Photosensitizer-Conjugated Hyaluronic Acid-Shielded Polydopamine

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Photosensitizer-Conjugated Hyaluronic Acid-Shielded Polydopamine Nanoparticles for Targeted Photo-mediated Tumor Therapy Jieun Han, Wooram Park, Sin-jung Park, and Kun Na ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01664 • Publication Date (Web): 11 Mar 2016 Downloaded from http://pubs.acs.org on March 17, 2016

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ACS Applied Materials & Interfaces

Photosensitizer-Conjugated Hyaluronic Acid-Shielded Polydopamine Nanoparticles for Targeted Photomediated Tumor Therapy Jieun Han‡, Wooram Park‡, Sin-jung Park, and Kun Na* Center for Photomedicine, Department of Biotechnology, The Catholic University of Korea, 43 Jibongro, Wonmi-gu, Bucheon-si, Gyeonggi do, 14662, Republic of Korea

‡

These authors contributed equally.

KEYWORDS: Photodynamic therapy (PDT), Photosensitizer, Polydopamine, Nanoparticles, Tumor therapy.

*Corresponding Authors Kun Na, Ph.D., Center for Photomedicine, Department of Biotechnology The Catholic University of Korea 43 Jibongro, Wonmi-gu, Bucheon-si, Gyeonggi do, 14662, Korea E-mail: [email protected]

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ABSTRACT Photodynamic therapy (PDT) is a widely used clinical option for tumor therapy. However, the clinical utilization of conventional small-molecule photosensitizers (PSs) for PDT has been limited by their low selectivity for disease sites, and undesirable photo-activation. To overcome these limitations, we demonstrated a tumor-specific and photo-activity-controllable nanoparticle photomedicine based on a combination of PS-biomacromolecule conjugates and polydopamine nanoparticles (PD-NP) for an effective tumor therapy. This novel photomedicine consisted of a PD-NP core shielded with a PS-conjugated hyaluronic acid (PS-HA) shell. The PD-NP and the PS-HA play roles as a quencher for PSs and a cancer targeting moiety, respectively. The synthesized PS-HA-shielded PD-NPs (PHPD-NPs) had a relatively narrow size distribution (approximately 130 nm) with uniform spherical shapes. In response to cancer-specific intracellular enzymes (e.g., hyaluronidase), the PHPD-NPs exhibited an excellent singlet oxygen generation capacity for PDT. Furthermore, an efficient photothermal conversion ability for photothermal therapy (PTT) was also shown in the PHPD-NPs system. These properties provide superior therapeutic efficacy against cancer cells. In mice tumor model, the photoactive restorative effects of the PD-NPs were much higher in cancer microenvironments compared to that in the normal tissue. As a result, the PD-NPs showed a significant antitumor activity in in vivo mice tumor model. The nanoparticle photomedicine design is a novel strategy for effective tumor therapy.

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INTRODUCTION Photodynamic therapy (PDT) has been regarded a successful clinical option for tumor treatment.1-3 The mechanism of PDT is based on the photoactivation of photosensitizers (PSs) under light irradiation.2 When an appropriate laser was irradiated, PSs can generate reactive oxygen species such as singlet oxygen species (1O2), which can kill tumor cells through direct/indirect pathway, damage vasculature surrounding tumor cells, as well as activate immunological responses against tumors.4 However, clinical applications of PS have been limited by their low selectivity for disease sites, undesirable photoactivation by sunlight and self-destruction under laser irradiation.5,

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Moreover, the activation by laser irradiation

consumes the available tissue oxygen and results in local hypoxia and decreased production of 1O2.7 In these regards, advanced PS system is required to compensate for the disadvantage of conventional PS. We developed a tumor-specific and photoactivity-controllable nanoparticle photomedicine for effective tumor therapy. To this end, the nanoparticles were prepared as core-shell nanocomposites consisting of polydopamine nanoparticle (PD-NP) cores surrounded by cancer-specific PS-conjugated hyaluronic acid (PS-HA) shells. Bioinspired PD-NP is originated from a dopamine-derived synthetic eumelanin polymer.8, 9 Since PD has excellent biocompatibility and biodegradability, PD and their derivatives have received attention as a suitable material for biomedical applications.10,

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Recently, PD-NP was utilized as a

photothermal therapeutic (PTT) agent for tumor treatment because of its unique characteristic such as strong light absorption and high photothermal conversion efficiency.12-14 Additionally, PD-NPs can be utilized as fluorescence quenchers due to their energy transfer and electron transfer properties.9,

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To provide cancer-targeting specificity, PS-conjugated HA was 3



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incorporated on the surfaces of the PD-NPs. HA, also called hyaluronan, is an anionic, nonsulfated polysaccharide composed of repeating d-glucuronic acid disaccharide and N-acetyld-glucosamine units.16-18 Because HA can specifically bind to specific receptors overexpressed in cancer cells, such as receptor for lymphatic vessel endothelial receptor-1 (LYVE-1), hyaluronic acid-mediated motility (RAHMM), and cluster determinant 44 (CD44).19 In this basis, HA has been frequently used as a targeting ligand in the development of nanoparticles for efficient delivery of anti-cancer therapeutics.18-20 As shown in Scheme 1, we hypothesized that when PS-HA is located on the surfaces of PDNPs, the energy transfer from the PSs to the PD-NP core is more efficient, resulting in the quenching of not only fluorescence emissions, but also singlet oxygen generation (SOG) of the PSs. However, in the tumor environment, the PS-HA-shelled PD-NPs (denoted as PHPDNPs) specifically bind to HA receptors and become internalized via HA receptor-mediated endocytosis. The PS-HA shells on the PD-NPs are degraded by hyaluronidase (HAase), a cancer-selective enzyme (this enzyme is abundant in the tumor environment and plays key role for tumor proliferation and metastasis),18, 21 thereby separating the PSs and the PD-NPs. This results in the complete restoration of fluorescence and SOG of the PSs inside the target cancer cells. Additionally, the laser absorptive properties of the PD-NPs could utilize the heat form laser irradiation to enhance the therapeutic efficacy of the PHPD-NPs through synergistic actions between PDT and PTT. Although utilization of various NPs for the combination of the PDT and PTT has been reported,22-24 the NPs used in those studies were non-degradable and non-biocompatible inorganic nanocomposites. In this regards, our design of PHPD-NPs consist of biodegradable PD and HA would be highly beneficial for in vivo applications. 4


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EXPERIMENTAL METHODS Synthesis of PS-HA conjugates. PS-HA conjugates were synthesized via a two-step process following our previous reports (Figure S1, Supporting Information).25,

