Indocyanine Green-Encapsulated Hybrid Polymeric Nanomicelles for

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Indocyanine Green-Encapsulated Hybrid Polymeric Nano-Micelles for Photothermal Cancer Therapy Wei-Hong Jian, Ting-Wei Yu, Chien-Ju Chen, Wen-Chia Huang, Hsin-Cheng Chiu, and Wen-Hsuan Chiang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b00963 • Publication Date (Web): 18 May 2015 Downloaded from http://pubs.acs.org on May 21, 2015

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Indocyanine Green-Encapsulated Hybrid Polymeric Nano-Micelles for Photothermal Cancer Therapy Wei-Hong Jian, Ting-Wei Yu, Chien-Ju Chen, Wen-Chia Huang, Hsin-Cheng Chiu* and Wen-Hsuan Chiang* Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 300, Taiwan. E-mail: [email protected] (W.-H. Chiang) ; [email protected] (H.-C. Chiu).

Abstract Indocyanine green (ICG), an FDA approved medical near-infrared (NIR) imaging agent, has been extensively used in cancer theranosis. However, the limited aqueous photo-stability, rapid body clearance and poor cellular uptake severely restrict its practical applications. To overcome these problems, the ICG-encapsulated hybrid polymeric nano-micelles (PNMs) were developed in this work through the co-association of the amphiphilic di-block copolymer, poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-b-PEG), and hydrophobic electrostatic complexes composed of ICG molecules and branched poly(ethylenimine) (PEI). The ICG-encapsulated hybrid PNMs featured a hydrophobic PLGA/ICG/PEI core stabilized by hydrophilic PEG shells. The encapsulation of electrostatic ICG/PEI complexes into the compact PLGA-rich core not only facilitated the ICG loading but also promoted its aqueous optical stability. The effects of the chain length of PEI in

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combination with ICG on the physiochemical properties of PNMs and their drug leakage were also investigated. PEI10k (10k Da) could form highly robust and dense complexes with ICG, thus prominently reduced ICG outflow from the PNMs. The results of in vitro cellular uptake and cytotoxicity studies revealed that the ICG/PEI10k-loaded PNMs significantly promoted the cellular uptake of ICG by HeLa cells due to their near-neutral surface, and thereby augmented the NIR-triggered hyperthermia effect in destroying cancer cells. These findings strongly indicate that the ICG/PEI10k-loaded PNMs have great potential to attain an effective cancer imaging and photothermal therapy.

Introduction Over the past decades, photothermal therapy (PTT) for cancer treatment has received much attention due to its several advantages such as minimal invasion, easy implementation and selective localized treatment.[1-5] Different from the traditional cancer chemotherapy with severe adverse effects, the imaging-guided PTT employs photo-absorbers to generate hyperthermia (above 42 oC) in target tumor tissues by remotely controlled photo-irradiation, leading to thermal ablation of cancer cells with significantly reduced toxic side effects. The NIR irradiation ranging from 700~1100 nm exploited in PTT is much preferred owing to its low tissue absorption, high tissue penetration, minimal autofluorescence, and low phototoxicity.[6-8] In this regard, various NIR-absorbing materials including carbon nanotubes,[9,10] graphene oxide,[11,12] gold nanoconstructions[13,14], and palladium nanosheets[15]

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have been extensively utilized as PTT agents due to their high optical extinction in the NIR wavelength range. Nevertheless, the clinical applications of these nanomaterials are seriously limited by their non-biodegradability and potential long-term toxicity. Indocyanine green (ICG), an amphiphilic tricarbocyanine dye, is the only U.S. Food and Drug Administration (FDA) approved NIR clinical imaging agent.[16-18] It can convert NIR light to generate heat for PTT.[16-20] Nevertheless, the applications of ICG in cancer imaging and therapy is restricted by its liability to aggregation and degradation in aqueous solution, poor aqueous photo-stability, lack of target specificity and quick body clearance (t1/2 ≈ 2~4 min).[16,17,20] To address these issues, taking advantage of the inherent enhanced permeability and retention (EPR) effect of tumor vessels, various nanoparticle-based

ICG

delivery

systems

have

been

developed.[16-25]

Among

them,

poly(lactic-co-glycolic acid) (PLGA)-constituted nanovehicles have emerged as one of the most promising carriers for ICG delivery because PLGA is biodegradable, biocompatible, and FDA-approved. Patel et al. reported the application of the ICG-loaded PLGA nanoparticles, prepared by the double emulsion technique using poly(vinyl alcohol) as the emulsifier.[22] Shao and co-workers fabricated the ICG-encapsulated PLGA nanoparticles covered by folic acid (FA)-modified poly(ethylene glycol) (PEG) for active targeting of cancer cells over-expressing FA receptors.[21] However, these delivery systems are too large (above 200 nm) to avoid reticuloendothelial clearance and exhibit low ICG loading efficiency (below 50 %) and/or loading content (ca. 0.2 wt %), and substantial burst release of ICG in aqueous solutions, revealing the difficulty of lading the amphiphilic

