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Biomaterials 35 (2014) 6037e6046

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Biomaterials

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Improving drug accumulation and photothermal efficacy in tumordepending on size of ICG loaded lipid-polymer nanoparticles

Pengfei Zhao a,1, Mingbin Zheng a,b,1, Caixia Yue a, Zhenyu Luo a, Ping Gong a,Guanhui Gao a, Zonghai Sheng a, Cuifang Zheng a, Lintao Cai a,*aGuangdong Key Laboratory of Nanomedicine & Shenzhen Key Laboratory of Cancer Nanotechnology, Institute of Biomedicine and Biotechnology,Chinese Academy of Sciences, Shenzhen 518055, PR ChinabDepartment of Chemistry, Guangdong Medical College, Dongguan 523808, PR China

a r t i c l e i n f o

Article history:Received 7 March 2014Accepted 6 April 2014Available online 26 April 2014

Keywords:Indocyanine greenLipid-polymer nanoparticleEndocytosisBiodistributionPhotothermal therapy

* Corresponding author.E-mail address: lt.cai@siat.ac.cn (L. Cai).

1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.biomaterials.2014.04.0190142-9612/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

A key challenge to strengthen anti-tumor efficacy is to improve drug accumulation in tumors throughsize control. To explore the biodistribution and tumor accumulation of nanoparticles, we developedindocyanine green (ICG) loaded poly (lactic-co-glycolic acid) (PLGA) -lecithin-polyethylene glycol (PEG)core-shell nanoparticles (INPs) with 39 nm, 68 nm and 116 nm via single-step nanoprecipitation. TheseINPs exhibited good monodispersity, excellent fluorescence and size stability, and enhanced temperatureresponse after laser irradiation. Through cell uptake and photothermal efficiency in vitro, we demon-strated that 39 nm INPs were more easily be absorbed by pancreatic carcinoma tumor cells (BxPC-3) andshowed better photothermal damage than that of 68 nm and 116 nm size of INPs. Simultaneously, thefluorescence of INPs offered a real-time imaging monitor for subcellular locating and in vivo metabolicdistribution. Near-infrared imaging in vivo and photothermal therapy illustrated that 68 nm INPs showedthe strongest efficiency to suppress tumor growth due to abundant accumulation in BxPC-3 xenografttumor model. The findings revealed that a nontoxic, size-dependent, theranostic INPs model was builtfor in vivo cancer imaging and photothermal therapy without adverse effect.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Indocyanine green (ICG), a tricarbocyanine dye with substantialabsorption and fluorescence in the near-infrared wavelength re-gion (NIR) and effective photothermal response, has been widelyutilized as a probe for diagnostic and therapeutic applications [1,2].However, the applications of ICG in photothermal therapy and NIRimaging were restricted due to unstable optical properties, quickdegradation and clearance in living body [3]. Various nanocarriershave been developed to encapsulate ICG and implement itsenhanced penetration and retention (EPR) effect and fluorescencestability in vivo [4,5].

Cancer nano-therapeutics is expected to solve limitation ofconventional drug delivery system, such as improving drug accu-mulation in tumor tissue [6]. Recently, great progress has beenachieved in improving drug pharmacokinetics, biodistribution anddrug penetration in tumor tissue through versatile nanocarriers,

considering multiple factors such as size, shape, surface charge andwater solubility [7]. Therein, the size of nanomedicines shows acritical effect on passive targeting, and accumulation in tumor,whichwould influence their therapeutic efficacy [8,9]. Additionally,the size control is also important in poorly permeable tumors likepancreatic carcinoma and colon carcinoma, which would inducelimited drug distribution in tumors [6,10,11]. It has been provedthat inorganic and polymer NPs with big size only accumulatednear the vasculature while NPs with small size could rapidly diffusethroughout tumor matrix and provide a better penetration effect[12e16]. Therefore, it was necessary and urgent to validate the sizecontrol for improving drug biodistribution in vivo and EPR effectwith enhanced therapeutic efficacy.

In this study, we developed ICG-loaded polymer-lipid NPs (INPs)with three different hydrodynamic dimensions of 39 nm, 68 nm,and 116 nm via single-step self-assembly, which integrated near-infrared imaging and photothermal therapy properties of ICG.Their physiochemical characters were systematically evaluated.In vitro endocytosis, subcellular localization, in vivo metabolic dis-tribution and accumulationwere directly observed utilizing the NIRfluorescence of ICG. The cytotoxic effects of different INPs based on

mailto:lt.cai@siat.ac.cnhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.biomaterials.2014.04.019&domain=pdfwww.sciencedirect.com/science/journal/01429612http://www.elsevier.com/locate/biomaterialshttp://dx.doi.org/10.1016/j.biomaterials.2014.04.019http://dx.doi.org/10.1016/j.biomaterials.2014.04.019http://dx.doi.org/10.1016/j.biomaterials.2014.04.019

P. Zhao et al. / Biomaterials 35 (2014) 6037e60466038

drug uptake were also investigated and compared in BxPC-3 cells.Moreover, the photothermal efficacy of three types of INPs to BxPC-3 xenograft tumors was evaluated in vivo.