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HA was first

acetylated and dissolved in organic solvent (e.g., DMSO) for further chemical reaction. PS was then incorporated into the acetylated HA (AC-HA) through a carbodiimide reaction. Briefly, into HA (500 mg; 0.08 mmol, Bioland, Cheonan, Korea) dissolved in 10 mL of formamide (Sigma-Aldrich, Yongin, Korea), pyridine (55 µL; 0.6 mmol, Sigma-Aldrich, Yongin, Korea) and acetic anhydride (50 µL; 0.5 mmol, Sigma-Aldrich, Yongin, Korea) were added. The reaction mixture was stirred for an additional 12 h via vigorous stirring at room temperature. The resulting solution was purified by dialysis using a 1,000 Da MWCO dialysis membrane (Spectrum Laboratories, Rancho Dominguez, CA, USA) against distilled water for 3 days. The final product was lyophilized and obtained as a white powder. For the conjugation of PS with AC-HA, pheo-a (5 mg, 8.4 mmol, Frontier Scientific, Salt Lake City, UT) in DMSO was added drop wise into a solution of AC-HA (50 mg) in anhydrous dimethyl sulfoxide (DMSO, 10 mL, Sigma-Aldrich, Yongin, Korea). After stirring for 1 h, 1,3dicyclohexyl carbodiimide (DCC, 2.6 mg, 12.6 mmol, Sigma-Aldrich, Yongin, Korea) and 4dimethylaminopyridine (DMAP, 1.5 mg, 12.6 mmol, Sigma-Aldrich, Yongin, Korea) were added. The solution was stirred for 24 h then, dialyzed for 3 days using a dialysis membrane (MWCO 1,000 Da) against distilled water. The final products were obtained by lyophilization and analyzed by 1H-NMR (Bruker 500 MHz NMR Spectrometer, Bruker, Germany, Figure S2, Supporting Information). Synthesis of PD-NPs. PD-NPs were synthesized by the chemical oxidation of dopamine hydrochloride, as presented in previous reports.8, 27 Briefly, into dopamine hydrochloride (90 5



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mg, 0.47 mmol, Sigma-Aldrich, Yongin, Korea) dissolved in 45 mL of deionized water, 475 µL (0.475 mmol) of 1 N NaOH solution was added drop wise. The reaction solution was stirred and their color gradually changed to dark brown over reaction time. The final product PD-NPs were collected by centrifugation (14,000 rpm, 15 min) with the Vivaspin concentrator tubes (exclusion limit 10,000 Da, Sartorius Stedim Biotech, Göttingen, Germany) and the collected solution was rinsed with deionized water several times. The polydopamine contents of the PD-NPs were measured using ultraviolet−visible spectrometry at 280 nm (UV-2450; Shimadzu, Japan). Preparation of PS-HA-shielded PD-NPs (PHPD-NPs). PHPD-NPs were synthesized by iron-mediated coordination chemistry (Figure S3, Supporting Information).28, 29 Fe3+-PD-NPs were first synthesized, and then the PS-HA was incorporated onto the surface of Fe3+-PD-NPs as shielding materials. To synthesize the Fe3+-PD-NPs, 40 µL of iron(III) chloride (FeCl3, Sigma-Aldrich, Yongin, Korea) solution (100 mg/mL) were mixed with 4 mL of the PD-NPs solution (5 mg/mL) and stirred for 24 h. The resulting Fe3+-PD-NPs were purified by dialysis (MWCO 1,000 Da) against distilled water. Finally, to prepare the PHPD-NPs, 5 mL of the purified Fe3+-PD-NPs solution (5 mg/mL) was mixed with 5 mL of the PS-HA solution (5 mg/mL, deionized water) under vigorous stirring. Characterization of PHPD-NPs. The particle sizes of the as-synthesized nanoparticles were measured by DLS (Nano-ZS, Malvern Instruments, Ltd., UK). The morphology of nanoparticles was monitored using field emission scanning electron microscopy (FE-SEM, Hitachi, Japan). Measurement of photothermal efficacy. To confirm the photothermal effect, a 1-mL aliquot 6


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of 0.5 mg/mL nanoparticle solution was exposed to a 670 nm laser (fiber optic-coupled laser system, Laser Lab®, Korea) with an output power of 50 mW for 800 sec. The temperature of the solution was measured by a fiber optic temperature sensor probe (Photon Control, Inc., Canada) and recorded by a fiber optic temperature converter (Photon Control, Inc., Canada). Stability test of the PHPD-NPs in various conditions. To evaluate the stability of PHPD-NPs in salt-rich conditions, PHPD-NPs in 100 µL of a 300 mM sodium chloride (NaCl, SigmaAldrich, Yongin, Korea) solution were loaded onto a 96-well plate and incubated for different periods. The plate was gently shaken at 100 rpm in a water bath at 37 °C. To investigate the changes in photo-activity of the PHPD-NPs in the presence of cancer cells, MDA-MB-231 cells (obtained from Korean Cell Line Bank) were seeded on 96-well plates. After 24 h, culture media were removed and the cells were treated with 100 µL of serum free (SF) culture medium containing the PHPD-NPs for different periods of time (10 min, 30 min, 1 h, 2 h and 5 h). For comparison, 100 µL of SF culture medium containing the PHPD-NPs were incubated in 96-well plates without cells under the same conditions. The changes in fluorescence intensity were measured using a plate reader (Tecan Genios, Durham, NC, USA) at a 650 nm excitation wavelength and a 675 nm emission wavelength. The fluorescent images were taken by in vivo image station (IVIS, Kodak, New Haven, CT, USA). To assess the change in photo-activity of the PHPD-NPs due to cancer-specific intracellular enzyme (e.g., HAase), the PHPD-NPs were incubated with hyaluronidase type Ⅱ (10 U/mL). The PHPD-NPs with HAase solution was gently shaken at 100 rpm in a water bath at 37 °C for 5 h. Then, the generation of singlet oxygen from pheo-a was quantitatively evaluated using a fluorescence probe (i.e., singlet oxygen sensor green (SOSG, Molecular Probes, Eugene, OR, USA)) at a concentration of 2 mM. Each sample was irradiated with 50 mW from a 670 nm 7



