Liposomal Indocyanine Green for Enhanced ... - ACS Publications

Letter www.acsami.org

Liposomal Indocyanine Green for Enhanced Photothermal Therapy Hwan-Jun Yoon,†,‡,⊥ Hye-Seong Lee,†,‡,⊥ Ji-Young Lim,‡,⊥ and Ji-Ho Park*,‡,§,⊥,# ‡

Department of Bio and Brain Engineering, §Program of Brain and Cognitive Engineering, ⊥Institute for Health Science and Technology, and #Institute for the Nanocentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea S Supporting Information *

ABSTRACT: In this study, we engineered liposomal indocyanine green (ICG) to maximize its photothermal effects while maintaining the fluorescence intensity. Various liposomal formulations of ICG were prepared by varying the lipid composition and the molar ratio between total lipid and ICG, and their photothermal characteristics were evaluated under near-infrared irradiation. We showed that the ICG dispersity in the liposomal membrane and its physical interaction with phospholipids were the main factors determining the photothermal conversion efficiency. In phototherapeutic studies, the optimized formulation of liposomal ICG showed greater anticancer effects in a mouse tumor model compared with other liposomal formulations and the free form of ICG. Furthermore, we utilized liposomal ICG to visualize the metastatic lymph node around the primary tumor under fluorescence imaging guidance and ablate the lymph node with the enhanced photothermal effect, indicating the potential for selective treatment of metastatic lymph node. KEYWORDS: cancer therapy, fluorescence, indocyanine green, liposome, photothermal therapy

P

ICG has been limited due to poor optical stability, weak photothermal capability, and short blood residence time. Furthermore, ICG binds to plasma proteins, including albumin and globulin in blood, travels through the bloodstream by hitchhiking, and is rapidly cleared in the liver.17,36,37 This protein binding dependency also restricts the extension of ICG use to disease-specific therapeutic applications. To overcome these limitations, a great deal of effort has been made to encapsulate ICG in various nanoparticle formulations, including liposomes, micelles, and polymeric nanoparticles.38−42 These nanoparticle formulations generally provide ICG with improved fluorescence intensity, prolonged blood residence time, protein binding independence, and easy modification for cellular delivery.43−45 Attention has been paid to liposomal formulations of ICG in the biomedical field due to their biocompatibility and easy surface modification.40,44,46−49 However, there have been limited efforts to maximize the photothermal effects of ICG-loaded nanoparticles, including liposomal ICG, for therapeutic applications. In this study, we engineered liposomal ICG to maximize its photothermal effects while maintaining the fluorescence intensity. Various liposomal formulations of ICG were prepared by varying the lipid composition and molar ratio between total lipid and ICG, and their fluorescence and photothermal

hotothermal therapy (PTT) is one of the most efficient therapies that can induce necrosis of specific malignant lesions with minimal invasiveness and side effects compared with other therapeutic modalities.1−6 Irradiation in the nearinfrared (NIR) region is particularly beneficial for PTT because NIR light penetrates tissues relatively deeply with minimal cytotoxicity due to the low photon absorption of endogenous biomolecules in this range of wavelengths.7−10 In PTT, photothermal agents are localized at the site of disease, and exposed to NIR light to generate cytotoxic heat. Among the many photothermal agents available, gold-based3,10−13 and carbon-based nanomaterials14−16 have been extensively studied in cancer therapy due to their high photothermal conversion efficiency and preferential tumor accumulation via enhanced permeability and retention (EPR) effects.17 However, clinical applications of these inorganic material-based photothermal agents have been limited because of issues with their long-term biosafety. Indocyanine green (ICG), which was granted approval by the United States Food and Drug Administration (US FDA) in 1959, has been widely used clinically as an NIR fluorescent dye for many diagnostic applications, such as angiography in ophthalmology,18,19 monitoring of liver function and splanchnic perfusion,20−22 perfusion-related analysis of tissues and organs,23−25 sentinel lymph node biopsy,26−29 and diagnosis of rheumatic diseases.30 ICG has also attracted attention in the biomedical field due to its phototherapeutic capability with NIR laser irradiation.31−35 However, phototherapeutic application of © 2017 American Chemical Society

