Supercritical fluid-assisted fabrication of Indocyanine green

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Supercritical fluid-assisted fabrication of Indocyanine green-encapsulated silk fibroin nanoparticles for dual-triggered synergistic cancer therapy Biao-Qi Chen, Ranjith Kumar Kankala, Geng-Yi He, Da-Yun Yang, GuoPing Li, Pei Wang, Shi-Bin Wang, Yu Shrike Zhang, and Ai-Zheng Chen ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018

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ACS Biomaterials Science & Engineering

Supercritical indocyanine

fluid-assisted

fabrication

green-encapsulated

silk

of

fibroin

nanoparticles for dual-triggered cancer therapy Biao-Qi Chen,†,‡ Ranjith Kumar Kankala,†,‡,§ Geng-Yi He,‡,§Da-Yun Yang,⊥ Guo-Ping Li,‡ Pei Wang,‡ Shi-Bin Wang,‡,§Yu Shrike Zhang,*,|| and Ai-Zheng Chen,*,‡,§ ‡

Institute of Biomaterials and Tissue Engineering, Huaqiao University, Xiamen 361021, P. R.

China. §

Fujian Provincial Key Laboratory of Biochemical Technology, Xiamen 361021, P. R. China

⊥Fujian

Key Laboratory for Translational Research in Cancer and Neurodegenerative Diseases,

Institute for Translational Medicine, School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350108, China ||Division

of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital,

Harvard Medical School, Cambridge, MA 02139, USA.

Mailing Addresses: Institute of Biomaterials and Tissue Engineering, Huaqiao University, 668, Jimei Avenue, Xiamen 361021, People’s Republic of China. Email: [email protected] (A. Z. Chen) Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA 02139, USA. Email: [email protected] (Y. S. Zhang) †

These authors contributed equally to this work 1 ACS Paragon Plus Environment

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ABSTRACT

Despite the success and advantages over traditional chemotherapeutic strategies, photothermal therapy (PTT) suffers from certain limitations such as poor stability and low therapeutic efficacy of PTT agents in vivo, and their affinity loss during the multi-step synthesis process of delivery carriers, among others. To address these limitations, we designed a stable, biocompatible, and dual-triggered formulation of indocyanine green (ICG)-encapsulated silk fibroin (SF) (ICG-SF) nanoparticles using the supercritical fluid (SCF) technology. We demonstrated that ICG encapsulation in SF through this environmental-friendly approach has offered excellent photothermal stability, the pH-responsive release of ICG from SF specifically in the tumor acidic environment and its substantial activation with near-infrared (NIR) light at 808 nm significantly enhanced the PTT efficiency. In vitro and in vivo photothermal experiments have shown that these ICG-SF nanoparticles were capable of devastating tumor cells merely under light-induced hyperthermia. Together, these results have suggested that the biocompatible ICG-SF nanoparticles prepared by the SCF process resulted in high PTT efficiency, and may have a great potential as a delivery system for sustained cancer therapy.

KEYWORDS: Photothermal therapy, pH-sensitive release, Supercritical fluid technology, Cancer, Hyperthermia

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INTRODUCTION In the past few decades, various light-induced therapeutic strategies have become potential alternatives to conventional approaches in the healthcare field due to their attractive properties such as high selectivity, minimal invasiveness, biocompatibility, and preferential localization of photosensitizers resulting in minimal adverse effects.1-3 Photothermal therapy (PTT) is one amongst them, in which the photosensitizers i.e., PTT agents, act by transforming the absorbed light energy at a specified wavelength window to heat to exhibit the therapeutic effects.4-8 Recently, there has been a great interest in developing near-infrared (NIR)-induced thermotherapy against cancer due to the excellent tissue penetration, high efficiency, and minimal invasiveness of NIR.9-10 Indocyanine green (ICG), is a United States Food and Drug Administration (FDA)-approved NIR (>750 nm) fluorescence dye for various biomedical applications. It is of particular interest in biomedical imaging, diagnosis, and therapeutics due to its attractive properties such as significant light-to-heat transformation efficacy, and excellent biocompatibility, among others.4-5, 11-12

However, the clinically approved ICG still suffers from a few limitations such as poor

aqueous stability and intestinal absorption, concentration-dependent aggregation in vivo resulting in poor intrabody recirculation, poor plasma half-life (t1/2 = ~3-4 min), prone to photobleaching, and lack of target specificity.6,

11, 13

To address these issues, ICG has been delivered using

different nanocarriers with improved stability and targeting efficacy such as mesoporous silica nanoparticles (MSNs), layered double hydroxides (LDHs), liposomes, and polymeric carriers (PCs), among others.4-5, 12, 14-17