2. Experimental section

2.1. Chemicals and materials

The following chemical reagents and materials were used in our experiment.PLGA (MW ¼ 5000e15,000 Da, [lactide acid]: [glycolic acid] ¼ 50:50), indocyaninegreen (ICG), 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT),hematoxylin and eosin were obtained from SigmaeAldrich (USA). Soybean lecithinand 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-maleimide (poly-ethylene glycol 2000) (DSPE-PEG) were obtained from Avanti (USA). Hoechst 33258,Calcein-AM, Alexa Fluor� 488 Annexin V and Propidium Iodide (PI) were obtainedfrom Invitrogen (USA). Amicon ultra-4 centrifugal filter with a molecular weightcutoff of 10 kDa was purchased from Merck Millipore (USA). RMPI 1640 medium,fetal bovine serum, trypsin EDTA, penicillin and streptomycin were obtained fromHyclone (USA).

2.2. Assembly of ICG loaded lipid-polymer nanoparticles (INPs) with three differentsizes

We synthesized three types of INPs through a single-step nanoprecipitationmethod. PLGA was dissolved in acetonitrile with concentrations of 2.5 mg/mL,lecithin/DSPE-PEG was dissolved at concentrations of 1 mg/mL in 4 wt% ethanolaqueous solution, and ICG was dissolved in ultrapure water at concentrations of3 mg/mL. The lecithin/DSPE-PEG solution was heated to 65 �C to ensure all lipidingredients were in liquid phase. Meanwhile, ICG aqueous solution of proper initialconcentration was added to PLGA acetonitrile solution and the solution was mixedadequately. Then, PLGA/ICG solution was added into preheated solution drop-wiseunder stirring (200 r/min). The final mixture was further stirred for 3 h at 65 �C.An Amicon Ultra-4 centrifugal filter was used to wash the solution for thrice toexclude the organic solvents and free molecules. The mass ratio of PLGA: DSPE-PEG:lecithinwas 8: 0.8: 1.2 for INP-1 synthesis. Themass ratiowas adjusted to 12: 0.8: 1.2for INP-2 synthesis, and 16: 0.8: 2.4 for INP-3 synthesis. 1 mL ICG was added duringthe assembly of three INPs.

2.3. Physiochemical characterization of three types of INPs

Size, surface charge, PDI and size distribution of all INPs was acquired using aDLS detector Malvern Zetasizer (Nano ZS, Malvern, USA) at 25 �C. The morphologyand structure were obtained through transmission electron microscope (F20,TECHNI, USA). The TEM samples were prepared by placing a drip of INPs solution at aconcentration of 10 mg/mL onto a 300-mesh copper grid (Zhongjingkeyi TechnologyCo. Ltd., Beijing), dropping a drip of 2 wt% phosphotungstic acid for negative stainingand then drying the sample at room temperature overnight. The primary particlesize was acquired through opportunity sampling on the copper grid. Gatan Micro-scopy Suite software was used to handle and choose acquired images.

The EE and LE of ICG loaded in INPs was detected as follows: isolating fresh NPsfrom aqueous suspension medium by ultracentrifuge (25,000 r/min, 30 min)(OptimaTM MAX-XP, Beckman, USA). Non-loaded ICG in the suspending liquid wasremoved. Then the concentration of ICG loaded in INPs was determined by UV/visspectrometer (Lambda25, PerkineElmer, USA). The EE and LE are acquired throughthe following formula: EE (%) ¼ (weight of loaded drug)/(weight of initially addeddrug) � 100; LE (%) ¼ (weight of loaded drug)/(total weight of NPs) � 100.

2.4. Fluorescence stability of INPs in vitro

Fluorescence spectra of free ICG and three types of INPs were determined byfluorescence spectroscopy (F900, Edinburgh Instruments Ltd., UK) with emissionwavelength at 740 nm. The ICG concentration of free ICG and three INPs were 1 mg/mL and the FL was determined at predetermined time within 30 d.

2.5. In vitro photothermal efficiency

1mL of PBS, free ICG (containing 20 mg/mL ICG), INP-1(containing 20 mg/mL ICG),INP-2 and INP-3(containing 20 mg/mL ICG) was added into different wells of 24-wellplate. Using 1.6 W/cm2 laser to irradiate the 5 samples for 8 min simultaneously, thetemperature changes of each group were recorded by an infrared thermal imagingcamera (Ti27, Fluke, USA).

