Construction and Evaluation of a Targeted Hyaluronic Acid

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Construction and evaluation of targeted hyaluronic acid nanoparticle/ photosensitizer complex for cancer photodynamic therapy Shi Gao, Jingjing Wang, Rui Tian, Guohao Wang, Liwen Zhang, Yesen, Li, Lu Li, Qingjie Ma, and Lei Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09331 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 6, 2017

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ACS Applied Materials & Interfaces

Construction and evaluation of targeted hyaluronic acid nanoparticle/photosensitizer complex for cancer photodynamic therapy Shi Gao1,ǂ, Jingjing Wang2,ǂ, Rui Tian3, Guohao Wang2, Liwen Zhang2, Yesen Li2, Lu Li1, Qingjie Ma1,*, Lei Zhu4,* 1. China-Japan Union Hospital, Jilin University, Changchun 130033, China; 2. State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen 361005, China; 3. Department of Ophthalmology Second Hospital, Jilin University, Changchun, Jilin 130033, China; 4. Department of Surgery, Emory University School of Medicine, Atlanta, GA 30322, United States. *To whom correspondence should be addressed. E-mail: [email protected] (L.Z.) and [email protected] (Q.M.). ǂ These authors contributed equally to this work.

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Abstract Photodynamic therapy (PDT) is a novel treatment modality that is under intensive preclinical investigations for a variety of diseases, including cancer. Despite extensive studies in this area, selective and effective photodynamic agents that can specifically accumulate in tumors to reach a therapeutic concentration are limited. Although recent attempts have produced photosensitizers complexed with various nanomaterials, the tedious preparation steps and poor tumor efficiency of therapy hampers its utilization. Here, we developed a CD44-targeted nanophotodynamic agent by physically encapsulating a photosensitizer, Ce6, into a hyaluronic acid nanoparticle (HANP), which were hereby designate as HANP/Ce6. Its physical features and capability for photodynamic therapy were characterized in vitro and in vivo. Systemic delivery of HANP/Ce6 resulted in its accumulation in a human colon cancer xenograft model. The tumor/muscle ratio reached 3.47 ± 0.46 at 4 h post-injection (h p.i.) as confirmed by fluorescence imaging. After treatment, tumor growth after HANP/Ce6 with laser irradiation (0.15 W/cm2, 630 nm) was significantly inhibited by 9.61 ± 1.09-fold compared to tumor control groups, which showed no change in tumor growth. No apparent systemic and local toxic effects on the mice were observed. HANP/Ce6mediated tumor growth inhibition was accessed and observed for the first time by 18Ffluoro-2-deoxy-D-glucose ([18F]FDG) positron emission tomography (PET) as early as one day after treatment, and persisted for 14 days within our treatment time window. In sum, our results highlight the imaging properties and therapeutic effects of the novel HANP/Ce6 theranostic nanoparticle for CD44-targeted PDT cancer therapy that may 2


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be potentially utilized in the clinic. This HANP system may also be applied for the delivery of other hydrophobic photosensitizers (PSs), particularly those that could not be chemically modified. Keywords: Photodynamic therapy, nanomedicine, hyaluronic acid nanoparticle, positron emission tomography; targeted therapy, [18F] FDG. INTRODUCTION Photodynamic therapy (PDT) is a relative new treatment approach for a variety of diseases

1-2

. This procedure has been approved by US Food and Drug Administration

(FDA) for the treatment of patients with esophageal cancer, Barrett's esophagus, and non-small cell lung cancer3. PDT utilizes a photosensitizer (PS) to generate cytotoxic reactive oxygen species (ROS) that induce cell death and tissue destruction under light activation4-5. Compared to conventional chemotherapy and radiotherapy, PDT demonstrates less systemic toxicity because it only affects locally with specific light illumination. Although photothermal therapy (PTT) can also ablate cancer cells locally, it induces severe damage of the skin surface

6-7

. To date, the clinical applications of

PDT are largely hindered by its poor biocompatibility and the lack of tumor targetability of the PSs8. Thus, the development of a novel PDT agent that exhibits ideal in vivo behavior and enhances tumor accumulation is warranted. Evaluation of therapeutic responses is another challenge in cancer drug development9-10. It has been reported that early therapeutic response assessments can improve the cure rate, reduce mortality, as well as circumvent the associated high costs and side effects of inefficient treatments9-12. Because PDT has the capacity to weakly 3