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laser source for 800 sec. The fluorescence emission intensity of the SOSG (λex = 494 nm, λem = 534 nm) was measured by fluorescence spectroscopy (RF-5301; Shimadzu, Japan). The fluorescent images of the PHPD-NPs incubated with or without HAase were taken by the IVIS system. Cell culture. The MDA-MB-231 cell line overexpressing CD44 receptor was cultured in Roswell Park Memorial Institute-1640 (RPMI-1640, Invitrogen Corp., Carlsbad, CA, USA) medium containing 10 % fetal bovine serum and 1 % penicillin/streptomycin. The NIH3T3 cell line (obtained from Korean Cell Line Bank) was used as a control cell line and was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen Corp., Carlsbad, CA, USA) with the same supplements. The cells were maintained at 37 °C at 100 % humidity and 5 % CO2. In vitro cellular internalization of PHPD-NPs. To study the cellular internalization of PHPD-NPs, MDA-MB-231 cells (2 × 105 cells/well) were seeded in 35 mm2 cell culture dishes and incubated (37 °C, 5 % CO2) overnight before experiments. After 24 h, the cultures were rinsed twice with Dulbecco’s phosphate buffer saline (DPBS, Invitrogen Corp., Carlsbad, CA, USA). Then, the cultures were treated with PHPD-NP solutions at different concentrations (equivalent to PD-NPs doses ranging from 0 µg/mL to 640 µg/mL) in SF culture medium. The cellular uptake of PHPD-NPs was quantified by flow cytometry (BD FACS Canto II, BD Biosciences, San Jose, CA, USA) and the data was analyzed by FACS Diva software (BD Biosciences, San Jose, CA, USA). After incubation for 4 h, the cells were washed with cold DPBS twice, collected using a trypsin and resuspended in DPBS. 10,000 cells were counted for each sample. Fluorescent signal from the cells was detected under logarithmic settings (FL4; λem = 670 nm). To visualize subcellular localizations of the PHPD8


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NPs, MDA-MB-231 cells were treated with the PHPD-NPs at different concentrations of PHPD-NPs (equivalent to PD-NPs doses ranging from 0 µg/mL to 640 µg/mL) in SF culture medium for 4 h at 37 °C. The cells were then washed twice with DPBS and fixed with 4 % paraformaldehyde. And then the cells were mounted with a mounting solution (Fluoroshield™ with DAPI, Sigma-Aldrich, Yongin, Korea). The fluorescence images of cells and subcellular localization of PHPD-NPs were observed using a confocal laser scanning microscope (Zeiss LSM 710Meta, Oberkochen, Germany) and analyzed using LSM Image Browser software (Zeiss, Oberkochen, Germany). Evaluation of in vitro cancer targeting efficiency of PHPD-NPs. To confirm the cancer cell targeting capacity of the PHPD-NPs, MDA-MB-231 cells (CD44 high-expressing) and NIH3T3 cells (CD44 low-expressing) were seeded in cell culture dishes and incubated (37 °C, 5 % CO2) overnight before experiments. After 24 h, cells were rinsed twice with DPBS. Then, the cultures were treated with free pheo-a or PHPD-NPs in SF culture medium. After incubation for 4 h, the cellular internalization of the nanoparticles was quantified by using the flow cytometry system as described above. To visualize subcellular localizations of the PHPD-NPs, MDA-MB-231 cells and NIH3T3 cells were treated with PHPD-NPs at a previously optimized concentration (640 µg/mL) in SF culture medium for 4 h at 37 °C. The cells were then washed twice with DPBS and fixed with 4 % paraformaldehyde. After mounting as described above, the fluorescence images were obtained using the confocal laser scanning microscope system as described above. In vitro cytotoxicity test. The cytotoxicities of PD-NPs and PHPD-NPs were evaluated by the cell counting kit-8 assay (CCK-8, Dojindo Molecular Technologies, Gaithersburg, MD). Briefly, MDA-MB-231 cells were seeded in 35 mm2 cell culture dish and cultured (37 °C, 5 % 9



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CO2) for 24 h. The PD-NPs or the PHPD-NPs in SF culture medium were treated for 4 h. The cells were then rinsed twice with DPBS and maintained with fresh culture medium for 12 h. Then, a CCK-8 solution was treated to the cells, and the cultures were incubated for 2 h. The absorbance intensity was measured at 450 nm using the microplate reader. In vitro phototoxicity test. To confirm the cytotoxicity of PHPD-NPs under laser irradiation, CCK-8 and Live/Dead assays were performed. MDA-MB-231 cells were seeded in 35 mm2 cell culture dishes and incubated (37 °C, 5 % CO2). After 24 h, samples in SF culture medium were treated for 4 h. After washing twice with DPBS, the cells were irradiated with a 670 nm laser (fiber optic-coupled laser system, Laser Lab®, Korea). After an overnight incubation, to perform the CCK-8 assay, a CCK-8 solution was treated to the cells for 2 h. The absorbance intensity was analyzed as described avobe. For the Live/Dead assay, the cells were treated with calcein AM and EthD-1 following manufacturer’s protocol. The live and the dead cells were stained green and red, respectively. The cells images were obtained by using a fluorescence microscope (Axio Imager D2, Carl Zeiss, Thornwood, USA). In vitro phototoxicity test under hypoxic condition. To confirm cytotoxicity under hypoxic condition, MDA-MB-231 cells were seeded in 35 mm2 cell culture dishes and incubated in a hypoxia chamber (BioSpherix, Ltd., Lacona, NY) with a 2 % O2 concentration which was controlled by a compact oxygen controller (BioSpherix, Ltd., Pro:OX). After 24 h, the cells were treated with free pheo-a or PHPD-NPs in oxygen-free SF culture medium for 4 h. After washing twice with oxygen-free DPBS, the cells were irradiated with a 670 nm fiber opticcoupled laser system (LaserLab®, Korea). After overnight incubation, CCK-8 assay was performed as following described above. All above processes were conducted in the hypoxia chamber. 10


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Evaluation of in vivo tumor targeting specificity of the NPs in tumor-bearing mice. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea (Republic of Korea) in accordance with the “Principles of Laboratory Animal Care”, NIH publication no. 85-23, revised in 1985. Five-week-old male BALB/c nude mice (Orient Bio, Seongnam, Korea) were used for the imaging study. To develop a tumor model, MDA-MB-231 cells (1 × 105 cells) were xenografted by subcutaneous injection into male BALB/c nude mice. When the tumors volume reached to approximately 10 mm3, the PHPD-NPs (equivalent to dose of 0.1 mg/kg of pheo-a) were subcutaneously injected into the tumor and normal flank regions. The images were obtained using the IVIS system as described above. Evaluation of in vivo tumor growth inhibition of the NPs in tumor-bearing mice. When the tumors volume reach to approximately 10 mm3, solutions of PBS, PD-NPs, PS-HA, or PHPD-NPs (equivalent to dose of 5.0 mg/kg of pheo-a) were intratumorally injected into the tumor of mice (n=4). After 16 h, tumors were irradiated by the laser (dose of laser, 100 J/cm2) with 670 nm fiber optic-coupled laser system (LaserLab®, Korea). The tumor sizes were measured by a caliper and the tumor volume was calculated following the formula (1). (1) = ℎ × ℎ /2 , (a≦b) The body weights were measured by a digital scale. The normalized tumor volumes were calculated as V/V0 (V0 is the initial tumor volume at starting point). To assess histological analysis, the tumors were explanted from the mice at 6 h after injection. The tumors were embedded in paraffin block and then sectioned with 5 µm thick slices. The sectioned tissue slices were stained with hematoxylin and eosin (H&E) and a terminal transferase dUTP nick11



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end labeling (TUNEL) assay kit according to the manufacturer's protocol.

