formulation that effectively delivers NIR dye IR780 by NLCs using oral administration in mice. IR780 was loaded in NLCs (IR780@ NLCs), and its stability and photothermal properties were evaluated under various conditions in vitro. Oral absorption, biodistribution, and tumor accumulation were assessed in mice bearing CT-26 mouse colon tumors (Figure 1). Lastly, the PTT was evaluated in vivo. Our results show successful use of orally administered IR780@NLCs and its potential in noninvasive PTT.

2. MATERIALS AND METHODS 2.1. Materials. IR780 iodide and sodium carboxymethyl cellulose (CMC-Na) were purchased from Sigma-Aldrich (St Louis, MO, USA). Trilaurin was purchased from Tokyo Chemical Industry (Tokyo, Japan). Soybean lecithin was purchased from Tywei Pharmaceutical Co., Ltd. (Shanghai, China). Labrafac CC was purchased from Gattefosse (France). The human epithelial colorectal adenocarcinoma cell line (Caco-2) and mouse colon cancer cell line (CT-26) were purchased from Shanghai Institute of Cell Biology (Shanghai, China). DAPI was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Unless otherwise stated, all other reagents were purchased from Nanjing Wanqing Chemical Glassware Instrument Company and used as received. 2.2. Preparation of IR780@NLCs. IR780@NLCs were prepared by a solvent evaporation method.31,32 Briefly, soy lecithin (260 mg), trilaurin (20 mg), Labrafac CC (70 mg), and IR780 (8 mg) were dissolved in 40 mL of ethanol/dichloromethane mixture (4/1, v/v). A film was formed on the surface of a round-bottom flask by evaporating the solvents on a rotary evaporator under vacuum at 37 °C. The dried film was then hydrated at 40 °C, ultrasonicated at 100 W, and extruded through 0.22 μm of cellulose nitrate membrane to remove free dye and to obtain IR780@NLCs. Dispersion of IR780 in CMC-Na was also B


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2.7. Statistical Analysis. Statistical analysis was performed by two-sided Student’s t test for two groups and one-way analysis of variance for multiple groups. A value of P < 0.05 was considered statistically significant.

set to 745/808 nm. Fluorescence images were acquired in anesthetized animals at different time points, ranging from 1 to 72 h after oral administration. IVIS Living Imaging Software was used for the analysis of the amount of IR780 in tissues. Therapeutic study started when the tumors reached ∼200 mm3. The mice were divided into groups (six mice per group) and treated with (i) PBS, (ii) PBS + NIR laser, (iii) IR780@CMC-Na, (iv) IR780@ CMC-Na + NIR laser, (v) IR780@NLCs, and (vi) IR780@NLCs + NIR laser. All samples were administered via oral gavage (6.5 mg/kg IR780). The day of administration was designated as day 0. After 24 h, the tumors were exposed to the NIR laser irradiation (2 W/cm2) for 1 min. Treatment administrations and laser irradiation were repeated on days 2 and 3. Tumor sizes and mouse body weights were recorded every other day. Tumor volume (V) was calculated as V = d2 × D/2, where D and d are the longest and shortest diameter of the tumor, respectively.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of IR780@NLCs. In this study, IR780@NLCs were prepared by a solvent evaporation method (Figure 1B). We used NLCs based on soy lecithin, trilaurin, and Labrafac CC.38 The formulation consists of a solid lipid matrix of the lecithin and trilaurin with spatially incompatible liquid lipid (Labrafac CC). This results in particles with increased drug loading capacity.39 We selected these NLCs components also for their simplicity and costeffectiveness to allow rapid clinical translation of the developed formulations. A control formulation in which IR780 was dispersed in CMC-Na was also prepared and used in the study. CMC-Na is a common excipient used to disperse poorly soluble drugs intended for oral administration. We first examined basic physicochemical properties of IR780@NLCs using TEM, dynamic light scattering (DLS), and UV−vis spectroscopy (Figure 2). Electron microscopy revealed that IR780@NLCs were formed as spherical nanoparticles with sizes ranging from 120 to 300 nm (Figure 2A). Hydrodynamic size of the IR780@NLCs measured by DLS confirmed the data obtained from TEM with average particle diameter of 170 nm and polydispersity of 0.057 (Figure 2B). The control IR780@CMC-Na formulation contained aggregated particles with high polydispersity. We next compared the absorption spectra of IR780@NLCs diluted in PBS and free IR780 dissolved in DMSO (Figure 2C). The strong observed absorption in IR780@NLCs confirmed successful loading and solubilization of the dye. The absorption was similar to that of free IR780 dissolved in DMSO. Because of the extensive aggregation, absorption spectra of IR780@CMC-Na dispersion could not be reliably measured.