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inhibit tumor growth, the evaluation of PDT treatment responses early on is vital for personalized cancer management. Traditionally, methods for assessing therapeutic effects are based on changes in tumor size and clinical symptoms, although an effective treatment might also cause physiological and biochemical changes in the tumor itself prior to the observation of changes in tumor size 13-14. However, traditional methods for the assessment of therapeutic effects do not provide real-time information and are relatively inaccurate. Positron emission tomography (PET) allows the dynamic and quantitative observation of in vivo biochemical changes at the molecular level 12, 15-17. It is noninvasive and widely used in oncologic applications for the detection of early symptoms of tumorigenesis prior to the development of gross anatomical evidence, thus facilitating early diagnosis and treatment options18. For example, 18F-fluoro-2-deoxyD-glucose ([18F]FDG) is a glucose analog that is taken up by tumor cells via glucose transporters, which are overexpressed in tumors, thereby providing the possibility of imaging cancer cells by using PET17, 19. Although [18F]FDG PET is an effective way of monitoring cancer chemotherapeutic response17, 20-21, studies using PET in monitoring the light-triggered therapeutic strategy such as PDT, especially for nanomaterial-based PDT, are limited. Hyaluronic acid (HA) is a hydrophilic glycosaminoglycan constituent of extracellular matrices that is composed of N-acetylglucosamine and D-glucuronic acid disaccharide units

6, 22

. It naturally exists in the human body, has various biological

functions, and participates in numerous pathways to induce cell adhesion, migration, and proliferation by binding with specific cell-surface receptors such as cluster 4


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determinant 44 (CD44)23-24. When chemically conjugated with a hydrophobic moiety such as 5β-cholanic acid (5β-CA), the HA-5β-CA complex can self-assemble into a reel of thread-like nanoparticles (HANPs), with a hydrophilic surface and hydrophobic caves within, thereby providing the possibility of ligand modification and poor biocompatible agent encapsulation4, 6, 23, 25. Several studies have shown that using HAbased nanoparticles may be utilized as effective vehicles for the tumor phototherapy. Despite their effectiveness in tumor accumulation26 or ablation 27-29, these designs have been limited to in vitro studies or require complex synthesis steps. In the present study, we constructed a simple tumor targeting nanocomplex by directly loading Ce6 into HANPs (Scheme 1a and Figure S1). The HANP/Ce6 complexes accumulated in tumors via active CD44 targeting and passive enhanced permeability retention (EPR) effects after intravenous administration (Scheme 1b), which were observed by fluorescent imaging. Upon the accumulation of HANP/Ce6 complexes in the tumor, PDT was conducted using low-power laser irradiation (630 nm, 0.15 W/cm2). To assess early tumor response, we applied ([18F]FDG) PET before and after PDT. Our results showed that this simple HANP/Ce6 complex can effectively inhibit tumor growth without inducing organ toxicity. Accordingly, tumor uptake of [18F]FDG in the PDT-treated group significantly decreased as early as 1 day after treatment compared to the control groups before visible changes in tumor size were observed, thereby allowing rapid optimization of treatment parameters. Herein, we present a simple and promising [18F]FDG PET-guided biodegradable photodynamic cancer treatment agent. This HANP platform can be further extended to other PS 5



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delivery systems that could not be modified in terms of covalent conjugations. Materials and methods Reagents. Sodium hyaluronate (MW = 2.344 × 105 Da) was purchased from Lifecore Biomedical (Chaska, MN, USA). 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and chlorin e6 (Ce6) were obtained from J&K Chemical Company (Beijing, China). 5β-Cholanic acid (CA), tetrabutylammonium hydroxide (TBA), and propidium iodide (PI) were procured from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and antibiotics were purchased from PAA (Chalfont St Giles, UK). HT29 (colon cancer) and NIH-3T3 (mouse embryonic fibroblast) cells were obtained from ATCC (Manassas, VA, USA). Eppendorf tubes (1.5 mL), six-well chambers, 96-well flat-bottomed plates, and cell culture dishes were purchased from JET BIOFIL (Guangzhou, China). All other chemicals were of analytical grade and used without further purification. Preparation of HANP/Ce6 complexes. The HANP/Ce6 complexes was prepared using a high-pressure homogenizer (D3-L, PhD Technology LLC, MN, USA) as previously described6. HA and 5β-cholanic acid conjugate were synthesized in the presence of EDC and NHS. To load Ce6 into HANP, a Ce6 solution was dissolved in 1 mL DMSO with sonication for 15 min, and then slowly added into an HANP solution (5 mg/mL in H2O) and homogenized for 10 min. The resulting mixture was centrifuged at 4,000 rpm for 10 min to precipitate free Ce6 and the supernatant was carefully collected. The yielding solution was further washed by water for 3 times in an ultrafiltration tube to remove DMSO and free Ce6 followed by a 4 hours dialysis. Final 6


