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Hybrid curcumin–phospholipid complex-near-infrared dye oral drug delivery system to inhibit lung metastasis of breast cancer

Authors Liu Y, Huang P, Hou X, Yan F, Jiang Z, Shi J, Xie X, Shen J, Fan Q, Wang Z, Feng N

Received 8 January 2019

Accepted for publication 12 March 2019

Published 7 May 2019 Volume 2019:14 Pages 3311—3330

DOI https://doi.org/10.2147/IJN.S200847

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Dr Mian Wang

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Ying Liu, Peiwen Huang, Xuefeng Hou, Fei Yan, Zifei Jiang, Jiangpei Shi, Xingmei Xie, Junyi Shen, Qiangyuan Fan, Zhi Wang, Nianping Feng

School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, People’s Republic of China

Background: Oral route of administration is preferred for treating breast cancer, especially when continued disease management with good tolerability is required; however, orally administered chemotherapeutics combined with near-infrared (NIR) dyes are hindered by the low bioavailability, insufficient for the desired therapeutic efficacy. In this study, we developed a hybrid self-microemulsifying drug delivery system for co-loading curcumin–phospholipid complex and NIR dye IR780 (CUR/[email protected]), to achieve combined phototherapeutic and chemotherapeutic effects against lung metastasis of breast cancer.
Methods: CUR/[email protected] were characterized. The efficacy against breast cancer metastasis was evaluated by photothermal and photodynamic assessment, cytotoxicity, invasion, and migration in metastatic 4T1 breast cancer cells in vitro, and in vivo oral bioavailability study in rats and pharmacodynamics studies in tumor-bearing nude mice.
Results: CUR/[email protected] improved oral bioavailability of curcumin and IR780 in rats compared with curcumin and IR780 suspensions. CUR/[email protected] exhibited remarkable photothermal and photodynamic effects in vitro. In metastatic 4T1 breast cancer cells, CUR/[email protected] combined with localized NIR laser irradiation induced significant cytotoxicity and inhibited invasion and migration of 4T1 cells, an outcome attributable to cumulative effects of IR780-induced hyperthermia and pharmacological effects of curcumin. In orthotopic 4T1 tumor-bearing nude mice, combination of oral administration of CUR/[email protected] with local NIR laser irradiation inhibited tumor progression and suppressed lung metastasis.

Keywords: curcumin, IR780, oral drug delivery, lung metastasis of breast cancer, photo/chemotherapy

Introduction

Metastatic breast cancer is a global, life-threatening neoplastic disease, with lung and bone metastases occurring in approximately 60% of breast cancer patients.1 Poor prognosis of metastatic breast cancer is illustrated by an overall 5-year survival rate of approximately 27%.2 Therefore, inhibiting tumor growth and metastasis and achieving long-term disease management remains a challenge in breast cancer therapy. Multiple treatment modalities for metastatic breast cancer have been developed, including chemotherapy,3 radiotherapy,4 phototherapy (photothermal and photodynamic therapy),5,6 gene therapy,7 and combination therapy.8,9 Photothermal therapy is attractive because of its non-invasive nature, light-triggered anticancer effects, and spatial/temporal selectivity. Additionally, compared to radiotherapy, phototherapy inflicts less damage to surrounding tissues and the patient body. Photothermal agents convert light to thermal energy by absorbing near-infrared (NIR) radiation, using hyperthermia to kill tumor cells.10 In contrast, photosensitizers, upon exposure to specific light wavelengths, generate ROS, leading to tumor cell apoptosis.11 IR780 is a stable, highly fluorescent NIR dye used in phototherapy and capable of producing hyperthermia and ROS. IR780 is transported by organic anion transporter polypeptides and can therefore accumulate in many tumor types including breast cancer and achieve tumor targeting, a feature beneficial for oral IR780 delivery.1215 Phototherapy was effective in inhibiting proliferation and inducing tumor cell death at the primary lesion. However, after tumor recurrence, in general, survival rate of patients decreases greatly. Combination of chemo- and photothermal therapy was more effective than either therapy alone.1618 Given IR780’s potent photothermal efficacy and suitability for oral administration, the dye is expected to exhibit improved therapeutic efficacy against metastatic breast cancer when combined with oral chemotherapeutics. More importantly, the combination is expected to reduce the administration dose of IR780, which is great beneficial to reduce the potential toxicity of IR780 at high dose in vivo.

Curcumin is the main active ingredient of Curcuma longa and potentially suppresses breast cancer progression. Curcumin has shown promising effects in inhibiting cancer angiogenesis, cell migration, and invasion by interacting with NF-κB,19 a key regulatory molecule of cancer progression and metastasis. Additionally, curcumin modulates the expression of multiple cell signaling proteins, such as NF-κB, COX-2, and MMP920 and inhibits epithelial–mesenchymal transition.21 Despite great promise of curcumin in metastatic breast cancer treatment, hydrophobicity in alkaline conditions, photodegradable, poor absorption, rapid metabolism and elimination, short half-life, and low and variable bioavailability have hindered its clinical application.2225

Recently, oral delivery of anticancer agents has gained increasing attention. Compared to intravenous administration, oral delivery of anticancer agents is non-invasive and cost-effective; it simplifies therapeutic procedures, improves patient life quality and compliance, and offers good tolerability.26 Additionally, it is well suited for maintenance treatment and prolonged disease control.27 However, oral delivery systems are in challenged by physiological and biochemical barriers and by physiochemical properties of anticancer drugs.28 To overcome these issues, numerous nanocarriers have been rationally designed in the last decade,2932 improving bioavailability and enhancing therapeutic efficacy.

