On-Demand Versatile Prodrug Nanomicelle for Tumor-Specific

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Biological and Medical Applications of Materials and Interfaces

On-Demand Versatile Prodrug Nanomicelle for Tumor-Specific Bioimaging and Photothermal-Chemo Synergistic Cancer Therapy Yujie Su, Yuan Liu, Xiangting Xu, Jianping Zhou, Lin Xu, Xiaole Xu, Dun Wang, Min Li, Kerong Chen, and Wei Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11349 • Publication Date (Web): 17 Oct 2018 Downloaded from http://pubs.acs.org on October 17, 2018

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ACS Applied Materials & Interfaces

On-Demand Versatile Prodrug Nanomicelle for Tumor-Specific Bioimaging and Photothermal-Chemo Synergistic Cancer Therapy Yujie Su 1, a, Yuan Liu 1, a, Xiangting Xu a, Jianping Zhou *, a, Lin Xu a, Xiaole Xu b Dun Wang *, c Min Li a, Kerong Chen a, Wei Wang *, a

a

State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China

Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China b Department c

of Pharmacology, Nantong University Pharmacy College, Nantong, China

Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of

Education, Shenyang Pharmaceutical University, Shenyang 110016, China

Author information 1

These authors contributed equally to this work

*

Corresponding Authors

E-mail:

[email protected]

(W.

Wang),

[email protected]

[email protected] (J. Zhou).

ACS Paragon Plus Environment

(D.

Wang),

ACS Applied Materials & Interfaces 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

Abstract Photothermal therapy (PTT) was a promising approach for antitumor application, while regrettably restricted by available photothermal agents. Physical entrapment of organic near-infrared dyes into nano-systems were extensively studied to reverse the dilemma. However, problems were still stayed, such as drug bursting and leakage. We developed here an amphiphilic prodrug conjugates by chemically modifying indocyanine green derivative (ICG-COOH) and paclitaxel (PTX) to hyaluronic acid (HA) backbone for integration of photothermal-chemotherapy and specific tumor imaging. The prepared ICGHA-PTX conjugates could self-assemble into nanomicelles to improve the stability and reduce systemic toxicity of the therapeutic agents. The high local concentration of ICGCOOH in nanomicelles resulted in fluorescence self-quenching, leading no fluorescence signal being detected in circulation. When the nanomicelles reached the tumor site via EPR effect and HA mediated active targeting, the over-expressed esterase in tumor cells ruptured the ester linkage between drugs and HA, achieving tumor targeted therapy and specific imaging. A series of in vitro and in vivo experiments demonstrated that the easy prepared ICG-HA-PTX nanomicelles with high stability, smart release behavior and excellent tumor targeting ability showed formidable synergy in tumor inhibition, which provided new thoughts in developing organic near-infrared dyes based multifunctional delivery system for tumor theranostics.

Keyword: prodrug nanomicelle, esterase-sensitive, carboxylated indocyanine green,


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tumor-specific imaging, cancer photothermal-chemotherapy

1. Introduction Regardless of the great success in cancer therapy, malignancy is still threatening people's health and will be the top killer of modern society 1. The traditional tumor therapeutic approaches, including surgery, chemotherapy and radiotherapy, have been stuck in bottleneck for their serious side effects and inefficiency in receiving favorable prognosis

2-3.

The emerging photothermal therapy (PTT), which employs certain near-

infrared lights to excite photothermal agents and converts light energy to heat to kill cancer cells 4, provides a new tool to improve tumor therapeutic outcome, as well as a supplement to conventional therapy. It offers many advantages such as high efficiency and minimal invasiveness 5-7. However, most of the exploited photothermal agents are stifled for further clinical application, since they are unstable to ambience or nonbiodegradable in vivo accompanying with long-term toxicity

8-9.

Therefore, there are insistent demands for

available photothermal therapeutic substances possessing superior biocompatibility and high light-thermal conversion efficiency. Indocyanine green (ICG) is the only FDA-approved NIR dye mainly applying to diagnostic imaging

10.

The favorable biocompatibility and light-thermal conversion

efficiency of ICG makes it an attractive photothermal agent for PTT. As direct administration of ICG suffered from self-aggregation in physiological fluid, non-specific binding to serum proteins and rapid elimination from the body


11-12,

a great number of


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studies had tried to entrap ICG to the interior core of nanoparticles

13-14.

Although these

strategies received considerable progress, physical entrapment of drugs into the core of nanoparticles inevitably resulted in quick release of drugs

15-16

and made nanoparticles

unstable 17. Polymer-drug conjugates have demonstrated great potentials in drug delivery

18-19,

which could self-assemble to nanoparticles by incorporating amphiphilic components. Comparing to the physical manner, the prodrug nanoparticles linked by chemical bonds preserved reliable stability and could be designed as on-demand drug delivery system with minimized drug leakage. Hyaluronic acid (HA), a naturally and negatively charged polysaccharide with strong hydrophilicity, was appropriate to conjugate with hydrophobic ICG to form amphiphilic prodrug. The innate affinity of HA towards CD44 receptor which was overexpressed in cancer cells 20-21 made the HA not only a vector, but also a targeting ligand for tumor specific drug delivery. However, monotherapy show incapability in controlling tumor progression, since tumor possesses complex mechanisms to avoid damage, subsequently accompanying with drug-resistance

22-24.

To appreciate the therapeutic efficacy, we proposed that paclitaxel

(PTX), a widely used anticancer drug, could be taken as another hydrophobic component connected to HA-ICG conjugates. Other than preparing PTX to Taxol, delivering PTX by biocompatible HA would prevent anaphylaxis and achieve tumor targeting to reduce systemic toxicity. The combination of chemotherapy and PTT might achieve synergy while PTT increased the sensitivity of tumor cells towards PTX, and PTX eliminate the sublethal


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cells survived from PTT 25-26. Herein, we successfully developed an amphiphilic prodrug conjugates by covalently linking indocyanine green derivative (ICG-COOH) and PTX to HA through ester bond, forming ICG-HA-PTX conjugates（Scheme 1）. The fabricated ICG-HA-PTX could selfassemble to nanomicelles which encapsulated ICG and PTX in the hydrophobic core and showed HA as hydrophilic shell for tumor targeting. After reaching the tumor site, the overexpressed esterase disrupted the ester bonds

27-30

leading rapid release of ICG and PTX,

accordingly recovering aggregation-caused quenched fluorescence of ICG and the antitumor activity of PTX. Comparing to free drugs, the prepared nanomicelles significantly reduced blood clearance of drugs and improved the therapeutic efficacy. The strategies to incorporate photothermal agents into delivery system for tumor theranostics were received growing attention and have made great progress

31-32.