Received: December 30, 2016 Accepted: February 2, 2017 Published: February 2, 2017 5683

DOI: 10.1021/acsami.6b16801 ACS Appl. Mater. Interfaces 2017, 9, 5683−5691

Letter

ACS Applied Materials & Interfaces Table 1. Lipid and ICG Compositions and Physicochemical Properties of Liposomal ICG Used in This Study lipid and ICG composition (molar ratio) liposomea

DSPC

S2 S4 S8 S16 S32 S62 S128 P2 P4 P8 P16 P32 P64 P128 M2 M4 M8 M16 M32 M64 M128 O2 O4 O8 O16 O32 O64 O128

950 950 950 950 950 950 950

DPPC

DMPC

DOPC

PEG−PE

ICG

hydrodynamic sizeb (nm)

polydispersity indexb

surface chargec (mV)

950 950 950 950 950 950 950

50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50

2 4 8 16 32 64 128 2 4 8 16 32 64 128 2 4 8 16 32 64 128 2 4 8 16 32 64 128

146.1 141.9 167.6 143.1 146.4 145.9 136.0 138.8 129.7 154.2 134 135.5 132.7 120.9 120.3 112 122.7 114 115.4 129 97.7 121.2 119.9 111.7 124.2 127.3 130.2 135.6

0.138 0.125 0.171 0.099 0.065 0.107 0.195 0.199 0.083 0.111 0.128 0.124 0.131 0.333 0.159 0.134 0.118 0.113 0.146 0.201 0.329 0.103 0.13 0.112 0.112 0.107 0.131 0.153

−30.5 −34.3 −30.0 −25.7 −23.3 −32.1 −23.6 −28.1 −33.6 −28.6 −28.4 −28.9 −21.6 −20.3 −29.5 −28.7 −27.2 −23.7 −20.1 −20 −21.1 −25.6 −27.9 −25.1 −20.7 −21.7 −28.6 −20.8

950 950 950 950 950 950 950 950 950 950 950 950 950 950

a The letter identifier before the number indicates the phospholipid type used as a base lipid. The number after the letter identifier designates the initial molar ratio of ICG to 1000 total lipids used to prepare the liposomal ICG. bMean hydrodynamic sizes and polydispersity indexes of the liposomes based on dynamic light scattering measurements (n = 3). cMean surface charges of the liposomes based on zeta-potential measurements (n = 3).

intensively by size-exclusion chromatography and dialysis to remove ICG molecules in the solution and bound to the liposomal surface. Hydrodynamic size, surface charge, and polydispersity index were similar in all liposomal formulations except for some liposomal ICGs prepared with the initial ICG ratio of 128, which showed increased polydispersity index (0.333 for P128 and 0.329 for M128) probably because of their aggregation mediated by excess ICG molecules exposed to the liposomal surface. The final ratio of total lipid and ICG in the liposomal ICG was determined for comparison with the initial ratio (Figure 1a). The final ratio of total lipid and ICG in the DOPC-based liposomal ICG was similar to the initial ratio, whereas those in other liposomal ICGs were somewhat reduced compared with the initial ratios. The DOPC-based liposomes composed of unsaturated phospholipids seemed to package more ICG molecules in the liposomal structure due to their multilayered membrane compared with other liposomes composed of saturated phospholipids (Figure S1), in agreement with previous findings.40 In addition, all liposomal ICGs showed linear correlations between the initial and final ratios until the initial ICG ratio reached 16. These results suggested that the initial ICG ratio should be chosen carefully for preparation of liposomal ICG considering the ICG loading capacity in the liposomal structure. We next investigated how the phospholipid type and the initial ICG ratio influence the fluorescence as well as