Self-microemulsifying drug delivery systems (SMEDDSs) have been intensively investigated to improve oral bioavailability of poorly water-soluble drugs.31,32 However, few oral nanocarriers carrying chemotherapeutics and NIR dyes and combining the benefits of chemo- and phototherapy have been investigated for metastatic breast cancer treatment. IR780 and curcumin may interact via π–π stacking, facilitating the accommodation of IR780 in SMEDDS, which improves their oral absorption and bioavailability. Curcumin acts as a chemotherapeutic, inhibiting the progression of metastatic breast cancer, whereas IR780, after NIR irradiation, exerts photothermal effects and promotes ROS generation. In this study, we evaluated the potential of CUR/[email protected] as an oral drug delivery system for metastatic breast cancer treatment using in vitro cell models and xenograft tumor nude mice models.

Methods

Materials

Curcumin (purity>98%) was purchased from Dalian Meilun Biotechnology Co. Ltd (Dalian, People’s Republic of China). IR780 (2-[2-[2-chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cyclohexen-1-yl] ethenyl]-3,3-dimethyl-1-propylindolium iodide) was purchased from Sigma Aldrich (St. Louis, MO, USA). Soya bean lecithin (S100) were obtained from A.V.T. Pharmaceutical Co. Ltd (Shanghai, People’s Republic of China). Oleoyl macrogol-6/polyoxyl-6 glycerides (Labrafil® M1944 CS), PEG-40 hydrogenated castor oil (Cremophor RH40®), and caprylocaproyl macrogol-8 glycerides (Labrasol®) were kindly gifted by Gattefossè, Gennevilliers, France. Chemicals and solvents were of analytical or chromatographic grade. 4T1 and Caco-2 cell lines were supplied by the Institute of Biochemistry and Cell Biology (Shanghai, People’s Republic of China).

Preparation and characterization of CUR/[email protected]

Curcumin, IR780, and phospholipids were dissolved in ethanol. After stirring at 37°C for 3 hrs, the solvent was evaporated using a rotary evaporator. The obtained complex was mixed with Labrasol, before adding Labrafil M1944 CS and Cremophor RH40 at 37°C and stirring the mixture until a transparent liquid formed. To study the droplet size, size distribution and ζ-potential of CUR/[email protected], 100 μL of prepared CUR/[email protected] were slowly dropped into the 10 mL 37°C water, stirring with a speed of 100 rpm to form microemulsions. CUR/[email protected] microemulsions samples were tested using a Zetasizer detector to determine droplet size, size distribution, and ζ-potential.33,34

Cumulative in vitro release of curcumin and IR780

Release profiles of curcumin and IR780 from CUR/[email protected] were investigated using the dialysis bag method. CUR/[email protected] containing 1.0 mg of curcumin was loaded into the dialysis bag, which was incubated in 1.0 mL PBS (pH 6.8) and shaken at 100 rpm and 37°C. At predetermined time intervals, 500 μL of the release medium were collected and replaced with the same volume of fresh PBS (pH 6.8). The released curcumin was measured by HPLC, whereas IR780 content was determined using UV-Vis spectrophotometry. All experiments were performed in triplicate.

Photothermal properties of CUR/IR780 @SMEDDS in vitro

CUR/[email protected] was diluted with ultrapure water to the final IR780 concentrations of 30 and 50 µg/mL. Samples (1 mL) were added to a 24-well plate, with 1 mL of deionized water and curcumin–phospholipid complex-loaded self-microemulsifying drug delivery system ([email protected]) used as controls. The wells were irradiated (808 nm, 0.8 W/cm2) for 5 mins and the temperature was recorded at 30 s intervals using a thermal probe. All experiments were performed in triplicate.

To investigate thermogenic capacity, thermal photos were taken during laser irradiation. CUR/[email protected] was diluted with ultrapure water to the final IR780 concentration of 50 µg/mL. Samples and [email protected] as negative controls were irradiated (808 nm, 0.8 W/cm2) for 5 mins and thermal infrared images were captured every 30 s using an infrared-seek thermal imaging camera.

Cellular uptake

Cellular uptake of SMEDDS was investigated using confocal laser scanning microscopy. As curcumin fluoresces, no additional fluorescent indicators were used. Caco-2 or 4T1 cells were cultured in glass-bottom cell culture dishes at a density of 1×104 cells per well. After overnight culture, the cells were treated with 1 mL of CUR/[email protected] (containing 6 µg/mL curcumin and 3 µg/mL IR780) for 1, 2, and 4 hrs at 37°C. Afterward, the cells were repeatedly washed with PBS, fixed with 4% paraformaldehyde for 30 mins, and stained with DAPI. After washing three times with PBS, the cells were visualized using a TCS SP8 confocal system, at the following wavelengths: 405 nm (DAPI), 488 nm (curcumin excitation), 560–620 nm (curcumin emission), 638 nm (IR780 excitation), and 700–800 nm (IR780 emission).

In vitro anti-metastatic effects in 4T1 cells

Cell proliferation analysis

4T1 cells were seeded in a 96-well plate at the density of 1×104 cells per well. After 24-hr incubation, the cells were treated with control (untreated), control+NIR, blank SMEDDS, [email protected], IR780, [email protected]+NIR, CUR/[email protected], and CUR/[email protected]+NIR for 12 hrs, with each treatment repeated in six wells. After washing the cells three times with PBS, the media were replaced with MTT solution (5 mg/mL) for 4 hrs. After dissolving formazan crystals in DMSO, cell viability was determined using a microplate reader at 570 nm.

