Hybrid Polypeptide Micelles Loading Indocyanine Green for Tumor

Indocyanine green (ICG) is a near-infrared (NIR) fluorescence dye for extensive applications; however, it is limited for further biological applicatio...
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Hybrid Polypeptide Micelles Loading Indocyanine Green for Tumor Imaging and Photothermal Effect Study Lei Wu, Shengtao Fang, Shuai Shi, Jizhe Deng, Bin Liu, and Lintao Cai* Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, PR China ABSTRACT: Indocyanine green (ICG) is a near-infrared (NIR) fluorescence dye for extensive applications; however, it is limited for further biological application due to its poor aqueous stability in vitro, concentration-dependent aggregation, rapid elimination from the body, and lack of target specificity. To overcome its limitations, ICG was encapsulated in the core of a polymeric micelle, which self-assembled from amphiphilic PEG-polypeptide hybrid triblock copolymers of poly(ethylene glycol)-b-poly( L -lysine)-b-poly( L -leucine) (PEG-PLL-PLLeu), with PLLeu as the hydrophobic core and PEG as the hydrophilic shell. The ICG was associated with the hydrophobic core via hydrophobic interaction and also the hydrophilic heads through electrostatic attractive interaction. Compared with free ICG, PEG-PLL-PLLeu-ICG micelles significantly improved quantum yield and fluorescent stability. The cellular uptake experiments showed that PEG-PLL-PLLeu-ICG micelles have a high cellular uptake rate. And the in vivo experiments revealed the excellent passive tumor targeting ability and long circulation time of PEG-PLL-PLLeu-ICG. The above results indicated the broad prospects of PEG-PLL-PLLeu-ICG application in the fields of tumor diagnosis and imaging. In addition, temperature measurements under NIR laser irradiation and in vitro photothermal ablation studies proved the potential application of PEG-PLL-PLLeu-ICG in tumor photothermal therapy.

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INTRODUCTION Compared with conventional resections, photothermal therapies have numerous benefits such as minimal invasion, relatively simple execution, and particular direct heating in the tumor.1,2 Furthermore, the use of near-infrared (NIR) light in the range of 700−1100 nm for photothermal therapy is particularly attractive because living tissues display negligible absorption and autofluorescence in this NIR wavelength region.3−6 Therefore, developing a dual-functional NIR probe which can be used for both optical imaging and photothermal therapy is very important. Indocyanine green (ICG) is the only nearinfrared (NIR) fluorescence dye approved by the United States Food and Drug Administration (FDA) for human use and has a wide range of applications in imaging7−9 and photothermal therapy10−14 due to its unique photomechanical, photochemical, and photobiological properties.15 However, several physicochemical characteristics such as lack of target specificity, poor aqueous stability in vitro, and short plasma half-life limit the application of ICG.16 In order to overcome these limitations, some attempts have been made, including the encapsulation of ICG within various protein, membrane, or micelle systems and the combination of ICG to several charged polymers.17−19 Moreover, some ICG-containing poly(lactic-co-glycolic acid) PLGA nanoparticles20,21 and nanoparticle-assembled capsules (NACs)22−24 presented improved stability and prolonged plasma half-life. However, all these ICG nanostructures displayed equivalent or even lower fluorescence intensity as compared with free ICG. Recently, © 2013 American Chemical Society

cationic micelles self-assembled from amphiphilic cationic copolymers have been developed as novel gene vectors for DNA or siRNA delivery. And phospholipid micelles have been used as novel lipid based carriers for water insoluble drugs.25,26 Also synthetic polypeptides, which own similar component and structure to natural proteins, have been widely used in drug and gene delivery, tissue engineering, sealants, and so on27 for their biocompatibility and biodegradability. For example, a new type of amphiphilic PEG-polypeptide hybrid triblock copolymer of poly(ethylene glycol)-b-poly(L-lysine)-b-poly(L-leucine) (PEGPLL-PLLeu) was previously reported as an efficient gene delivery system by our group.28 The self-assembled PEG-PLLPLLeu micelles effectively promoted cellular uptake and miraculously regulated the cellular uptake efficiency by altering the length of PLL and PLLeu segments. In the present study, a kind of ICG-encapsulated micelle (PEG-PLL-PLLeu-ICG) was developed based on the noncovalent self-assembly of PEG-PLL-PLLeu and ICG (Figure 1). The physicochemical properties were characterized by dynamic laser scattering (DLS), transmission electron microscopy (TEM), and fluorescent measurements. The cellular uptake experiments, performed on human nonsmall cell lung cancer cells (H460 cells), revealed the intracellular localization of Received: April 18, 2013 Revised: July 25, 2013 Published: July 26, 2013 3027