2.6. In vitro drug release of INPs

The drug release velocity of three INPs was determined through the followingprocedure: 1 mL of three different INPs was taken into three bag filter segments(MW ¼ 10,000) which were submerged into 2 L of phosphate-buffered saline (PBS,pH ¼ 7.4) as dissolution media respectively. Then keep shaking the INPs in bag filterin PBS at 37 �C, and obtaining the dialysate of three types of INPs at predeterminedtime points to calculate the amount of released drug, while adding the same volumeof fresh PBS back onto a shaker to continue further study. The ICG concentration ofdialysate was measured by Lambda25 UV/vis spectrometer, as described above.

2.7. Tumor cells

In situ pancreatic carcinoma cells (BxPC-3) were used for cell studies. Cells weregrown in RMPI 1640 culture medium containing 10% fetal bovine serum, 1% peni-cillin and 1% streptomycin at 37 �C under a circumstance containing 5% CO2.

2.8. In vitro cellular uptake

2 � 104 BxPC-3 cells were seeded into 8-well chambered coverglasses (Lab-Tek,Nunc, USA) in 200 mL of medium. After 24 h culturing at 37 �C under a circumstancecontaining 5% CO2, the medium was replaced by new medium with three types ofINPs containing 10 mg/mL ICG. After 2 h incubation, the cells were washed for thriceand fixed with PBS. Finally, the cells were observed by confocal laser scanning mi-croscope (TCS SP5, Leica, Germany). The fluorescence was imaged at lEx 365 nm fornucleolus location and lEx 633 nm for ICG.

The cell uptake was further quantitatively evaluated by Flow cytometry. BxPC-3cells were seeded into 8-well plates (1 � 105 cells per well) in 1 mL medium. After24 h, the original medium was removed and new medium with three types of INPscontaining 10 mg/mL ICG was added into each well, and medium without INPs wasused as negative control. After 2 h incubation, the cells were harvested by 0.05%trypsin EDTA. The FL was detected by FC (Accuri C6, BD, USA).

2.9. In vitro photothermal therapy

Seeding BxPC-3 cells into 96-well plate (1�104 cells/well) in 100 mL of medium.The plates were settled at 37 �C for 24 h so that the cells could adhere to the bottomof plates. To explore the photothermal therapeutic efficacy, the medium wasreplaced by newmedium containing INPs with different ICG concentrations, and themediumwithout INPs was used as positive control. The cells were incubated withinthe medium containing INPs for 24 h and then the medium was removed andwashed by PBS. Fresh medium without INPs was added and the plates were thenplaced on the digital dry bath incubator (AccublockTM, Labnet, USA) so that thetemperature of the wells remained at 37 �C before and during laser irradiation.Considering that the maximum temperature of INPs solution became stable afterlaser irradiation for 5min, the cells were irradiated with a 1.6 W/cm2 808 nm laserfor 5 min for photothermal therapy. The cell viability was evaluated usingMTTassay.

The cytotoxicity of photothermal treatment was also evaluated by Calcein-AM/PI dying. BxPC-3 cells were seeded into 24-well plates (4 � 104 cells/well) in 400 mLmedium. After 24 h incubation at 37 �C, the original medium was replaced by newmediumwith INPs containing 80 mg/mL ICG, and themediumwithout INPs was usedas control. The cells were irradiated with laser for 5 min using the same procedure.Then cells were washed with PBS and stained with Calcein-AM for live cell visual-ization andwith PI for dead/late apoptosis cell visualization abided bymanufacture’ssuggestion (Invitrogen). The results were obtained through biological inverted mi-croscope (IX71, Olympus, Japan) by eliminating the fluorescence interference of ICG.

2.10. Animals and tumor model

Female BALB/c nude mice (6 weeks old and weighted 18e20 g) were purchasedfrom Slac Laboratory Animal Co. Ltd (Hunan, China). All mice received care incom-pliance with the guidelines outlined in the Guide for the Care and Use of LaboratoryAnimals. The procedures were stated by Shenzhen Institutes of Advanced Tech-nology, Chinese Academy of Sciences Animal Care and Use Committee. To obtaintumor model, 3 � 106 BxPC-3 cells were injected to the mice through subcutaneousinjection. 2 weeks after post-inoculation, the xenograft tumor model with lowpermeability was built. Tumor volume was calculated as A � B2/2, where A and Bcorrespond to the max and minor axes of tumor.