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product was freeze dried overnight. Characterization of HANP/Ce6 complexes. The amount of loaded Ce6 in the HANP/Ce6 complexes were determined using a UV-VIS spectrometer by measuring absorbance of free Ce6. The samples were analyzed based on a standard curve of free Ce6. Briefly, Ce6 was dissolved at different concentrations (1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.2 mg/mL, and 0.00825 mg/mL). We observed a linear relationship between the concentration of Ce6 and its absorbance using a highperformance liquid chromatography (HPLC) system with a reverse-phase C-18 column (5 μm, 120 Å, 250 mm × 4.6 mm) using 5% to 65% acetonitrile containing 0.1% TFA versus distilled water containing 0.1% TFA over 30 min at a flow rate of 1 mL/min. The fluorescence intensity of the HANP/Ce6 and free Ce6 was measured with a fluorescence spectrophotometer using an excitation wavelength of 403 nm. Dynamic light scattering was used to measure the size distribution and the zeta potential of the HANP/Ce6 complexes. Detection of reactive oxygen species (ROS). HT29 cells were cultured in McCoy’s 5A medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37oC in a CO2 incubator. The HT29 cells (density: 1 × 105 cells/well) were seeded onto 96well plates and incubated in a complete medium for 24 h at 37°C. Then, the medium was then replaced with fresh culture medium containing HANP/Ce6 and free Ce6 at different concentrations (40 µg/mL, 20 µg/mL, 10 µg/mL, 5 µg/mL, and 2.5 µg/mL) and then incubated for 4 h at 37°C. Then, fresh culture medium containing 10 μM Dichlorodihydrofluorescindiacetate (DCFH-DA) was added, and cells were incubated 7



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for another 20 min. DCFH-DA is a widely used probe for detecting intracellular ROS. It is cell-permeable non-fluorescent agent and can be hydrolyzed intracellularly to the fluorescent DCFH carboxylate anion

5, 23

. After incubation, the cells were irradiated

with a 630 nm laser at a power of 100 mW/cm2 for 300 s, and fluorescence detection of DCF was performed with the fluorescence microplate reader, which presents the amount of intracellular ROS. Cellular uptake assay. The HT29 and NIH3T3 cells were respectively cultured in McCoy’s 5A and DMEM medium, each containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37oC in a CO2 incubator. The HT29 cells (3 × 105 cells/well) and NIH3T3 cells (1 × 105 cells/well) were seeded onto six-well plates and incubated in complete medium for 24 h at 37°C. Then, the medium was replaced with fresh culture medium containing HANP/Ce6 and free Ce6 (20 µg/mL) and incubated for 4 h at 37°C. To verify the specificity of HA binding with CD44, free HA (1 mg/mL) was added into the cell cultures 30 min before exposure to HANP/Ce6. After washing with PBS (pH 7.4) for thrice, the cells were fixed in cold ethanol at -20°C for 15 min. Then, the cells were stained with DAPI for 10 min in the dark and then imaged using a laser scanning confocal fluorescence microscope (Leica, Germany). In Vitro Detection of Singlet Oxygen (1O2) molecules. Singlet oxygen (1O2) was quantified

using

a

1

commercial

O2

kit

that

contained

2',7'-

dichlorodihydrofluorescindiacetate (DCFHDA), which originally nonfluorescent but becomes fluorescent after 1O2 oxidation. To verify 1O2 generation potency, the HT29 cells were incubated with the HANP/Ce6 complexes and free Ce6 molecules for 4 h. 8