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Figure 1. (A) Chemical structure of the PEG-PLL-PLLeu copolymer. (B) Diameter distribution of the PEG-PLL-PLLeu-ICG micelles by DLS. (C) Schematic illustration of the PEG-PLL-PLLeu-ICG micelle and chemical structure of ICG. (D) TEM image of the PEG-PLL-PLLeu-ICG micelle. water method was adopted. Briefly, PEG-PLL-PLLeu and ICG were dissolved in DMSO at room temperature with 4 h magnetic stirring. After that the mixture was dialyzed against water and the ICGencapsulated micelles self-assembled. By adjusting the ratio of PFGPLL-PLLeu and ICG, the PEG-PLL30-PLLeu40-ICG5:1 micelles (abbreviated as 1:30:40/ICG5:1) were prepared. The particle size and zeta potential of 1:30:40/ICG5:1 were measured by using a NanoZS ZEN3600 (Malvern Instruments) instrument at 25 °C. ICG Encapsulation Efficiency. To determine the encapsulation efficiency (EE) of the micelles, a certain volume of dialyzed 1:30:40/ ICG5:1 solution was diluted 100 times with DMSO with the purpose of destroying the micellar structure and thus releasing the encapsulated ICG adequately. The concentration of ICG in the diluted solution was determined by Edinburgh F900 fluorescence spectroscopy with emission at 815 nm and excitation at 780 nm. The EE is expressed according to the following formula: EE (%) = (weight of encapsulated ICG/weight of initially added ICG) × 100 (%). Spectroscopic Measurements and Fluorescent Stability. The fluorescence spectra was recorded on both freely dissolved ICG and PEG-PLL-PLLeu-ICG probe using a fluorescence spectrometer (F900, Edinburgh Instruments Ltd., U.K.) with an excitation wavelength of 780 nm. The aqueous stability of dark stored ICG and PEG-PLLPLLeu-ICG probe at 25 °C was investigated by measuring the fluorescence intensity each week over a period of 5 weeks. Dynamic Light Scattering (DLS) Measurement and Morphology. The size and zeta potential of both ICG free and ICG loaded PEG-PLL-PLLeu micelles were measured by dynamic light scattering (DLS) using a Malvern ZS90 instrument equipped with a 532 nm laser at a scattering angle of 90°. The measurements were performed at 25 °C after diluting the samples to an appropriate concentration with ultrapure water. Also the morphology of both ICG free and ICG loaded micelles was observed by TEM (JEM-1230, Japan) with an accelerating voltage of 200 kV. One drop of the diluted micellar solution, originating from both micelles, respectively, was placed on a 400 mesh copper grid and then stained with 2% (w/V) phosphotungstic acid.

micelles. In addition, tumor targeting studies were carried out by using tumor-bearing mouse models.

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MATERIALS AND METHODS

Materials. The following chemicals and reagents were used in our experiments: O-(2-aminoethyl)-O′-(2-methyl) polyethylene glycol (PEG-NH2, Mw = 2000) and branched polyethyleneimine (PEI25k, Mw = 25 000) were purchased from Sigma-Aldrich (St. Louis, MO). LLeucine (LLeu) and ε-benzyloxycarbonyl-L-lysine (LLZ) were purchased from GL Biochem (Shanghai, China) and recrystallized three times from ethyl acetate. Triphosgene was purchased from J&K Scientific and recrystallized from diethyl ether before using. Hydrogen bromide, 33 wt % solution in glacial acetic acid, was purchased from ACROS Organics. Tetrahydrofuran (THF) and n-hexane were provided by Shanghai Chemical Reagent China and dried with sodium before using. N,N′-Dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) were provided by J&K Scientific and distilled under reduced pressure before using. Other reagents were of analytic grade and used as received. Milli-Q ultrapure water was used in all experiments. Preparation of PEG-PLL-PLLeu Micelle. The amphiphilic PEGPLL-PLLeu hybrid polypeptide copolymers used in this study were synthesized according to the ring-opening polymerization method reported in the literature.28 By adjusting the feed ratio of PEG-NH2, LLZ-NCA, and LLeu-NCA to 1:30:40, the PEG-PLL30-PLLeu40 copolymers were synthesized. Subsequently, micelles were formed by dissolving the PEG-PLL-PLLeu copolymers in aqueous media at a concentration of 1 mg/mL with overnight stirring and then 1 h of sonication. Cell Culture. Human nonsmall cell lung carcinoma (H460) cells were obtained from Shanghai Cell Biology Institute of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 μg/mL), in 5% CO2 at 37 °C. Preparation and Characterization of ICG-Encapsulated Micelles. To prepare ICG-encapsulated micelles, the dialysis against 3028