Wound healing assay

Wound healing assay was performed to investigate the effect of CUR/[email protected] on migratory potential of 4T1 cells, as previously described.35 Briefly, 8×105 cells were seeded in 6-well culture plates and cultured overnight. Wounds were generated with a 200 μL pipette tip and the cells were treated with control, control+NIR, IR780, [email protected], IR780+NIR, CUR/[email protected], and CUR/[email protected]+NIR with curcumin and IR780 concentrations of 10 and 5 μg/mL, respectively. Cell images were captured. The wound area was calculated using ImageJ software.

In vitro migration and invasion assay

4T1 cells were seeded in a 6-well culture plate and cultured for 24 hrs. The cells were treated with control, control+NIR, IR780, [email protected], IR780+NIR, CUR/[email protected], and CUR/[email protected]+NIR (curcumin, 6 μg/mL; IR780, 3 μg/mL). After 12 hrs, the cells were washed twice with PBS and the NIR group was exposed to laser irradiation (808 nm, 0.8 W/cm2). The cells were suspended in serum-free culture medium and in vitro transwell migration and invasion assays were performed, as previously described.36 Briefly, 1×105 cells were seeded to the top chambers of 24-well transwell inserts without a gel-coated membrane for the transwell migration assay, whereas for the invasion assay, 1×105 cells were seeded in the top chamber with a Matrigel-coated membrane, supplemented with no serum medium. In both assays, the lower chambers were filled with 600 μL medium supplemented with serum. After a 24-hr incubation, cells remaining in the top chamber were removed with a cotton swab. The cells that migrated through the filters into the lower wells were fixed with 90% ethanol for 20 mins, stained with crystal violet for 20 mins, and photographed. For quantification, the crystals were dissolved in 35% acetic acid solution and the samples were analyzed using a microplate reader.

ROS detection in vitro

Photodynamic efficacy of CUR/[email protected] was evaluated by determining ROS production in 4T1 cells, as previously described.37 1×104 4T1 cells were seeded into a glass-bottom culture dish. After 24-hr incubation, the cells were treated with CUR/[email protected] (curcumin, 10 μg/mL; IR780, 5 μg/mL) containing H2DCFDA (40 mM). Blank culture medium was used as control. The cells were incubated for 4 hrs at 37°C, rinsed with PBS, and irradiated using a NIR laser (808 nm, 0.8 W/cm2) for 5 mins per well. Control cell groups received no irradiation and were not treated with SMEDDS. Fluorescence images were immediately observed using confocal laser scanning microscopy.

In vivo bioavailability

The animals were housed under standard laboratory conditions and given access to sterile food and clean water ad libitum. All animal experiments were conducted and approved by Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine. The experimental procedures were in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. Tumor-free Sprague Dawley rats (200±20 g) were randomly divided into CUR/[email protected], curcumin suspensions, IR780 suspensions (n=5). Curcumin and IR780 suspensions were prepared by dispersing curcumin and IR780 in 0.5% carboxymethyl cellulose solution. The animals were orally administered a single dose of 75 mg/kg curcumin and 10 mg/kg IR780. Blood samples were collected from the orbital vein at 15, 30, 60, 90, 120, 240, 360, 480, 720, 1,440, 2,880 mins after oral administration and centrifuged at 1,800 g for 5 mins. Curcumin and IR780 content in the serum samples were separately determined using LC-MS and Varioskan Flash Spectral Scanning multimode plate reader. LC-MS was performed on an Ultimate 3000 UPLC system (Thermo, USA) using an Hypersil GOLD C18 column (2.1 mm×50 mm, 3 µm, Thermo, USA). The analysis was achieved with gradient elution using (A) acetonitrile and (B) water (containing 0.5% formic acid) as the mobile phase (A:B=68:32, v/v). A TSQ triple-quadrupole tandem mass spectrometer (Thermo, USA) in positive HESI mode was used. The conditions are as follows: capillary voltage, 2,800 V; vaporizer temperature, 80°C; capillary temperature, 350°C. Selective reaction monitor (SRM) of the transitions of m/z 367.1→272.9 for curcumin. For collision-induced dissociation, argon was used as the collision gas at a flow rate of 0.20 mL/min.

In vivo imaging and biodistribution studies

In order to evaluate tumor retention of CUR/[email protected] and biodistribution, 4T1 breast cancer models were constructed in BALB/c nude mice (20±2 g). Tumor xenografts were imaged at the tumor size of approximately 500 mm3. The mice were orally administered CUR/[email protected] and IR780 suspension at IR780 dose of 6.5 mg/kg; mice receiving normal saline were used as control. After 1, 2, 4, 6, 8, 24, and 48 hrs, in vivo imaging using IVIS imaging system was performed. After 48 hrs, the mice were sacrificed and biodistribution of IR780 in mice organs was evaluated.

In vivo therapeutic efficacy against metastatic breast cancer

4T1 cells (1×106) were directly injected into the second pair of mammary pads of 4-week-old BALB/c nude mice to establish breast cancer models. 4T1 tumor-bearing mice were randomly divided into seven groups after tumors reached approximately 150 mm3 (n=7) and were orally administered different formulations (control, IR780, CUR, IR780+NIR, CUR/[email protected], [email protected], and CUR/[email protected]+NIR) every 2 days for 16 days with dose of 50 mg/kg curcumin and 5 mg/kg IR780. Tumor volume and mice body weight were recorded. Mice tumors were irradiated (0.8 W/cm2 at 808 nm for 5 mins with the area of irradiation spot of 66 mm3) 24 hrs after each treatment. During irradiation, the temperature of tumors in control+NIR, IR780+NIR, and CUR/[email protected]+NIR groups was recorded every 30 s and the tumors were photographed using a thermal camera. On day 16, the mice were sacrificed and the pulmonary metastatic nodules were photographed and counted for the in vivo anti-metastatic assay. Tumors and organs were fixed with 10% paraformaldehyde and embedded into paraffin. The tissue blocks (superficial and deep tissue regions) were cut and stained with H&E and analyzed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay).