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ICG molecules into the hydrophobic PLGA-rich cores of nanoparticles. Cai’s group has recently demonstrated that the size of ICG-loaded nanoparticles (INPs), prepared by co-assembling of lecithin, DSPE-PEG and PLGA via the one-step nanoprecipitation, can be controlled by adjusting the mass ratio of PLGA to lecithin in feed.[23] The INPs showed excellent colloidal and fluorescence stability and enhanced photothermal conversion ability compared to free ICG. In spite of these progresses, it remains a great challenge to endow ICG carriers with high ICG loading efficiency/capacity, low premature payload leakage, and suitable size (ca. 50~150 nm) for EPR effect. In order to enhance the intracellular ICG delivery of PLGA-based nanovehicles, we herein report the fabricatation of ICG/poly(ethylenimine) (PEI)-encapsulated hybrid polymeric nanomicelles (PNMs) by a simple one-step nanoprecipitation. Via the formation of hydrophobic complexes with PEI, the ICG loading efficiency and aqueous photo-stability of the polymeric micelles self-assembled from the amphiphilic PLGA-b-PEG copolymer were prominently promoted. The effects of chain length of PEI on the physiochemical properties and ICG leakage of the ICG-encapsulated hybrid PNMs were investigated. Furthermore, the in vitro cellular uptake of ICG-encapsulated hybrid PNMs by HeLa cells and their photothermal-triggered cytotoxicity were also evaluated.

Experimental Section Materials. ICG was purchased from Chem-Impex (USA). Maleimide-PEG-NH2 (M.W. = 5k Da) was acquired from Jenkem (Beijing, China). PLGA (M.W. = 49k Da, [LA]:[GA] = 75:25),

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N-Hydroxysuccinimide 4-dimethylaminopyridine

(NHS) (DMAP)

and and

N,N′-dicyclohexylcarbodiimide

(DCC),

3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl

tetrazolium

bromide (MTT) were purchased from Sigma-Aldrich (USA). Branched PEI10k (M.W. = 10k Da) and PEI1.8k (1.8k Da) were obtained from Alfa Aesar (USA). Dulbecco's modified Eagle medium (DMEM), Hoechst 33342, propidium iodide (PI) and fetal bovine serum (FBS) were purchased from Invitrogen. CDCl3 used in 1H-NMR measurements was obtained from Cambridge Isotope (MA, USA). Deionized water was produced from Milli-Q Synthesis (18 MΩ, Millipore). All other chemicals were reagent grade and used as received. Synthesis of PLGA-b-PEG Copolymer. The PLGA-b-PEG copolymer used in the work was synthesized by the conjugation of the carboxyl end of PLGA with the amine group of maleimide-PEG-NH2 via DCC/DMAP-mediated aminolysis, as illustrated in Figure 1a. PLGA (1.0 g, 2.04×10-5 mol), maleimide-PEG-NH2 (153 mg, 3.06×10-5 mol), DCC (8.4 mg, 4.08×10-5 mol), DMAP (4.98 mg, 4.08×10-5 mol) and NHS (4.7 mg, 4.08×10-5 mol) were dissolved in chloroform (6 mL). The reaction was carried out under stirring at 25 oC for 3 days, followed by the repeated filtration to remove dicyclohexylcarbodiurea, the byproduct. Upon solvent removal under vacuum at 40 oC, DMSO (10 mL) was added. The solution was then dialyzed (Cellu Sep MWCO 50000) against deionized water for 5 days to eliminate the residual reactants and DMSO. The final product was attained by lyophilization and characterized by 1H-NMR, using CDCl3 as the solvent.

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(a)

(b)

Figure 1. (a) Synthetic route and (b) 1H-NMR spectrum of PLGA-b-PEG copolymer.

Preparation of ICG/PEI-Encapsulated Hybrid PNMs. The ICG/PEI-encapsulated PNMs were prepared by one-step nanoprecipitation. PLGA-b-PEG (6.0 mg), ICG (0.6 mg) and either the branched PEI10k or PEI1.8k (0.6 mg) were dissolved in DMSO (0.5 mL). The ratio of the number of the amines from PEI to the number of the sulfonates from ICG (N/S ratio) was fixed at 9.0. The mixture was added dropwise into pH 6.0 phosphate buffer (ionic strength 0.01 M, 3.5 mL) under stirring. The solution was gently stirred in dark at 25 oC for 1 h and then equilibrated for 30 min. The ICG/PEI-loaded PNM solution was dialyzed (Cellu Sep MWCO 12000~14000) against pH 7.4 phosphate buffer to remove unloaded ICG and DMSO. For comparison, the pristine PNMs,