RESULTS AND DISCUSSION Preparation of PHPD-NPs. The PHPD-NPs were prepared through a facile two-step process. First, the PD-NPs were synthesized using a previously reported protocol through the polymerization reaction of dopamine monomer under an basic condition.13, 27 The synthesized PD-NPs showed a relatively narrow size distribution (approximately 86 nm) with uniform spherical shapes (Figure 1a). Second, to improve the tumor targeting specificity and introduce PSs for PDT in this system, the surfaces of the PD-NPs were modified with PS-conjugated HA. The PS-HA complex was synthesized using low-molecular weight HA (5,800 Da) following our previously reported procedure (Figures S1, S2 and Table S1, Supporting Information).25, 26 Because PD-NPs can form stable complexes through coordination effects with various metal ions, such as Fe3+, Mn2+, and Cu2+, Fe3+ ions were incorporated onto the surface of PD-NPs by mixing the as-synthesized PD-NPs suspension in an FeCl3 solution.8, 28, 30

The resultant Fe3+-PD-NPs were then decorated with the PS-HA through coordination

bonding between the Fe3+ ions on PD-NPs and the carboxyl groups of the PS-HA complex (Figure S3, Supporting Information) without the assistance of any extrinsic chemical agents (e.g., carbodiimide coupling). As shown in Figure 1b, when PS-HA was coated on the surfaces of the PD-NPs, the NPs had an average diameter of approximately 130 nm and maintained uniform spherical shapes. The successful formation of these PHPD-NPs was confirmed by Fourier transform infrared (FT-IR) spectroscopy (Figure S4, Supporting Information). The FT-IR spectrum of the PD-NPs showed strong absorption bands at 3740 12


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cm−1, 1520 cm−1, and 1280 cm−1, characterizing the O-H stretch, the C-C stretch (aromatic ring) and the C-N stretch (aromatic amine) of the PD-NPs, respectively. After shielding with PS-HA, the new absorption bands at 2980 cm−1 and 1080 cm−1 belonging to the O–H stretch (carboxyl group) and the C-O stretch (carboxyl acids, esters) appeared, indicating the presence of PS-HA on the surfaces of the PD-NPs. Physico-chemical characterization of PHPD-NPs. In this study, laser source was chosen with 670 nm. Because the PS (pheo-a) can be specifically activated by the 670 nm laser source and the PD-NPs exhibits higher light absorption at 670 nm, compared to that at 808 nm.13 Nevertheless, use of NIR laser source should be considered in the future study, regarding the light tissue penetration issue.31 To characterize the photoactivity of the PHPDNPs, the changes in fluorescence intensity were examined under various conditions (e.g., high salt concentrations, different cell media, and the presence of cells) as functions of time. The fluorescence signals were completely quenched in both high salt (300 mM) and cell culture media conditions regardless of the incubation time. However, when the PHPD-NPs were incubated in the presence of cells with culture media, the fluorescence intensity of the PHPD-NPs recovered depending on the incubation time (Figures 1c and d). Similar result was observed in the presence of a cancer-selective enzyme (i.e., HAase type Ⅱ) in different concentration without cancer cells (Figure S5, Supporting Information). ROS generation, which is majorly responsible for cancer cell killing effect, is a key factor used in estimating the PDT efficacy. In this regard, the singlet oxygen generation (SOG) of the PHPD-NPs was subsequently assessed using the HAase with laser (670 nm) irradiation. Consistent with observations from the fluorescence experiments, depending on the laser intensity, the PHPDNPs treated with HAase (10 Units/mL) exhibited significantly increased SOG over the non13



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enzyme-treated control group (Figure 1e). These results indicate that the PHPD-NPs formed stable complexes in a fluorescence-quenched state under physiological conditions. The fluorescent capabilities of the nanocomplexes were restored under intracellular conditions due to the enzyme-mediated decomposition of PS-HA on the surfaces of the PD-NPs. Furthermore, to assess the photothermal effect of PHPD-NPs, the temperature changes of the PHPD-NPs and PD-NPs suspensions (10 mg/mL) were measured under laser irradiation with a 670 nm external laser source (output power, 50 mW) for 800 sec (Figure 1f). The temperatures of the PHPD-NPs and PD-NPs suspensions increased with irradiation time up to approximately 47 °C. The temperature increased more rapidly in the PD-NPs suspension than that of the PHPD-NPs suspension, which could be attributed to the temperature insulation effect of PS-HA on the surfaces of the PD-NPs. However, a slight increase of temperature (only 2.8 °C increased) was observed in the control group (deionized water) under the same experimental conditions. These results clearly indicate that the PHPD-NPs exhibit not only an excellent singlet oxygen generation for PDT, but also a photothermal conversion capability for PTT. Additionally, successful degradation of the PHPD-NPs (Figure S6, Supporting Information) appeared in the existence of hydrogen peroxide (H2O2), which is abundant in physiological environment as well as is related to various disease.32 This result suggests that the PHPD-NPs were biodegradable in physiological condition.13 In vitro cellular internalization behavior of PHPD-NPs. To investigate the cancer cellspecific internalization of the PHPD-NPs, the cellular internalization behavior of the PHPDNPs were monitored in two different cell lines by flow cytometry and confocal microscopy. Two cell lines, NIH3T3 (CD44 receptor negative) and MDA-MB-231 (CD44 receptor positive) cells, were used in this study. The concentration of the PHPD-NPs for this 14