Preliminary safety evaluation in mice

BALB/c mice were randomly divided into two groups (n=5) and orally received saline (as control) or CUR/[email protected] with dose of 50 mg/kg curcumin and 5 mg/kg IR780 every 2 days for 16 days. On day 16, the mice were sacrificed to collect blood samples, stomach, and intestines for further blood biochemistry analyses, hematological analysis, and histological H&E staining.

Statistical analysis

Data are expressed as means±SD of at least three independent experiments. Statistical analysis was performed using Student’s t-test and one-way ANOVA for pairs of samples. p-values <0.05 were considered statistically significant.

Results and discussion

Preparation and characterization of CUR/[email protected]

In this study, an oral SMEDDS-based nanocarrier was developed for co-loading of curcumin and IR780 (Figure 1A), with the purpose of achieving combined photo- and chemotherapy of metastatic breast cancer. Curcumin–phospholipid complexes were prepared by solvent evaporation as previously described,38 based on interaction of phospholipids with curcumin in an aprotic solvent. Drug-phospholipid complexes are formed via electrostatic interactions, hydrogen bonds, and van der Walls forces.39 Complex formation was confirmed by Fourier transform infrared spectroscopy (FTIR) spectroscopy (Figure1B). Curcumin exhibited characteristic hydroxyl stretching band at 3,504 cm−1, which weakened considerably in the complex spectrum. Additionally, phospholipid showed peaks at 1,249 cm−1 (P=O stretching), which shifted to 1,254 and 1,284 cm−1 in curcumin–phospholipid physical mixture and complex spectra, respectively, possibly because of the complexation between the hydroxy and the P=O group. Curcumin–phospholipid complex has been previously reported.40,41 Whether IR780 can form a complex with phospholipids or only interacts with curcumin was initially unclear; however, in this study, IR780 did not form a complex with the phospholipid (Figure S1). Curcumin structure incorporates an extensive aromatic π-electron system (Figure 1C), which allows strong π-π stacking interactions with aromatic molecules, whereas IR780 features a rigid cyclohexenyl ring in the heptamethine chain.42 Thus, we propose that curcumin co-assembles with IR780 via π–π stacking interactions. To verify this hypothesis, we analyzed fluorescence quenching by comparing the changes in the emission spectra of curcumin and IR780 with changes in compound concentration. As shown in Figure 1E and F, fluorescence intensity of curcumin and IR780 gradually decreased with increasing IR780 and curcumin concentrations, indicating fluorescence quenching and suggesting the existence of interactions between curcumin and IR780.

Figure 1 (A) Schematic illustration of preparation of the curcumin–phospholipid complex and IR780-loaded self-microemulsifying drug delivery systems. (B) FTIR spectra of curcumin (CUR), phospholipid (PC), physical mixture of CUR and PC (CUR-PC physical mixture), and CUR-PC complex. (C) Chemical structures of CUR and IR780. (D) Droplet size of CUR/[email protected] Insert: TEM image of CUR/[email protected] (upper panel) and photograph of blank SMEDDS, [email protected], and CUR/[email protected] (lower panel). (E) Fluorescence emission spectra of CUR in the presence of different IR780 concentrations. (F) Fluorescence emission spectra of IR780 in the presence of different CUR concentrations. (G) UV-Vis absorption spectra of CUR, phospholipids, IR780, and CUR/[email protected]

The SMEDDS formulation was designed by determining solubility and constructing a pseudoternary phase diagram. Labrafil M1944 CS, Cremophor RH40, and Labrasol (2:1:1, w/w/w) were selected as the oil phase, surfactant, and co-surfactant, respectively, as these excipients dissolved both curcumin and IR780 and showed promising self-microemulsifying efficiency. Additionally, in the absence of curcumin–phospholipid complex, IR780 loaded into the blank SMEDDS failed to form a stable microemulsion, indicating the significance of interaction between curcumin and IR780 in CUR/IR780 formation. Furthermore, the interaction between curcumin and IR780 in SMEDDS was also revealed by the absorption spectra of CUR/[email protected] (Figure 1G). Due to the interference of phospholipids on the curcumin absorption determination (as shown in the inset figure at the upper right corner), it is difficult to judge the absorption peak shift of curcumin in the spectrum of CUR/[email protected] compared with the raw curcumin. However, compared with raw IR780, CUR/[email protected] exhibited clear red shift of the absorption peak of IR780 from 784 to 792 nm, suggesting the presence of interaction between curcumin and IR780 in SMEDDS.

The prepared CUR/[email protected] particles were spherical and displayed a narrow size distribution, as shown in the transmission electron microscopy image (Figure 1D). Hydrodynamic size of CUR/[email protected] dispersed in deionized water determined by dynamic light scattering was 38.26±0.25 nm (Figure 1D), suitable for oral administration and absorption through the gastrointestinal tract. Self-emulsifying time of the formulation was <3 mins. Drug loading increased droplet size compared to particle sizes of drug-free preparations and the final formulation was further optimized for use in this study.