Figure 2. Characterization of IR780@NLCs. (A) Representative TEM images of IR780@NLCs. (B) Size distribution of IR780@NLCs determined by DLS. (C) Absorption spectra. (D) Solution temperatures after the exposure to laser irradiation (n = 3).

Figure 3. Stability of IR780@NLCs at different pH and in the presence of gastric and small intestine enzymes. Relative absorption of free IR780 and IR780@NLCs at different time points and different pH: (A) pH = 1, (B) pH = 2, (C) pH = 7. (D) Changes in particle size of IR780@NLCs at different time points and pH values. Changes in particle size (E) and relative UV−vis absorption (F) after treatment with pepsin and trypsin. C


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Figure 5. Cellular uptake of IR780@NLCs by CT-26 cells. (A) Cell uptake determined by flow cytometry at 2 h postincubation with different IR780@NLCs concentrations. (B) Time course of the cell uptake in cells incubated with 6 μg/mL IR780@NLCs. (C) Confocal fluorescence images of CT-26 cells incubated with 6 μg/mL IR780@ NLCs for 1, 2, and 3 h. Figure 4. Cellular uptake and cytotoxicity of IR780@NLCs in Caco-2 cells. (A) Cell uptake determined by flow cytometry at 2 h postincubation with different IR780@NLCs concentrations. (B) Time course of the cell uptake in cells incubated with 6 μg/mL IR780@ NLCs. (C) Confocal fluorescence images of CT-26 cells incubated with 6 μg/mL IR780@NLCs for 1, 2, and 3 h. Cell viability of Caco-2 cells incubated with different concentrations of IR780@NLCs for 2 h (D) and 24 h (E).

from acidic to slightly alkaline. The pH in the stomach is ∼1.5−3.0, while the pH of the small intestine is ∼7.0.33 As demonstrated in Figure 3A−C, the absorption of IR780@ NLCs remained nearly unchanged within the 5 h experiment at pH 1, 2, and 7. In contrast, the absorption of free IR780 decreased significantly. Monitoring pH-induced changes in the fluorescence intensity of IR780@NLCs and free IR780 showed similar improvement in stability of the NLCs-formulated IR780 (Figure S2A). After establishing improved photostability, we then evaluated the effect of pH on the size of IR780@NLCs nanoparticles. The only small change in size was observed at pH 2, while no changes were seen at pH 1 and 7 (Figure 3D and Figure S2B). In addition to the changes in pH, enzymes in the GI tract may compromise the stability of the NLCs.40 We determined the stability of IR780@NLCs in the presence of important GI enzymes. As shown in Figure 3E, the size of NLCs exhibited almost no change in the presence of pepsin and a mixture of pepsin and trypsin. Interestingly, a decrease in the absorption of IR780 was observed in the presence of enzymes in the case of both free dye and IR780@NLCs. Nevertheless, the dye formulated in the NLCs showed improved stability in the presence of the enzymes when compared with the free dye. These results suggest that IR780@NLCs possess favorable stability in the simulated GI tract environment. The protective function of NLCs will be of great significance for the oral delivery of IR780. 3.3. Cytotoxicity and Cellular Uptake of IR780@NLCs in Caco-2 Cells. Safety of orally given formulations is vital for their practical use. To predict the interactions of IR780@NLCs