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experiment was optimized at 640 µg/mL based on a preliminary study (Figure S7, Supporting Information). The expression level of CD44 receptor in NIH3T3 and MDA-MB-231 cells was measured by flow cytometry (Figure S8, Supporting Information). As shown in Figure 2a, the cellular uptake of the PHPD-NPs was much higher in the MDA-MB-231 cells compared to the NIH3T3 cells. However, free pheo-a showed relatively lower cellular uptake capabilities in both cell lines, which may be due to the passive diffusion-mediated uptake mechanism of free pheo-a. The confocal microscopy images were well-matched with the flow cytometry results (Figure 2b). The fluorescence signals (red colored) of the PHPD-NPs in the MDA-MB-231 cells were significantly higher than those in the NIH3T3 cells. The results of CD44 receptor-mediated cellular uptake enhancement of the HA-shielded NPs was consistent with our previous observations,6,

19, 33, 34

which suggests that the PHPD-NPs specifically

targeted cancer cells overexpressing HA receptors. Additionally, we found that the cellular internalization of the PHPD-NPs was more enhanced at 37 °C than at 4 °C in the MDA-MB231 cells (Figure S9, Supporting Information), which indicates that the PHPD-NPs internalize into the cells through an energy-dependent endocytosis pathway.35, 36 In vitro therapeutic efficacy of PHPD-NPs. To assess the cytotoxicity of the PHPD-NPs, the viabilities of the MDA-MB-231 cultures treated with the PHPD-NPs were investigated for different concentrations of PHPD-NPs and were compared with cultures treated with only PD-NPs using a Cell Counting Kit-8 (CCK-8, Figure 3a). The cultures treated with PHPDNPs demonstrated a significantly lower toxicity even at high NP concentrations (> 1 mg/mL), whereas the cytotoxicity of PD-NPs increased dependent on the NP concentration. The exact cytotoxic mechanism of the PD-NPs remains unknown. We speculated that because the PDNPs can randomly bind with the cellular membrane due to their strong binding affinity, the 15



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cellular membrane could be damaged by reduced membrane mobility, resulting in cytotoxicity. However, because the reactive surface is shielded by the PS-HA complexes, the PHPD-NPs can be uptaken by the cell via CD44 receptor-mediated endocytosis without undesirable cellular damage. To further verify the therapeutic efficacy of the PHPD-NPs, an in vitro cytotoxicity test was carried out using the CCK-8 and Live/Dead assay under laser irradiation. MDA-MB-231 cells were incubated with the PHPD-NPs, followed by irradiation at 50 mW from a 670 nm laser source for a predetermined irradiation period (Figure 3b). The PHPD-NPs showed significantly enhanced cytotoxicity depending on the laser irradiation power (from 0.5 to 3.0 J/cm2). The results of the live/dead assay showed a similar trend with those of the CCK-8 assay (Figure 3c). The number of dead cells (red fluorescence) was increased in the group treated with PHPD-NPs under laser irradiation (3.0 J/cm2). This result can be ascribed to the restored photo-activity of the PSs cleaved from the PHPD-NPs by intracellular enzymes and the additional PTT effect of the polydopamine core, which led to an efficient cancer cell killing effect. It is worth note that the PHPD-NPs under laser irradiation exhibited most substantial cytotoxicity compared to that of PD-NPs and PS-HA at the same condition (Figure S10, Supporting Information). Interestingly, in between the PDNPs and the PS-HA, the PS-HA showed much higher toxicity against cancer cells. These results indicate that the PDT effect plays a more crucial role in the photo-toxicity mechanism of the PHPD-NPs than the PTT effect. In addition, hypoxia (low-oxygen condition), a common characteristic of the tumor microenvironment,37, 38 hindered the therapeutic efficacy (Figure S11, Supporting Information) of PDT. This would greatly restrict the potential applications of PHPD-NPs in the clinic.37, 39, 40 To evaluate the therapeutic efficacy of PHPDNPs under hypoxic conditions, we compared the cytotoxicities of free pheo-a and PHPD-NPs

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against MDA-MB-231 cells under hypoxia (Figure 3d). Free pheo-a showed negligible cytotoxicity even at high laser power, whereas the PHPD-NPs retained their therapeutic activity in the low oxygen condition. Because the hypoxic environment only prevents the generation of singlet oxygen species and does not affect heat production, this result can account for the assistance of PTT effect produced by the PHPD-NPs under laser irradiation. In vivo tumor-specific photo-activity restoration of PHPD-NPs. To evaluate the feasibility of utilizing PHPD-NPs for in vivo applications, we performed animal experiments using mice models bearing an MDA-MB-231 tumor. PHPD-NPs were subcutaneously injected into normal and tumor-bearing regions. The tumor specificities of the PHPD-NPs were continuously monitored by in vivo image station (Figure 4a). The fluorescence signals from the tumor site were steadily increased over time (till 5 h post-injection), whereas in the normal tissue, a slight fluorescence signal increase was observed during the experimental period. The fluorescence signal was quantified as a region of intensity (ROI) value (Figure 4b). The signal intensity change was an approximately 3.2-fold increase in the tumor site at 5 h after-injection when compared with that of the normal region. These results indicate that the PHPD-NPs were able to selective target and localize to cancer cells in vivo. Because tumor cells exhibit a significant faster endocytosis rate compared to that of normal cells,6, 41, 42 we believe that the PHPD-NPs could be rapidly uptaken by the cancer cell through CD44 receptor-mediated endocytosis as mentioned above. The subsequent photoactivation of the PHPD-NPs leads to the enzymatic degradation by intra-cellular HAase. Based on these results, the PHPD-NPs were found to be promising nanoparticle photomedicines capable of cancer-specific photo-activation In vivo anti-tumor efficacy of PHPD-NPs. Finally, to evaluate the anti-tumor efficacy, we 17



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carried out an in vivo tumor inhibition study with PHPD-NPs. The mice tumor models divided into four groups of 4 mice per group were intratumorally administered with a single dose of PBS, PD-NPs, PS-HA, and PHPD-NPs and irradiated by a 670 nm laser (laser power, 100 J/cm2). Notably, the tumors of mice treated with photo-activatable samples (i.e., PD-NP, PS-HA, and PHPD-NPs) exhibited a slower growth rate compared with that of the PBStreated control group (Figure 5a). Especially, the tumors in the two groups treated with PSHA and PHPD-NPs showed the most significant tumor growth suppression with similar trend, suggesting that the PDT effect is dominant in the in vivo tumor growth inhibition mechanism of PHPD-NPs. This result is consistent with the in vitro cytotoxicity result (Figure S9, Supporting Information). We speculate that the low effectiveness of PTT in the tumor growth inhibition study could be attributed to the limited laser power dose. According to previous reports,13, 38 PTT requires much higher laser power than that used in PDT. As shown in Figure 5b, there was no body weight change during a 14-day treatment period. Furthermore, the tumors were harvested for histological investigation such as hematoxylin and eosin (H&E) and TUNEL staining (Figure 5c). As expected, the tumor cells in PBS-treated control exhibited characteristic morphology with distinctive membrane and nuclear structures in the broad tissue area. However, severely destroyed tumor cells were observed in the tumors in the two groups treated with PS-HA or PHPD-NPs: a considerable reduction of the number of cancerous cells was observed after treatment, as well as an increase of the number of TUNEL-positive tumor cells was detected in both treated groups. These results indicate the treatment effectively suppress the tumor growth through the inhibition of proliferation and induction of apoptosis. These findings are well matched with the tumor growth inhibition result.