Different from the

previous studies, the ICG-HA-PTX nanomicelles took full advantage of ICG-COOH in both PTT and fluorescence imaging application with no need to construct complex structure. The system integrated multiple theranostics function with enhanced stability and on-demand release behavior in an easy to fabricate prodrug conjugates, providing available approach for clinical application. A series of in vitro and in vivo experiments demonstrated that the nanomicelles effectively suppressed tumor growth by synergetic photothermalchemotherapy and exhibited admirable tumor fluorescent imaging ability.



Scheme 1. Schematic illustration of preparation of ICG-HA-PTX nanomicelles and tumortargeted delivery of PTX and ICG-COOH. An amphiphilic prodrug conjugates was synthesized by linking ICG-COOH and PTX to HA through ester bond, forming ICG-HAPTX conjugates. The ICG-HA-PTX could self-assemble to nanomicelles in liquid. After i.v. administration, nanomicelles were specifically accumulated in tumor through EPR effect and HA-mediated affinity and internalized into cytoplasm. The high concentrations of esterase in cytoplasm disrupted the ester bonds, leading rapid release of ICG and PTX, accordingly recovering aggregation-caused fluorescence quenching of ICG and the antitumor activity of PTX for photothermal-chemotherapy and specific tumor imaging.

2. Materials and methods 2.1 Materials


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Sodium hyaluronate (MW 7KDa) was purchased from Freda Biochem Co., Ltd. (Jinan, Shandong, China). Paclitaxel were obtained from Chongqing Meilian Pharmaceutical Co., Ltd. (Chongqing, China). ICG-COOH was kindly donated by Shenyang Pharmaceutical University. Pyrene, N, N'-carbonyldiimidazole (CDI), 4-dimethylaminopyridine (DMAP) and thiazolylblue tetrazolium bromide (MTT) were purchased from Aladdin Reagent Database Inc. (Shanghai, China). Esterase from porcine liver was purchased from SigmaAldrich. Newborn calf serum was obtained from Sijiqing Hangzhou bioengineering company (Zhejiang, China). DMEM high glucose media, penicillin, streptomycin and trypsin were purchased from and GIBCO Co., Ltd. (Grand Island, NY, USA), respectively. 4',6-diamidino-2-phenylindole (DAPI) was obtained from Beyotime Institute of Biotechnology Co., Ltd. (Shanghai, China). Annexin V-FITC/PI cell apoptosis assay kit was purchased from Jiangsu Kaiji biological technology Limited by Share Ltd. (Jiangsu, China). All other chemical agents were of chromatographic or analytical grades and used without further purification. 2.2 Synthesis of ICG-HA and HA-PTX conjugates ICG-HA and HA-PTX conjugates were synthesized by esterification reactions. For synthesis of ICG-HA, 21.15 mg (0.03 mmol) of ICG-COOH and 75.84 mg (0.47 mmol) of CDI were dissolved in DMF and stirred at room temperature (R.T.) for 0.5 h under nitrogen. The resulted solution was added drop-wise to 60 mg (0.16 mmol of units) of HA and 57.12 mg (0.47 mmol) of DMAP dissolved in DMF for another 14 h reaction. For synthesis of HA-PTX, 60.00 mg (0.16 mmol of units) of HA and 77.83 mg (0.48 mmol) of CDI were



dissolved in DMF for 0.5 h of agitation. Then, the resulted solution was reacted with 25.62 mg (0.03 mmol) of PTX and 58.64 mg (0.48 mmol) of DMAP dissolved in DMF for 24 h at R.T. The ICG-HA and HA-PTX conjugates were obtained by lyophilization after dialysis of the reaction mixtures against DMF and deionized water, successively. 2.3 Synthesis of ICG-HA-PTX conjugates The synthesis of ICG-HA-PTX conjugate was performed by esterification reaction between carboxyl groups of ICG-HA and hydroxyl group of PTX. In brief, 68.75 mg (0.16 mmol of units) of ICG-HA (molar grafting rates of ICG-COOH was 7.19%) and 79.92 mg (0.496 mmol) of CDI dissolved in DMF was agitated for 1 h to activate the carboxyl group of ICG-HA. The activated ICG-HA solution was then dropped into DMF dissolving 27.33 mg (0.032 mmol) of PTX and 60 mg (0.496 mmol) of DMAP. After another 24 h of agitation, the reaction mixture was transferred to a dialysis bag, dialyzed against DMF and deionized water, and lyophilized for further use. 2.4 Formulation of nanomicelles ICG-HA, HA-PTX and ICG-HA-PTX nanomicelles were prepared by sonicationlyophilization methods. Briefly, 10 mg of HA-ICG, HA-PTX and ICG-HA-PTX conjugates were respectively dispersed in 5 ml water and stirred for 5 min. Subsequently, the dispersions were sonicated at 130 W with a probe-type ultrasonicator for 30 min in ice bath. The ultrasound probe was set to working 2 s with the interval of 4 s. The final nanomicelles were obtained by filtering the sample solution through a 0.22μm membrane and lyophilized.


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2.5 Characterization of conjugates and nanomicelles The chemical structures of ICG-HA, HA-PTX and ICG-HA-PTX were first characterized by 1H-NMR spectroscopy (Avance™ 600, Bruker, Germany, 300 MHz and D2O). FT-IR spectroscopy was also performed to verify the structure of ICG-HA-PTX. The grafting efficiency of PTX on ICG-HA was estimated by UV spectroscopy. The apparent molecular weight and apparent molecular weight distribution of the conjugates were determined by gel permeation chromatography (GPC) equipped with ERC-7515A refractive index detector using an OHpak SB-803 HQ column and THF as a mobile phase at elution rate of 0.8 mL/min. For characterizing nanomicelles, dynamic light scattering (DLS) was employed to determine hydrodynamic diameter and zeta potential of HA-ICG, HA-PTX and ICG-HA-PTX nanomicelles (Nano ZS-90, Malvern instruments, UK, 25℃ with 90 °

scattering angle). The morphology of ICG-HA-PTX nanomicelles was

examined by transmission electron microscope system (TEM, Hitachi, Japan, 80 kV). Furthermore, Critical micelle concentrations (CMC) of HA-ICG, HA-PTX and ICG-HAPTX nanomicelles were measured by employing a hydrophobic fluorescent probe, pyrene 33.