Next, we assessed the photothermal properties of the IR780@ NLCs. The particles were dissolved in PBS and exposed to a laser irradiation (808 nm, 1 W/cm2) for 5 min, and the solution temperature was recorded every 10 s (Figure 2D). Our results showed superior photothermal effect of the IR780@NLCs as documented by rapid temperature increase and the ability to reach 56 °C during the irradiation period. As a control, free IR780 was dissolved in DMSO and added to PBS, resulting in a formation of unstable suspension, which nevertheless could produce noticeable photothermal effect, although the temperature increase was not as pronounced as that observed with IR780@NLCs. The IR780@CMC-Na, with its extensive aggregation, showed similar photothermal effect as the free dye suspension, further confirming the benefits of IR780@ NLCs. Control PBS showed negligible heating under the same experimental conditions. The observed heating with IR780@ NLCs was high enough to cause significant hyperthermiainduced effects in biological systems. 3.2. Stability of IR780@NLCs. The pH stability of oral delivery systems is crucial for their survival during transport in the GI tract. The pH environment along the GI tract varies D


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Figure 6. Cytotoxicity of IR780@NLCs in CT-26 cells. (A) Cell viability of CT-26 cells treated with different concentrations of IR780@NLCs and exposed to a laser for 3 min (n = 3). (B) Apoptotic cell death detection using DAPI staining. ***P < 0.005, vs IR780@NLCs group.

Figure 7. Plasma concentration-vs-time profiles in tumor-free mice treated with intravenous injection (A) of IR780@NLCs and free IR780 and oral administration (B) of IR780@NLCs and IR780@CMC-Na. iv: intravenous injection. po: oral administration (n = 5).

with intestinal epithelial cells in vivo, we first evaluated the ability of Caco-2 cells to internalize the particles. The cellular uptake was studied using flow cytometry and confocal laser scanning microscopy. Incubation of Caco-2 cells with increasing concentrations (1−6 μg/mL) of IR780@NLCs (drug loading ∼1.2 wt %) for 10, 20, 30, and 120 min resulted in increased cellular uptake (Figure 4A and Figure S3A). When the particle concentration was kept constant at 1, 3, and 6 μg/mL, the cell uptake continued to increase during the 2 h observation period (Figure 4B and Figure S3B). The flow cytometry results were corroborated by confocal microscopy (Figure 4C). Strong cytoplasmic distribution of the red fluorescence of IR780 was seen at the latter stages of incubation with Caco-2 cells. Importantly, despite the high cellular uptake and broad intracellular distribution, we observed no significant cytotoxicity associated with the 2 h incubation with either IR780@NLCs or free IR780 control (Figure 4D). Even after we extended the incubation to 24 h, only a small decrease in cell viability was observed at the highest concentration tested (7.5 μg/mL; Figure 4E). The fact that similar decrease of cell viability was observed for both IR780@NLCs and IR780 suggests that potential adverse effects are due to the dye itself and not the NLCs or any of the components used in the nanoparticle formulation. 3.4. Phototoxicity of IR780@NLCs in CT-26 Colon Cancer Cells. After confirming the safety of the IR780@NLCs in Caco-2 cells, we focused our attention on the anticancer potential of the formulations in CT-26 colon tumor cells. We first determined the ability of the cancer cells to internalize the particles using flow cytometry and confocal microscopy as above. As expected, enhanced IR780 uptake was observed at higher particle concentrations (Figure 5A and Figure S4B) and following longer incubation times (Figure 5B and Figure S4A).

As in Caco-2 cells, confocal microscopy revealed effective time- and dose-dependent uptake and cytoplasmic, but not nuclear, distribution of IR780 in the CT-26 cells (Figure 5C and Figure S5). The cytotoxicity of IR780@NLCs caused by its photothermal effects was tested in the CT-26 cells by MTT assay. Without laser irradiation, neither IR780@NLCs nor free IR780 exhibited any appreciable cytotoxicity up to a dye concentration of 8 μg/mL (Figure 6A). However, after laser irradiation, the cytotoxicity of both treatments increased greatly. IR780@NLCs showed stronger photothermal cell killing ability than free IR780. To further confirm the results of the MTT assay, we also used DAPI staining to assess nuclear fragmentation in the cells. As shown in Figure 6B, combined exposure to the laser and increasing concentrations of IR780@NLCs for 2 h markedly increased nuclear fragmentation−a hallmark of apoptosis.36 Taken together, IR780@NLCs showed promising photothermal effect and induced cell apoptosis in vitro, providing rationale for the subsequent in vivo studies. 3.5. In Vivo Biodistribution and Pharmacokinetics of IR780@NLCs in Mice. In previous studies, free IR780 was used as a control in studies with micelles and nanoparticles given by intravenous injection, and it showed very limited antitumor efficiency due to insufficient tumor accumulation and poor solubility in aqueous solutions.17,22 Our study aimed to evaluate the feasibility of oral administration of IR780@NLCs for antitumor PTT and in vivo imaging. Our pharmacokinetics study (Figure 7A,B and Figure S6A,B) showed that the oral administration of IR780@NLCs resulted in a 5.2-fold higher peak concentration (Cmax) when compared with the control IR780@CMC-Na (6.33 vs 1.21 μg/mL). The area under the plasma concentration−time curve (AUC), E