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CONCLUSIONS In this study, we present tumor-specific and photoactivity-controllable nanoparticle photomedicines for effective tumor therapy. This novel photomedicine consists of polydopamine nanoparticle (PD-NP) cores shielded with a photosensitizer-conjugated hyaluronic acid (PS-HA) coating. The PD-NPs and the PS-HA function as a quencher for PSs and a cancer-targeting moiety, respectively. In response to a cancer-specific intracellular enzyme, the prepared PS-HA-coated PD-NPs (PHPD-NPs) exhibited an excellent singlet oxygen generation for PDT. Furthermore, an efficient photothermal conversion ability for photothermal therapy (PTT) was also shown in the PHPD-NPs system. This provides superior therapeutic efficacy against cancer cells. The tumor microenvironment specific photoactivation of the PD-NPs were confirmed using an in vivo mice tumor model. Finally, the PD-NPs were found to have an effective anti-tumor capability in in vivo study. These properties indicate that the PHPD-NP is a new promising approach for successful photodynamic tumor therapy. ASSOCIATED CONTENT Supporting Information Additional schematic illustrations, chemical synthetic routes, 1H-NMR analyses, and in vitro cell experiment results are available free of charge at http://pubs.acs.org.

Corresponding Author *Prof. Kun Na 19



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Email: [email protected] Center for Photomedicine, Department of Biotechnology, The Catholic University of Korea, 43 Jibongro, Wonmi-gu, Bucheon-si, Gyeonggi do, 14662, Korea

Author Contributions ‡

These authors contributed equally. The manuscript was written through contributions from

all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported by the Strategic Research through the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (no. 2011– 0028726).

REFERENCES (1) Agostinis, P.; Berg, K.; Cengel, K. A.; Foster, T. H.; Girotti, A. W.; Gollnick, S. O.; Hahn, S. M.; Hamblin, M. R.; Juzeniene, A.; Kessel, D. Korbelik, M.; Moan, J.; Mroz, P.; Nowis, D.; 20


Page 20 of 33

Page 21 of 33

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


Piette, J.; Wilson, B. C.; Golab, J. Photodynamic Therapy of Cancer: an Update. CA. Cancer. J. Clin. 2011, 61, 250-281. (2) Dolmans, D. E.; Fukumura, D.; Jain, R. K. Photodynamic Therapy for Cancer. Nat. Rev. Cancer. 2003, 3, 380-387. (3) Zhang, D.; Wu, M.; Zeng, Y.; Wu, L.; Wang, Q.; Han, X.; Liu, X.; Liu, J. Chlorin e6 Conjugated Poly(dopamine) Nanospheres as PDT/PTT Dual-Modal Therapeutic Agents for Enhanced Cancer Therapy. ACS. Appl. Mater. Interfaces. 2015, 7, 8176-8187. (4) Juarranz, A.; Jaén, P.; Sanz-Rodríguez, F.; Cuevas, J.; González, S. Photodynamic Therapy of Cancer. Basic Principles and Applications. Clin. Transl. Oncol. 2008, 10, 148-154. (5) Choi, Y.; Weissleder, R.; Tung, C. H. Selective Antitumor Effect of Novel ProteaseMediated Photodynamic Agent. Cancer. Res. 2006, 66, 7225-7229. (6) Park, W.; Park, S. J.; Na, K. The Controlled Photoactivity of Nanoparticles Derived from Ionic Interactions between a Water Soluble Polymeric Photosensitizer and Polysaccharide Quencher. Biomaterials. 2011, 32, 8261-8270. (7) Wang, S.; Huang, P.; Nie, L.; Xing, R.; Liu, D.; Wang, Z.; Lin, J.; Chen, S.; Niu, G.; Lu, G.; Chem, X. Single Continuous Wave Laser Induced Photodynamic/Plasmonic Photothermal Therapy Using Photosensitizer-Functionalized Gold Nanostars. Adv. Mater. 2013, 25, 30553061. (8) Ju, K.-Y.; Lee, Y.; Lee, S.; Park, S. B.; Lee, J.-K. Bioinspired Polymerization of Dopamine to Generate Melanin-Like Nanoparticles Having an Excellent Free-RadicalScavenging Property. Biomacromolecules. 2011, 12, 625-632. 21



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

(9) Qiang, W.; Li, W.; Li, X.; Chen, X.; Xu, D. Bioinspired Polydopamine Nanospheres: a Superquencher for Fluorescence Sensing of Biomolecules. Chem. Sci. 2014, 5, 3018-3024. (10) Ju, K. Y.; Lee, S.; Pyo, J.; Choo, J.; Lee, J. K. Bio-Inspired Development of a DualMode Nanoprobe for MRI and Raman Imaging. Small. 2015, 11, 84-89. (11) Liu, Y.; Ai, K.; Lu, L. Polydopamine and Its Derivative Materials: Synthesis and Promising Applications in Energy, Environmental, and Biomedical Fields. Chem. Rev. 2014, 114, 5057-5115. (12) Lin, L.S.; Cong, Z.X.; Cao, J.B.; Ke, K.M.; Peng, Q.L.; Gao, J.; Yang, H.H.; Liu, G.; Chen, X. Multifunctional Fe3O4@Polydopamine Core-Shell Nanocomposites for Intracellular mRNA Detection and Imaging-Guided Photothermal Therapy. ACS. Nano. 2014, 8, 38763883. (13) Liu, Y.; Ai, K.; Liu, J.; Deng, M.; He, Y.; Lu, L. Dopamine-Melanin Colloidal Nanospheres: an Efficient Near-Infrared Photothermal Therapeutic Agent for in vivo Cancer Therapy. Adv. Mater. 2013, 25, 1353-1359. (14) Zhang, R.; Su, S.; Hu, K.; Shao, L.; Deng, X.; Sheng, W.; Wu, Y. Smart micelle@ Polydopamine Core–Shell Nanoparticles for Highly Effective Chemo–Photothermal Combination Therapy. Nanoscale. 2015, 7, 19722-19731. (15) Medintz, I. L.; Stewart, M. H.; Trammell, S. A.; Susumu, K.; Delehanty, J. B.; Mei, B. C.; Melinger, J. S.; Blanco-Canosa, J. B.; Dawson, P. E.; Mattoussi, H. Quantum-Dot/Dopamine Bioconjugates Function as Redox Coupled Assemblies for in vitro and Intracellular pH Sensing. Nat. Mater. 2010, 9, 676-684.