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Spurred by recent progress in fabricating various smart carriers for drug delivery, stimuliresponsive nanomaterials have become one of the most efficient carriers that deliver a drug in spatial-, temporal- and dosage-controlled fashions, while undergoing physical or chemical modification in response to the external stimuli.18-19 It is evident that most of the solid tumors possess lower pH (i.e., 4.5-6.0) compared to the surrounding physiological fluids, which can be used as one of the external biological stimuli for the delivery of therapeutic cargos. In addition, the nanoparticles as drug delivery carriers can gain access to the tumor tissue through enhanced permeation and retention (EPR) effect and enter the cancer cells through endocytosis. Moreover, these carriers end up in the lysosomal acidic environment (low pH ~4.5) where they are exposed to proteolytic enzymes that trigger the drug release from the stimulus-responsive polymeric constructs.20 Implementation of such devices requires the use of biocompatible materials that are susceptible to a specific physical incitement in response to the pH such as functional groups that undergo protonation and a hydrolytic cleavage and a (supra)molecular conformational change.21 Thus, engineering a multifunctional design with this stimuli-responsive material is an essential prerequisite in fabricating therapeutic devices that can significantly perform therapeutic duties. This can be achieved by designing materials with hierarchical structures across several length scales or by embedding active molecules at the point of material formation.22 Silk fibroin (SF) is one such biodegradable natural protein extracted from the Bombyx mori (B. mori) caterpillar, and has been widely used in various biomedical applications such as tissue engineering, drug delivery system, and filters, due to its biocompatibility, optical transparency, and robust mechanical properties.23-26 As the major component of fibroin, the fibroin heavy chain is a considerably large protein comprising N-terminal and C-terminal hydrophilic domains and twelve highly repetitive glycine-alanine-rich regions flanked by internal hydrophilic blocks.27 In 4 ACS Paragon Plus Environment

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this framework, the fibroin N-terminal domain (FibNT) undergoes a pH-responsive conformational transition at pH less than 6.0 from random coils to β-sheets, resulting in the delivery of loaded therapeutic cargo.27-28 The biocompatibility of SF and its remarkable material properties have allowed the development of a diverse spectrum of material formats that can be utilized for a broad range of biomedical applications such as sponges, fibers, films, and micro-, and nano-spheres.23-24,

27, 29

Numerous studies have demonstrated the preparation of SF

nanoparticles such as poly(vinyl alcohol) blends (particle size range 300 nm to 10 μm), emulsification (>6 μm), capillary microdot printing (25−140 nm), salting leaching (486−1200 nm), supercritical CO2 (50−300 nm),25, 29 and organic solvent precipitation (35−170 nm). Some of these studies have examined the ability of SF in its nanoparticulate forms to entrap and release the drugs.23-25,

27-30

Amongst them, the SCF technology has attracted great interest from

researchers in the past decade for fabricating the polymers and other therapeutic actives due to its environmentally benign nature and economically promising character.25,

31-32

In addition, this

technology offers numerous advantages over other conventional processes such as acceptable limits of organic residues in the end product, single step fabrication, uniform size distribution of particles, enables the control over the particle morphology by altering the critical conditions, among others.28, 33-36 In an attempt to accomplish the fabrication of versatile dual-triggered design with high therapeutic efficacy and desirable biodegradation, herein, we designed ICG-encapsulated SF nanoparticles using a single step, environmental-friendly supercritical anti-solvent (SAS) process (Figure 1). The SF nanoparticles not only protects the ICG from plasmatic proteins to control the elimination rate of ICG in the physiological medium, but also enhances the PTT efficiency by its pH-responsive drug release. Furthermore, in vitro and in vivo experiments were performed 5 ACS Paragon Plus Environment


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systematically to evaluate the performance of the ICG-SF nanoparticles as biodegradable and biocompatible PTT agents in cancer therapy.

Figure 1. Schematic illustration showing the outline of preparation of ICG-SF nanoparticles by the SAS process and dual-triggered cancer therapeutics.

EXPERIMENTAL SECTION Materials. ICG, and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP, 99%) were purchased from Sigma-Aldrich (Santa Barbara, USA). Protease X IV from Streptomyces griseous, phosphate buffered saline (PBS, pH-7.4), fetal bovine serum (FBS), high glucose- Dulbecco’s modified Eagle medium (HDMEM), minimum essential medium (MEM), trypsin-ethylenediaminetetraacetic acid (TrypsinEDTA), Cell Counting Kit-8 (CCK-8), calcein AM, propidium iodide (PI), and 4',6-diamidino-2phenylindole (DAPI) were obtained from Life Technologies of Thermo Fisher Scientific 6 ACS Paragon Plus Environment

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(Waltham, USA). CO2 of 99.9% purity was purchased from the Rihong Air Products Co., Ltd. (Xiamen, China). SAS process. In the SAS process, the CO2 fed from the cylinder was rapidly cooled down to around 3 °C by a cooler to ensure the liquefaction of the fluid before it entered the pump, thus avoiding cavitation. A heat exchanger was used to preheat the liquefied CO2 to the desired operating temperature after it left the pump head. The CO2 was then delivered by a high-pressure meter pump to the high-pressure vessel. When the desired pressure and temperature at the supercritical state of CO2 were achieved, a steady flow of CO2 was maintained, and the system pressure was controlled by adjusting a downstream valve and maintained the constant pressure. In addition, the precipitation temperature was regulated by placing the high-pressure vessel in a gas bath.29 Fabrication of ICG-SF nanoparticles. In our previous study, a 24 full factorial design of SCF-assisted particle fabrication indicated that the decrease of the concentration as well as flow rate of the SF solution and operational temperature resulted in the reduced particle size as well as particle size distribution of SF nanoparticles.29 Motivated by such interesting results, we fabricated ICG-encapsulated SF nanoparticles using the single-step SAS process, following the reported procedure (Figure S1).29 Intact cocoons from B. mori were used as predominant source of fibroin. SF was extracted by a standard degumming process by following the reported procedure.37 Herewith, the SF solution (20 mL) was prepared by dissolving the SF dry powder in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP, 99%), and then different theoretical amounts of ICG (2, 4, 6% w/w of SF) was added to the mixture separately. Subsequently, 0.5% (w/v) of ICG-SF solution in HFIP was sprayed 7 ACS Paragon Plus Environment