characteristics were evaluated with NIR light. We examined how the ICG dispersity in the liposomal membrane and its physical interaction with phospholipids influenced the photothermal conversion efficiency of liposomal ICG. We then tested the phototherapeutic effects of engineered liposomal ICG in cultured cells and spheroids in vitro as well as in tumors in vivo and compared the results with those of free ICG. Furthermore, we utilized liposomal ICG to visualize the sentinel lymph node around the primary tumor under fluorescence imaging guidance, and subsequently ablated the lymph node harboring metastatic tumor cells with the enhanced photothermal effect. ICG is an amphiphilic molecule consisting of a lipophilic polyaromatic polyene group and a hydrophilic sulfonate group. Liposomal formulations can be used to incorporate ICG molecules in the membrane in monomeric form because the lipophilic part of ICG is inserted into the phospholipid bilayer, whereas the hydrophilic part faces the aqueous environment. Various formulations of liposomal ICG were prepared by varying the lipid composition and molar ratio between total lipid and ICG (Table 1). 1,2-dilauroyl-sn-glycero-3-phosphocholine (12:0 phosphatidylcholine (PC), DLPC), and 1,2didecanoyl-sn-glycero-3-phosphocholine (10:0 PC) were excluded in this study because our preliminary experiments indicated that liposomal ICG prepared with these phospholipids induced severe cellular toxicity. Briefly, liposomal ICG was synthesized by the hydration-extrusion method, and washed 5684


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ACS Applied Materials & Interfaces

Figure 1. Preparation and characterization of liposomal indocyanine green (ICG). (a) Comparison of final molar ratio of total lipid and ICG encapsulated in the liposomal ICG and initial molar ratio of lipid and ICG used to prepare the liposomal ICG. The ideal ratio indicates the ideal liposomal ICG encapsulating all lipids and ICG used for liposome preparation. (b) Relative fluorescence intensity of various liposomal ICG preparations and free ICG (λex = 785 nm and λem = 820 nm) (c) Photothermal properties of various liposomal ICG preparations with an initial ICG ratio of 4, and free ICG upon 808 nm laser irradiation. (d). Single oxygen production of various liposomal ICGs prepared with the initial ICG ratio of 4, and free ICG upon 808 nm laser irradiation. Data represent averages ± standard error of the mean (SEM) (n = 3; ***P < 0.001 by Student ttest).

with free ICG. The photothermal conversion efficiency of M4 was determined to be ∼8.99%, which was twice as efficient as free ICG showing ∼3.37%. In the photodynamic experiments, we found that all liposomal ICGs prepared with the initial ICG ratio of 4 exhibited negligible singlet oxygen production upon NIR laser irradiation compared with free ICG (Figure 1d), suggesting that substantial amount of singlet oxygen produced from the ICG incorporated in the liposomal membrane seems to be absorbed by nearby phospholipids and ICG molecules.50,51 The hydrodynamic size and fluorescence of liposomal ICG were preserved for a longer period compared with those of free ICG (Figure S3). After NIR irradiation, liposomal and free ICG showed significantly reduced fluorescence due to photodegradation of ICG (Figures S4a and S4b). Liposomal ICG retained its structure without significant size change during irradiation (Figure S4c). Taken together, these results suggest that the M4 formulation is the liposomal ICG optimized for both fluorescence imaging and photothermal therapy. We next conducted experiments to examine how the dispersity of ICG molecules incorporated in the liposomal membrane influences fluorescence and photothermal properties. It has been demonstrated that individual ICG binds immediately to plasma albumin once administered into the bloodstream.52 To mimic this albumin binding, the ICG solution was mixed with different concentrations of FBS solutions. The absorbance and fluorescence spectra of mixture solutions revealed that the ratio of absorbance at 800 nm to that at 740 nm and fluorescence intensity increased with FBS concentration (Figure S5a−c). In addition, the photothermal