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ICG-loaded PNMs and ICG/PEI10k-encapsulated PNMs with various N/S ratios (4.5 and 13.5) were also prepared accordingly. Characterization of Pristine PNMs and ICG/PEI-Encapsulated Hybrid PNMs. Measurements of the hydrodynamic diameter, size distribution (polydispersity index, PDI) and zeta potential of pristine PNMs and ICG/PEI-loaded PNMs in pH 7.4 phosphate buffer were conducted on a Malvern ZetaSizer Nano Series instrument. The results shown herein represent an average of at least triplicate measurements. The morphology of ICG/PEI-encapsulated hybrid PNMs was examined by transmission electron microscopy (TEM) (HT7700, Hitachi, Japan). TEM samples were prepared by depositing a few drops of the PNM solution on a 300-mesh copper grid covered with carbon and then negatively stained with uranyl acetate solution (5.0 wt%) for 40 s. The samples were dried at 25 oC for 3 days before measurement. The UV/Vis absorption spectra of free ICG and ICG/PEI-encapsulated PNMs in phosphate buffered saline (PBS) were obtained over different time intervals using a UV/Vis spectrophotometer (U2900, Hitachi, Japan). ICG Encapsulation Efficiency. To determine the ICG loading level, a prescribed amount of ICG/PEI-encapsulated PNM solution was freeze-dried and then dissolved in DMSO for complete disruption of micelle structure and ICG release. The absorbance of ICG encapsulated was measured at 794 nm. The loading efficiency (LE) and loading capacity (LC) of ICG were calculated as following formulas: LE (%) = (weight of loaded ICG/weight of initially added ICG) × 100%.

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LC (%) = (weight of loaded ICG/weight of ICG/PEI-encapsulated PNMs) × 100%. In Vitro ICG Leakage Profiles. The in vitro ICG leakage of ICG/PEI-encapsulated PNMs was evaluated by the dialysis technique (Cellu Sep MWCO 12000~14000). The ICG/PEI-loaded PNM solution containing 15 µM ICG (3.0 mL) was dialyzed (Cellu Sep MWCO 12000~14000) against PBS (pH 7.4) and succinic acid buffer (pH 5.0) (ionic strength 0.15 M, 60 mL), respectively, at 37 oC. At different time intervals, the internal sample solution (pH 5.0 or 7.4) was withdrawn and the maximum ICG absorbance was determined. The sample solution was placed back into the dialysis tube after analysis. The cumulative ICG leakage (%) was calculated by the following equation: Cumulative ICG leakage (%) = ((Initial ICG absorbance - ICG absorbance at different time points)/Initial ICG absorbance) × 100%. The results presented herein represent an average of triplicate measurements. Temperature Measurement under NIR Laser Irradiation. ICG/PEI-encapsulated PNMs and free ICG in PBS (1.0 mL) were irradiated separately by NIR laser of 808 nm (1.25 W/cm2) for 6 min. The solution temperature and infrared thermographic map were recorded during NIR irradiation by using an infrared thermal imaging camera (Thermo Shot F20, NEC Avio Infrared, Germany). The temperature profiles of the ICG/PEI10k-encapsulated PNM solutions containing 20 µM ICG treated with NIR laser irradiation of different power densities were also monitored. In Vitro Cellular Uptake. HeLa cells (2×105 cells/well) in 24-well culture plates were incubated with free ICG species and ICG/PEI-encapsulated PNMs, respectively, at an ICG concentration of 10

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µM at 37 oC for 3 h. DMSO (0.65 mL) was then added for cell disruption. An in vivo imaging system (Xenogen IVIS Spectrum) was utilized to collect the fluorescence signals of ICG (745 nm excitation and 810 nm filter). The cellular uptake was further studied by confocal laser scanning microscopy (CLSM) (Zeiss LSM 780, Jena, Germany). HeLa cells (2×105 cells/well) were seeded in 6-well plate containing 22 mm round glass coverslips and cultured overnight. The cells were then incubated with free ICG and ICG/PEI-loaded PNMs, respectively, at an ICG concentration of 10 µM for 3 h. Upon washing twice with PBS and fixing with 4 % formaldehyde, the cells were stained with Hoechst 33342 for 15 min, and the slides were rinsed three times with PBS. The fluorescence images were obtained at the excitation wavelengths of 405 and 633 nm for Hoechst and ICG, respectively. In Vitro Photothermal Efficacy. HeLa cells (105 cells/well) were seeded in a 24-well plate and incubated at 37 oC for 24 h in DMEM containing 10 % FBS and 1 % penicillin. The medium was then replaced with 0.5 mL of fresh medium containing either free ICG or ICG/PEI10k-encapsulated PNMs at varying ICG concentrations and the cells were incubated for additional 24 h. After being washed twice with PBS, the cells were detached by trypsin-EDTA and centrifuged. The collected cell pellet colloids were then dispersed in DMEM (20 µL) and irradiated by 808 nm laser (1.25 W/cm2) for 5 min. Following the addition of DMEM (0.98 mL), the laser-treated cells were reseeded in a 96-well plate and incubated for additional 24 h. Afterward, 200 µL MTT (0.25 mg/mL) was added into each well, followed by additional 4 h incubation at 37 oC. After discarding the culture medium by trypsin-EDTA, DMSO (0.98 mL) was added into each well to dissolve the precipitate and the

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absorbance of resulting solution at 570 nm was measured with a FLUOstar OPTIMA microplate reader. The cell viability (%) was calculated by the following formula:

Cell viability % =

× 100

where Asample and Acontrol are the absorbance of the sample and the control, respectively. The cytotoxicity of photothermal treatment was also assessed by Hoechst/PI staining. HeLa cells (3×105 cells/well) in 6-well plate were incubated with either free ICG or ICG/PEI10k-loaded PNMs (ICG concentration = 10 µM) for 24 h and irradiated by NIR 808 nm laser (1.25 W/cm2) for 5 min at a selected area. Finally, the cells were double stained with Hoechst 33342 and propidium iodide (PI), and then analyzed on a Nikon ECLIPSE Ti-U inverted microscope.