2.6 Optical properties and light stability Fluorescent spectra of free ICG-COOH and ICG-HA-PTX nanomicelles were obtained using a fluorescence spectroscopy with a 780 nm excitation light. The fluorescent intensities at 842 nm of both samples were recorded. To explore the photothermal properties, free ICG-COOH and ICG-HA-PTX nanomicelles at different concentrations



were exposed to an 808 nm (1.0 W/cm2) laser for 5 min, respectively. At predetermined time point, region maximum temperatures of solutions were monitored using an infrared thermal imaging camera. PBS buffer was set as negative control. To examine the light stabilities, free ICG-COOH and ICG-HA-PTX nanomicelles dispersed in deionized water were exposed to natural light for 0, 12, 24, 48, and 72h. At each time point, the absorbances at 791 nm of the samples were obtained using TU-1800 UV/Vis spectrophotometer (Beijing China). The sample colors of free ICG-COOH and ICG-HA-PTX nanomicelles were also recorded after 72 h exposure. 2.7 In vitro drug release and recovery of aggregation-caused quenched fluorescence In vitro drug release was determined in absence or presence of esterase in PBS buffer (pH 7.4 and pH 5.5) and 10% serum. In brief, ICG-HA-PTX nanomicelles dispersion at a concentration of 0.5 mg/mL were prepared in multiple and incubated with or without 30 U/mL of esterase at 37℃ under continuously shaking (100 rpm). At predetermined time points, one sample was taken out and centrifuged at 10000 rpm for 10 min to collect precipitate. For ICG-COOH, the precipitate was dissolved in DMF and measured by fluorescence spectrophotometry. For PTX, the precipitate was dissolved in acetonitrile and measured by UV-Vis spectrophotometer. The drug cumulative release rate was calculated as: Cumulative release rate (%) = (Amount Total drug - Amount Unleased drug) / Amount Total drug. The recovery of aggregation-caused quenched fluorescence of ICG-HA-PTX nanomicelles was investigate under the same condition as drug release study. ICG-HAPTX nanomicelles dispersion at a concentration of 53.7 μg/mL were incubated with or


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without 30 U/mL of esterase under continuously shaking (100 rpm) at 37℃ and measured by fluorescence spectrophotometry at predetermined time points. The fluorescence intensities were then plotted versus time. 2.8 Cell culture Human breast cancer cell line MCF-7, mouse fibroblast cell line NIH3T3 and human umbilical vein endothelial cell line HUVEC were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were maintained in DMEM medium with high glucose, supplemented with 10% (v/v) inactivated fetal bovine serum (FBS), 100U/mL penicillin and 100mg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37℃. 2.9 In vitro cellular uptake and intracellular trafficking MCF-7 cells seeded in 24-well plates were co-incubated with free ICG-COOH or ICGHA-PTX nanomicelles at various concentrations for cellular uptake for 1, 2 and 4 h, respectively. As for competitive binding assays, cells were pretreated with HA polymer (5 mg/mL) for 4 h prior to incubation with ICG-HA-PTX nanomicelles. After cellular uptake, the cells were washed thrice with PBS buffer to remove the extracellular free ICG-COOH or ICG-HA-PTX nanomicelles and additional incubated for 8 h for ICG-COOH release. The intracellular fluorescences were quantitated using flow cytometry (BD FACS Calibur, USA) and observed under confocal laser scanning microscope (CLSM, Leica TCS SP5, Germany) after staining cells with DAPI. For intracellular trafficking study, cells exposed to 40 μg/mL of ICG-HA-COOH for



30 min were continued to incubate for different time. Cells were then fixed, stained with Lyso Tracker Green and DAPI, and observed by CLSM. 2.10 In vitro therapeutic efficacy study To evaluate the chemotherapeutic efficacy of ICG-HA-PTX nanomicelles, MCF-7 cells seeded into 96-well plates were co-incubated with free PTX, HA-PTX and ICG-HAPTX nanomicelles at various PTX concentrations for 24 h. To determine the photothermal therapeutic efficacy of ICG-HA-PTX nanomicelles, cells were co-incubated with free ICGCOOH, HA-ICG and ICG-HA-PTX nanomicelles at various ICG concentrations for 24 h with or without an 808 nm laser irradiation. For the laser irradiation groups, the cells were exposed to 1.0 W/cm2 laser for 5 min at 4 h after the co-incubation beginning. Then, all the resulted cells were subjected to a standard MTT assay and the absorbances were measured at 570 nm using a microplate reader (EL800, BIO-TEK Instruments Inc., USA). NIH3T3 cell with negative CD44 expression 34 and low esterase activity 35 was chosen as model of normal cells to perform the MTT assay. Untreated cells were set as control with 100% viability while wells with only medium were set as blank. The cell viability was calculated as: Cell viability (%) = (Asample - Ablank/Acontrol - Ablank) × 100% The therapeutic induced cell apoptosis was also investigated. MCF-7 cells seeded in 24-well plates were divided into 5 groups: (1) PBS (laser-), (2) PBS (laser+), (3) HA-PTX (containing 24 μg/mL of PTX, laser-), (4) HA-ICG (containing 20 μg/mL ICG-COOH, laser+), (5) ICG-HA-PTX (containing 20 μg/mL of ICG-COOH and 24 μg/mL of PTX, laser-). After 4 h co-incubation with various formulations, cells in laser groups were