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through a specially designed nozzle into the high-pressure vessel at a flow rate of 0.5 mL/min. The ICG-SF solution mixture (0.5 mL/min) and supercritical carbon dioxide (SC-CO2, 30 g/min) were pumped into the high-pressure vessel at constant pressure/temperature (10 MPa, 35 °C) through two different nozzles. In the end, fresh SC-CO2 was used to wash the product for approximately 30 min to remove the residual organic solvent at constant operating conditions. The high-pressure vessel was then slowly depressurized and the product was eventually collected. Physical characterizations. The surface morphology of ICG-SF nanoparticles was elucidated by FE-SEM (S-4800, Hitachi, Japan) and TEM (H-7650, Hitachi, Japan). The samples were absorbed onto the conducting resin and then sprayed with gold under vacuum conditions before SEM observation. For TEM observation, a copper micro-grid supporting a carbon-coated film was used as a sample holder. The aqueous solution of the samples prepared by ultrasonication was deposited onto the sample holder and dried at room temperature. Before the TEM observation, the ICG-SF nanoparticles were negatively stained, using phosphotungstic acid. The physicochemical properties of the ICGSF nanoparticles were characterized by FTIR spectroscopy, and TGA. FTIR spectra of ICG-SF nanoparticles were recorded on a Bruker Alpha spectrometer using dried KBr pellet method. The degradation behavior of ICG-SF nanoparticles was examined using a TGA (Q5000, TA instruments, America) from ambient temperature to 800 °C at a heating rate of 10 °C/min under pure nitrogen purge at a flow rate of 50 mL/min. Furthermore, DSC was used for the evaluation of the thermodynamic properties of the materials before and after the fabrication process. Particle size and zeta potential of SF as well as ICG-SF nanoparticles were determined by DLS (Zetasizer Nano-ZS Malvern Instrument, Worcestershire, U.K.) in dd-H2O, unless otherwise


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stated. Refractive indices of 1.33 for dd-H2O and 1.60 for protein were taken for computation of particle size. The drug loading (DL) and encapsulation efficiencies (EE) were further determined by using ultraviolet-visible (UV-vis) spectroscopy (λmax- 780 nm). The DL and EE were calculated according to equations listed below: DL (%) = W1 /W2 x 100

(1)

EE (%) = (W1-W3)/W1 x100

(2)

where W1, W2, and W3 represent the total weights of ICG after SAS processing, ICG-SF nanoparticles, and unbounded ICG after SAS processing, respectively. NIR laser-induced hyperthermia and optical properties. NIR-induced hyperthermia or light-to-heat conversion efficiency of ICG was evaluated using the following procedure. First, ICG-dispersed samples (1.5 mL) were placed in a quartz cuvette and they were then irradiated with a fiber-coupled NIR laser (MDL-N-808 nm-10 W, New Industries Optoelectronics Technology Co., Ltd. Changchun, China) at a power density of 1.5 W cm -2 for 6 min. It is mandatory that the laser spot should be adjusted such that it covers the entire surface of the cuvette. Eventually, the real-time thermal imaging was obtained and then the maximum temperature was recorded using an infrared thermal imaging camera (Tis65, Fluke, USA). In addition, the fluorescence spectra of free ICG and ICG-SF NPs were recorded using a fluorescence spectrometer (F900, Edinburgh Instruments Ltd, UK) with an excitation wavelength of 740 nm. In vitro release and degradation. 9 ACS Paragon Plus Environment


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To investigate the pH-responsive behavior of SF, ICG-encapsulated SF nanoparticles (2 mg) were dispersed in the buffer solutions (6 mL) that were adjusted to various pH values simulating the tumor acidic environment and physiological fluids (i.e., pH-4.8, 6.0, and 7.4). The ICG-SF nanoparticles were dispersed in the respective buffer solutions and then, the tubes were placed in a shaker maintained at 60 rpm and 37 °C. At predetermined intervals, aliquots of the samples were collected after centrifuging the tubes at 10,000 g for 10 min. Further, the amount of released ICG in the supernatant was determined by UV-vis spectroscopy. In addition to the delivery efficiency, we have investigated the pH-responsive degradation behavior of SF in the acidic conditions in vitro simulating the endosomal/lysosomal environment of the tumor cells. ICG-SF nanoparticles (10 mg) (n=3 per group and time point) were dispersed in 10 mL of PBS solution (pH-4.8) containing protease XIV (1.0 mg mL-1), in comparison to PBS alone (pH-7.4) as a negative control, and the samples were then incubated at 37 °C. The samples were centrifuged and the PBS was replenished at regular intervals. At predetermined time intervals, the ICG-SF nanoparticles were centrifuged (10,000 g for 10 min), and the solids were lyophilized and weighed. The residual weight percentage of the sample for different degradation time (0-5 weeks) was determined by the following formula: Residual weight (%)=Wa x 100/Wb, where Wa represents the dry weight of the sample after degradation and Wb represents the initial weight of the sample. In vitro cytotoxicity assay. The biocompatibility of the designed formulation was evaluated in various human cell cultures, i.e., normal liver cells (HL-7702), normal breast epithelial cells (MCF-10A), breast cancer cells 10 ACS Paragon Plus Environment