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Figure 2. (A) Fluorescence spectra for 1:30:40/ICG5:1 micelles and free ICG, respectively, dissolved in DMSO and H2O with an ICG concentration of 4 μg/mL. (B) Fluorescence stability test of 1:30:40/ICG5:1 and free ICG aqueous solution stored in the dark at 25 °C. (C) Aqueous stability of 1:30:40/ICG5:1 in aqueous solution stored in the dark at 25 °C. (D) Drug release profiles of 1:30:40/ICG5:1 in phosphate-buffered saline (PBS, pH = 7.4) containing 5% FBS at 37 °C. In Vitro Release Profile Study. To study the in vitro release profile of PEG-PLL-PLLeu-ICG micelles, 1 mL of 1:30:40/ICG5:1 aqueous solution (containing 20 μg/mL ICG) was infused into a dialysis tube (M.W. 3500) and dialyzed against phosphate-buffered saline (PBS, pH = 7.4) containing 5% FBS at 37 °C under continuous shaking in the dark. At each predetermined time point over a period of 160 h, the ICG concentration in the dialysate was measured by fluorescence spectroscopy. The total volume of dialysis medium was maintained at 50 mL through the test. Temperature Measurement during NIR Irradiation. A volume of 1 mL of 1:30:40/ICG5:1 (containing 4 μg/mL ICG), free ICG (containing 4 μg/mL ICG), and PBS were, respectively, added to centrifuge tubes and irradiated with a 808 nm laser at 1 W/cm2 for 5 min. Temperatures and infrared thermographic maps were recorded in 60 s intervals with a Fluke Ti27 infrared thermal imaging camera. Cellular Uptake and Cytotoxicity Study. H460 cells were seeded in 8-well plates (2 × 104 cells/well) and cultured with PEGPLL-PLLeu-ICG micelle or free ICG in RPMI-1640 medium containing 5% FBS at 37 °C for 4 h. After washing twice with PBS, cells were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min and then counterstained with DAPI. The uptake of PEG-PLL-PLLeu-ICG micelles was measured using a Beckman Coulter Quanta SC flow cytometer (Beckman Coulter, Brea, CA). The in vitro cytotoxicity was evaluated using Cell Counting Kit-8 (CCK-8) assay.29 H460 cells were seeded in a 96-well plate (Costar Corning, Rochester, NY) at 3 × 103 cells/well in 100 μL of culture medium and cultured, respectively, with 0−120 μg/mL of the ICG free micelles and 1:30:40/ICG5:1 micelles at 37 °C for 24 h. Untreated cells were used as the blank control. After that, CCK-8 assay was carried out to evaluate the cell viability. The absorbance was measured at 450 nm using a microplate Bio-Rad reader (Thermo Fisher Scientific, Waltham, MA). The cell viability was calculated according to the following formula: cell viability (%) = (ODexp − ODblank)/(ODcontrol − ODblank) × 100%, and five replicates were analyzed for each sample. In order to real-time visualize the “off−on” process, H460 cells were seeded in 24-well plates (2 × 104 cells/well) and cultured, respectively, with 1:30:40/ICG5:1 (containing 4 μg/mL ICG) and free ICG (4 μg/ mL) in RPMI-1640 medium containing 5% FBS at 37 °C for various periods (2, 4, 6, and 8 h). After that, the cell medium was refreshed, and the real-time visualization of “off−on” process was performed on a