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exposed to an 808 nm laser (1.0 W/cm2) for 5 min. Cells were continuously cultivated for another 20 h, followed by staining with AnnexinV-FITC and PI in dark for 10 min. The apoptotic cells were then detected by flow cytometry. To further reveal the therapeutic induced cell apoptosis on protein level, cells treated as above mention were harvested and performed by standard western-blot analysis to detect the Caspase-3, Bax and Bcl-2 expression. The bands were then analyzed with an Odyssey Scanning System (LI-COR Inc., USA) and normalized to GAPDH expression. 2.11 Animals and tumor models Female BALB/c nude mice (18-20 g) were housed under aseptic conditions in small animal isolators with free access to food and water. All care and procedures of animals were approved by the University Ethics Committee for the use of experimental animals. To establish tumor xenograft models, MCF-7 cell suspensions (1×106 cells) were subcutaneously injected into the flank of nude mice. A caliper was used to measure tumor sizes, and tumor volume (mm3) was calculated as (tumor major axis) × (tumor minor axis)2/2. 2.12 In vivo specific tumor imaging The in vivo imaging studies were performed when average tumor volume reached about 100 mm3. PBS buffer, free ICG-COOH and ICG-HA-PTX nanomicelles (0.2 mg of ICG-COOH/kg) were intravenously injected into mice (n=5), respectively, and fluorescence images were acquired by an in vivo imaging system (FX PRO, Kodak, USA) using a 780 nm exciting light at predetermined time points. After 12 h post-injection, mice



were sacrificed, and various tissues including heart, liver, spleen, kidney and tumor were extracted for ex vivo imaging for quantitative analysis using the same imaging system. 2.13 In vivo antitumor efficacy The tumor-bearing mice with average tumor volume around 100 mm3 were divided into six groups treating with (1) PBS (laser -), (2) PBS (laser +), (3) HA-ICG nanomicelles (laser +), (4) HA-PTX nanomicelles, (5) ICG-HA-PTX nanomicelles (laser -) and (6) ICGHA-PTX nanomicelles (laser +), respectively, at a dose of 50 μg of ICG-COOH or/and 115 μg of PTX per mouse. The injections were performed at day 0, 2 and 4 while the laser irradiations applied for 10 min were performed at day 1, 3 and 5. The temperature changes of tumors during the irradiation were monitored by an infrared thermal imaging camera (Ti27, Fluke, USA). The tumor volumes and body weights of mice were inspected every 2 days until day 14. After the therapeutic period, mice were sacrificed. Tumors were excised and sectioned for TUNEL assay. The apoptosis was expressed as fold change of TUNELpositive tumor cells in the treated tumors by those in untreated tumors. 2.14 Statistical analysis The results are expressed as the mean ± standard error. Statistical comparisons between the two groups were performed using the two-tailed, unpaired Student’s-test. Statistical significance was indicated as *P < 0.05, **P < 0.01 or ***P < 0.001.

3. Results and discussions 3.1 Synthesis and characterization of ICG-HA, HA-PTX and ICG-HA-PTX conjugates


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HA possesses many reactive groups such as hydroxyl, carboxyl, N-acetyl amino and reducing end, which can be utilized for chemical modification 36. The synthesis procedures of ICG-HA-PTX conjugates were depicted in Fig. 1a. ICG-HA was firstly synthesized by esterification reaction between carboxyl of ICG-COOH and C6’-hydroxyl of HA in the presence of activator CDI and super acylating catalyst DMAP. Subsequently, esterification was reapplied between C2’-hydroxyl of PTX 37 and carbonyl of HA-ICG to form ICG-HAPTX conjugates. As showed in Fig. 1b, the chemical structures of ICG-HA, HA-PTX and ICG-HA-PTX were revealed by 1H-NMR. For ICG-HA, the characteristic peaks of HA at 4.43 and 4.52 ppm (-OCHC2), and at 1.98 ppm (-COCH3), ICG-COOH’s peaks at 7.7-8.5 ppm (Ar-H) and at 6.5-7.0 ppm (C=C) were all observed in 1H-NMR spectrum of ICGHA, which suggested the successful connection of ICG-COOH and HA. In 1H-NMR spectrum of HA-PTX, peaks of HA at 4.43 and 4.52 ppm (-OCHC2), and at 1.98 ppm (COCH3), peaks of PTX at 7.5-8.3 ppm (Ar-H) and at 6.3-6.7 ppm (-CHO-) were all appeared, verifying the successful synthesis of HA-PTX. The molar grafting rates of ICGCOOH in ICG-HA and PTX in HA-PTX were calculated by corresponding peak areas in 1H-NMR

spectra and determined as 17.67% and 15.75%, respectively. In 1H-NMR

spectrum of ICG-HA-PTX, the characteristic peaks of ICG-COOH and PTX at 7.3-8.3 ppm (Ar-H) and at 6.1-6.7 ppm (-CHO-) were overlapped and appeared in the 1H-NMR spectrum of ICG-HA-PTX conjugates. As showed in Fig. 1c, the chemical structure of ICG-HA, and ICG-HA-PTX were also determined by FT-IR spectra. Comparing to peaks at 1642.3 cm-1 (-CONH-), 3391.6 cm-1 (-NH-) and 2898.6 cm-1 (-CH3) in HA, new



characteristic peaks attributed to ICG-COOH were detected in ICG-HA at 1529.3 cm-1 and 1509 cm-1 (C=C), which conformed the formation of ICG-HA conjugates. In FT-IR spectrum of ICG-HA-PTX, peaks attributed to HA at 1646.5 cm-1 (-CONH-), 3402.5 cm-1 (-NH-) and 2923.6 cm-1 (-CH3), peaks attributed to ICG-COOH at 1580.2 cm-1 and 1481.1 cm-1 (C=C), and peaks attributed to PTX at 709.0 cm-1 (-CH), 1735.1 cm-1 and 1705.4 cm1

(-CO-) were all detected in FT-IR spectrum of ICG-HA-PTX, conforming the formation

of ICG-HA-PTX conjugates. To reveal the modification of PTX to ICG-HA, UV-Vis spectroscopy was also employed to determine the amount of PTX in ICG-HA-PTX conjugates at 227 nm. As showed in Fig. 1d, no obvious absorption peak was detected in HA. The absorption peaks of PTX at 227 nm and of ICG-COOH at 793nm were both appeared in ICG-HA-PTX, verifying the successful synthesis of the conjugates. The final grafting rate of PTX and ICG-COOH in ICG-HA-PTX conjugates was figure out as 21.11% and 9.31% in weight, respectively. The GPC spectra in Fig. 1e revealed that the number average molar mass (Mn) and polydispersity index (PDI) of HA, ICG-HA, HA-PTX and ICG-HA-PTX were 7.12 g/mol with 1.16, 9.63 g/mol with 1.25, 9.72 g/mol with 1.22 and 10.7 g/mol with 1.21, respectively. The increased average molar mass of ICG-HA, HAPTX and ICG-HA-PTX comparing to HA was attributed to the connection of drugs to HA backbone, which was also in accord with the molecular density determined by 1H-NMR spectra and UV-Vis spectroscopy.