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(MCF-7), and cervical cancer cells (HeLa). These cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, USA) and were cultured under the recommended conditions. The viability of cells was quantitatively determined using the CCK-8 assay. Cells were incubated with the media (200 µL) containing different concentrations of ICG-SF nanoparticles (at equivalent ICG concentrations of 0, 5, 10, 20 and 50 ppm, respectively). Eventually, the absorbance at 450 nm was determined on a microplate spectrophotometer (Varioskan Flash 4.00.53, Finland) and the cell viability was normalized to the control group and calculated using the following formula. Cell viability (%) = (mean of absorbance values of treatment group/mean of absorbance values of control) × 100. In vitro photothermal efficacy. The human cancerous cells, MCF-7 and HeLa cells, were used to evaluate the PTT efficacy of ICG-SF nanoparticles. The PTT efficacy was determined both qualitatively and quantitatively using fluorescence microscopy and CCK-8 assay, respectively. After a wash with PBS, the cells were incubated with ICG-SF nanoparticles (ICG concentrations of 0, 5, 10, and 20 μg mL-1) for 4 h at 37 °C and then the respective treatments were illuminated with the 808-nm laser (1.5 W cm-2) for 5 min by adjusting the laser spot such that it fully covered the area of the respective well. After irradiation, the incubation of cells was continued for another 12 h. Then, the cells were rinsed with PBS, and co-stained with calcein AM and propidium iodide (PI) for 30 min. Afterwards, the cells were given a wash with PBS and examined by an Olympus IX71 inverted fluorescence microscope. Moreover, the cell viability was estimated by the CCK-8 assay. (see In vitro cytotoxicity assay section). 11 ACS Paragon Plus Environment


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In vitro cellular uptake study. The delivery efficiency of the SF nanoparticles was investigated using the cellular internalization study through tracking the auto-fluorescent ICG in confocal laser scanning microscopy (CLSM). MCF-7 cells were incubated with the media containing ICG (10 μg mL-1) or its equivalent concentration in the SF nanoparticles. After 2 and 4 h of incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde solution for 20 min. Further, the nuclei were stained using DAPI and the excess stain was washed with PBS. Eventually, the fluorescence of ICG in the cells were observed by CLSM (Leica TCS SP5, GER, λEX - 638 nm for ICG). In vivo photothermal experiments. Balb/c nude mice (weighing 20-30 g) were used as a model to investigate the PTT efficacy of the designed ICG-SF formulation. All experimental protocols utilizing animals were performed according to the Experimental Animal Ethics Committee of Fujian Medical University following the guidelines of the National institute of Health Animal Care and the Animal Management Rules of the Ministry of Health of the People’s Republic of China. The animals were given utmost human care and no mice were expired during the course of the experiment. To establish the tumors in situ, 1×107 cells suspended in 100 µL of PBS were subcutaneously injected into the right hind leg armpit of each mouse. After attainment of the tumor volume of approximately 200 mm3, the mice were randomly divided into 4 groups (n=6 per group) of negative control (saline solution treatment), SF nanoparticles treatment, ICG solution treatment, and ICG-SF nanoparticles treatment, and the doses were injected into the mice. At 4 h post-injection, the entire region of the tumor was irradiated with the 808-nm NIR laser (1.5 W cm-2) for 5 min. The temperature of the tumors and the infrared thermographic maps were simultaneously monitored 12 ACS Paragon Plus Environment

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by an infrared thermal camera (Tis65, Fluke, USA). The tumor volume was calculated according to the formula: volume (V) = (tumor length) × (tumor width)2/2. In addition, the tissue morphology of cancer and the major organs such as heart, lung, liver, lung, and kidney of the sacrificed mice was examined by stationing them with hematoxylin and eosin to detect the efficacy of PTT, and biocompatibility in vivo. Simultaneously, the weight of the mice was also recorded regularly at each time point.

RESULTS As shown in Figure 2A, B and S2, the field emission-scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) observations indicated that the SAS process resulted in uniform-sized spherical particles of ICG-SF with smooth surfaces. Further, the particle size was calculated by statistical analysis (Figure 2C) of 200 nanoparticles from the SEM image, which represented an average mean diameter of 99.43±27.03 nm. In addition, the average hydrodynamic diameter of the ICG-SF nanoparticles was obtained from dynamic light scattering (DLS) measurements, which gave an average size of 165.9±27.87 nm (Figure S3), indicating slightly larger size than that determined by SEM due to a small extent of aggregation during the measurement.38 However, the size of the ICG-SF nanoparticles is within the acceptable range and the particles were adequately suspended, indicating their applicability for efficient cell internalization through the EPR effect for cancer therapeutics.38 The ICG-SF nanoparticles had an overall moderate negative charge, resulting in a zeta potential of -38.60±2.41 mV in water. Further, the loading of ICG in SF was determined by UV-vis-NIR measurements. The absorption spectrum of ICG-SF nanoparticles in the UV-vis-NIR wavelength range (λ=200-1000 nm) displayed the characteristic absorption peak of ICG, indicating the successful encapsulation of ICG in the SF. Moreover, it was evident from the spectrum of ICG-SF that the characteristic 13 ACS Paragon Plus Environment