photothermal and photodynamic properties of liposomal ICGs. It has been demonstrated that the fluorescence intensity (at 820 nm) and absorbance ratio (800 nm:740 nm) of ICG molecules are diminished due to intermolecular quenching when they undergo self-aggregation.44 A similar pattern was observed in our results where fluorescence intensity and absorbance ratio of liposomal ICGs were both decreased from the initial ICG ratio of 4 (Figure 1b and Figure S2a, b). These results suggest that ICG molecules in the liposomal formulations prepared with initial ICG ratios of 2 and 4 are preferentially incorporated into the liposomal membrane in a well-dispersed state, while those with initial ICG ratios >4 are likely to encapsulate excess ICG molecules in aggregated form in the aqueous core, as well as in the membrane. Thus, liposomal formulations prepared with an initial ICG ratio of 4 are likely to contain the maximum amount of ICG molecules in the membrane without significant aggregation. Photothermal and photodynamic properties of liposomal ICGs were measured upon NIR laser irradiation using an IR thermographic camera and singlet oxygen sensor green, respectively. Among various liposomal formulations, the highest photothermal effect was observed in the DMPC-based liposomal ICGs prepared with initial ICG ratios of 2 and 4 (M2 and M4, respectively), presumably due to monodisperse incorporation of ICG molecules in the membrane (Figure 1c and Figure S2c). However, the M4 formulation could be a more efficient liposomal structure than M2 because it requires half the quantity of liposomes to induce the same photothermal effect compared with M2. Importantly, M4 also showed superior fluorescence and photothermal properties compared 5685


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Figure 2. In vitro phototoxicity of liposomal ICG in HeLa cancer cells. (a) Cellular phototoxicity of various liposomal ICGs prepared with the initial ICG ratio of 4 upon 808 nm laser irradiation for 3 min. (b) Cellular phototoxicity of liposomal ICGs, free ICG, and mixture of liposome and ICG upon 808 nm laser irradiation for 3 min. (c) Cellular uptake of liposomal and free ICG after incubating with cells for 5 min. The cells treated with liposomal or free ICG (red) were imaged using confocal fluorescence microscopy. Nuclei were stained with Hoechst (blue). Scale bar indicate 5 μm. Data represent averages ± SEM (n = 3, *P < 0.05, **P < 0.01 by student t-test).

ICG could also be engaged with the thermal conductivity of phospholipids. A previous study indicated that heat transfer occurred more efficiently across membranes comprised of phospholipids with shorter carbon chains, compared with those with longer carbon chains.54 Thus, the heat generated from the incorporated ICG molecules could be transferred more efficiently to the surrounding medium during laser irradiation in the M4 formulation with shorter carbon chains. In addition, the photothermal heating generated from ICG incorporated into an unsaturated phospholipid membrane such as DOPC could increase the membrane fluidity and induce selfaggregation of ICG molecules in the membrane, lowering the photothermal conversion efficiency. Thus, we reconfirmed that the M4 formulation has many advantages for efficient photothermal heating over other liposomal formulations. We next examined the phototherapeutic ability of liposomal ICG with cancer cells in vitro. Liposomal formulations with an initial ICG ratio of 4 were mainly tested for in vitro phototherapeutic experiments due to their superior photothermal capability over other formulations. HeLa cells were coincubated with liposomal or free ICG for 5 min and irradiated using an NIR laser for 3 min. The treated cells were then rinsed and incubated further for 24 h to induce phototherapeutic effects. Their viability was evaluated by MTT, Live/Dead, and LDH assays. As expected, the M4 formulation showed the highest phototherapeutic effect against cancer cells (Figure 2a and Figure S7). We also examined the impact of monomeric incorporation of ICG molecules into the