Release of curcumin and IR780 from SMEDDS was analyzed in vitro without laser irradiation. Because of limited solubility of curcumin in PBS, 50% ethanol was used as the release medium to provide the sink condition. In the first 3 hrs, 41.6% curcumin and 55.1% IR780 was released from CUR/[email protected], whereas approximately 89.9% of curcumin (Figure 2A) and 92.1% of IR780 (Figure 2B) was released from SMEDDS in 24 hrs. To get clearly understand the release and stability in stomach, in future, it would be beneficial to study the release of SMEDDS in acidic pH, and reveal its behavior in the stomach.

Figure 2 In vitro release profiles of curcumin (CUR) (A) and IR780 (B) in PBS (pH 6.8). (CD) In vitro photothermal behavior of CUR/[email protected]: (C) temperature change curves of CUR/[email protected] containing different concentrations of IR780 after NIR irradiation (808 nm, 0.8 W/cm2). [email protected] and deionized water were used as control. (D) IR thermal images of deionized water and CUR/[email protected] during 5-min laser irradiation. Data are represented as means±SD (n=3).

Photothermal and photodynamic effects of CUR/[email protected]

In order to evaluate photothermal properties of CUR/[email protected], we monitored temperature changes upon laser irradiation (808 nm, 0.8 W/cm2). As shown in Figure 2C, negative control (deionized water) and [email protected] showed a temperature increase of approximately 4°C. In contrast, in aqueous dispersions of CUR/[email protected] at different concentrations, temperature increased gradually and reached a plateau for the concentration of 30 μg/mL, increasing by approximately 15°C after 5-min irradiation (Figure 2C). In addition, the rate of temperature increase in aqueous dispersions of CUR/[email protected] rapidly increased with higher concentrations of the preparation, corresponding to higher IR780 content.

Furthermore, photos and thermal images of CUR/[email protected] were taken to observe the changes in temperature during exposure to 808 nm laser irradiation. As shown in Figure 2D, pseudo-color signals in the SMEDDS group grew stronger with irradiation time, indicating the increase in temperature, whereas almost no change in pseudo-color signals was observed in the control group. Taken together, these results suggest promising NIR photothermal capacity of CUR/[email protected]

In order to investigate ROS generation potential of CUR/[email protected] upon exposure to NIR laser irradiation, we detected intracellular ROS generation using a redox-sensitive probe H2DCFDA in 4T1 cells. No fluorescence signal was found in cells in control PBS and CUR/[email protected] groups. However, green fluorescence was observed in cells treated with CUR/[email protected] under NIR irradiation (Figure 4A, Figure S2), indicating ROS production. After NIR exposure, IR780 produces reactive molecules and the generated ROS possibly mediate photo-induced cytotoxicity.43 Therefore, we attempted to correlate ROS generation and cell viability. CUR/[email protected] treatment with exposure to NIR irradiation exhibited the highest cytotoxicity, consistent with high ROS production. This observation implies that ROS generated by CUR/[email protected] upon laser irradiation may induce cellular death, in agreement with a previous report.44 Additionally, in the ROS detection assay, CUR/[email protected] treatment without NIR irradiation showed very light fluorescence compared to the control group (Figure 4B), which might be caused by intrinsic ROS generation capacity of curcumin.

In vitro cellular uptake and cytotoxicity analysis

Cellular uptake was evaluated in Caco-2 and 4T1 cells. As shown in confocal images in Figure 3, Figure S3, and Figure S4, fluorescence signal was shown in both cell lines. Red IR780 signal was distributed in cytoplasm, in line with a previous report.37 Additionally, we evaluated the effects of NIR laser irradiation on cellular uptake in 4T1 cells. After incubation with NIR laser irradiation for 5 mins, cellular uptake increased, which may be attributed to improve movement and diffusion of nanoparticles in culture media at higher temperatures. Cellular uptake was also determined by flow cytometry. As shown in Figure 4C, NIR irradiation improved cellular uptake, in agreement with a previous report of photothermal effect promoting nanocarrier endocytosis and intracellular accumulation.45

Figure 3 Confocal images of cellular uptake for CUR/[email protected] in Caco-2 cells and 4T1 cells.

Figure 4 (A) Confocal images of ROS generation by CUR/[email protected] after NIR irradiation (808 nm, 0.8 W/cm2, 5 mins) in 4T1 cells. (B) Quantitative analysis of ROS generation by PBS (control), CUR/[email protected], and CUR/[email protected] after NIR irradiation (808 nm, 0.8 W/cm2, 5 mins) in 4T1 cells. (C) In vitro cellular uptake of control, CUR/[email protected], and CUR/[email protected]+NIR. ** p<0.01. (D) Cell viability of 4T1 cells incubated with IR780, [email protected], CUR/[email protected], IR780+NIR (808 nm, 0.8 W/cm2, 5 mins), and CUR/[email protected]+NIR. (E) Quantitative analysis in 4T1 cells: (a) wound healing assay, (b) transwell migration assay, and (c) invasion assay. Photographs of the transwell migration assay (F) and invasion assay (G) with 4T1 cells treated with control, IR780, [email protected], CUR/[email protected], control+NIR, IR780+NIR, and CUR/[email protected]+NIR. Data are represented as means±SD (n=5).