Results and Discussion Synthesis and Characterization of PLGA-b-PEG copolymer. The PLGA-b-PEG copolymer employed in this work was synthesized by the DCC/DMAP-mediated aminolysis of acid terminated PLGA and maleimide-PEG-NH2. The gel permeation chromatography profile of the resultant PLGA-b-PEG shows a unimodal curve (polydispersity of ca. 1.35) (Figure S1). The composition of PLGA-b-PEG di-block copolymer was characterized by 1H-NMR. Based on the integral ratio of the characteristic proton signals from the methyl groups (1.55 ppm) of PLGA and the ethylene groups (3.61 ppm) of PEG, a high conjugation efficiency (over 96 %) of PLGA with PEG was attained in this study (Figure 1b).

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Preparation and Characterization of Pristine PNMs and ICG/PEI-Loaded Hybrid PNMs. In this work, through the one-step nanoprecipitation approach, pristine PNMs and ICG-loaded PNMs were prepared in aqueous solution of pH 7.4. Different from the nearly transparent PNM solution, a green precipitate was observed at the bottom of the ICG-loaded PNM solution, because amphiphilic ICG was not effectively entrapped into the hydrophobic PLGA core of PNMs due to its two anionic sulfonate groups. We postulated that it might be possible to improve the encapsulation of the negatively charged ICG into the solid PLGA core structure by forming hydrophobic electrostatic complexes with the positively charged PEI segments. To prove the feasibility of the concept, the PEI1.8k/ICG mixture with a N/S ratio of 9.0 in DMSO was added dropwise into phosphate buffer (pH 6.0). Substantial green precipitates were immediately observed (Figure 2a), indicating the formation of hydrophobic ICG/PEI ionic complexes. Based on the observation, PEI1.8k was utilized for the preparation of the ICG/PEI-loaded PNMs. Importantly, distinct from the visible phase separation of ICG/PEI complex from aqueous solution, the obtained ICG/PEI1.8k-loaded PNMs (N/S ratio = 9.0) were well suspended in aqueous phase (Figure 2a), suggesting that the ICG/PEI complexes could be effectively enclosed in the PNMs by associating with PLGA blocks via hydrophobic interactions. The hybrid cores thus constructed are further stabilized by the highly hydrated PEG outer shells (Figure 2b). In order to attain ICG delivery nanovehicles with high cargo loading capacity and the desired physiochemical properties, a series of ICG/PEI-encapsulated PNMs comprising PEI1.8k or PEI10k were prepared and characterized.

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(b)

(a)

(e)

1.6

ICG/PEI1.8k-loaded PNMs ICG/PEI10k-loaded PNMs Pristine PNMs

1.4

Absorbance (a.u.)

20

Intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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15

10

5

0 10

100

1000

(f)

Free ICG ICG/PEI10k-encapsulated PNMs ICG/PEI1.8k-encapsulated PNMs

1.2 1.0 0.8 0.6 0.4 0.2 0.0 500

600

Hydrodynamic diameter (nm)

700

800

900

Wavelength (nm)

Figure 2. (a) Photographs of aqueous solutions of (i) pristine PNMs, (ii) ICG-loaded PNMs, (iii) ICG/PEI1.8k mixtures and (iv) ICG/PEI1.8k-encapsulated PNMs. (b) A scheme showing the structure of ICG/PEI-encapsulated PNMs. TEM images of (c) ICG/PEI10k-loaded PNMs and (d) ICG/PEI1.8k-loaded PNMs. (e) DLS particle size distribution profiles of pristine PNMs, ICG/PEI10kand ICG/PEI1.8k-encapsulated PNMs. (f) UV/Vis absorption spectra of free ICG, ICG/PEI10k- and ICG/PEI1.8k-encapsulated PNMs in aqueous solutions.