Maestro in vivo imaging system. The groups treated without cells were used as control groups. In Vitro Photothermal Study. To assess photothermal cytotoxicity, H460 cells were incubated with PEG-PLL-PLLeu-ICG (containing 4−12 μg/mL ICG) and free ICG solution in 96-well microplates for 4 h and subsequently rinsed with PBS. The cells were irradiated with a 1 W/cm2 NIR laser for 5 min; as for the control group, the cells were not exposed to NIR laser. After 24 h incubation, CCK-8 assay was carried out to evaluate the cell viability. The absorbance was measured at 450 nm using a microplate Bio-Rad reader (Thermo Fisher Scientific, Waltham, MA). In order to conduct viability staining, H460 cells were incubated respectively with PEGPLL-PLLeu-ICG (containing 4−12 μg/mL ICG) and free ICG solution in 12-well microplates for 4 h and then rinsed with PBS. After 24 h incubation following NIR irradiation, medium was replaced with 1 mL PBS solution. Each well was treated with 4 μL calcein AM stock solution (1 mg/mL in DMSO) and continued to be incubated for 45 min at ambient temperatures. Subsequently, PBS was removed and 1 mL of propidium iodide (PI) solution (50% in PBS) was added to each well followed by 5 min incubation. After that, each well was rinsed 2 times (2 min each) with 3 mL of PBS. Finally, cells immersed in 3 mL of PBS were taken for fluorescence imaging by using a fluorescent microscopy (Olympus, BX41-32P02-FLB3, Japan). In Vivo Imaging and Biodistribution. All animal experiments were conducted in agreement with the “Principles of Laboratory Animal Care” (NIH publication no. 86-23, revised 1985). The guidelines of the Institute for Nutritional Science of Chinese Academy of Sciences were also respected. Female BALB/C nude mice (6−8 weeks old, weight around 20 g) were used as a tumor model. The mice were injected subcutaneously with 100 μL of NCI-H460 cell suspension containing 106 cells in the right flank. When the tumor volumes reached about 50 mm3, the nude mice were divided into two groups (n = 3): free ICG group (0.05 mg/kg) and 1:30:40/ICG5:1 micelles group (0.05 mg/kg ICG-equivalent for micelles). The samples were injected to nude mice via tail vein. The nude mice were sacrificed, respectively, 4 and 24 h after injection, and then the organs including heart, liver, spleen, lung, kidney, and tumor were analyzed using the Maestro in vivo imaging system with a 704 nm excitation wavelength and a 735 nm filter to collect the fluorescence signals. 3029

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Figure 3. (A) Infrared thermographic maps of 1:30:40/ICG5:1 at different time points under NIR irradiation recorded with an infrared thermal imaging camera. (B) Temperature rise profile of 1:30:40/ICG5:1 (containing 4 μg/mL ICG), free ICG (4 μg/mL), and PBS as a function of the irradiation time under 808 nm continuous laser irradiation with a power intensity of 1 W/cm2. (C) Temperature rise profile of 1:30:40/ICG5:1 with different ICG concentration (4, 8, and 12 μg/mL respectively) as a function of the irradiation time under 808 nm continuous laser irradiation with a power intensity of 1 W/cm2.

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RESULTS AND DISCUSSION Characterization of ICG-PEG-PLL-PLLeu Micelle. In the previous study, PEG-PLL-PLLeu triblock copolymers were dissolved in water and simultaneously formed self-assembled micelles. The hydrodynamic particle sizes of these micelles were well-tunable, indeed strongly correlated with the length of PLL and PLLeu segments.27 In the current study, negatively charged ICG was encapsulated into the hydrophobic core during the self-assembly process of PEG-PLL-PLLeu-ICG micelle, attributing to the hydrophobic effect (Figure 1). The hydrodynamic size of 1:30:40/ICG5:1 was 159.1 nm with good monodispersity (the PDI value of 1:30:40/ICG5:1 was 0.173), slighter higher than that of PEG-PLL-PLLeu. This size of PEGPLL-PLLeu-ICG micelle might benefit the passive targeting of drug delivery into tumors, according to the enhanced permeability and retention effect (EPR).30 The encapsulation efficiency (EE) of 1:30:40/ICG5:1 was 46.78%, as the ratio of encapsulated ICG to initial ICG mass. Fluorescent Property and Aqueous Stability. In aqueous solution, the concentrating of ICG in the core of PEG-PLL-PLLeu micelles may cause self-quenching to reduce the fluorescence quantum yields of ICG. However, when diluted with DMSO, the micelle was completely destroyed, bringing with the complete release of encapsulated ICG thus the possible recovery of fluorescent property. In order to verify this conclusion, we conducted the fluorescence tests of free ICG and 1:30:40/ICG (both at an ICG concentration of 4 μg/ mL), respectively, dispersed in H2O and DMSO. The results showed that fluorescence intensity of 1:30:40/ICG was very weak in H2O in comparison with in DMSO, indicating that ICG was successfully encapsulated in the micelles (Figure 2A). Concentration-dependent aggregation is a well-known phenomenon for ICG in aqueous solution. The van der Waals forces and hydrophobic interactions between ICG molecules can result in the formation of highly ordered aggregates and further induce self-quenching and spectral changes due to forming dimers or J-aggregates.16 To compare the stabilities of