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Figure 1. Synthesis procedures and spectra characterization. (a) Synthesis procedures of



ICG-HA-PTX conjugates. (b) 1H-NMR spectra of HA, ICG-HA, HA-PTX and ICG-HAPTX. (c) FT-IR spectra of HA, ICG-HA and ICG-HA-PTX. (d) UV-Vis spectra of HA, ICG-COOH, PTX, ICG-HA, HA-PTX and ICG-HA-PTX. (e) Representative gel permeation chromatograms of HA, ICG-HA, HA-PTX and ICG-HA-PTX.

3.2 Preparation and characterization of nanomicelles Because of the good amphipathicity of HA-ICG, HA-PTX and ICG-HA-PTX conjugates, nanomicelles could be simply prepared by ultrasonic treatment of conjugates in ice bath. The CMC value of each nanomicelle was first estimated by a conventional pyrene fluorescent method. Pyrene is a fluorescent substance, of which the fluorescent intensity is strengthen and exciting wavelength red shift from 373 nm to 383 nm at CMC of amphiphilic polymers 38-39. Fig. 2a presented the line chart to obtain CMC value of ICGHA-PTX conjugates and the CMC values of HA-ICG, HA-PTX and ICG-HA-PTX conjugates were 0.1117, 0.1172 and 0.0576 mg/mL, respectively. Comparing to HA-ICG and HA-PTX conjugates, the CMC value of ICG-HA-PTX was significantly decreased. This might attribute to the simultaneously connection of ICG-COOH and PTX to HA increased the hydrophobicity of conjugates, leading ICG-HA-PTX easier to form nanomicelles. The UV-Vis absorption of ICG-COOH and ICG-HA-PTX nanomicelles were showed in Fig. 2b. Comparing to free ICG-COOH, the absorption peak of ICG-HAPTX nanomicelles showed 10 nm of red shift with no other obvious change. It was presumably that the covalent linkage between ICG-COOH and HA leading aromatic


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conjugation resulted in this shift, which could be supported by previous researches

40-41.

The hydrodynamic diameter, polydispersity index (PDI) and zeta potential of nanomicelles were also detected and shown in Fig. 2c. The ICG-HA-PTX nanomicelles exhibited a narrow size distribution with PDI of 0.172 ± 0.011 and average particle size of 134.03 ± 4.17 nm. The negative charged nanomicelles with zeta potential of -26.3 ± 1.1 mV were more stable in vivo since they could avoid interaction with negative plasma component 42. The morphology of ICG-HA-PTX nanomicelles investigated by TEM was presented in Fig. 2d. It showed that ICG-HA-PTX nanomicelles possessed uniform dimensions about 130 nm with a spheroid structure. The dispersion stability of nanomicelles in present of 10% serum was shown in Fig. S1. No significant changes of particle size were detected within 48 h in present of 10% serum, accompanying with a slightly PDI increasing from 0.162 to 0.212. All results above confirmed the well formation of nanomicelles and indicated the potential capacities of the nanomicelles as drug delivery system. 3.3 Optical properties and light stability Fluorescent dyes are susceptible to light. Exposing to light usually leads degeneration of fluorescent dyes accompanying with fluorescence decay. It is necessary to increase the light stability of fluorescent dyes for biological application. As showed in Fig. 2f, the UVVis absorption of free ICG-COOH was dramatically dropped after 72 h exposure while the absorbance of ICG-COOH in ICG-HA-PTX nanomicelles was slightly decreased. The results demonstrated that ICG-COOH encapsulated in nanomicelles was more stable against light, probably due to the external HA shell isolating ICG-COOH from ambience.



Fig. 2e recorded the color change of free ICG-COOH and ICG-HA-PTX nanomicelles, which also verified the light stability of ICG-HA-PTX nanomicelles. Besides, it was reported that high concentration of ICG would self-quench to lose their fluorescence 43. As demonstrated in Fig. 2g, free ICG-COOH showed strong emission peak at 842 nm when dissolved with proper concentration. In contrast, ICG-HA-PTX nanomicelles under the same concentration of ICG-COOH showed no fluorescence. It could be inferred that the encapsulation caused a high local concentration of ICG-COOH which resulted in fluorescence self-quenching. This phenomenon also provided the prerequisite for in vivo tumor-specific imaging. The photothermal conversion efficiency of photothermal agents directly impacted the therapeutic outcome

44.

The photothermal effect of free ICG-COOH and ICG-HA-PTX

nanomicelles at different concentrations were evaluated under an 808 nm laser and presented in Fig. 2h and 2i. It was quite reasonable that the increments of temperature of both free ICG-COOH and ICG-HA-PTX nanomicelles were positive correlation with concentrations. However, comparing to free ICG-COOH, ICG-HA-PTX nanomicelles under the same concentration of ICG-COOH showed more evident temperature increase. The photothermal conversion efficiencies of free ICG-COOH and ICG-HA-PTX nanomicelles were calculated from Fig. S2 and determined as 49.68% and 68.73%, respectively. After suffered from 5 cycles of 808 nm irradiation, the photothermal conversion efficiencies of free ICG-COOH and ICG-HA-PTX nanomicelles dropped to 15.41% and 35.97%, which exhibited 68.98% and 47.66% decline comparing to that in the