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absorption range of ICG was broader (600-950 nm) compared to that of free ICG (600-850 nm), demonstrating the successful entrapment of ICG in the polymeric framework of SF (Figure 2D). To further evaluate the chemical functionalities of the design and confirmation of ICG loading in SF, we recorded Fourier-transform infrared (FTIR) spectra of SF and ICG-SF nanoparticles and the results were compared with the free ICG and unprocessed SF (Figure 2E). It was observed that the peaks attributed to ICG and SF were robust during the SCF treatment without structural changes and the successful encapsulation of ICG in the SF nanoparticles. For raw SF, the peaks at 1625 cm-1 (amide I), 1515 cm-1 (amide II), and 1263 cm-1 (amide III) were attributed to the βsheets while the peaks around 1706 cm-1 (amide I), 1554 cm-1 (amide II), and 1230 cm-1 (amide III) were assigned to random coil or α-helix or both.29 For SF nanoparticles after SCF treatment, the three characteristic peaks were attributed to the amide I, amide II, and amide III that occurred at 1651, 1526, and 1235 cm-1, respectively, confirming that the polymeric framework of SF was stable and a slight transformation from the β-sheet conformation to the α-helix or random coil conformation occurred during the SCF treatment. To this end, ICG-SF nanoparticles have resulted in the additional peaks at 1113 cm-1 and 1012 cm-1. Moreover, the peak at 1411 cm-1 was increased. These spectra explicitly revealed that the SF and ICG were robust during the SCF treatment without structural changes and the successful encapsulation of ICG in the SF nanoparticles.


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Figure 2. Physical characterization elucidating the morphology and chemical functionalities of ICG-SF nanoparticles through various techniques. (A, B) SEM images of ICG-SF nanoparticles at different magnifications. Scale bars: (A) 1 µm and (B) 500 nm (captured after subjecting to ultrasonication for attaining better dispersion). (C) Statistical analysis illustrating the average particle diameter of ICG-SF nanoparticles (n=200) based on the SEM images and surface charge of the ICG-SF nanoparticles determined by zeta potential measurements, (D) UV-vis-NIR spectra of free ICG, SF nanoparticles, and ICG-SF nanoparticles, indicating the successful encapsulation of ICG in the SF nanoparticles, (E) FTIR spectra of raw SF, SF nanoparticles, ICG, and ICG-SF nanoparticle. (F) The loading and encapsulation efficiency of ICG in SF nanoparticles at the different theoretical loading amounts of ICG (2, 4, and 6% w/w of ICG-SF).

Furthermore, the thermal behavior of raw SF and ICG-SF nanoparticles was also analyzed by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) (Figure S4 and S5). The melting point at 232.2 °C for the raw SF powder was almost the same with SF nanoparticles (236.4 °C), exhibiting its inappreciable changes after the SCF treatment. Interestingly, the encapsulated ICG made the melting peak of SF nanoparticles appearing at a higher temperature of 240.7 °C, indicating a strong interaction between ICG and SF. 15 ACS Paragon Plus Environment


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Prior to the in vitro release study, the loading capacity and encapsulation efficiency of ICG in SF nanoparticles were determined. As shown in Figure 2F, the ICG loading amounts in the SF nanoparticles at theoretical loading amounts of 2, 4, and 6% were 1.8, 3.6, and 4.9%, respectively, indicating that there was a slight loss of drug during the SAS processing because of its poor solubility in SC-CO2. On the contrary, the encapsulation efficiency of ICG in the SF nanoparticles was reduced with the increase of theoretical ICG loading amounts. The drug (ICG) and polymer (SF) were dissolved in HFIP solvents and then mixed with SC-CO2, the drug and polymer were co-precipitated due to a higher supersaturation produced by diffusion of the organic solvent into the SC-CO2 and vice versa. The most interesting characteristic feature of this formulation is the stimuli-responsive release of ICG from the polymeric framework of SF. To evaluate the pH-triggered release of ICG from SF, we performed the release study of this formulation in buffers adjusted to various pH values simulating the physiological fluid (pH-7.4) and the acidic environment of the tumor (pH-4.8 and 6.0). The released amounts of ICG at pH 4.8 were comparatively higher than those at pH-7.4, indicating that the SF could potentially serve as a natural biopolymer with the inherent ability to respond to changes in pH for triggered drug release (Figure 3A, B). We further studied the in vitro degradation behavior of the ICG-SF nanoparticles, by suspending the composites in PBS supplemented with the protease XIV (1.0 mg mL-1), a predominant constituent of the lysosomal environment responsible for degrading most of the accumulated substances. Figure 3C depicts that the ICG-SF nanoparticles were degraded by protease XIV, while maintaining the stability in PBS until 7 weeks with 90% of residual weight intact. Further, the biodegradation of ICG-SF incubated with protease XIV at 0 week (i), 1st week (ii), 4th week


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(iii), and 7th week (iv) was confirmed by the corresponding SEM images (Figure 3D), demonstrating the effective degradation of SF in the simulated lysosomal environment.

Figure 3. In vitro release rate and biodegradation behavior of ICG-SF nanoparticles. ICG release from SF was investigated by suspending the nanoparticles in various pH values, (A) after 24 h, and (B) after 10 days. (C) Graphical representation illustrating the residual weight percentage of ICG-SF nanocomposites after exposed to simulated lysosomal environment with time and their (D) corresponding real-time SEM images captured at (i) 0th week, (ii) 1st week, (iii) 4th week, and (iv) 7th week.