2.11. In vivo imaging and biodistribution studies

When tumor volumes of mice reached to 50 mm3, free ICG (600 mL, containing200 mg/mL ICG), INP-1 (600 mL, containing 200 mg/mL ICG), INP-2 (600 mL, containing200 mg/mL ICG) and INP-3 (600 mL, containing 200 mg/mL ICG) was added into 1.5 mLcentrifuge tubes. Mice bearing BxPC-3 cells were divided into 4 groups (three micein each group) and 200 mL of each sample was injected into nudemice via tail vein sothat the ICG dosage in mice was 2 mg/kg. Another three mice were injected with200 mL PBS through the same operation as control group. ICG biodistribution anal-ysis was obtained at indicated time after injection using the ex/in vivo imagingsystem (Maestro, USA) with a 704 nm excitation wavelength and 735 nm filter tocollect the fluorescence. The mice were sacrificed immediately and major organsincluding heart, liver, spleen, lung, kidneys and tumors were collected for in vitroimaging using the same imaging system.

2.12. Measuring photodynamic efficiency through temperature

The protocol was exactly identical with in vivo biodistribution studies until thetail vein injection. Then the tumor location of each mouse was irradiated by the808 nm laser with strength of 0.8 W/cm2 for 10 min after 24 h intravenous injection.The temperature of tumors was determined by infrared thermal imaging camera(Ti27, Fluke, USA) simultaneously.

Table 1Size distribution, surface charge, and drug encapsulating efficiency (EE) and loadingefficiency (LE) of INP-1, INP-2 and INP-3. The data were shown as mean� SD (n¼ 3).

Particle type Particle size Surface potential EE (%) LE (%)

INP-1 39.4 � 2.8 �53.6 � 0.3 28.41 � 0.05 6.71 � 0.01INP-2 67.9 � 4.0 �52.8 � 2.4 31.30 � 0.29 7.39 � 0.07INP-3 115.8 � 1.5 �50.9 � 0.6 39.30 � 0.19 6.73 � 0.03

P. Zhao et al. / Biomaterials 35 (2014) 6037e6046 6039

2.13. In vivo tumor photothermal treatment

After tumor model was performed and tumor sizes reached to 50 mm3, micewere divided into six groups (five per group). The mice were separately injectedwith 200 mL PBS, 200 mL PBS, 200 mL free ICG, 200 mL INP-1, 200 mL INP-2, and 200 mLINP-3. Total groups except the first one were irradiated by the 808 nm laser (0.8 W/cm2) for 10 min after drug squirt for 24 h, and the PBS-only group was set as control.The changes of tumor size and body weight of every mouse were recorded within 4weeks. Mice with tumor volumes exceeding 600 cm3 would be euthanatized ac-cording to animal protocol, and the slow-growth and malignance of this tumormodel.

To further survey the photothermal efficiency in vivo, appropriate size sectionsof tumors at 12 h and major organs (heart, liver, spleen, lung and kidney) at 28 dafter treatment were collected, and then were stained with hematoxylin-eosin(H&E) and examined by biological inverted microscope.

3. Results and discussion

3.1. Formulation and characterization of INPs

In order to investigate the size-dependent biodistribution innude mice and drug accumulation behavior of INPs in pancreascarcinoma (BxPC-3) xenograft tumors, three types of INPs (INP-1,INP-2 and INP-3) were successfully developed according to previ-ous works [17,18]. Afterward, diameter and surface potential ofthree INPs were measured by dynamic light scattering (DLS) andshown in Table 1. The average size of three INPs in water was39.4 nm, 67.9 nm and 115.8 nm respectively, and average zeta po-tential were �53.6 mV, �52.8 mV and �50.9 mV. Transmission

Fig. 1. Characterazation of different size ICG NPs of INP-1, INP-2 and INP-3. (A) TEM images oPBS, free ICG, INP-1, INP-2 and INP-3 under continuous laser irradiation. (C) Infrared thermoDrug-release curve of INP-1, INP-2 and INP-3 in PBS at 37 �C (n ¼ 3).

electron microscopy (TEM) was also utilized to investigate themorphology of three types of INPs. The images vividly demon-strated that INPs were divided into three types distribution andeach type of INPs exhibited an excellent monodispersity (Fig. 1A).The size of INPs was slightly small than corresponding DLS resultsbecause TEM images only gave the dehydrationmorphology of NPs,and DLS overestimate mean particle size due to high scatteringintensities of large objects [19,20].

The size stability of three different INPs was investigated by DLSwithin 4 weeks. The diameter of INP-1, INP-2 and INP-3 after 4weeks remained their size without evident variation (Fig. S1). ICGfluorescence intensity (FL) of free ICG and three types of INPs wasalso detected by fluorescence spectroscopy for 30 d to evaluate thefluorescence stability of ICG. The results showed that the fluores-cence of free ICG deceased to 13.4% compared to initial intensity,while the fluorescence of ICG in INP-1, INP-2 and INP-3 stillremained above 60% (Fig. S2). These data indicated the stability ofICG was apparently improved when it was isolated from sur-rounding environment.