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first round of irradiation, respectively, indicating that ICG-HA-PTX nanomicelles possessed better conversion stability towards free ICG-COOH. The differences might be interpreted theoretically. As indicated by UV-Vis absorption study, the maximum absorbance peak of ICG-HA-PTX nanomicelles red shift 10 nm comparing to free ICG-COOH from 791 nm to 801 nm, which was more consistent with 808 nm of the exciting laser, leading to the strengthened photothermal effect 45. Besides, the higher condensed concentration of ICG-COOH in ICG-HA-PTX nanomicelles resulted in higher energy efficiency and lower heat dissipation 46, which might be another reason for the enhanced photothermal conversion efficiency of ICG-HA-PTX relative to ICGCOOH. The decay of photothermal effect of ICG-COOH was attributed to the photobleaching and degradation of ICG-COOH in the surrounding environment. ICGCOOH entrapment into the core of nanomicelles reduced the extent of photodegradation of ICG-COOH in aqueous media

47

and isolated ICG-COOH from the surrounding

environment 46, 48, thereby improving the photothermal conversion stability of ICG-COOH. These results provided experimental basis for following PTT and fluorescence imaging studies, and indicated that ICG-HA-PTX nanomicelles with higher light stability and photothermal conversion efficiency than those of free ICG-COOH were valuable for biological application.




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Figure 2. Characterization of nanomicelles. (a) The line chart to obtain CMC value of ICGHA-PTX nanomicelles. (b) UV-Vis spectra of free ICG-COOH and ICG-HA-PTX nanomicelles. (c) Hydrodynamic diameter, PDI and zeta potential of various nanomicelles. (d) TEM image of ICG-HA-PTX nanomicelles. (e) Picture of free ICG-COOH and ICGHA-PTX nanomicelles exposed to light for 72 h. (f) UV-Vis absorption changes of free ICG-COOH and ICG-HA-PTX nanomicelles exposed to light for 72 h. (g) Fluorescence spectra of free ICG-COOH and ICG-HA-PTX nanomicelles. In vitro heating curves of (h) different concentrations of free ICG-COOH and (i) ICG-HA-PTX nanomicelles. Results were expressed as mean ± S.D. (n = 3).

3.4 In vitro drug release and recovery of aggregation-caused quenched fluorescence Drug delivery system responsive to tumor environment and capable of quickly releasing payload in the tumor site is necessary to improve drug efficacy and reduce side effects 49. The esterase concentrations in tumor cells are two to three orders of magnitude higher than those in the extracellular fluid 50. The ester bond between therapeutic agents and HA was not only designed as a linker, but also as an esterase-sensitive switch to release the drugs at tumor site. As showed in Fig. 3a and 3b, the release profiles of ICG-COOH and PTX from ICG-HA-PTX nanomicelles were investigated. Both drugs showed low release rates in normal physiological condition (pH 7.4 without esterase) with only 17.74% of ICG-COOH and 18.16% of PTX released within 48 h. However, the cumulative release rates of both drugs were dramatically increased in the present of esterase (pH 7.4 with 30



U/mL of esterase) 51-52, reaching 76.33% and 74.22%, respectively. These changes in drug release rates could be ascribed to the rupture of ester bond by esterase, leading the uncoupling of conjugates and then nanomicelles disassembly. In addition, the accumulative release rates of drugs from ICG-HA-PTX nanomicelles in present of 10% serum within 48 h were no significant differences from that of in PBS at pH 7.4, indicating the ICG-HAPTX nanomicelles was stable in biological media with small drug leakage. The cumulative release rates of drugs from nanomicelles within 48 h showed about 10% increasing in pH 5.5 comparing to that in pH 7.4 both in absence and presence of esterase. These data suggested that the nanomicelles were unstable under acidic conditions, which might attribute to the instability of ester bond in acid. This property of the nanomicelles was desired for tumor theranostics application since the weak acid environment at tumor site would further increase drug release. Fig. 3c revealed the recovery of quenched fluorescence of ICG-COOH. No fluorescence was detected at the initial time point as ICG-COOH encapsulated in the nanomicelles with high local concentration resulted in fluorescence quenching. ICG-HA-PTX nanomicelles exhibited a quick recovery of fluorescence of 85.7% in the present of esterase (pH 7.4 with 30 U/mL of esterase) within 24 h while only 18% in the absence of esterase. Similar results as drug release study were also received in 10% serum and acidic condition. Besides, Fig. S3 indicated that the 808 nm irradiation did not influence the drug release and recovery quenched fluorescence of the nanomicelles. The results demonstrated that ICG-HA-PTX nanomicelles could remain stable in normal tissues but rapidly released their preloads at esterase over-expressed tumor site to achieve tumor-


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specific therapy and imaging.

Figure 3. In vitro drug release studies and recovery of quenched fluorescence. Cumulative release curve of (a) ICG-COOH and (b) PTX at 37℃ in absence or presence of 30 U/mL of esterase. (c) Recovery of aggregation-caused quenched fluorescence of ICG-COOH in absence or presence of 30 U/mL of esterase. The experiment was performed in 10% serum, PBS at pH 7.4 and PBS at pH 5.5. Results were expressed as mean ± S.D. (n = 3).

3.5 In vitro cellular uptake and intracellular trafficking



ICG is the only NIR fluorescent probe approved by FDA. In cellular uptake assay, ICG-COOH could be employed as an indicator to investigate the amount of ICG-HA-PTX nanomicelles internalized by tumor cells. As showed in Fig. 4a and 4b, all experimental groups showed increased cellular uptake whether extending co- incubation time or increasing ICG-COOH concentration. These results demonstrated that the cellular uptakes in the experiments were in time and concentration dependent manners 53. Besides, larger quantities of ICG-HA-PTX nanomicelles were internalized into MCF-7 cells in contrast to free ICG-COOH under all tested concentrations and co-incubation time. When pretreatment with HA, the cellular uptakes of ICG-HA-PTX nanomicelles were significantly decreased, indicating that HA with affinity to CD44 receptor over-expressed in MCF-7 cells 54, played an important role in receptor-mediated active targeting. Fig. 4c presented the representative confocal images of cellular uptakes at 20 μg/mL of ICGCOOH for 4 h. The fluorescence intensity in cytoplasm of ICG-HA-PTX group was obviously stronger than that of free ICG-COOH group, and could be impaired by pretreating with HA, which was consistent with the quantitative data from flow cytometry showed in Fig. 4d. The results above declared that ICG-HA-PTX nanomicelles could be efficiently internalized by MCF-7 cells through HA mediated active targeting. A detailed investigation of intracellular trafficking was showed in Fig. 4e. After 30 min of cellular uptake, only weak red fluorescence was observed in cells. As time progresses, the red fluorescence was strengthened in some spots and co-located with the green fluorescence. The red fluorescence was further extended to a wider distribution as