To evaluate the photothermal stability of ICG and ICG-SF nanoparticles, different amounts of the ICG were dispersed in the aqueous solutions and exposed to NIR laser at 808 nm (power density: 1.5 W cm-2) for 6 min. The temperature of solution was then monitored as a function of time (Figure 4A, B). At a very low concentration of ICG (50 ppm), the solution temperature had increased by more than 25 °C after irradiation. In fact, the concentration, i.e., 50 ppm, in Figure 4B (the ICG-SF NPs group) represented the actual ICG concentration, which could make it easy 17 ACS Paragon Plus Environment


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to compare with the free ICG group at the same concentration of ICG. In contrast, the temperature of pure water or a solution containing SF nanoparticles had increased by only 1.5 °C, indicating that the ICG could efficiently convert the NIR light into thermal energy. However, the encapsulation of ICG in SF had no significant effect on its photothermal efficacy. Further, we investigated the influence of SF on the stability of ICG, by determining the optical properties of all the samples of ICG with similar conditions provided. ICG and ICG-SF nanoparticles with the same amount of ICG (20 ppm) were dispersed in water and exposed to air for 8 days and then their optical properties were examined at the predetermined time intervals (0, 2, 4, 6, and 8 days) after the 808-nm laser irradiation (1.5 W cm-2) for 6 min. From Figure 4C, it could be observed that the temperature of the ICG solution after irradiation was decreased with the increase of time. In the beginning (Day 0), the rise of temperature of the ICG solution in the presence of light was 14.9 °C to that of control (water) treatment in 6 min, but after 8 days the rise of temperature was only 9.4 °C due to the weak aqueous stability and photobleaching of ICG in vitro. Comparatively, the temperature increase of the ICG-SF sample after irradiation remained consistent for 6 days at 17 °C. After 8 days, this temperature difference of the ICG-SF nanoparticles solution slightly declined to 13.5 °C still much higher than that of the control, indicating that SF encapsulation could effectively prevent the photodegradation of ICG and maintain its optical characteristics in water (Figure 4D). The fluorescence signal of the ICG-SF nanoparticles was located the same maximum emission wavelength at 810 nm as in a solution of free ICG. However, the fluorescence intensity of ICGSF nanoparticles was more stable than that of the free ICG solution (Figure S6). The fluorescence intensity of the ICG-SF nanoparticles was decreased to 59.3% of its initial intensity, while the free ICG was rapidly decreased to 34.5% after 10 days of storage in water at the 18 ACS Paragon Plus Environment

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equivalent amount of ICG (i.e., 20 ppm) and exposed to air, which further confirmed the abovementioned results elucidating the photothermal stability (Figure 4) that SF encapsulation enhanced the stability of ICG.

Figure 4. Evaluation of photothermal stability under ambient conditions. Photothermal heating curves of the ICG and ICG-SF nanoparticles with the same amount of ICG, after irradiated with the 808-nm laser (1.5 W cm-2) for 6 min at (A, B) different concentrations of ICG and (C, D) different time intervals.

To evaluate the biocompatibility of the ICG-SF nanoparticles, we performed in vitro cytotoxicity study towards normal (HL-7702, MCF-10A) and tumor (MCF-7 and HeLa) cells using the Cell Counting Kit-8 (CCK-8) assay at different exposure times (24 and 48 h). The relative viability of the cells was recorded by incubating them with the ICG-SF nanoparticles at various concentrations (equivalent ICG concentration of 0, 5, 10, 20, and 50 ppm). Figure 5 depicts that the relative viability of all the cell lines was higher than 90% in various concentrations at different exposure times, indicating the high cytocompatibility of SF. These results confirmed 19 ACS Paragon Plus Environment


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that the low organic solvent residues in the eventual formulation of SF by the SAS process had no significant effect on the compatibility of the polymer indicating safety as the delivery carrier.

Figure 5. In vitro cytotoxicity study of ICG-SF nanocomposites. Relative viability of normal cells (HL-7702, and MCF-10A), and cancerous cells (MCF-7 and HeLa) after incubation with ICG-SF nanoparticles (ICG concentrations of 0, 5, 10, 20, and 50 ppm) for (A) 24 h and (B) 48 h.

The photothermal efficacy of the ICG-SF nanoparticles in ablating the cancer cells was investigated. After incubation with the nanoparticles for 4 h, the MCF-7 and HeLa cells were irradiated with the 808-nm laser (1.5 W cm-2) for 5 min and the cell viability was quantitatively assessed by the CCK-8 assay after 24 h of incubation. Figure 6A depicts that the ICG-SF nanoparticles restricted the growth of cancer cells in a dose-dependent PTT effect, indicating the complete ablation of cells at a concentration equivalent to 20 ppm of ICG in the presence of NIR laser. Furthermore, the viability of cells was qualitatively assessed by capturing their microscopic images after co-stained by calcein AM and PI visualizing the live cells with green fluorescence and dead cells in red, respectively (Figure 6B-F and Figure S7A-E). The results have drawn the similar conclusions of dose-dependent PTT efficacy of ICG. Meanwhile, the cells were irradiated with the same doses of NIR light in the absence of treatment with ICG-SF nanoparticles. However, the results have shown no significant reduction in cell viability and 20 ACS Paragon Plus Environment

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were similar to the control in the dark (absence of light) conditions, indicating that the NIR light itself was not harmful.

Figure 6. In vitro PTT efficacy of ICG-SF nanocomposites. (A) Relative viabilities of MCF-7 and HeLa cells after the treatment of ICG-SF at various amounts (equivalent to the concentration of ICG in the range of 0-20 μg mL-1), results are shown in mean ± standard deviations (SDs) of six parallel samples. Representative fluorescence images showing the viability of the MCF-7 cells stained with calcein AM (live cells, green fluorescence) and PI (dead cells, red fluorescence) after various treatments, (B) control in dark, (C) control in the presence of laser 808-nm laser (1.5 W cm-2) for 5 min, (D-F) ICG-SF nanoparticles in the presence of laser (D) ICG@5 μg, (E) ICG@10 μg, and (F) ICG@20 μg. Scale bars =50 μm.