The drug encapsulation efficiency (EE) and drug loading effi-ciency (LE) were important parameters for clinical application [17].The EE of ICG encapsulated in INP-1, INP-2 and INP-3 was 28.41%,31.30% and 39.30%; the LE was 6.71%, 7.39% and 6.73% (Table 1).These results exhibited increased EE due to enhanced polymerweight fraction [21] and similar LE of three types of INPs.

To estimate whether the photothermal efficiency of ICG wereaffected by NPs, the temperature changing profiles in vitro wasinvestigated by continuous laser irradiation. After 1.6 W/cm2 laserirradiation for 8 min, the maximum temperature of free ICG, INP-1,INP-2 and INP-3 aqueous solution rose to 56.0 �C, 57.1 �C, 57.3 �Cand 57.4 �C respectively, while PBS-only rose to 32 �C withcomparative unapparent increase (Fig. 1B). Moreover, themaximum temperature in each group turned stable after 5 minirradiation. The infrared thermo-graphic images proved that themaximum temperature of PBS, free ICG, INP-1, INP-2 and INP-3 at

f INP1, INP-2 and INP-3 (Scale bar ¼ 50 nm). (B) The maximum temperature profiles of-graphic images of PBS, free ICG, INP-1, INP-2 and INP-3 after irradiation for 8 min. (D)

Fig. 2. Endocytosis of INP-1, INP-2 and INP-3 by BxPC-3 cells. (A) Cellular localization of ICG after 2 h incubation with INP-1, INP-2 or INP-3 respectively. Blue represented thefluorescence of Hoechst 33258; red represented the fluorescence of ICG (Scale bar ¼ 30 mm). (B) Flow cytometry analysis of BxPC-3 cells endocytosis incubated with INP-1, INP-2 orINP-3 for 2 h. (C) Geometric mean fluorescence of BxPC-3 cells after incubation (*) P < 0.05, compared with INP-1; (**) P < 0.01, compared with INP-1 (n ¼ 3). (For interpretation ofthe references to color in this figure legend, the reader is referred to the web version of this article.)

P. Zhao et al. / Biomaterials 35 (2014) 6037e60466040

8 min was 31.9 �C, 55.9 �C, 57.4 �C, 56.8 �C, and 56.9 �C respectively(Fig. 1C). The temperature of INPs was rapidly increased at the first100 s. Moreover, the photothermal efficiency of INPs was evenslightly enhanced compared with free ICG at 5e8 min.

Drug release of three types of INPs was also explored due to itsimportance for drug efficiency and safety. ICG was released fromINP-1, INP-2 and INP-3 with a biphasic profile, where an initialburst release was seen in the first 12 h and more than 50% of ICGwere released, followed by a sustained and uniformed release

(Fig. 1D). These results demonstrated that the INPs showed similardrug release rates, which was also supported by Cabral’s work [9].

3.2. In vitro cell uptake

Tuning the size of NPs would greatly influence cell uptake[14,22]. The subcellular localization of ICG in BxPC-3 cells wasdetected by confocal microscopy. After 2 h incubation, highest NIRfluorescence signals could be observed in INP-1 group cells,

Fig. 3. Cell survivals of BxPC-3 cells after incubation and photothermal treatment. (A) Quantitative evaluation of cell survival treated with INP-1, INP-2 or INP-3 plus laser irra-diation. (*) P < 0.05, compared with INP-1; (**) P < 0.01, compared with INP-1 (n ¼ 4). (##) P < 0.01. (B) FL images of BxPC-3 cells after photothermal treatment. Viable cells werestained green with Calcein-AM, dead/later apoptosis cells were stained red with PI (Scale bar ¼ 50 mm). (For interpretation of the references to color in this figure legend, the readeris referred to the web version of this article.)

P. Zhao et al. / Biomaterials 35 (2014) 6037e6046 6041

illustrating that ICG could efficiently distribute into cells via INP-1transportation. The fluorescence signals in INP-2 group cells wasnot that sharp compared to INP-1 group, displaying a less ICGdistribution into cells. While only faint fluorescence signals couldbe observed in INP-3 group cells and illustrated only a few ICG wasobserved in BxPC-3 cells (Fig. 2A). To confirm the differences ofsubcellular localization in three groups, Flow cytometrywas furtherutilized. The results exhibitedmost amount of ICG were detected inBxPC-3 cells incubated with INP-1, while less ICG signals wereobtained in the cells incubated by INP-2 and INP-3 (72.79% and44.00% FL intensity of INP-1 group) (Fig. 2). It had been proven thatmost NPs were taken up by an endocytic process, and NPs of 20e50 nm were taken up more rapidly than larger particles [22e24].Therefore, INP-1 could bemost internalized by cells and the highestamount of ICG could accumulate in cells in INP-1 group. And it was

obvious to conclude that the size of INPs had a significant effect ondelivering drugs into BxPC-3 cells.