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Then, the cells were washed and then incubated with 1 μM of DCFHDA in PBS with 2% DMSO and irradiated with a 630-nm continuous laser at a power of 0.1 W/cm2 for 4 min. Fluorescence signals of the DCF were recorded 1 min after laser irradiation using a fluorescent microplate reader. The entire surface of the samples was adjusted to confirm that these were covered by the laser spot. The fluorescence intensity that is proportional to the amount of 1O2 was detected using a fluorescence spectrometer with a 488-nm excitation and 525-nm emission wavelength. Cell viability assays. HT29 cells (density: 1 × 104 cells/well) and NIH3T3 cells (density: 1 × 105 cell/well) were seeded onto six-well plates and incubated in complete medium for 24 h at 37°C. The next day, the cells were treated with the HANP/Ce6 complexes and free Ce6 molecules at different concentrations (40 µg/mL, 20 µg/mL, 10 µg/mL, 5 µg/mL, and 2.5 µg/mL) for 4 h at 37°C. After incubation, the cells were washed twice with serum-free medium and then irradiated with a 630-nm laser at 0.1 W/cm2 for 4 min and then transferred onto 96-well plates. In another group, the cells were washed twice with serum-free medium and without irradiation to verify cell viability in the dark. After incubation, cell viability with and without irradiation was evaluated by using the MTT assay. In vivo fluorescence imaging. The HT29 tumor models were generated by subcutaneous injection of 1 × 107 cells in l00 µL PBS into the right leg of athymic nude mice. When tumors grew to approximately 60 mm3 in size, HANP/Ce6 (5 mg/kg of equivalent amount of Ce6) and Ce6 were administered to the mice via tail vein injection (n = 3 per a group), respectively. 1 mg of Ce 6 was dissolved in 100 µL DMSO first 9



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and then equal amount of Kolliphor El was added. At last, 800 µL saline was added into the mixture. The solution is ready to use for intravenously injection. The fluorescent signals were observed using Carestream FX Pro at time points of 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h after intravenous injection of the HANP/Ce6 complexes. For the competitive study, 10 times excess amount of free HA (50 mg/kg) was injected 30 min prior HANP/Ce6 (5 mg/kg of equivalent amount of HA) was injected. The fluorescent signals were observed at 4 hours p.i. when tumors accumulate HANP/Ce6 at peak. For the biodistribution experiment, mice that underwent HANP/Ce6 or Ce6 treatment were sacrificed at 4 h p.i.. Tumors and major organs, including heart, liver, spleen, kidney, and muscle were collected for imaging using Carestream FX Pro. In vivo PDT. The tumor-bearing mice were randomly divided into the following five groups (n = 5/group): (1) Saline, (2) free Ce6 with laser, (3) free Ce6 without laser, (4) HANP/Ce6 with laser, and (5) HANP/Ce6 without laser. Mice in the treatment groups received HANP/Ce6 or free Ce6 (5 mg/kg of equivalent amount of Ce6) via tail vein injection, whereas the control mice received saline only. When the tumor size of the mice grew up to around 100 mm3 (n = 5 per group), PDT was conducted, wherein the tumors were irradiated with 630 nm laser at 150 mW/cm2 for 30 min at 4 h and 24 h p.i.. Tumor size was monitored every other day post treatment with a digital caliper, and tumor volume was calculated using the equation V = ab2 /2, where a is the longer diameter, and b is the shorter diameter. The body weights of all mice were measured every other day. In vivo PET imaging. PET scans and image analysis were performed using an Inveon 10


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microPET scanner (Siemens Medical Solutions). At predetermined time points before (Day 0) and after PDT treatment (Day 1, Day 7, and Day 14), each tumor-bearing mouse was received 3.7 MBq (100 mCi) of [18F] FDG in a volume of 100 μL saline via tail vein injection under isoflurane anesthesia. Five-minute static scans were acquired at 1 h after injection. The mice were fasted for 4 h prior to tracer injection and maintained under isoflurane anesthesia during the injection, accumulation, and [18F]FDG scan. Histological studies. For histological analysis, the animals were sacrificed two weeks after the treatments, and the tumors were isolated and then fixed in 4% formaldehyde at room temperature for 48 h for haematoxylin and eosin (H&E) analysis. The 8-μm tissue sections were stained with H&E using a standard protocol. All tissue sections were examined under a Leica confocal microscope. Statistical analysis. Statistical analysis was performed using one-way ANOVA, followed by a Bonferroni multiple comparison test. P < 0.05 was considered statistically significant. Results and discussion Preparation and Characterization of HANP/Ce6 complexes. HANPs were prepared as described in a previous report6, 23. Hydrophobic Ce6 was then loaded into the HANPs by using a high-pressure homogenizer. To determine optimal loading, different Ce6:HANP ratios, namely, 1:9, 1:4, and 2:3 (w/w) were assessed according to Figure S2. Table 1 shows that 97.83% of the Ce6 molecules was loaded into the HANPs when the HANP:Ce6 ratio was 1:4, which was then used in the subsequent analyses. The HANP/Ce6 complexes were confirmed by UV-visible-near infrared (UV-VIS-NIR) 11