effect also increased with FBS concentration (Figure S5d). Taken together, these results indicated that the dispersion of ICG molecules in aqueous solution is responsible for the enhanced fluorescence and photothermal heating. Furthermore, these results also support the suggestion that the M4 formulation has ICG molecules in relatively monomeric form in the membrane. Importantly, absorbance, fluorescence, and photothermal properties of M4 formulation did not change with FBS concentration, indicating that the ICG molecules are secured well in the liposomal membrane without plasma protein binding (Figure S6). We further studied why the M4 formulation showed superior photothermal effects over other liposomal formulations. From the viewpoint of thermodynamics, the heat capacity and thermal conductivity of the phospholipid could influence the photothermal heating of ICG molecules embedded in the phospholipid bilayer. It was reported that the heat capacity of phospholipids increases markedly when the ambient temperature approaches the phase transition temperature.53 On the basis of this previous observation, the heat capacity of DMPC phospholipids comprising the M4 formulation may not increase during laser irradiation because their phase transition temperature is already lower than room temperature. In contrast, the heat capacities of DPPC and DSPC phospholipids, composing other liposomal formulations, with phase transition temperatures higher than room temperature would increase as the ambient temperature approaches their transition temperature during laser irradiation. The photothermal effect of liposomal 5686


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Figure 3. In vitro phototoxicity of liposomal ICG in HeLa tumor spheroids. (a) Scanning electron microscopic images showing size and morphology of the spheroids prepared for different period of time. Spheroid diameter indicates 100 μm (b) Diameter of tumor spheroids prepared for different period of time. (c−e) Cellular phototoxicity in the (c) day 2, (d) day 4, and (e) day 6 spheroids treated with liposomal or free ICG upon 808 nm laser irradiation. Data represent averages ± SEM (n = 3, ***P < 0.001 by student t-test).

Figure 4. In vivo fluorescence-based localization and phototherapy of tumors using liposomal ICG. (a) In vivo whole-body near-infrared (NIR) fluorescence imaging of mice before and after intratumoral injection of free or liposomal ICG followed by NIR irradiation. Arrowheads indicate tumors implanted subcutaneously in the left flank. Red and green signals indicate mouse body autofluorescence (λex = 685 nm and λem = 700 nm) and ICG fluorescence (λex = 785 nm and λem = 800 nm), respectively. (b) Thermographic measurement of tumor surface temperatures over time during NIR irradiation after intratumoral injection of free or liposomal ICG. (c) Tumor volume change over time after intratumoral injection of free or liposomal ICG followed by NIR irradiation. Tumor volumes between day 0 and day 8 in the mice treated with M4 were not measured due to scar formation. (d) Mouse body weight change over time after phototherapy with free or liposomal ICG. (e) Survival of mice after phototherapy with free or liposomal ICG. Phototherapy was performed on day 0 for all mice in c−e. Animal experiment data represent averages ± SEM (n = 5; ***P < 0.001 by analysis of variance, ANOVA).

and ICG. The liposomal ICG showed greater cellular phototoxicity than the mixture; the majority of ICG molecules and their aggregates were randomly attached to the liposomal

membrane on the cellular phototoxicity. The cellular phototoxicity of liposomal ICG (M4) was compared to those of free ICG and a mixture of empty DMPC-based liposomes (M0) 5687


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Figure 5. Phototherapy of tumor-draining lymph nodes using liposomal ICG. (a) In vivo NIR fluorescence imaging of popliteal lymph nodes after peritumoral injection of free or liposomal ICG and subsequent NIR irradiation. Arrowheads indicate popliteal lymph nodes. (b) Thermographic measurement of popliteal lymph node surface temperatures over time during NIR irradiation after peritumoral injection of free or liposomal ICG. (c) Fluorescence images of popliteal lymph nodes after phototherapy with free or liposomal ICG. Apoptotic cells in the lymph nodes after phototherapy with liposomal or free ICG were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, red). Nuclei were stained with Hoechst (blue). Scale bars in the top and bottom panels indicate 500 and 100 μm, respectively.