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time extended from 2 h to 4 h, presenting an excellent overlap with green fluorescence. These phenomenons demonstrated that the internalized ICG-HA-PTX nanomicelles could be disaggregated in cells to recover the quenched fluorescence of ICG-COOH and illustrated the nanomicelles were internalized by lysosomal pathway. At 8 h after cellular uptake, the red fluorescence showed a relatively homogeneous distribution without colocalization with green fluorescence, indicating the released drugs escaping from lysosomes to cytoplasm. Such wide distribution of drugs might achieve better therapeutic efficacies. These results verified the intracellular process of ICG-HA-PTX nanomicelles for specific tumor imaging and photothermal-chemo therapy.




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Figure 4. In vitro cellular uptake and intracellular trafficking. Fluorescence intensities of free ICG-COOH and ICG-HA-PTX nanomicelles with or without HA polymer in MCF-7 cells (a) at 10 μg/mL for different incubation times and (b) at different concentrations for 4 h. Results were expressed as mean ± S.D. (n = 3), *P < 0.05, ***P < 0.001. (c) Representative confocal images of cellular uptakes at 20 μg/mL of ICG-COOH for 4 h incubation. (d) Flow cytometric profiles of various formulations in MCF-7 cells at 20 μg/mL of ICG-COOH for 4 h incubation. (e) Representative confocal images of intracellular trafficking of ICG-HA-PTX nanomicelles in MCF-7 cells. Green arrows indicated endosomes. Red arrows indicated the released ICG-COOH. Yellow arrows indicated colocalization of ICG-COOH and endosomes.

3.6 In vitro therapeutic efficacy study The cytotoxicities of different formulations towards MCF-7 cells were evaluated by MTT assay. Since the amounts of two drugs in nanomicelles were different, the cell viability assays were divided into two parts at various concentrations of PTX and ICGCOOH, respectively. As showed in Fig. 5a, as the concentration of PTX increased, all formulations exhibited enhanced toxicity to MCF-7 cells. Comparing to HA-PTX and ICGHA-PTX nanomicelles, free PTX resulted in slightly stronger influence in cell viability at high concentrations, which might be attributed to the toxicity of Cremphor EL used in free PTX formulation 55. The cytotoxicities between different formulations under the same PTX concentration showed no significant difference, indicating delivering PTX by the



nanomicelles would not affect the chemotherapeutic efficacy in vitro 56. The photothermal effect induced cytotoxicity was conducted at various ICG-COOH concentrations with or without 808 nm laser irradiation and presented in Fig. 5b. The cytotoxicities of ICG-HA group were higher than that of free ICG-COOH group, probably because the formation of nanomicelles enhanced the photothermal conversion efficiency and cellular uptake of ICGCOOH. Besides, the ICG-HA-PTX group with laser irradiation showed highest cytotoxicity towards other groups, indicating the synergy of chemotherapy and photothermal therapy. As indicated in Fig. S4a and S4c, both of HA-PTX and ICG-HAPTX nanomicelles showed significant weakened cytotoxicity to NIH3T3 cell and HUVEC comparing to free PTX, which was different from the results in cytotoxicity study of MCF7 cell. Fig. S4b and S4d revealed that NIH3T3 cell and HUVEC treated with nanomicelles under 808 nm laser irradiation also showed higher cell viabilities than that of MCF-7 cell. The reduced cytotoxicity of nanomicelles in normal cells might attribute to two main factors: the low CD44 expression on normal cells reduced the cellular uptake of nanomicelles and the low esterase activity hampered the drug release in cytoplasm. These data demonstrated that the ICG-HA-PTX nanomicelles were less toxicity towards normal cells comparing to tumor cell, exhibiting targeted cytotoxicity to some extent. To quantitatively investigate the apoptosis-inducing capacity of chemo-photothermal therapy, cell apoptosis assay was performed using flow cytometry. Annexin V-FITC and PI were used to stain live/viable apoptotic cells and dead/late apoptotic cells, respectively 57.

As indicated by Fig. 5c, negligible apoptotic cells were detected in control and simple


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laser irradiation groups, while all treatment groups containing drugs showed significant cell apoptosis. It was worth noting that the combination therapy group with PTX and ICGCOOH plus laser received highest cell apoptosis rate of 97.16%, suggesting that both PTX and ICG-COOH based photothermal therapy contributed to strong cell apoptosis and could achieve synergistic enhancement efficacy. The expression levels of apoptosis-related proteins were important indicators in cell apoptosis. Caspase-3 and Bcl-2 were the most concerned protein families in apoptosis regulation. The expression of Caspase-3 and Bax potently promotes apoptosis while expression of Bcl-2 results opposite effect. The ratio of Bax/Bcl-2 determines the fate of cell. As showed in Fig. 5d and 5e, cells suffered from monotherapy presented significant increase levels of both Caspase-3 and Bax. However, no significant differences were observed in Bcl-2 expression levels. When cells suffered from synergistic photothermalchemo therapy, the Caspase-3 and Bax showed further increase to 1.28 and 1.33 folds of control, respectively, meanwhile receiving a significant decrease in Bcl-2 expression level. Fig. 5f revealed that only the combination group showed a significant increase in ratio of Bax/Bcl-2. These changes in protein expression explained the underlying mechanism of the high efficiency of combination strategy in inducing apoptosis.



Figure 5. In vitro therapeutic efficacy study. (a) Cell viabilities of MCF-7 cells treated with various formulations at different concentrations of PTX for 24 h. (b) Cell viabilities of MCF-7 cells treated with various formulations at different concentrations of ICG-COOH for 24 h. (c) Flow cytometry analysis of cell apoptosis induced by various formulations. (d) Representative immunoblots and (e) quantification of Caspase-3, Bax and Bcl-2 proteins in MCF-7 cells after treated with different formulations for 24 h. (f) Quantification of the ratio of Bax/Bcl-2 in MCF-7 cells after treated with different formulations for 24 h.