The delivery efficacy of the SF nanoparticles in comparison to the uptake of free ICG molecules was investigated by visualizing them with the confocal laser scanning microscopy (CLSM) at different incubation times. As ICG is auto-fluorescent, it was easy to track the molecules without external labeling. Figure 7 depicts the homogenous distribution of ICG and ICG-SF nanoparticles in the cytosol of MCF-7 cells, which is in agreement with the previous report that 21 ACS Paragon Plus Environment


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demonstrated the binding efficiency of ICG mainly with the intracellular protein (glutathione Stransferase).4 After 2 h of incubation with ICG-SF nanoparticles, the fluorescence images showed that the red fluorescence was predominantly clustered at proximity to the cell nuclei compared to the treatment of free ICG, whose fluorescence was unstable in the physiological fluids. Subsequently, the ICG uptake efficiency of cells was gradually increased after 4 h, demonstrating the time-dependent internalization efficiency of ICG-SF. Although the uptake of free ICG was slightly increased with time, the fluorescence levels were lower than those in the case of ICG-SF nanoparticles due to the enhanced internalization of ICG by nanoparticles formulation. These results indicated that the increased accumulation of ICG from SF close to the nuclei would possibly improve its anti-proliferative effect upon light irradiation.


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Figure 7. In vitro cellular internalization study of ICG-SF in MCF-7 cells. Cells subjected to various treatments of ICG and ICG-SF at different exposure times were fixed using formaldehyde after 2 and 4 h of treatment, stained with DAPI, and the images were captured using CLSM. Scale bars =25 μm.

To evaluate the PTT efficiency of ICG-SF in vivo, we initially measured the intratumoral temperature in mice after the tumor region was injected with various samples of saline, SF nanoparticles, free ICG, and ICG-SF nanoparticles and irradiated by 1.5-W cm-2 NIR laser for 5 min. Figure 8A and B depicts that the tumors treated with ICG-SF nanoparticles showed a 23 ACS Paragon Plus Environment


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maximum temperature of 56.9 °C in a time-dependent manner, which was significantly higher than that of the free ICG molecules (46 °C), SF nanoparticles (39.8 °C), and control (39.7 °C) treatments. Indeed, this high intratumoral temperature had exceeded the damage threshold temperature required to induce the irreversible tissue damage.15 Further, the antitumor effect of the ICG-SF nanoparticles in vivo was investigated by continuing the treatment for 20 days. No significant weight loss of animals in all the treatment conditions was observed (Figure 8C). The PTT efficacy was determined eventually by various analyses such as measuring the volume of excised tumors, and histological examination of stained tumor tissues with hematoxylin and eosin. Figure 8D illustrates the volumes of excised tumors after various treatments in the presence of light. It is evident that the ICG-SF nanoparticles in the presence of light showed a significant reduction in the tumor size compared to the other treatment of SF nanoparticles, free ICG, and control, demonstrating the triggered release of ICG in the acidic pH and its subsequent activation in the presence of light. In addition, the other evidences of histological examination of MCF-7 tumor tissues are supportive to the above results showing the typical features of thermal damage, such as coagulative necrosis, abundant pykonosis, and considerable regions of karyolysis in tumors (Figure 8E). Finally, the excised major organs such as kidney, liver, spleen, lung, and heart of animals’ post-treatment were also subjected to histological examinations for determining any signs of metastasis or cell death (Figure 8F). DISCUSSIONS In this study, we have reported the fabrication of ICG-encapsulated SF nanoparticles by a singlestep, environmental-friendly method based on the SCF technology for dual-triggered cancer therapeutics. Conceptually, the designed ICG-SF formulation delivers the ICG specifically in the tumor cells via pH-triggered release behavior of SF and the NIR light-induced activation of ICG 24 ACS Paragon Plus Environment

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resulting in hyperthermia, which ablates the cancer cells. The synthesized nanocomposites were systematically characterized using various techniques for determining their morphological as well as physicochemical attributes. Next, the pH-responsive release, photothermal stability, biocompatibility, and degradation behavior of these composites were examined. Eventually, the PTT efficacy of ICG was investigated both in vitro using various cell cultures as well as in vivo with Balb/c nude mice as a model. In this design, the polymeric framework of SF predominantly offered a few benefits, such as the pH-responsive conformational transition favoring the release of ICG and subsequently enhancement in the PTT efficiency, and the protection from the plasma proteins to decrease the elimination rate of ICG in physiological conditions. In this SAS process, the solubility of ICG in HFIP was not very high, which resulted in a higher saturation ratio and a lower concentration, thus leading to its faster attainment of supersaturation and subsequent precipitation. These consequences resulted in the formation of large-sized nuclei that was soon encapsulated by SF. Consequently, with the increase of theoretical loading amounts of ICG, the resultant nuclei of ICG were formed separately and a few of them would not be coated by SF. More often, the traditional fabrication methods such as emulsification, salting leaching, or organic solvent precipitation, have been successful in encapsulating the positively charged, hydrophobic drug molecules in the SF framework. Comparatively, herewith, the SAS process allows single-step fabrication and convenient way for encapsulating the negatively charged, hydrophilic ICG molecules in SF that is difficult to obtain by traditional techniques.39-40 The responsiveness of SF to acidic pH might correspond to the following mechanisms: one is due to the pH-responsive conformational transition of FibNT in the SF polymeric framework from random coils to β-sheets at lower pH values, resulting in the loss of overall acidic surface properties and negative net charge; the other is that SF tends to oligomerize and destabilize itself 25 ACS Paragon Plus Environment