3.3. In vitro photothermal therapy

To explore its biomedical applications of INPs, the potentialtoxicity of INPs was first tested. The cytotoxicity of free ICG, emptyNPs and three INPs to BxPC-3 cells was quantitatively evaluatedthrough MTT assay. The cells treated with INPs containing even80 mg/mL ICG still performed favorable cell viability after 24 h in-cubation, which indicated that INPs below the concentration werenontoxic (Fig. S2).

Next, INPs was employed as photothermal agent for in vitrotumor cell therapy. BxPC-3 cells exhibited different survive ratewhen incubated with various ICG concentrations of INP-1, INP-2 or

Fig. 4. Biodistribution of INP-1, INP-2 and INP-3 in major organs and tumors of nude mice bearing BxPC-3 tumors after intravenous injection of PBS, free ICG, INP-2 or INP-3. (A) NIRfluorescence images of major organs and tumors of nude mice after 24 h pre-injection of PBS, free ICG, INP-2 or INP-3 respectively. (B) Total near-infrared FL of correspondingindividual organs and tumors after 24 h injection of INP-1, INP-2 or INP-3 respectively. (*) P < 0.05, compared with INP-2 (n ¼ 3). (C) Main potential reasons why INP-2 accumulatedthe highest amount of ICG in the tumor tissue.

P. Zhao et al. / Biomaterials 35 (2014) 6037e60466042

INP-3 for 24 h and then irradiated by 808 nm laser (1.6 W/cm2).BxPC-3 cells in INP-1 group were killed most efficiently at each ICGgradient concentration, and only 5.76% cells could survive afterphotothermal treatment when the ICG concentration reached to80 mg/mL (Fig. 3A). ICG concentration was a crucial factor for finalphotothermal efficiency [25], it was not surprising to indicate thatthe amount of ICG existed in cells determined the efficiency ofphotothermal treatment. The smaller size and better cell uptake ofINP-1 could evidently increase intracellular ICG concentration inBxPC-3 cells, thus significantly improved the photothermal effi-ciency when treated with same dosage of ICG and irradiation.

The efficiency of three types of INPs was further evaluatedthrough Calcein-AM and PI staining after 24 h incubation of threedifferent INPs and followed by laser irradiation (1.6 W/cm2). Viablecells were stained green with Calcein-AM, while dead or laterapoptosis cells were stained red with PI. In vitro treatment effi-ciency was directly visualized through the staining, and the imagesillustrated that laser irradiation alone was quite safe for BxPC-3cells. When ICG concentration increased to 80 mg/mL, cells in INP-1 group were almost entirely dead after 24 h incubation; nearly20% cells were still alive in INP-2 group and about 70% survival cells

in INP-3 group, which was consistent with the cytotoxicity resultsof photothermal therapy (Fig. 3B).

3.4. In vivo biodistribution

The biodistribution results of three different INPs were observedin BxPC-3 tumor bearing mice by ex/in vivo imaging system. ICG FLintensity was obtained after injected with PBS, free ICG, INP-1, INP-2 or INP-3 after 24 h Fig. 4A demonstrated that free ICG wasmetabolized and quickly excreted from living body and weak FLintensity could only be detected in liver after 24 h. This was becauseafter binding to plasma proteins, ICG was cleared away by initialhepatic localization and eventual total clearance through biliarytreewith minimal acute renal involvement [1,2,26], simultaneouslyshowed the inefficient passive targeting of free ICG.

However, our results demonstrated that three types of INPscould be reserved in living body for a longer time. In INP-1, INP-2and INP-3 groups, FL intensity of ICG could hardly be detected inheart, indicating the high biological safety of three INPs for heart.While ICG fluorescence could be detected in liver, spleen, lung andkidneys, which indicated that pulmonary endothelial cells uptake

Fig. 5. Temperature increasing profiles in BxPC-3 tumor tissue after in vivo photothermal treatment. (A) Maximum temperature profiles of xenograft tumors after tail vein post-injection of 200 mL PBS, free ICG, INP-2 or INP-3 plus further laser irradiation. (B) Infrared photothermal images of partial mice measured after tail vein injection and laser irra-diation. (*)P < 0.05, (**) P < 0.01.

P. Zhao et al. / Biomaterials 35 (2014) 6037e6046 6043

was also a pathway to remove INPs, except for hepatic clearanceeffect [2]. No statistical fluorescence difference was detected exceptin spleen, where the highest fluorescence intensity was found inINP-3 group. Since liver, spleen and lung were the organs of retic-uloendothelial system [2,27], and the total fluorescence in theseorgans of each INPs groupwere analogous, a similar biodistributioncould be deduced in three INPs groups. Therefore, these threedifferent INPs apparently extended ICG circulation time in livingbody, exhibited similar distribution, and provided a noninvasiveand visible method to analyze the biodistribution of NPs in vivo.