ICG both encapsulated in PEG-PLL-PLLeu micelle and freely dissolved in aqueous solution, the fluorescence intensities of both were tested over a period of 5 weeks. As a result, the micelle-encapsulated ICG lost approximate 30% fluorescence intensity, while the free-dissolved ICG rest only 1%, both darkly stored at 25 °C for 5 weeks. It was indicated that the PEG-PLLPLLeu-ICG micelle own better fluorescence stabilization. The aqueously dissolved micelles remained around 150−160 nm in size without any aggregation or precipitation over the test period, presenting their excellent aqueous stability (Figure 2C). The improved stability of PEG-PLL-PLLeu-ICG micelle can be attributed to the presence of PEG layer and the high charge density on its surface. These results also confirmed the previous research findings.28 As shown in Figure 2D, the in vitro release characteristics of ICG from 1:30:40/ICG5:1 were summarized in two release curves. During the first 10 h, a rapid release occurred with a cumulative release of 15%. The release characteristic was fitted into the Higuchi equation (Q = −0.2663 + 4.6518t1/2) (Figure 2D (b)),31 for the release was mainly attributed to the initial burst of free ICG in micelle. Subsequently, from 10 to 90 h, a relatively fast release presented with a cumulative release up to 72%, followed by a slow release stage until terminated at 76% (Figure 2D (a)). The release behavior from 10 h to the end 160 h was coherent with the single-exponential equation (y = 76.66(1 − exp(−0.033163t))), due to the micellar degradation and the subsequent release of ICG adsorbed and encapsulated internal the micelle. The results indicated that PEG-PLL-PLLeu-ICG micelle achieved the sustained release of ICG. These results demonstrated favorable release character of PEG-PLL-PLLeuICG micelles, which is a critical factor for the application in optical imaging and photothermal therapy. It can be concluded from Figure 2B and C that PEG-PLLPLLeu-ICG micelles were very stable with constant fluorescence property in aqueous solution at room temperature, in other words, the phenomena like incorporated ICG leaking out or micelle breaking up did not exist. Further analysis, combined with the subsequent in vivo imaging and biodistribution 3030