surface, but exhibited lower phototoxicity than free ICG (Figure 2b). We then observed cellular uptake of liposomal and free ICG using confocal fluorescence microscopy. Free ICG was internalized rapidly into cancer cells, while liposomal ICG rarely interacted with cells within the tested time period due to the antifouling PEG surface (Figure 2c and Figure S8). These results suggested that the free ICG internalized in the cell induced relatively high cellular phototoxicity by combining photothermal and photodynamic effects while the cellular phototoxicity of liposomal ICG was induced only by the photothermal heating produced in the medium. We next examined the phototherapeutic effects of free and liposomal ICG in a tumor spheroid model because it mimics the three-dimensional structure of tumor tissues in vivo. Liposomal ICG showed a significantly higher phototherapeutic effect in tumor spheroids compared with free ICG (Figures 3). Importantly, the phototherapeutic effect of liposomal ICG was not dependent on the size of the spheroids examined in this study. However, the phototherapeutic effect of free ICG decreased gradually with increasing spheroid size, likely because of the limited penetration of reactive oxygen species in large spheroids. These observations indicated that the photothermal effect induced by liposomal ICG can be distributed efficiently through multiple cell layers via thermal diffusion, whereas the photodynamic effect of free ICG is restricted only to the host cell. Taken together, these results suggest that liposomal ICG is

more advantageous for in vivo cancer phototherapy than free ICG due to its excellent photothermal capability. Having verified the effective phototherapeutic effects of liposomal ICG in the spheroid system, we next tested its feasibility in primary tumors in vivo. The P4 formulation that showed the second highest photothermal effect was also tested along with the M4 formulation and free ICG. Liposomal or free ICG was injected directly into the center of 4T1 tumors implanted into the left flank of each mouse. At 30 min postinjection, the tumors were irradiated for 20 min with a single dose of 808 nm irradiation; the temperature change in the tumor region was measured using an IR thermographic camera. The tumor volumes were measured over 3 weeks after laser irradiation. For fluorescence imaging-based tumor localization, the tumors were visualized using an NIR fluorescence imaging system immediately and 30 min after injection, and after laser irradiation. Fluorescence images revealed that both liposomal ICGs visualized tumor localization clearly with their fluorescence (Figure 4a). In contrast, the NIR fluorescence of free ICG was not detected in the tumor region, likely due to the relatively weak fluorescence intensity. The fluorescence signal of tumors injected with liposomal ICGs was significantly reduced after laser irradiation, indicating that the ICG molecules incorporated into the liposomal structure were activated photothermally and then degraded over time. In measurements of tumor temperature, the M4 formulation induced the most efficient photothermal heating during 5688


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be responsible for the high temperature localized in the lymph nodes. TUNEL analysis using histological samples indicated that liposomal ICG treatment induced substantial apoptosis throughout the lymph nodes compared with free ICG treatment (Figure 5c). Taken together, these observations suggest that liposomal ICG has great potential for treatment of metastatic lymph nodes by fluorescence detection of sentinel lymph nodes near the primary tumor and subsequent photothermal ablation. More importantly, the whole procedure, including photothermal treatment, could be monitored by NIR fluorescence-based imaging. In this work, liposomal ICG was engineered systematically to maximize its photothermal effects while maintaining the fluorescence intensity by adjusting the lipid composition and molar ratio between total lipid and ICG. Among the various liposomal ICG formulations tested in this study, the M4 formulation (DMPC-based liposomal ICG with the initial ICG ratio of 4) showed the most efficient photothermal capability. Importantly, we found that the ICG dispersity in the liposomal membrane and its physical interaction with thermosensitive phospholipids were responsible for enhanced photothermal conversion of liposomal ICG, which was not previously observed in various ICG-based photothermal nanoparticles.31,33−35,38,39,43,47 In phototherapeutic studies, the liposomal ICG exhibited greater phototoxicity in a mouse tumor model compared with free ICG and other liposomal ICGs. Furthermore, we demonstrated that the peritumorally injected liposomal ICG fluorescently visualized the sentinel lymph nodes near the primary tumor and subsequently induced substantial photothermal effects localized in these lymph nodes upon NIR irradiation, which would be potentially useful for selective engineering and ablation of metastatic lymph nodes. Liposomal ICG has great potential for clinical translation in oncology because it was prepared by self-assembly of FDAapproved components (phospholipid and ICG) via hydrophobic interactions, and exhibited superior NIR fluorescence imaging and photothermal capability over free ICG. Their potential clinical applications could be extended to superficial diseases sensitive to thermal treatment. Furthermore, therapeutic agents could be also encapsulated in the aqueous core of liposomal ICG to exert combined therapeutic effects with photothermal heating and chemotherapeutics. We believe that this work will facilitate the use of liposomal ICG with enhanced photothermal capability for many therapeutic applications.