Contemporarily, ICG FL intensity was detected in tumor of miceafter injected with INP-1, INP-2 and INP-3. Compared with the hi-erarchy of arterioles, capillaries and venules in normal blood ves-sels, tumor blood vessels were disorganized, irregularly shaped andleaky to obtain sufficient nutrition from blood [27,28]. This vascu-lature allowed preferential extravasation of circulating NPs. Andpolymersome have been used to encapsulate hydrophilic and hy-drophobic drugs for passive targeting of tumors, and demonstratedtumor passive-targeting of ICG was enhanced through EPR effectthrough the encapsulation of lipid/polymer NPs [27,29]. The high-est ICG FL intensity in tumor tissue was clearly observed in INP-2group, which indicated INP-2 possessed best passive-targeting ef-ficiency and highest ICG accumulation in tumor tissue.

In order to achieve efficient extravascular accumulation in tu-mor tissue, NPs must leave tumor blood vessel efficiently andmaintain retention in the tissues [6,30]. It was reported thatpancreatic carcinoma was a low-permeable tumor, whose mediandiameter of vessel pores in the sinusoids was around 110 nm [9,31].Since the size of INP-1, INP-2 and INP-3 was 39.4 nm, 67.9 nm and115.8 nm, the pores would obviously restrict the convection of INP-3 and it would only accumulate at or near the surface of tumorblood vessel. While INP-1 and INP-2 could easily pass through thevessel pores because their size was smaller than 100 nm.

Secondly, the particles diffused to tumor tissue must obtainefficient retention, and the retention was influenced by both cellmetabolism and lymphatic drainage. According to previous reports,

smaller particles could diffuse to the deep tissue of tumor muchfaster [9,32e34]. Decreased pO2 and hypoxic cells were alsodiscovered in the deep region of tumor [6], which meant thatsmaller particles would be metabolized by tumor cells morequickly. Moreover, smaller particles could more easily be clearedaway from tumor through lymphatic drainage and further entersentinel lymph node [35e38]. Therefore, INP-1, the particle withthe smallest size, was very easily to be metabolized by tumor cells,and then eliminated from tumor through lymphatic drainage afterthe diffusion from vessel (Fig. 4C). And due to relatively shallowerdiffusion and slower cell metabolism, INP-2 could be betterreserved in tumor tissue.

3.5. In vivo photothermal efficiency and treatments

After intravenous injection of PBS, free ICG, INP-1, INP-2 or INP-3through tail vein for 24 h, the tumor regionwas irradiated by 0.8W/cm2 near-infrared laser for 10 min. Then the temperature variationof tumor in 5 different groups was observed utilizing infraredthermal imaging camera. The maximum temperature of tumors ineach group was 41.1 �C, 41.6 �C, 46.9 �C, 50.1 �C and 44.1 �Crespectively (Fig. 5). The results indicated that when treated withINPs, tumors would probably acquire adequate temperature tocause destruction, while treated with PBS or free ICG would beinsufficient to achieve tumor destruction, owing to previous workthat efficient tumor cell destruction would be induced when tem-perature increased over 43 �C [39e41]. It demonstrated that thehighest accumulation in tumor could bring about the greatesttemperature increase in INP-2 group.

To identify the anti-tumor effect of passive targeted INPs tomice, BxPC-3 xenograft tumors were collected and stained withhematoxylin and eosin. In PBS plus laser and ICG plus laser groups,poorly differentiated histology and thick fibrotic tissue in theinterstitium was discovered, and no vasculature could be observedinside tumor cell nests (Fig. 6A), which was corresponded with thecharacteristics of BxPC-3 xenograft tumor tissue described in

Fig. 6. The photothermal efficacy of in vivo and histological assessments for the INPs. (A) Histological staining of removed tumors at 48 h after injection of PBS, free ICG, INP-1, INP-2or INP-3 plus laser irradiation (Scale bar ¼ 50 mm). (B) The growth curve of BxPC-3 xenograft tumors within 4 weeks in different groups (n ¼ 5). (C) Survival rate of mice bearingBxPC-3 tumors of disparate groups within 30 d after corresponding treatments. (D) H&E stained images of sliced major organs in PBS plus laser group and INP-2 plus laser group. (*)P < 0.05, (**) P < 0.01 (Scale bar ¼ 50 mm).