In vitro cytotoxicity of CUR/[email protected] in 4T1 cells was evaluated using the MTT assay (Figure 4D). Compared to IR780 treatment in the absence of NIR irradiation, IR780 under NIR irradiation significantly decreased 4T1 cell viability, indicating that NIR irradiation increased IR780 cytotoxicity. This result was consistent with previous findings46 and possibly associated with cytotoxicity caused by temperature increase and increased ROS production upon irradiation. Similarly, decreased cell viability was observed after CUR/[email protected] treatment combined with NIR irradiation, compared to cell viability after treatment with preparations in the absence of NIR irradiation. Furthermore, compared to [email protected], CUR/[email protected] demonstrated significantly increased cytotoxicity, indicating additive cytotoxic effects of curcumin and IR780. Improved cytotoxicity of IR780 combined with chemotherapeutics such as doxorubicin was previously attributed to enhance cytotoxicity of doxorubicin with temperature increase and higher heat sensitivity of tumor cells exposed to doxorubicin.46

CUR/[email protected] inhibits migration and invasion of 4T1 cells in vitro

To evaluate inhibition of breast cancer metastasis, we first investigated the effect of various formulations on 4T1 cell migration by wound healing assay (Figure 4E(a), Figure S5). Untreated cells and cells treated only with NIR irradiation migrated and filled the wound gap after 24 hrs (Figure 4E, panel (a)). In IR780 group, approximately 62% cell migration occurred, indicating that IR780 partially inhibits tumor cell migration, consistent with a previous report.7 Additionally, wound healing was significantly inhibited by [email protected] Combining IR780 and curcumin in SMEDDS (CUR/[email protected]) did not further inhibit migration, as shown in Figure 4E(a). We next examined the effects of NIR on cell migration. CUR/[email protected] significantly inhibited cell migration after NIR irradiation, compared to treatments in the absence of NIR, revealing the potential of photothermal treatments in cell migration inhibition.

Inhibitory effects of CUR/[email protected] on cell longitudinal migration were further confirmed by the transwell migration assay (Figure 4E(b) and F). As shown in Figure 4E(b), 72.5% and 49.6% of cells migrated from the upper to the lower chamber after free IR780 and CUR/[email protected] treatments, respectively. As expected, after NIR exposure, fraction of migrating 4T1 cells reduced to 35.8% and 12.1%, indicating that NIR enhanced migration inhibition.

As invasion is a hallmark of breast cancer metastasis, we evaluated the effects of CUR/[email protected] on the invasive behavior of 4T1 cells (Figure 4E(c) and G). Cell invasion through Matrigel was inhibited 48.5% by CUR/[email protected], suggesting that, in agreement with the results of the migration assays, CUR/[email protected] reduced the invasive capacity of 4T1 cells. NIR irradiation greatly enhanced the effect (cell invasion rate of 6.1%). It has been reported that curcumin may suppress migration and invasion of breast cancer cells by modulating multiple signaling pathways, 4749 attributable to effects on expression of NF-κB p105, COX-2, and MMP9. In addition, anti-invasion effect of free IR780 to 4T1 cells was similar with the previous report,50 suggesting the contribution of phototherapy on suppressing the tumor cell migration. Taken together, these findings indicate that CUR/[email protected] markedly inhibited migration and invasion of 4T1 cells, implying that CUR/[email protected] could potentially suppress breast cancer metastasis in vivo.

In vivo bioavailability

As enhanced bioavailability is a major condition for improving therapeutic efficacy, we compared CUR/[email protected] bioavailability with that of suspensions prepared by dispersing curcumin and IR780 in 0.5% CMC-Na. Plasma concentration-time profiles for gavage administration of CUR/[email protected] and suspensions in rats are shown in Figure 5A and B, whereas pharmacokinetic parameters are listed in Table 1. Significant differences in Cmax, Tmax, and AUC were observed between the groups. CUR/[email protected] showed the highest Cmax and AUC, with relative bioavailability of 743.7% and 307.0% for curcumin and IR780, respectively, indicating that CUR/[email protected] significantly improved oral absorption of curcumin and IR780. Additionally, MRT of CUR/[email protected] was significantly longer than that of suspensions, indicating a longer retention time of SMEDDS in systemic circulation. IR780 was reported to be rapidly cleared from systemic circulation,51 whereas SMEDDS formulations would allow accumulation of IR780 at the primary tumor and metastatic sites.

Table 1 Pharmacokinetic parameters of curcumin (CUR) and IR780 after oral administration of CUR/[email protected], CUR suspensions, or IR780 suspensions in rats (n=5). Data are expressed as mean±SD.

Figure 5 In vivo bioavailability and distribution study. (A) Plasma concentration-time curve of curcumin (CUR) in rats after oral administration of CUR suspensions and CUR/[email protected] (B) Plasma concentration-time curve of IR780 in rats after oral administration of IR780 suspensions and CUR/[email protected] Data are represented as means±SD (n=5). (C) Representative fluorescence images of rats treated with IR780 dispersions and CUR/[email protected] at predetermined time points. (D) Representative fluorescence images of isolated tumors and organs, including the gastrointestinal tract.