3.7 In vivo tumor-specific imaging and pharmacokinetic study Drug delivery system capable of targeting tumor tissue and releasing their cargo on


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demand was desirable, which could achieve enhanced therapeutic efficacy and avoid systemic toxicity

58.

The in vivo tumor-specific imaging of ICG-HA-PTX nanomicelles

was investigated and shown in Fig. 6a. Mice injected with free ICG-COOH consistently showed a primary accumulation in liver with no fluorescence at tumor site. After intravenous injection, the fluorescent intensity of free ICG-COOH in liver rapidly weakened to a great extent as time moves forward, showing the half-time of free ICGCOOH in vivo was extremely short out of non-specific binding with plasma proteins and degradation 46, 59. In contrast, mice in ICG-HA-PTX nanomicelles groups exhibited strong fluorescence signal in tumor with on signal in other tissues. Even at 12 h after injection, the fluorescence signal was still clearly detected in tumor, suggesting an enhanced and specific accumulation of ICG-HA-PTX nanomicelles in tumor. As shown in Fig. 6b and 6c, tumors as well as major organs were excised for ex vivo imaging for quantitative analysis at 12 h post-injection. It demonstrated that free ICGCOOH was non-specific accumulated in all examined organs with highest level in liver, followed by kidney, spleen, lung and heart. However, the ICG-HA-PTX nanomicelles only exhibited strong fluorescence in tumor. These results could be explained theoretically. The free ICG-COOH with no targeting ligand was hard to reach the tumor parenchyma with rapid body clearance and low blood perfusion at tumor. Differently, ICG-HA-PTX nanomicelles with proper dimension for EPR effect and HA shell specifically bind to CD44 receptors were in favor of tumor accumulation. Furthermore, the entrapped ICG-COOH in the core of nanomicelles showed no detectable fluorescence in normal organs due to ICG-



COOH aggregation-caused quenching but exhibited strong fluorescence signal at tumor site after the nanomicelles were disassembled by excess esterase of tumor cells. It was reasonable to conclude that ICG-HA-PTX nanomicelles were the promising drug delivery system for tumor-specific therapy and imaging. The pharmacokinetic curves of free ICG-COOH, ICG-HA nanomicelles and ICG-HAPTX nanomicelles in plasma were investigated and shown in Fig. S5. ICG-HA and ICGHA-PTX exhibited slower reduction of concentrations towards free ICG-COOH at all monitored points, demonstrating the nanomicelles with reduced blood clearance. The pharmacokinetic parameters of each formulation were summarized in Table S1. The Cmax, AUC0-t, T1/2 and MRT were all significantly increased in nanomicelles groups comparing to those in free ICG-COOH group, suggesting the prolonged half time of ICG-COOH and enhanced stability were achieved by encapsulating ICG-COOH into nanomicelles. Pharmacokinetic parameters in ICG-HA-PTX group were slightly higher than those in ICG-HA group, which might be attributed to the difference in particle size that affected the in vivo behavior. These data confirmed the ability of ICG-HA-PTX nanomicelles in improving pharmacokinetic behavior of ICG-COOH for tumor therapy and imaging.


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Figure 6. In vivo tumor-specific imaging studies of ICG-HA-PTX nanomicelles. (a) Representative time-lapse NIR fluorescence images of MCF-7 bearing nude mice. (b) Representative ex vivo NIR fluorescence images of tumors and tissues separated from MCF-7 bearing nude mice at 12 h post injection. (c) Quantification analysis of fluorescence intensities in tumor and tissues at 12 h post injection. Results were expressed as mean ± S.D. (n = 5), ***P < 0.001.

3.8 In vivo synergistic antitumor efficacy NIR light exhibits high tissue penetration, enabling high absorption by ICG-COOH molecules in tumor tissue

60.

Temperatures at tumor sites during radiation were major



Page 36 of 46

indicator of PTT and directly related to therapeutic efficiency. Therefore, the local temperatures of tumors under 808 nm laser radiation were first recorded and presented in Fig. 7a. Mice injected with free ICG-COOH appeared as slight temperature increase to 32.9℃ which was far below the required treatment temperature. In contrast, a sharp rise of temperature to 50.2℃ and 53.3℃ was observed in ICG-HA and ICG-HA-PTX groups, respectively, of which the temperature was high enough for tumor ablation

61.

Such

difference between free ICG-COOH and nanomicelles groups were probably attributed to the high tumor accumulation of nanomicelles, and indicated the high efficiency of ICGHA-PTX as a PTT agent for in vivo tumor ablation. However, PTT alone usually remained sub-lethal tumor cells, failing to eliminate tumor thoroughly. The combination therapy with chemotherapy provided a promising approach to solve the problem. To elucidate the in vivo synergistic efficacy of photothermal-chemotherapy, MCF-7 tumor bearing nude mice were intravenously injected with various formulations containing comparable amounts of therapeutics. As shown in Fig. 7b, laser irradiation alone had no obvious influence on tumor growth. In contrast, monotherapy groups showed moderate tumor growth inhibition with tumor inhibitory rate ranging from 40.06% to 59.07%. The maximum inhibition rate of 98.84% was received in combination therapy group of ICG-HA-PTX nanomicelles plus laser. In Fig. 7d. the average tumor weights in BPS group was about 2 folds of that in monotherapy groups. The lightest tumor weight was observed in combination therapy group, which were consistent with the results of relative tumor volumes. Similar trend between groups was also received


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in TUNEL assay showed in Fig. S6. The highest level of TUNEL-positive cells was observed in photothermal-chemotherapy group with 20.05 times of apoptotic cell comparing to control, indicating the synergy of chemotherapy and photothermal therapy in tumor inhibition. As shown in Fig. 7c, the body weight of mice in therapeutic groups were gained steadily, illustrating the formulations did not affect the life qualities of mice. It was worth noting that mice receiving no treatment exhibited relatively slow increase in body weight relative to therapeutic groups. This might be attributed to the enlarged tumor which affected health of mice. The safety of ICG-HA-PTX nanomicelles was further evaluation by hemolysis test. As showed in Fig. 7S, comparing to deionized water, no significant hemolysis (

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