P. Zhao et al. / Biomaterials 35 (2014) 6037e60466044

earlier references [9e11]. According to such structure, tumor cellnests were probably the barrier against the penetration of drugsand nanocarriers [11], and destroying nests structure was essentialto inhibit tumor growth and relapse. In Fig. 6A, the BxPC-3 tumortissue in PBS plus laser group showed the same features comparedwith control group, which indicated that a moderate and safe laserirradiation (0.8 W/cm2) was offered without local toxicity orevident systemic toxicity. Meanwhile, the tumor cells in three INPsgroups exhibited coagulative necrosis and pyknosis. Particularly,the histological features in INP-2 group proclaimed an irreversibledestruction to tumor nests and blood vessels.

The efficiency of photothermal treatment was further investi-gated in nudemice bearing BxPC-3 tumors. The results showed thattumors treated with PBS, PBS plus laser or free ICG plus laser grewrapidly, straightly confirming the laser irradiation and free ICGalmost had no effect on tumor growth. While the growths of

tumors treated with INP-1 and INP-3 were strongly inhibited dur-ing the first week after treatment, but then relapsed and grewrapidly. However, the tumors treated with INP-2 plus laser in thisgroup were successfully suppressed. Notably, the tumor growthwith INP-2 plus laser treatment was much more efficient than thatof INP-1 or INP-3 plus laser treatment, even though the survive rateof mice within INP-1 or INP-3 groups were improved in a short-term (Fig. 6B and C). Our results demonstrated that adequate ICGaccumulation in tumor tissuewith INP-2 plus laser treatment couldtrigger sufficient temperature to obtain complete remission oftumors.

Side-effect of anti-tumor therapy is greatly concernedthroughout clinical applications, and it proved that the loss ofweight was an indicator for treatments-induced toxicity [17]. On28 d, theweight of mice bearing BxPC-3 tumors increased 1.9w8.9%in the control groups treated with PBS or PBS plus laser, and the

P. Zhao et al. / Biomaterials 35 (2014) 6037e6046 6045

weight of nudemice bearing BxPC-3 tumors in other groups treatedwith ICG plus laser or INPs plus laser increased 4.8w15.6% (Fig. S5).These results indicated all treatments were nontoxic in ourexperiment. In the meantime, the major organs of mice treatedwith INP-2 plus laser were collected for histological staining tocompare with normal organs. No noticeable abnormality wasobserved in heart, liver, spleen, lung and kidneys. The resultsindicated that photothermal therapy of INPs was tolerable and safein vivo.

4. Conclusions

We successfully fabricated ICG-loaded lipid-polymer nano-particles with three size distributions (39 nm, 68 nm and 116 nm)using single-step nanoprecipitation method. NIR fluorescence im-aging and photothermal therapeutic efficiency of ICG was perfectlyintegrated in these three types of INPs for cancer theranostics.These three types of INPs exhibited excellent size stability andfluorescence stability, and enhanced temperature responsecompared to free ICG. Size could not only obviously influenceendocytosis and further photothermal efficiency of INPs in vitro, butalso affect their tumor accumulation and photothermal therapeuticefficiency in vivo. INP-2 showed the strongest efficiency to suppresstumor growth in BxPC-3 xenograft tumors. Hence, we offered anontoxic and size-dependent theranostic model for passive tar-geting and NIR imaging guided photothermal therapy with mini-mized adverse effect.

Acknowledgments

The authors gratefully acknowledge the support from the Na-tional Natural Science Foundation of China (Grant No. 81071249,81171446 and 21375141), Natural Science Foundation of Guang-dong Province of China (Grant No. 9478922035-X003399), ChinaPostdoctoral Science Foundation (20090450587), GuangdongInnovation Reasearch Team of Low-cost Healthcare. Science andTechnology Project of Shenzhen (CXB201005250029A,JC201005270326A, JC201104220242A, JC201005260247A).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2014.04.019.

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Improving drug accumulation and photothermal efficacy in tumor depending on size of ICG loaded lipid-polymer nanoparticles1 Introduction2 Experimental section2.1 Chemicals and materials2.2 Assembly of ICG loaded lipid-polymer nanoparticles (INPs) with three different sizes2.3 Physiochemical characterization of three types of INPs2.4 Fluorescence stability of INPs in vitro2.5 In vitro photothermal efficiency2.6 In vitro drug release of INPs2.7 Tumor cells2.8 In vitro cellular uptake2.9 In vitro photothermal therapy2.10 Animals and tumor model2.11 In vivo imaging and biodistribution studies2.12 Measuring photodynamic efficiency through temperature2.13 In vivo tumor photothermal treatment

3 Results and discussion3.1 Formulation and characterization of INPs3.2 In vitro cell uptake3.3 In vitro photothermal therapy3.4 In vivo biodistribution3.5 In vivo photothermal efficiency and treatments

4 ConclusionsAcknowledgmentsAppendix A Supplementary dataReferences

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