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As shown in TEM images (Figure 2c and d), the ICG/PEI10k- and ICG/PEI1.8k-encapsulated PNMs have a well-dispersed spherical shape. The particle size of ICG/PEI10k-encapsulated PNMs (ca. 80 nm) is appreciably smaller than that of the ICG/PEI1.8k counterparts (ca. 105 nm). The DLS data illustrate that both PNMs loaded with ICG/PEI10k and ICG/PEI1.8k have a mono-modal size distribution in aqueous solution (Figure 2e) and the mean hydrodynamic diameter (Dh) (ca. 104.9 nm) of the former is smaller comparing with the latter (134.3 nm) (Table 1). Since it has been reported that long branched PEI segments are apt to form more stable and compact ionic complexes with negatively charged molecules (e.g. siRNA and DNA),[26,27] such a difference in particle size can be ascribed to the varying compact degrees of the hybrid hydrophobic cores composed of PLGA segments and ICG/PEI complexes of different PEI molecular weights. It should be noted that the particle sizes of ICG/PEI-encapsulated PNMs analyzed by DLS are somewhat larger than those observed by TEM owing to the transition of PNMs from swollen state (DLS) to dried state (TEM).[28,29,30] The zeta potential of the PNMs upon the encapsulation of the ICG/PEI complexes was shifted from -11.1 to 1.0 mV, indicating that the surface of ICG/PEI-loaded PNMs is close to neutral presumably due to the partial protonation of the amines of uncomplexed PEI segments at the PNM surfaces and the presence of the outer PEG segments capable of significantly shielding the positive charges of protonated amine groups.[31,32] On the other hand, as shown in Figure 2f, a significant red shift from 776 nm to 802 nm of the feature absorption peak of ICG was observed as the ICG/PEI complexes were confined within the

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PNMs. This result indicates that the microstructure of ICG is considerably affected by its strong binding with PEI within the PNMs. Such a red-shift in maximum absorption of ICG combined with chitosan via electrostatic interaction was observed as well.[13] Both ICG/PEI10k- and ICG/PEI1.8k-encapsulated PNMs have a high drug loading efficiency (ca. 70 %) and a loading content (ca. 6.0 wt%), as presented in Table 1. Compared to the previously developed PLGA-based ICG nanocarriers suffering from a rather low ICG loading efficiency and a large particle size,[21,22,24] the ICG/PEI-loaded PNMs in this work not only have a suitable size for EPR effect but also display considerably enhanced ICG loading capacity. The particle size and ICG loading efficiency of ICG/PEI-loaded PNMs were found to be governed by the N/S ratio in feed (Figure S2 and Table S1). For example, changing the N/S ratio from 9.0 to 4.5 led to a significant increase in particle size and a concomitant reduction in ICG loading efficiency for the ICG/PEI10k-encapsulated PNMs. It is presumed that the structure of ICG/PEI complexes formed at N/S ratio of 4.5 is loose and unstable due to incomplete complexation. The loose ICG/PEI complexes in the hybrid cores are not able to adequately prevent cargo outflow during the purification of PNMs. When more PEI10k segments were added by increasing N/S ratio from 9 to 13.5, on the other hand, the particle size of the ICG/PEI10k-loaded PNMs decreased with an unchanged ICG loading efficiency. These results imply that most of the ICG molecules can couple with PEI10k segments at N/S ratios of 9.0 and 13.5 to form stable and dense complexes. In order to maintain high ICG loading efficiency as well as avoid cytotoxicity associated with the long branched

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PEI10k segments, the ICG/PEI10k-encapsulated PNMs prepared with a N/S ratio of 9.0 were employed for the subsequent experiments.

Table 1. Physiochemical properties, drug loading efficiency and capacity of ICG/PEI-loaded PNMs and pristine PNMs.

Sample Pristine PNMs

ICG/PEI1.8k-loaded PNMs

ICG/PEI10k-loaded PNMs

a)

ZP: Zeta potential;

b)

LE: loading efficiency

c)

LC: loading content

a)

ZP (mV)

b)

LE (%)

c)

Dh (nm)

PdI

LC (wt %)

135.8 ± 2.9

0.16 ± 0.04

-11.1 ± 1.1

-

-

134.3 ± 10.5

0.18 ± 0.05

1.3 ± 0.1

69.0 ± 1.7

5.9 ± 0.1

104.9 ± 9.7

0.16 ± 0.02

1.8 ± 0.3

72.8 ± 2.0

6.2 ± 0.2

Optical and Colloidal Stability of ICG-Encapsulated Hybrid PNMs. Since the prolonged photo-stability of ICG in aqueous phase is a prerequisite for effective cancer theranosis, the optical stability of the ICG/PEI-loaded PNMs in PBS (I = 0.15 M) at 37 oC was assessed by monitoring the variation in absorbance over time. Based on the UV/Vis absorption spectra of ICG/PEI-loaded PNMs and free ICG (Figure S3), the maximum absorbance of ICG at each preset time is normalized to that at the beginning. As shown in Figure 3a, the normalized absorbance of free ICG in PBS was