22


Page 22 of 33

Page 23 of 33

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


(16) Choi, K. Y.; Chung, H.; Min, K. H.; Yoon, H. Y.; Kim, K.; Park, J. H.; Kwon, I. C.; Jeong, S. Y. Self-Assembled Hyaluronic Acid Nanoparticles for Active Tumor Targeting. Biomaterials. 2010, 31, 106-114. (17) Lapcík, L.; Lapcik, L.; De Smedt, S.; Demeester, J.; Chabrecek, P. Hyaluronan: Preparation, Structure, Properties, and Applications. Chem. Rev. 1998, 98, 2663-2684. (18) Yim, H.; Park, W.; Kim, D.; Fahmy, T. M.; Na, K. A Self-Assembled Polymeric Micellar Immunomodulator for Cancer Treatment Based on Cationic Amphiphilic Polymers. Biomaterials. 2014, 35, 9912-9919. (19) Park, W.; Kim, K. S.; Bae, B. C.; Kim, Y. H.; Na, K. Cancer Cell Specific Targeting of Nanogels from Acetylated Hyaluronic Acid with Low Molecular Weight. Eur J Pharm Sci. 2010, 40, 367-375. (20) Lee, C. S.; Na, K. Photochemically Triggered Cytosolic Drug Delivery Using pHResponsive

Hyaluronic

Acid

Nanoparticles

for

Light-Induced

Cancer

Therapy.

Biomacromolecules. 2014, 15, 4228-4238. (21) Stern, R. Hyaluronidases in Cancer Biology. Semin. Cancer. biol. 2008, 18, 275-280. (22) Shi, Z.; Ren, W.; Gong, A.; Zhao, X.; Zou, Y.; Brown, E. M. B.; Chen, X.; Wu, A. Stability Enhanced Polyelectrolyte-Coated Gold Nanorod-Photosensitizer Complexes for High/Low Power Density Photodynamic Therapy. Biomaterials. 2014, 35, 7058-7067. (23) Zeng, L.; Pan, Y.; Tian, Y.; Wang, X.; Ren, W.; Wang, S.; Lu, G.; Wu, A. DoxorubicinLoaded NaYF4:Yb/Tm–TiO2 Inorganic Photosensitizers for NIR-Triggered Photodynamic Therapy and Enhanced Chemotherapy in Drug-Resistant Breast Cancers. Biomaterials. 2015, 23



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

57, 93-106. (24) Zhang, L. e.; Zeng, L.; Pan, Y.; Luo, S.; Ren, W.; Gong, A.; Ma, X.; Liang, H.; Lu, G.; Wu, A. Inorganic

Photosensitizer Coupled Gd-Based Upconversion Luminescent

Nanocomposites for in vivo Magnetic Resonance Imaging and Near-Infrared-Responsive Photodynamic Therapy in Cancers. Biomaterials. 2015, 44, 82-90. (25) Li, F.; Bae, B. C.; Na, K. Acetylated Hyaluronic Acid/Photosensitizer Conjugate for The Preparation of Nanogels with Controllable Phototoxicity: Synthesis, Characterization, Autophotoquenching Properties, and in vitro Phototoxicity against HeLa Cells. Bioconjugate Chem. 2010, 21, 1312-1320. (26) Li, F.; Na, K. Self-Assembled Chlorin e6 Conjugated Chondroitin Sulfate Nanodrug for Photodynamic Therapy. Biomacromolecules. 2011, 12, 1724-1730. (27) Yan, J.; Yang, L.; Lin, M. F.; Ma, J.; Lu, X.; Lee, P. S. Polydopamine spheres as Active Templates for Convenient Synthesis of Various Nanostructures. Small. 2013, 9, 596-603. (28) Holten-Andersen, N.; Harrington, M. J.; Birkedal, H.; Lee, B. P.; Messersmith, P. B.; Lee, K. Y. C.; Waite, J. H. pH-Induced Metal-Ligand Cross-Links Inspired by Mussel Yield SelfHealing Polymer Networks with Near-Covalent Elastic Moduli. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 2651-2655. (29) Ju, K.-Y.; Lee, J. W.; Im, G. H.; Lee, S.; Pyo, J.; Park, S. B.; Lee, J. H.; Lee, J.-K. BioInspired, Melanin-Like Nanoparticles as a Highly Efficient Contrast Agent for T 1-Weighted Magnetic Resonance Imaging. Biomacromolecules 2013, 14, 3491-3497. (30) Enochs, W. S.; Petherick, P.; Bogdanova, A.; Mohr, U.; Weissleder, R. Paramagnetic 24


Page 24 of 33

Page 25 of 33

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


Metal Scavenging by Melanin: MR Imaging. Radiology. 1997, 204, 417-423. (31) Cui, S.; Yin, D.; Chen, Y.; Di, Y.; Chen, H.; Ma, Y.; Achilefu, S.; Gu, Y. In vivo Targeted Deep-Tissue Photodynamic Therapy Based on Near-Infrared Light Triggered Upconversion Nanoconstruct. ACS nano. 2012, 7, 676-688. (32) Cave, A. C.; Brewer, A. C.; Narayanapanicker, A.; Ray, R.; Grieve, D. J.; Walker, S.; Shah, A. M. NADPH Oxidases in Cardiovascular Health and Disease. Antioxid. Redox Signaling. 2006, 8, 691-728. (33) Li, F.; Park, S. J.; Ling, D.; Park, W.; Han, J. Y.; Na, K.; Char, K. Hyaluronic AcidConjugated Graphene Oxide/Photosensitizer Nanohybrids for Cancer Targeted Photodynamic Therapy. J. Mater. Chem. B. 2013, 1, 1678-1686. (34) Park, S. J.; Park, W.; Na, K. Photo-Activatable Ternary Complex Based on a Multifunctional Shielding Material for Targeted shRNA Delivery in Cancer Treatment. Biomaterials. 2013, 34, 8991-8999. (35) Kang, B.; Chang, S.; Dai, Y.; Yu, D.; Chen, D. Cell Response to Carbon Nanotubes: Size‐Dependent Intracellular Uptake Mechanism and Subcellular Fate. Small. 2010, 6, 23622366. (36) Zhu, J.; Liao, L.; Zhu, L.; Zhang, P.; Guo, K.; Kong, J.; Ji, C.; Liu, B. Size-Dependent Cellular Uptake Efficiency, Mechanism, and Cytotoxicity of Silica Nanoparticles toward HeLa Cells. Talanta. 2013, 107, 408-415. (37) Gordijo, C. R.; Abbasi, A. Z.; Amini, M. A.; Lip, H. Y.; Maeda, A.; Cai, P.; O'Brien, P. J.; DaCosta, R. S.; Rauth, A. M.; Wu, X. Y. Design of Hybrid MnO2‐Polymer‐Lipid 25