In vivo fluorescence imaging and biodistribution analysis

To analyze in vivo distribution and verify anti-metastatic potential of CUR/[email protected], in vivo fluorescence imaging was performed. IR780 fluorescence was recorded at predetermined time points after oral administration of CUR/[email protected] Whole animal fluorescence imaging showed that the control IR780 group produced almost no obvious fluorescence signals after 1 hr (Figure 5C). In contrast, strong fluorescence was observed in the group treated with CUR/[email protected], likely due to the rapid absorption of IR780 loaded in SMEDDS (Figure 5D). Fluorescence intensity decreased gradually and no obvious fluorescence was observed in IR780 suspension group after 48 hrs because of IR780 systemic clearance. In contrast, in animals treated with SMEDDS, fluorescence persisted longer, and was detectable even after 48 hrs. Passive IR780 tumor accumulation was further verified by fluorescence imaging of excised tissues (Figure 5D). IR780 tumor accumulation was significantly higher in animals treated with CUR/[email protected] CUR/[email protected] group showed improved distribution of fluorescence in the gastrointestinal tract, especially at the jejunum, which is proposed as the main absorption site for IR780, indicating higher IR780 amounts available for systemic absorption in this group. This result supports the whole animal fluorescence imaging; CUR/[email protected] treatment maintained stronger and longer-lasting IR780 fluorescence than that of control. Combined with the results of pharmacokinetic studies, these data indicate that CUR/[email protected] achieved improved in vivo IR780 bioavailability.

Photothermal efficacy of CUR/[email protected] in vivo

Photothermal efficacy was evaluated in 4T1 tumor-bearing mice to further demonstrate the potential of CUR/[email protected] in the treatment of metastatic breast cancer. IR images were taken after 5-min laser irradiation and corresponding temperatures were recorded. As shown in Figure 6A and B, in IR780 and CUR/[email protected] groups temperature rapidly increased for the first 120 s, before leveling off. Tumor temperature reached 50°C and 60°C in the two groups, respectively, with 60°C sufficient for tumor ablation. Only slight changes in temperature were observed in the control group. These results suggest that CUR/[email protected] possesses potent in vivo photothermal efficacy.

Figure 6 In vivo antitumor effects. (A) Photothermal images of 4T1 tumor-bearing nude mice treated with PBS, IR780, and CUR/[email protected] under NIR laser irradiation. (B) Tumor temperature-time curve after NIR irradiation (n=3). (C) Photographs of tumors and 4T1 tumor-bearing nude mice treated with control, IR780, CUR, IR780+NIR, CUR/[email protected], [email protected], and CUR/[email protected]+NIR 16 days after administration. (D) (a) Tumor volume in nude mice treated with control, IR780, CUR, [email protected], CUR/[email protected], IR780+NIR, and CUR/[email protected]+NIR; **p<0.01. (b) Body weight change of tumor-bearing mice. Data are represented as mean±SD (n=7).

In vivo therapeutic efficacy

In vivo therapeutic efficacy of CUR/[email protected] against metastatic breast cancer was assessed on 4T1 tumor-bearing mice. As shown in Figure 6C, tumor volumes of animals treated with free IR780, free curcumin, and IR780 combined with NIR irradiation showed no significant differences compared with the control group, in which tumor volumes increased rapidly, reaching 6–10 times starting volumes by day 16 (Figure 6D(a)). These results indicated that these three treatments failed to achieve desirable antitumor effects, possibly because of poor oral absorption and low bioavailability of the compounds. In the same experiment, CUR/[email protected] showed enhanced tumor inhibition compared with [email protected], indicating that IR780 contributes to antitumor effects when introduced into SMEDDS. The most remarkable tumor inhibition effects were observed in mice receiving CUR/[email protected] combined with NIR irradiation, in which gradual decrease in tumor size was observed until day 8. After day 8, tumor recurrence was observed in three mice in this group, however, tumor volumes remained lower than initial volumes. Taken together, these data suggest that CUR/[email protected] treatment combined with NIR irradiation showed remarkable antitumor efficacy compared with chemotherapy or phototherapy alone. No notable body weight loss was observed in any group during the 16 days of treatment (Figure 6D(a)), indicating a lack of significant physiological toxicity of the treatments at the dose used (Figure 6D(b)).

Although oral IR780 loaded nanostructured lipid carrier designed for photothermal therapy has been reported previously, how to further improve the therapeutic efficiency and assure safety remains worthy of investigation. Firstly, given the limited penetration depth of NIR dye, sole phototherapy may be ineffective to the inner section of the tumor especially when the tumor becomes larger. Therefore, it is rational to understand the better therapeutic efficiency in CUR/[email protected] than the pure IR780 by NIR treatment. Combination of photothermal therapy with chemotherapy is more valuable in the treatment of metastatic breast cancer. Secondly, one of the limitations for IR780 application is its acute toxicity (1 mg/kg to mice by intravenous administration52). To assure the safety of IR780, dose reduction is desirable. Compared to previously reported IR780 nanostructured lipid carrier designed for photothermal therapy, CUR/[email protected] demonstrated superior effects at a reduced dose (IR780 5 mg/kg vs 6 mg/kg) and lower NIR power density (0.8 W/cm2 vs 2 W/cm2) due to improved IR780 absorption and facilitated chemotherapeutic effect.

To further demonstrate cellular antitumor activity of CUR/[email protected], histological examination and apoptosis assays were performed. As deeper segments of tumor tissues could be shielded from photothermal treatment because of limited light penetration,5 both superficial and deep tumor tissue regions were H&E stained (Figure 7A). Control group tumors showed numerous tumor cells with preserved tissue structure. Free IR780 treatment combined with NIR irradiation was not cytotoxic to tumor cells in deeper tissue regions, consistent with a previous report.30 After CUR/[email protected] treatment in the absence of NIR irradiation, notable tumor tissue structure and invasion of tumor cells to soft tissues were observed in superficial tissue sections, whereas considerable necrosis and infiltration of inflammatory cells occurred in deep tissue regions. In contrast, CUR/[email protected] treatment combined with NIR irradiation resulted in no observable tumor tissue sections, with local inflammatory cell infiltration occurring in both superficial and deep tissue regions. As shown in Figure 7B, apoptosis was very rarely observed in control groups, whereas in IR780 and curcumin group slight apoptosis was observable in superficial and deep tissue sections, respectively. In [email protected], CUR/[email protected], and CUR/[email protected]+NIRand IR780+NIR group, apoptosis was significantly more pronounced (Figure 7B). The CUR/[email protected] group presented cavities in superficial tissue structures, possibly caused by high temperatures resulting from the photothermal effect, with considerable degree of apoptosis occurring in deep tissues. These results illustrate improved antitumor effects of combined photo- and chemotherapy.