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experiments, demonstrated that ICG can be well encapsulated in PEG-PLL-PLLeu micelles and transported to tumor site by passive targeting and then it would be slowly released, avoiding highly bound to nonspecific plasma proteins and leading to rapid elimination from the body with a half-life of 3−4 min. Temperature Measurements under NIR Irradiation. We investigated the photothermal effect and temperature increment in PBS, including free ICG and 1:30:40/ICG5:1 solution at an ICG concentration of 4 μg/mL under 808 nm laser irradiation for 5 min. Figure 3A shows the thermographic maps of 1:30:40/ICG5:1 solution at different time points under NIR irradiation recorded with a Fluke Ti27 infrared thermal imaging camera. At an irradiation power density of 1 W/cm2, the temperature of 1:30:40/ICG5:1 solution finally rose to 43 °C, while the temperature of free ICG and PBS solutions just rose to 27.5 and 23 °C respectively (Figure 3B). In the first 120 s, the temperature increased rapidly and then further rose steadily to the maximum temperature at 5 min after NIR irradiation. The PEG-PLL-PLLeu-ICG formulation had a higher temperature response than free ICG under NIR irradiation, similar to the previously reported ICG-loaded lipid NPs, which were more efficient in producing a NIRdependent temperature increase than ICG alone.32 In order to verify the potential of 1:30:40/ICG5:1 as the photothermal agent, 1:30:40/ICG5:1 solutions at ICG concentrations of 4, 8, and 12 μg/mL were exposed to the 808 nm NIR laser at a power density of 1 W/cm2 for 5 min. An obvious concentration dependent temperature increase was observed for 1:30:40/ ICG5:1 solutions under laser irradiation (Figure 3C). In Vitro Cellular Uptake and Cytotoxicity Study. Cellular uptake of PEG-PLL-PLLeu-ICG micelles and free ICG was elucidated using confocal microscopy after incubation of H460 cells with 1:30:40/ICG5:1 (containing 4 μg/mL ICG) and free ICG (4 μg/mL) at 37 °C for 4 h followed by washing respectively. DAPI stained H460 cells exhibited bright blue fluorescence under confocal microscopy, while PEG-PLLPLLeu-ICG probes exhibited bright red (Figure 4A). Apparently, the uptake level of 1:30:40/ICG5:1 was higher than that of free ICG. The red probes are seen internalized extensively in the cytoplasmic region. The enhanced cellular uptake might be partially due to intensive hydrophobic interaction of PLLeu segments with cell membranes.33 For another reason, the positively charged micelles associated with the net negatively charged cell membranes, and then were internalized by macropinocytosis. The results clarified that PEG-PLL-PLLeu-ICG probes were internalized readily by the H460 cells in comparison to the control ones and could be easily used in targeted delivery systems. We tested the potential toxicity of PEG-PLL-PLLeu-ICG micelles on H460 cells. The CCK-8 assays were carried out to determine the relative viabilities of H460 cells after being respectively incubated with PEG-PLL-PLLeu and 1:30:40/ ICG5:1 at various concentrations (0−120 μg/mL) for 24 h. As for the two materials, no significant cytotoxicity was observed on H460 cells at concentrations below 40 μg/mL (Figure 4B). At a relative high concentration of 40−120 μg/mL, PEG-PLLPLLeu showed some cytotoxicity on H460 cells, as the viability decreased to 70%. Similarly, PEG-PLL-PLLeu-ICG slightly inhibited the viability of H460 cells when the concentration was higher than 40 μg/mL. “Off−On” Nanoprobes for in Vivo Imaging and Biodistribution. The real-time visualization of “off−on” process of 1:30:40/ICG5:1 (containing 4 μg/mL ICG) in

Figure 4. (A) In vitro cellular uptake confocal images of 1:30:40/ ICG5:1 (4 μg/mL of ICG) after 4 h incubation. (B) Relative growth rate (RGR) of H460 cells incubated with 1:30:40/ICG5:1 and 1:30:40 respectively at the concentrations of 0−120 μg/mL, as measured by the CCK-8 test (n = 5).

H460 cells is shown in Figure 5A. The fluorescence intensity of 1:30:40/ICG5:1 incubated with H460 cells was getting stronger over time and reached strongest at 8 h. However, there was no fluorescence signal captured when 1:30:40/ICG5:1 exposed without cells, owing to the fluorescence quenching of ICG encapsulated intensively in the core of PEG-PLL-PLLeu. In

Figure 5. (A) Fluorescence images of 1:30:40/ICG5:1, respectively, exposed with and without cells at different time points. (B) Fluorescence images of organs and tumors in NCI-H460 tumorbearing mice at 4 and 24 h after the injection of 1:30:40/ICG5:1 and free ICG, respectively. The in vivo imaging studies were replicated in three individual groups of mice. 3031

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Figure 6. Cell survival of H460 cells after potothermal treatment. (A) Microscopy images of calcein AM (green, live cells) and propidium iodide (red, dead cells) costained H460 cells with and without 808 nm laser irradiation. (B) Relative growth rates (RGR) of H460 cells, intubated with 1:30:40/ICG5:1 and free ICG (both of 4, 8, 12 μg/mL equivalent to ICG), respectively, were measured by the CCK-8 tests pre- and post-5-min photothermal treatment (n = 5). (C) Cell viabilities of H460 cells, treated with 1:30:40/ICG5:1 and free ICG, respectively (both of 4 μg/mL equivalent to ICG), were assessed at 0, 2.5, 5, and 10 min post the NIR laser irradiation with an intensity of 1 W/cm2 (n = 5).