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Figure 8. In vivo fluorescence imaging and biodistribution of IR780 in CT-26 tumor-bearing mice. (A) In vivo IR780 fluorescence images in CT-26 tumor-bearing mice at different time after oral administration of IR780@NLCs and IR780@CMC-Na. (B) Organ distribution of IR780 determined based on ex vivo fluorescence intensity. (C) Ex vivo fluorescence images of major organs and tumors. (D) IR780 fluorescence imaging in tumor sections following oral administration of IR780@CMC-Na and IR780@NLCs. ***P < 0.005, vs IR780@CMC-Na group (n = 3).

Quantification of the fluorescence in the tumors and other tissues further confirmed increased oral bioavailability of the dye when given as IR780@NLCs. Furthermore, strong IR780 fluorescence was also observed in tumor tissue sections of mice treated with IR780@NLCs (Figure 8D) but not in mice treated with IR780@CMC-Na. 3.6. In Vivo Photothermal Therapy by Oral Administration in a Subcutaneous Tumor Model. After we confirmed improved oral bioavailability and effective tumor accumulation of IR780, we studied the antitumor PTT efficacy of IR780@NLCs. First, we confirmed that the amount of IR780 that accumulates in the tumors following oral delivery of IR780@NLCs is sufficient to facilitate local temperature increase after laser irradiation. We recorded the temperature in the tumor region as described previously.41,42 On the basis of the biodistribution and pharmacokinetics results, we measured tumor temperature 24 h after oral administration. The average tumor temperature following laser irradiation increased rapidly in the IR780@NLCs group (Figure 9A). We were able to reach nearly 50 °C, which is well-above the temperatures expected to cause substantial tumor cell damage.37 In contrast, the tumor temperatures achieved with the control IR780@CMC-Na and PBS were only slightly elevated to ∼41 °C under the same laser

which corresponds with the in vivo therapeutic effect, was 20.8 times higher in case of IR780@NLCs than IR780@CMCNa (334.73 vs 16.10 μg/mL h). In comparison to the free IR780 or IR780@CMC-Na, the IR780@NLCs also exhibited significantly prolonged half-life in the plasma (t1/2) in both intravenous (from 16.4 to 9.4 h) and oral (from 41.1 to 24.2 h) administration. These data indicate that the NLCs significantly improved the pharmacokinetics properties of IR780 after the oral administration. We will conduct direct head-to-head comparison with free IR780 (i.v.) in our future antitumor studies. To analyze biodistribution and tumor accumulation, IR780@ NLCs and control IR780@CMC-Na were orally administered in Balb/c mice bearing the CT-26 tumors. In vivo fluorescence images were recorded at 12, 24, and 48 h after administration. As shown in Figure 8A,B, significantly increased tumor accumulation was observed for IR780@NLCs when compared with IR780@CMC-Na. The tumor and major organs were harvested 48 h postadministration and imaged ex vivo. As shown in Figure 8C, IR780@CMC-Na was mostly retained in the stomach and small intestine after administration, suggesting poor systemic absorption of the dye. In contrast, the mice given IR780@NLCs showed increased systemic absorption and accumulation of the dye in the distant tumor and the liver. F