irradiation compared with the P4 formulation and free ICG, and the temperature was maintained above 50 °C up to 15 min with the tested ICG concentration and laser dose (Figure 4b). The photothermal effect of free ICG was somewhat improved in the tumors compared with the in vitro results because the protein binding in vivo facilitates effective dispersion. In the phototherapeutic results, the M4-treated tumors disappeared completely by external observation within 10 days after irradiation, whereas all other tumors, including those not exposed to laser irradiation, continued to grow without significant reduction (Figure 4c and Figure S9a). Particularly, black scars were observed on all M4-treated tumors and removed naturally around 8−10 days after irradiation. For all of the treatments tested in this study, no significant loss of body mass was observed because of liposome or ICG injection, laser irradiation, or tumor ablation (Figure 4d and Figure S9b). The survival benefit of liposomal ICG-directed tumor ablation was also assessed over the course of 50 days of observation. All M4treated mice survived at 50 days with no evidence of tumor regrowth, whereas most of the other mice had to be euthanized before 50 days (Figure 4e and Figure S9c). Mirroring the phototherapeutic results in the spheroids, the M4 formulation showing the enhanced photothermal effect also exhibited the greatest phototherapeutic outcome in the tumor model, along with the capability of NIR fluorescence-based tumor localization. Finally, we further assessed the potential of liposomal ICG to photothermally ablate sentinel lymph nodes harboring metastatic tumor cells. Metastatic lymph nodes are dominantly incised near the primary tumor during physical surgery, but excessive excision including surrounding normal tissues for complete elimination of the metastatic region often results in unwanted side effects, making it difficult to recover fully after physical surgery.55,56 Recent studies demonstrated that photothermal ablation of sentinel lymph nodes with other nanomaterials can inhibit metastasis to remote organs.57−59 Thus, selective treatment of metastatic lymph nodes is needed to reduce the undesirable effects and prevent systemic metastasis. For a metastatic lymph node model, metastatic cancer cells were engrafted into the rear footpads of mice to induce migration of tumor cells to the popliteal lymph node. For fluorescence imaging-based sentinel lymph node localization, free or liposomal (M4) ICG was injected peritumorally into the hock, and the popliteal lymph nodes were imaged using the NIR fluorescence imaging system at 1-h postinjection. Fluorescence images revealed selective accumulation of free and liposomal ICG in the popliteal lymph nodes after peritumoral injection, and higher fluorescence signals were observed in the lymph nodes treated with liposomal ICG (Figure 5a). As observed in the primary tumor, the fluorescence signal in the lymph node disappeared after irradiation indicating that ICG molecules localized in the lymph nodes were activated well by NIR irradiation from outside the body of the mouse. For photothermal ablation, the popliteal lymph nodes were irradiated for 20 min with a single dose of 808 nm irradiation at 1 h postinjection of liposomal or free ICG while measuring the temperature change in the lymph node region, and histological analysis of treated lymph nodes was performed at 1-day postirradiation. As expected, the photothermal effect in the liposomal ICG-treated lymph nodes was significantly higher than those in the free ICG-treated and untreated lymph nodes (Figure 5b). The enhanced lymph node accumulation and photothermal capability of liposomal ICG are both thought to

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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.6b16801.

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Experimental methods and Figures S1−S9 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ji-Ho Park: 0000-0002-0721-0428 Author Contributions †

H.-J.Y. and H.-S.L. contributed equally to this work.

Notes

The authors declare no competing financial interest. 5689


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ACKNOWLEDGMENTS This work was supported by the Basic Science Research Programs through the National Research Foundation funded by the Ministry of Science, ICT & Future Planning, Republic of Korea (NRF-2015R1A1A1A05001420 and NRF2015R1A2A2A04005760).

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