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at pH values less than 6.0.27-28, 40 In addition, the ICG attains less negative charge when exposed to acidic environment due to its amphipathic nature and impulse itself out. Also, the low stability of ICG-SF nanoparticles in the acidic pH loosens the framework and facilitates the release of ICG (Figure 3A, B).27 In general, SF is considered as a biocompatible polymer, which has been widely used in the preparation of therapeutic devices for biomedical applications including scaffolds for tissue engineering and vehicles for drug delivery.28-29, 34 However, the preparation strategy may reduce its compatibility due to exposure to organic solvents or applying high shear stress leading to altered polymeric framework, which may further result in undesired biological responses. Our results confirmed that the low organic solvent residues in the eventual formulation of SF by the SAS process had no significant effect on the compatibility of the polymer indicating safety as the delivery carrier (Figure 5). Biodegradation is an essential attribute of theranostic nanomedicine to be addressed for its utilization in diverse biomedical applications as most of the nanoconstructs result in reduced biodegradation subsequently leading to biosafety risk due to long-term accumulation. While SF as an FDA-approved biomaterial is defined as non-degradable material by the United States Pharmacopeia (USP) because it retains greater than 50% of its tensile integrity after 60 days post-implantation in vivo.41 However, as a protein, SF can be proteolytically degraded and resorbed in vivo over a longer time period (e.g., typically within a year).42 Recently, remarkable efforts have been devoted to addressing this critical issue of biodegradation of SF-based constructs that include fibers, films, and scaffolds for various applications.43 However, no correlative investigation has reported the biodegradation behavior of SF nanoparticles yet. Herewith our results confirmed that these composites have exhibited effective degradation of SF 26 ACS Paragon Plus Environment

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in the simulated lysosomal environment. Finally, the photothermal efficacy of the pH-responsive ICG-SF nanoparticles in ablating the cancer cells was investigated by in vitro and in vivo experiments. Our results proved that ICG acted as an anti-proliferative agent in the presence of NIR light, providing a strong evidence for anti-proliferative efficacy due to PTT on various cell lines in vitro (Figure 6A) as well as inhibiting tumors in vivo (Figure 8), possibly due to the enhanced cellular internalization efficiency (Figure 7) and photothermal stability (Figure 4). As expected, we found no signs of any atypical histological features in tissues of all groups, indicating that the formulation was highly biocompatible and no tumor cell metastasis was observed.



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Figure 8. In vivo photothermal efficiency of ICG-SF nanoparticles. A) Infrared thermographic maps and B) time-dependent temperature curves in the MCF7 breast tumor-bearing nude mice irradiated by the 808-nm laser. (C) Body weight of nude mice recorded during 20 days after various treatments. D) i) Graphical representation of tumor volume with respect to time in days after treatment of ICG-SF nanoparticles and corresponding pictures of excised tumors of mice treated with samples for 16 d, ii) saline, iii) SF nanoparticles, iv) ICG, and v) ICG-SF nanoparticles. E) Histological staining of the excised tumors after injection of i) saline, ii) SF nanoparticles, iii) free ICG, and iv) ICG-SF nanoparticles under laser irradiation (scale bar: 100 µm). F)


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H&E-stained tissue cross-sections of major organs (heart, liver, spleen, lung and kidney) from mice treated with various samples of saline, SF nanoparticles, ICG and ICG-SF nanoparticles. Scale bars = 50 μm.

CONCLUSIONS In this study, we successfully fabricated the SF nanoparticles that encapsulated the ICG in its polymeric framework by a single-step, environmental-friendly SCF method, which exhibited the pH-responsive release characteristics and enhanced the photothermal stability of ICG in the physiological fluids with appropriate degradation behavior. In addition, the highly biocompatible ICG-SF formulation successfully ablated the tumor cells both in vitro and in vivo in the presence of light through NIR-induced hyperthermia. Hence, the combinatorial therapeutics utilizing a biocompatible formulation with pH- and NIR light-responsive properties could be a promising strategy for photothermal cancer therapy.

Supporting Information Graphical illustration showing the setup of the SAS process and the mechanism of the particle fabrication, transmission electron microscopy, dynamic light scattering, thermogravimetric analysis, differential scanning calorimetry, fluorescence spectra and calcein AM/PI assay in Hela cells.

AUTHOR INFORMATION



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Corresponding authors: ∗ E-mail addresses: [email protected] (A. Z. Chen), [email protected] (Y. S. Zhang) Notes The authors declare no conflicts of interest with this work. ACKNOWLEDGMENT Financial supports from National Natural Science Foundation of China (U1605225, 31570974 and 31470927), Public Science and Technology Research Funds Projects of Ocean (201505029), Promotion Program for Young and Middle-aged Teacher in Science and Technology Research of Huaqiao University (ZQN-PY107) and Subsidized Project for Cultivating Postgraduates’ Innovative Ability in Scientific Research of Huaqiao University are gratefully acknowledged.

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For Table of Contents Use Only

Supercritical indocyanine

fluid-assisted

fabrication

green-encapsulated

silk

of

fibroin

nanoparticles for dual-triggered cancer therapy Biao-Qi Chen,†,‡ Ranjith Kumar Kankala,†,‡,§ Geng-Yi He,‡,§ Da-Yun Yang,⊥ Guo-Ping Li,‡ Pei Wang,‡ Shi-Bin Wang,‡,§ Yu Shrike Zhang,*,|| and Ai-Zheng Chen,*,‡,§


Supercritical fluid-assisted fabrication of Indocyanine green

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