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Figure 9. Antitumor PTT activity of IR780@NLCs following oral administration in CT-26 colon cancer model. (A) Tumor temperature changes after laser exposure. (B) H&E stained tumor sections after PTT. (C) Tumor volume after oral administration of PBS, IR780@CMC-Na, and IR780@NLCs with or without laser. (D) Body weight changes over the treatment period. (E) H&E stained sections of the heart, liver, spleen, lung, and kidney from untreated healthy mice and treated mice with IR780@NLCs after PTT. *P < 0.05, vs IR780@CMC-Na group (n = 6).

liver enzymes alkaline alanine transaminase (ALT) and aspartate transaminase (AST) were measured to further assess liver toxicity, and blood urea nitrogen (BUN) was used to evaluate potential kidney toxicity (Figure S7B−D). No significant differences in the levels of any of the measured biomarkers were observed between IR780@NLCs and control groups. Overall, these findings suggest that the PTT with IR780@NLCs is a safe therapeutic modality. Previous reports that described oral administration of NIR dyes only focused on using the dye to trace transport of delivery systems through the GI tract, but none of them attempted to achieve systemic absorption and delivery.33,34 The main innovation of this study rests in the ability of our nanoparticles to be given orally and to be combined with local laser irradiation. The nanoparticles are prepared from safe and wellvalidated components, and thus this noninvasive character of the developed therapeutic intervention may bring advantages in the ability to translate the nanoparticles into a clinical use for the treatment of multiple types of cancers compatible with PTT modality based on what we call “oral-imaging and oral PTT”.

irradiation conditions. Such a modest increase in the local tumor temperature was not expected to be sufficient to damage the tumor cells.37 These findings were corroborated by histology examination of the hematoxylin and eosin (H&E)stained tumor sections, which revealed significant necrosis of the tumors in the IR780@NLCs group but not in any of the control groups (Figure 9B). Antitumor efficacy of the PTT with IR780@NLCs was then studied. As shown in Figure 9C, combination of laser treatment with IR780@NLCs significantly suppressed the rate of tumor growth several days after laser irradiation. In fact, three of six mice in the IR780@NLCs group showed complete tumor regression by the PTT. None of the control groups showed any significant antitumor effect, confirming the superiority of our IR780@NLCs formulation. Lastly, we evaluated if treatment with the IR780@NLCs has any potential toxic side effects. Body weight change was used to detect the overall treatment-induced toxicity.43 The body weights of all six treatment groups were monitored throughout the whole experiment. No obvious weight changes were observed in any of the groups (Figure 9D). The animals also showed no noticeable variation in the relative weight of any of the major organs (heart, liver, spleen, lung, and kidney; Figure S7A). Histological analysis of the main organs using H&E staining further confirmed safety of the treatment with IR780@NLCs, as there were no pathological changes compared with the untreated group (PBS; Figure 9E). The levels of

4. CONCLUSION We have successfully developed a new photothermal therapeutic platform based on orally administered IR780@NLCs. We found that IR780@NLCs were stable in the simulated GI tract environment. Superior photothermal cytotoxicity to cancer cells was clearly demonstrated under laser irradiation G


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compared with free IR780. In addition, IR780@NLCs exhibited high tumor accumulation and the ability to fluorescently image tumor tissue. After imaging, IR780@NLCs-treated mice showed excellent PTT effect for tumor ablation under laser irradiation, without producing any appreciable off-target toxicity. These results support the potential of IR780@NLCs as a safe theranostic platform for image-guided PTT by oral administration. The reported formulation represents a noninvasive method of delivery that may further enhance the potential of PTT in human medicine.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b07425. Biodistribution of free near-infrared dye; changes in IR780 fluorescence of IR780@NLCs and particle size at different time and pH values; flow cytometry data for IR780@NLCs uptake in Caco-2 and CT-26 cells; confocal images of CT-26 cells incubated with IR780@ NLCs; pharmacokinetics parameters obtained following intravenous injection and oral administration; weight of mouse organs; and quantification of ALT, AST, and BUN in the plasma of the mice. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (D.O.) *E-mail: [email protected]. (K.W.) *E-mail: [email protected]. (M.S.) Author Contributions #

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported in part by the China National Science Foundation (Nos. 81373983 and 81573377) and in part by the Changjiang Scholar program.

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REFERENCES

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Oral Nanostructured Lipid Carriers Loaded with Near-Infrared Dye for

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