Figure 7 (A) Histological examination (scale 200 μm) and (B) TUNEL analysis of tumor tissues treated with control, IR780, CUR, [email protected], CUR/[email protected], IR780+NIR, and CUR/[email protected]+NIR 16 days after administration. “Surface” and “Deep” labeled rows represent H&E images of superficial and deep tumor tissue regions, respectively. (a) TUNEL assay images and (b) quantitative analysis of fluorescence intensity in apoptotic cells (mean±SD, n=5).

To evaluate the efficacy of CUR/[email protected] in inhibiting lung metastasis, we determined the number of pulmonary metastatic nodules and histopathologically analyzed lung sections from 4T1 tumor-bearing mice (Figure 8A). IR780 and curcumin treatment induced approximately onefold decrease in the number of metastatic nodules, compared to control (Figure 8C). Enhanced inhibition was observed in all SMEDDS groups (p<0.05), possibly because SMEDDS improved in vivo absorption of curcumin and IR780. Additionally, metastatic nodules were rarely observed in the lungs of CUR/[email protected]+NIR group mice; the strongest anti-metastatic effects were observed in this group. H&E staining (Figure 8B) indicated that smaller areas of metastasis foci were observed in the SMEDDS groups compared to free curcumin and IR780 groups, with CUR/[email protected] group showing the smallest foci area (Figure 8D), consistent with the nodule counting results. These findings indicate that CUR/[email protected] combined with NIR irradiation achieved the highest inhibition of 4T1 tumor metastasis among the treatments studied.

Figure 8 (A) Photographs and (B) histological examination of pulmonary metastatic nodules in nude mice treated with control, IR780, CUR, [email protected], CUR/[email protected], IR780+NIR, and CUR/[email protected]+NIR. (C) Number of pulmonary metastatic nodules in nude mice after treatments. (D) Pulmonary metastatic area of nude mice after treatments. Data are represented as mean±SD of five animals. ** p<0.01.

Preliminary safety evaluation in mice

In addition to monitoring mice body weight, we evaluated histopathological, biochemical, and hematologic parameters. H&E staining indicated no significant signs of organ damage to heart, liver, spleen, and kidney in treated mice, compared to organs of control animals (Figure S6). Gastrointestinal tract histopathological findings of CUR/[email protected] and control group are shown in Figure 9A. No obvious squamous epithelial cell proliferation, prominent keratinization, or obvious lesions were observed in the stomach. In jejunum and ileum, the shape of intestinal villi was unaltered and no congestion, proliferation, heteromorphosis, or inflammatory infiltration was observed. These results indicate that CUR/[email protected] lacks gastrointestinal toxicity. Aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and total cholesterol levels (Figure 9B) suggested no liver or kidney dysfunctions. Additionally, no significant changes in white blood cell, red blood cell, and neutrophil levels were observed in CUR/[email protected] animals compared to levels in the control group. These results suggest negligible side effects of CUR/[email protected] in vivo, possibly because of preferential accumulation of IR780 and curcumin in tumors and low treatment dose used in this study.

Figure 9 (A) Histological examination of stomach, jejunum, and ileum. (B) (a) Hematological and (b) biochemical parameters of liver and kidney function in mice treated with control and CUR/[email protected] (means±SD, n=5).

Conclusion

In summary, CUR/[email protected] improved cellular uptake in Caco-2 cells and enhanced oral bioavailability of curcumin and IR780 in rats. Additionally, CUR/[email protected] showed enhanced cytotoxicity and inhibited migration and invasion of 4T1 cells when combined with NIR irradiation. Improved therapeutic effect of the designed SMEDDS was also demonstrated in vivo, in 4T1 tumor-bearing nude mice, suggesting potential benefits of this formulation in treating metastatic breast cancer. These results suggest CUR/[email protected] as a safe and promising addition to combined cancer therapy approaches, suitable for oral administration.

Acknowledgment

This work was supported by the National Natural Science Foundation of China [Grant No. 81773913 and 81303232].

Disclosure

The authors report no conflicts of interest in this work.

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Supplemental materials

Figure S1 FTIR spectra of (A) Raw IR780, (B) PC, (C) physical mixture of IR780 and PC, and (D) IR780 PC formed by the phospholipid complex preparing process.

Figure S2 Confocal images of ROS generation by control under NIR irradiation (808 nm, 0.8 W/cm2, 5 mins) and [email protected] in 4T1 cells.

Figure S3 Confocal images of cellular uptake for CUR/[email protected] in Caco-2 cells.

Figure S4 Confocal images of cellular uptake for CUR/[email protected] in (A) 4T1 cells and (B) 4T1 cells under NIR irridiation (808 nm, 0.8 W/cm2, 5 mins).

Figure S5 Photographs of the wound healing assay with 4T1 cells treated with control, IR780, [email protected], CUR/[email protected], control+NIR, IR780+NIR, and CUR/[email protected]+NIR.

Figure S6 Histological examination of heart, liver, spleen, and kidney in tumor-bearing nude mice treated with control and CUR/[email protected]+NIR.

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