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remarkably decreased within 7 days due to its self-aggregation and degradation. Similar results were also observed elsewhere.[13,19] In contrast, the change in ICG absorbance of ICG/PEI-encapsulated PNMs with time was appreciably lowered, in particular for ICG/PEI10k-encapsulated PNMs, signifying that the encapsulation of the ICG/PEI complexes in PNMs could adequately prevent self-aggregation and degradation of ICG in aqueous phase. Furthermore, over a period of 7 days, the ICG/PEI10k-loaded PNMs in PBS maintained unvaried particle size and size distribution (Figure 3b), demonstrating that the ICG/PEI10k-loaded PNMs exhibited excellent aqueous photo-stability and colloidal stability, probably owing to their compact inner ICG/PEI10k complexes in resisting the disruption of electrostatic interactions under high salt concentration. The ICG/PEI1.8k-encapsulated PNMs, in contrast, showed appreciable enlarged particle size and broadened size distribution (Figure 3b), indicating the ICG/PEI1.8k-encapsulated PNMs became swollen possibly due to the impairment of the loose ionic ICG/PEI1.8k complexes by salt ions. The colloidal stability of ICG/PEI-encapsulated PNMs in DMEM containing 10 % FBS was also evaluated by DLS measurement. Notably, no significant change in particle size was observed within 24 h for the ICG/PEI10k- or ICE/PEI1.8k-loaded PNMs (Figure 3c and d), indicating that the outer PEG segments of PNMs could avoid inter-particle aggregation in FBS-containing environment. Interestingly, the signals corresponding to serum proteins (ca. 10 nm) in ICG/PEI1.8k-loaded PNM suspension became undetectable after 3 h due to their adsorption to PNMs. The signals, on the other hand, in the ICG/PEI10k-loaded PNM solution remained detectable after at least up to 9 h incubation,

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implying that the ICG/PEI10k-loaded PNMs have a superior capability in resisting non-specific protein adsorption compared to the ICG/PEI1.8k-loaded PNMs. Furthermore, the colloidal size of the ICG/PEI10k-encapsulated PNMs upon large-volume dilution with PBS remained essentially unchanged

(Figure

S4).

Based

on

these

results,

it

is

anticipated

that

the

robust

ICG/PEI10k-encapsulated PNMs can better preserve structural integrity in blood circulation after parenteral administration to avoid rapid clearance of ICG from the body.

(a)

Dh of ICG/PEI1.8k-loaded PNMs

350

PDI of ICG/PEI10k-loaded PNMs 0.4 PDI of ICG/PEI1.8k-loaded PNMs

80 300 70 60 50 40

ICG/PEI10k-loaded PNMs ICG/PEI1.8k-loaded PNMs Free ICG

30 0

1

2

3

4

0.3

250 200

0.2

150 0.1 100 5

6

7

0

1

12 10

18 0h 3h 9h 24 h

16

(d)

14

Intensity (%)

(c)

2

3

4

5

6

7

8

Time (day)

Time (day)

14

0.5

Dh of ICG/PEI10k-loaded PNMs

(b)

PDI

90

400

Dh (nm)

Normalized absorbance (%)

100

Intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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8 6 4 2

12

0h 3h 9h 24 h

10 8 6 4 2

0

0 1

10

100

1000


1

10

100

1000


Figure 3. (a) Normalized absorbance of free ICG and ICG/PEI-encapsulated PNMs in PBS at different time intervals. (b) Particle size and size distribution variation of ICG/PEI10k- and ICG/PEI1.8k-encapsulated PNMs in PBS at 37oC as a function of time. DLS particle size distribution profiles of (c) ICG/PEI10k-loaded PNMs and (d) ICG/PEI1.8k-loaded PNMs suspended in DMEM

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containing 10 % FBS at different time intervals. In Vitro ICG Leakage Study. In order to test if the undesired premature cargo leakage from ICG/PEI-loaded PNMs occurs, the in vitro ICG leakage was evaluated by dialysis. Compared to rapid diffusion of free ICG species (over 70 %) across the dialysis tube at pH 7.4 over a period of 12 h, the ICG leakage rates of ICG/PEI-encapsulated PNMs at the same pH are significantly slower (Figure 4). Obviously, the hydrophobic PLGA-rich core acting as a barrier can efficiently inhibit ICG efflux

from

PNMs

under

the

simulated

physiological

conditions.

Furthermore,

for

ICG/PEI-encapsulated PNMs, significant lower ICG leakages were attained at pH 5.0 relative to pH 7.4 as a result of the enhanced electrostatic attraction between ICG species and PEI segments in weak acidic environment where the protonation of PEI was further increased. This could prevent ICG from biodegradation in acidic organelles such as endosomes and lysosomes after the ICG/PEI-loaded PNMs are internalized by cancer cells.[19] Importantly, consistent with the results of optical property and colloidal stability studies (Figure 3a and b), the lower ICG leakages for the ICG/PEI10k-loaded PNMs compared to that for the ICG/PEI1.8k-encapsulated PNMs at both pH 7.4 and 5.0 further verify that the dense hybrid core comprised of the compact ionic ICG/PEI10k complexes and the hydrophobic PLGA segments can effectively suppress ICG outflow. Based on these results, it can be concluded that the ICG/PEI10k-loaded PNMs are promising for cancer imaging and PTT.