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

Nanoparticles with Tunable Oxygen Generation Rates and Tumor Accumulation for Cancer Treatment. Adv. Funct Mater. 2015, 25, 1858-1872. (38) Park, W.; Bae, B. C.; Na, K. A Highly Tumor-Specific Light-Triggerable Drug Carrier Responds to Hypoxic Tumor Conditions for Effective Tumor Treatment. Biomaterials. 2016, 77, 227-234. (39) Hayashi, K.; Nakamura, M.; Miki, H.; Ozaki, S.; Abe, M.; Matsumoto, T.; Kori, T.; Ishimura, K. Photostable Iodinated Silica/Porphyrin Hybrid Nanoparticles with Heavy‐Atom Effect for Wide‐field Photodynamic/Photothermal Therapy Using Single Light Source. Adv. Funct. Mater. 2014, 24, 503-513. (40) Meric-Bernstam, F.; Mills, G. B. Overcoming Implementation Challenges of Prsonalized Cancer Therapy. Nat. Rev. Clin. Oncol. 2012, 9, 542-548. (41) Moore, A.; Marecos, E.; Bogdanov Jr, A.; Weissleder, R. Tumoral Distribution of LongCirculating Dextran-Coated Iron Oxide Nanoparticles in a Rodent Model 1. Radiology. 2000, 214, 568-574. (42) Mosesson, Y.; Mills, G. B.; Yarden, Y. Derailed Endocytosis: An Emerging Feature of Cancer. Nat. Rev. Cancer. 2008, 8, 835-850.

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(Scheme 1)

Scheme 1. Schematic illustration of a tumor-specific and photoactivity-controllable nanoparticle photomedicine. (a) Preparation process of photosensitizer-hyaluronic acid conjugates shelled polydopamine nanoparticles (PHPD-NPs) and their photo-activity on-off control, (b) chemical structure of polydopamine nanoparticles (PD-NPs) and photosensitizerhyaluronic acid (PS-HA) conjugates, and (c) cancer-specific cellular internalization of PHPDNPs and their photo-activity restoration by cancer specific enzyme (e.g., hyaluronidase, HAase): under laser irradiation, the ROS generated from PS and the heat from PD-NPs can effectively kill cancer cells.

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(Figure 1)

Figure 1. Physico-chemical characterization of PHPD-NPs. (a and b) The size distribution and SEM images of (a) PD-NPs and (b) PHPD-NPs (scale bar: 100 nm). (c and d) In vitro photo-activity measurement of PHPD-NPs: (c) fluorescence images of PHPD-NPs in (1) high salt condition and (2) cell medium and (3) cell medium with cells as function of the incubation time. (d) Quantitative analysis of fluorescence intensity change of PHPD-NPs in cell medium with cells over the incubation time (n=3). (e) Evaluation of singlet oxygen generation of PHPD-NPs with or without hyaluronidase (HAase, 10 units/mL) under laser irradiation (n=3). (f) Temperature elevation of deionized water (D.W.), PD-NPs, and PHPDNPs as a function of irradiation time (n=3).

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(Figure 2)

Figure 2. In vitro cellular internalization of PHPD-NPs. (a) Flow cytometry quantification of the cellular internalization of free pheo-a and PHPD-NPs against NIH3T3 and MDA-MB231 cells: (1) non-treated control cells, (2) free pheo-a treated cells, and (3) PHPD-NPs treated cells. (b) Confocal laser scanning microscopy images of NIH3T3 and MDA-MB-231 cells treated with PHPD-NPs (scale bar: 20 µm).

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(Figure 3)

Figure 3. In vitro cytotoxicity evaluation of PHPD-NPs under various conditions. (a) Cell viability of MDA-MB-231 cells treated with PD-NPs or PHPD-NPs at different PD-NPs concentrations (incubation time: 4 h). (b) Cell viability of MDA-MB-231 cells treated with PHPD-NPs under different laser irradiation power. (c) Live/Dead assay of MDA-MB-231 cells treated with PHPD-NPs with or without laser irradiation at 3.0 J/cm2 power. (d) Cell viability of MDA-MB-231 cells treated with free pheo-a or PHPD-NPs in the hypoxic condition under different laser irradiation power.

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(Figure 4)

Figure 4. In vivo tumor target specificity of PHPD-NPs in tumor-bearing mice. (a) In vivo fluorescence images of nude mice bearing MDA-MB-231 tumors treated with PHPDNPs (0.1 mg/kg of pheo-a) through subcutaneous injection on the upper flank (normal region) and the lower flank (tumoral region): ⅰ) 10 min post-injection, ⅱ) 2 h post-injection and ⅲ) 5 h post-injection images. (b) Quantitative analysis of fluorescence restoration ratio of PHPD-NPs at the tumoral region in MDA-MB-231 tumor-bearing mice: ROItum corresponds to the fluorescent region of interest (ROI) value in the tumoral region and ROInor corresponds to the fluorescent ROI value in the normal region (n=3).

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(Figure 5)

Figure 5. In vivo tumor growth inhibition efficacy of PHPD-NPs. (a) Tumor growth inhibition after intratumoral injection of PBS, PD-NPs, PS-HA, or PHPD-NPs with laser irradiation (n = 4). (b) Body weight measurement during a 14-day treatment period. (c) Histological analyses of the tumor sections from PBS, PD-NPs, PS-HA, or PHPD-NPstreated tumor-bearing mice with laser irradiation. The tissue sections were analyzed for H&E and TUNEL staining via immunohistochemistry (Scale bar = 20 µm).

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For Table of Contents Use Only

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Photosensitizer-Conjugated Hyaluronic Acid-Shielded Polydopamine

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