environment, also to the rapid aggregation and clearance from the body.35 Preliminary Study of in Vitro Photothermal Effect. We evaluated the feasibility of PEG-PLL-PLLeu-ICG as a photothermal agent for in vitro cancer cell ablation under laser irradiation. Herein H460 cells were incubated with 1:30:40/ ICG5:1 and free ICG respectively for 4 h and then exposed to an 808 nm laser at 1 W/cm2 for 5 min. Subsequently, the cells were stained with both calcein AM and propidium iodide (PI). Figure 6A shows fluorescent images of calcein AM stained live cells (green) and propidium iodide (PI) stained dead cells (red) captured with a microscope. Under excitation, clear regions of live cells (green) and dead cells (red) were observed, and the majority of H460 cells were killed with laser irradiation when the concentration of 1:30:40/ICG5:1 was higher than 4 μg/mL (equivalent to ICG). In contrast, the cells treated with either 1:30:40/ICG5:1 or free ICG alone, as well as free ICG with laser irradiation, showed negligible death rate. As the concentration of 1:30:40/ICG5:1 increased from 4 to 12 μg/mL (equivalent to ICG), the red zone to mean cell death enlarged. We further quantified their photothermal cytotoxicity on H460 cells using a CCK-8 assay (Figure 6B, C). As the concentration of 1:30:40/ICG5:1 increased, more cells were killed by laser irradiation (Figure 6B). This increase may be attributed to increased amounts of probes internalized into cancer cells. When the concentration was higher than 4 μg/mL, the viability of H460 cells treated with 1:30:40/ICG5:1 was significantly

order to visualize PEG-PLL-PLLeu-ICG in vivo, biodistribution studies were performed on NCI-H460 tumor xenografted mice intravenously injected with 1:30:40/ICG5:1 and free ICG respectively. Figure 5B showed the fluorescence and intensity distributions as a function of time for free ICG and 1:30:40/ ICG5:1 delivered systemically via tail vein injections. Obviously, for nude mice treated with free ICG, the fluorescence spots appeared in liver and kidneys at 4 h post injection, showing dominant uptake of ICG in liver and kidneys. High levels of 1:30:40/ICG5:1 uptake were detected in the tumor and kidneys after intravenous injection, and tumor uptake increased as a function of time (Figure 5B). However, at 24 h postinjection, no detectable signal was recorded from the free ICG. One of the properties of solid tumors is their enhanced permeation and retention (EPR) effect, which allows a several hundred nanometer compound to extravagate from blood circulation into the vicinity of tumor, and then remain trapped and unable to return to the blood flow.34 The results were good evidence for the high efficiency of tumor targeting of PEG-PLL-PLLeuICG, and also proved that free ICG tends to be accumulated in reticulo-endothelial systems (RES) after intravenous injection, and be excreted slowly by the hepatic excretion pathways. PEGPLL-PLLeu-ICG probes extended the circulation time and protected the encapsulated ICG from degradation. The relatively short fluorescent lifetime of free ICG was attributed to the fluorescence quenching of free ICG in physiological 3032

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Biomacromolecules

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decreased to 20%, which was measured after 5 min irradiation of 808 nm at 1 W/cm2. Whereas cells with free ICG incubation were affected far less than those with 1:30:40/ICG5:1 even after laser exposure. The CCK-8 assays were also performed to quantify the relative cell viabilities of different time points after NIR laser irradiation (Figure 6C). For all cases, cells retained their viability when only irradiated by laser. When PEG-PLLPLLeu-ICG (containing 4 μg/mL of ICG) was added, cell viability decreased significantly under laser irradiation. It was determined that as the irradiation time was prolonged, more cells incubated with 1:30:40/ICG5:1 were killed by the NIR laser irradiation. The results indicated potential application of PEG-PLL-PLLeu-ICG for photothermal therapy.

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CONCLUSIONS The present research proposed well-defined PEG-PLL-PLLeuICG probes by entrapping ICG within PEG-PLL-PLLeu through electrostatic adsorption and hydrophobic interaction. The 1:30:40/ICG probes presented excellent optical properties and in vitro release profile of ICG. The in vivo evaluations illustrated exact tumor localization and prolonged circulation time of 1:30:40/ICG, indicating an attractive fluoroprobe for tumor diagnosis and targeted imaging. In addition, good heating performance under NIR laser irradiation and in vitro photothermal property indicated the feasibility of PEG-PLLPLLeu-ICG as a photothermal agent for tumor photothermal therapy.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-755 86392210. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (Grant No. 81071249, Grant No. 20905050), the Guangdong Innovation Team of Low-cost Healthcare, the Science and Technology Key Project of Guangdong (2009A030301010), the Shenzhen Science and Technology Program (Grant No. JC201005270326A), and the “Hundred Talents Program” of Chinese Academy of Sciences.

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dx.doi.org/10.1021/bm400839b | Biomacromolecules 2013, 14, 3027−3033