18


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Cumulative ICG leakage (%)

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120

ICG/PEI10k-loaded PNMs at pH 7.4 ICG/PEI10k-loaded PNMs at pH 5.0 ICG/PEI1.8k-loaded PNMs at pH 7.4 ICG/PEI1.8k-loaded PNMs at pH 5.0 Free ICG at pH 7.4

100 80 60 40 20 0 0

4

8

12

16

20

24

Time (h)

Figure 4. Cumulative ICG leakage profiles of ICG/PEI-loaded PNMs at pH 5.0 and 7.4 at 37 oC, using the dialysis technique. For comparison, diffusion of free ICG across the dialysis tube at pH 7.4 is included. Photothermal Effect of ICG-Encapsulated Hybrid PNMs Triggered by NIR Laser Irradiation. Since the photothermal conversion capability of ICG molecules plays a key role in the efficacy of PTT, the temperature change of the ICG/PEI10k-loaded PNM solution during NIR laser irradiation (808 nm, 1.25 W/cm2) was monitored using an infrared thermal imaging camera. As revealed in Figure 5a, under 2 min NIR laser irradiation, the ICG/PEI10k-loaded PNM dispersion and free ICG solution (ICG concentration = 10 µM) exhibited the appreciably temperature elevation (ca. 10 oC), whereas PBS remained a nearly constant temperature. This illustrates that the ICG molecules bound with PEI segments within the PNMs still preserve the original photothermal conversion property. Increasing the ICG concentration from 10 µM to 30 µM, the laser-triggered temperature rise of the ICG/PEI10k-loaded PNM solution was further augmented by more than 22 oC. Due to the irreversible photo-bleaching and thermal degradation of ICG exposed to NIR irradiation, the photo-triggered 19


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hyperthermia of ICG-containing solution after 2 min NIR irradiation declined appreciably. Such a decreased ICG-based photothermal conversion ability with prolonging NIR irradiation time was also reported elsewhere.[4] Moreover, the photothermal conversion effects of the ICG/PEI10k-loaded PNMs were dependent on the laser power density (Figure 5b). Obviously, the use of higher laser power density can increase the photothermal conversion capability of ICG. Such a hyperthermia produced by NIR irradiation of ICG/PEI10k-encapsulated PNMs could lead to irreversible damages to neoplastic cells.

Inceased temperature (oC)

35 30

ICG/PEI10k-loaded PNMs (10 µM ICG) ICG/PEI10k-loaded PNMs (20 µM ICG) ICG/PEI10k-loaded PNMs (30 µM ICG) Free ICG (10 µM) PBS

(a)

25

PBS

ICG/PEI10k-loaded PNMs

20 15 10 5 0 0

50

100

150

200

250

300

350

400

Laser irradiation time (s)

Increased temperature (oC)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 29

25

(b)

20

2

1.25 W/cm 2 0.8 W/cm 2 0.5 W/cm

15 10 5 0 0

50

100 150 200 250 300 350 400

Laser irradiation time (s)

Figure 5. (a) Effect of ICG concentration on temperature change of ICG/PEI10k-encapsulated PNMs under NIR laser irradiation (808 nm, 1.25 W/cm2). The infrared thermographic maps of PBS and the ICG/PEI10k-encapsualted PNMs (30 µM ICG) under NIR irradiation are also included. (b) Effect of

20


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NIR laser power density on temperature change of ICG/PEI10k-encapsulated PNMs (20 µM ICG) in PBS. In Vitro Cellular Uptake. The cellular uptake of ICG/PEI-loaded PNMs by HeLa cells was assessed by IVIS and CLSM. Figure 6a shows the quantity of ICG uptake by HeLa cells based on fluorescence image and intensity. Cells without ICG treatment were used as the negative control. Compared to the control, the ICG fluorescence detected is indicative of the cellular uptake of free ICG and ICG/PEI-encapsulated PNMs. The fluorescence signals of ICG from HeLa cells treated with ICG/PEI1.8k- and ICG/PEI10k-loaded PNMs are 2.9 and 4.1 folds higher than that from cells incubated with free ICG, suggesting that the ICG/PEI-loaded PNMs are capable of effectively promoting the intracellular ICG transport. A significant reduction in ICG fluorescence intensity was observed from HeLa cells incubated at 4 oC with all ICG-containing formulations (Figure S5), indicating that the cellular uptake of ICG in either free drug or nanoparticles is achieved via the energy-dependent endocytosis pathway. As shown by the CLSM images in Figure 6b, HeLa cells incubated with ICG/PEI-loaded PNMs displayed substantially higher fluorescence intensity in the cytoplasm region compared to free ICG-treated HeLa cells, demonstrating that the endocytosis of ICG/PEI-loaded PNMs was more efficient than that of free ICG. The phenomenon can be attributed to the intense interactions between cell membranes and the ICG/PEI-loaded PNMs featuring a near-neutral surface, whereas free ICG-based aggregates are repulsive to the cell membrane due to their negatively-charged surface. Such an enhanced intracellular ICG delivery achieved by

21


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nanovehicles was also reported elsewhere.[20,33]

(a)

1E9

Total flux (p/s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1E8

ICG /PE

Fre ICG e IC /P E I10k G I1.8k -loa l oad ded ed P PNM NM s s

Cel ls a

lone

(b)

Figure 6. (a) NIR fluorescence images and intensity of ICG molecules from HeLa cells incubated with free ICG, ICG/PEI10k- and ICG/PEI1.8k-encapsulated PNMs (ICG concentration = 10 µM), at 37 o

C for 3 h. (b) CLSM images of HeLa cells treated with free ICG, ICG/PEI10k- and

22


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ICG/PEI1.8K-loaded PNMs (ICG concentration = 10 µM) at 37 oC for 3 h. Cell nuclei were stained with Hoechst 33342. Scale bars are 10 µm. In Vitro Photothermal Therapy. The cytotoxic effect of photo-triggered hyperthermia on HeLa cells treated with ICG/PEI10k-loaded PNMs or free ICG was evaluated. Without NIR laser irradiation, the viability of HeLa cells treated with either free ICG or ICG/PEI10k-encapsulated PNMs maintained rather high, confirming that ICG molecules and the PNMs are virtually nontoxic to HeLa cells (Figure 7a). Notably, upon NIR irradiation, the cytotoxicity of ICG/PEI10k-loaded PNMs was more pronouncedly increased than that of free ICG. Since NIR laser irradiation alone did not lead to a decline in HeLa cells viability (data not shown), the observed increase in cytotoxicity can be attributed to the NIR irradiation-induced hyperthermia mediated by ICG/PEI10k-loaded PNMs. A similar result was also observed in the fluorescence staining of live/dead cells, where most of HeLa cells incubated with ICG/PEI10k-loaded PNMs with NIR irradiation presented PI-positive staining due to extensive cell death (Figure 7b). The results can be ascribed to the following two reasons. First, in comparison with free ICG, ICG/PEI10k-loaded PNMs were presumably internalized by HeLa cells in a more effective manner, leading to a higher intracellular ICG concentration and a more profound photo-triggered hyperthermia effect. Second, the ICG/PEI10k-loaded PNMs can remarkably reduce the leakage and biodegradation of ICG in the acidic environments of endosomes/lysosomes, thereby better preserving the photothermal conversion ability. Based on these findings, it can be concluded that the ICG/PEI10k-loaded PNMs developed in this work are promising for the

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imaging-guided cancer PTT. 120

(b)

(a)

ICG/PEI10k-loaded PNMs Free ICG

100

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 60 40 20 0

1 2.5

5

10 20

w/o NIR irradiation

1 2.5

5

10 20

w/ NIR irradiation

ICG concentration (µM)

Figure 7. (a) Cell viability of HeLa cells incubated with ICG/PEI10k-loaded PNMs and free ICG for 24 h with and without NIR laser irradiation (5 min). (b) Fluorescence images of HeLa cells treated with free ICG or ICG/PEI-loaded PNMs with and without laser irradiation. An ICG concentration of 10 µM was used. Viable cells are Hoechst 33342-positive and PI-negative, whereas dead cells are Hoechst 33342-positive and PI-positive.

Conclusions In this work, the theranostic nano-micelles featured by a hydrophobic PLGA/ICG/PEI hybrid core surrounded by the highly hydrated PEG shells were attained by means of the co-association of hydrophobic electrostatic ICG/PEI complexes and PLGA-b-PEG copolymers in aqueous phase. The robust hybrid cores constructed of dense ICG/PEI10k complexes and PLGA blocks not only facilitate ICG loading but also enhance its aqueous optical stability. The resulting ICG/PEI10k-loaded PNMs showed several outstanding properties including (1) the superior optical and colloidal stability, (2) significantly reduced ICG leakage, (3) promoted intracellular ICG transport and photothermal 24


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cytotoxicity, and thus exhibit great potential for practical applications in cancer imaging and PTT. Acknowledgements This work is supported by the Ministry of Science and Technology (NSC 102-2218-E-007-007 and MOST 103-2221-E-007-023), Taiwan.

Supporting Information Size exclusion chromatography profiles of PLGA-b-PEG and maleimide-PEG-NH2, DLS size distribution profiles of the ICG/PEI10k-loaded PNMs with different N/S ratios in aqueous solution, DLS size distribution profiles of the ICG/PEI10k-encapsulated PNMs (N/S ratio = 9) upon PBS dilution, UV/Vis absorption spectra of free ICG and ICG/PEI-loaded PNMs in PBS (37 oC) at different time intervals, physiochemical properties, drug loading efficiency and capacity of ICG/PEI10k-loaded PNMs with varied N/S ratios, NIR fluorescence intensity of ICG molecules from HeLa cells incubated with free ICG, ICG/PEI10k- and ICG/PEI1.8k-encapsulated PNMs at 4 oC for 3 h. This information is available free of charge via the Internet at http://pubs.acs.org/.

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Table of Contents graphic Indocyanine Green-Encapsulated Hybrid Polymeric Nano-Micelles for Photothermal Cancer Therapy

NIR irradiation

PTT

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Indocyanine Green-Encapsulated Hybrid Polymeric Nanomicelles for

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