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absorbance readings. Furthermore, the HANP/Ce6 complexes that were prepared using the optimal 1:4 ratio showed extensive dispersal in different physiological buffers such as fetal bovine serum (FBS), cell medium containing 10% FBS, PBS, and water (Figure 1, upper panel), whereas free Ce6 immediately precipitated in buffer (Figure 1, lower panel), indicating that HANPs indeed improve hydrophobic Ce6 solubility and stability. To further confirm the construction of HANP/Ce6, the absorbance spectrum of this complex was scanned and compared to that of free Ce6. Figure 1b shows that the characteristic Ce6 peaks were also detected in the HANP/Ce6 complex mixtures at wavelengths of 404 nm and 656 nm. Figure 1c shows the fluorescent spectrum of the complexes, which was significantly quenched due to the aggregation of Ce6 in the hydrophobic caves of the HANPs. To further confirm the encapsulation of Ce6, the diameter size of the HANPs and the HANP/Ce6 complexes was measured by dynamic light scattering (DLS), which showed that the diameter of the HANPs increased from 190.5 ± 17.5 nm before Ce6 encapsulation to 227.1 ± 11.5 nm after Ce6 encapsulation (Figure 1d). The 37-nm difference may be attributable to the encapsulation of Ce6 into HANP. These findings were indicative of the successful encapsulation of Ce6 into the HANPs. Other hydrophobic PSs can also be loaded into the HANP system, particularly those that cannot be covalently modified. Cell Targetability of the HANP/Ce6 complexes. Prior to the drug delivery experiment, we first examined the tumor cell targetability of the HANP/Ce6 complexes on CD44positive (HT29)28, 30 and -negative (NIH3T3)28, 31 cells by fluorescence labeling. We used cells with different levels of CD44 expression as described in a previous study 32. 12


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HA has the capability to bind to CD44, which is overexpressed in most cancer cells, and thus the recognition of CD44 increases the accumulation of the HANP/Ce6 complexes and ultimately, drug delivery. Figure 2 shows that strong Ce6 fluorescence signals were observed in the cytoplasm of CD44-positive HT29 cells after 4 h of incubation. On the other hand, weak fluorescent signals, representing free Ce6 molecules, were observed intracellularly, indicating their limited entry into cells compared to the HANP-mediated cell internalization of the HANP/Ce6 complexes. Fluorescence was also detected after Ce6 was released from the HANP/Ce6 complexes in the lysosomes via hyaluronidase-2 degradation33 (Figure S3), but quenched in vitro. On the other hand, few fluorescent signals were observed in the NIH3T3 cells in the HANP/Ce6- and Ce6-treated groups due to the low expression of CD44. To verify the specific binding of HA to CD44, a competitive binding study was performed by adding 10 times the excess amount of HA to the HT29 cells to block CD44 prior to the addition of the HANP/Ce6 complexes. Few fluorescent signals were detected, indicating that the CD44-mediated entry of HANP/Ce6 was ligand-specific. The strong CD44 binding of HANPs to cancer cells and their EPR effect on tumors indicate that the HANP/Ce6 complexes are an ideal candidate for tumor theranostics. In addition, incubating cells with the HANP/Ce6 complexes did not alter their cellular morphology, which is indicative of the biocompatibility of these nanocomplexes. Evaluation of ROS Generation via HANP/Ce6 Complexes In Vitro. To evaluate the PDT effect in vitro, ROS generation was measured in HANP/Ce6- and Ce6-treated cells. Figure 3a shows that a high amount of ROS was detected in HANP/Ce6-treated cells, 13



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which in turn increased the accumulation of HANP/Ce6 complexes in cells. On the other hand, a low amount of ROS was detected in Ce6-treated cells, which may be attributable to their non-targetability, ultimately resulting in the entry of a minimal amount of Ce6 molecules into the cells. We next performed an MTT assay to assess the phototoxicity of HANP/Ce6 and free Ce6 molecules. Figure 3b shows that the HANP/Ce6 complexes and free Ce6 molecules without laser irradiation treatment groups presented negligible cytotoxicity levels and over 80% of the cells remained alive at a concentration of 40 µg/mL (the equivalent amount of Ce6). However, cell viability decreased when a 630-nm laser was used in irradiating the HANP/Ce6-treated cells, which followed a dose-dependent manner. Only 13.05% ± 1.43% of the HANP/Ce6treated cells remained alive at a concentration of 40 µg/mL (equivalent amount of Ce6). Because of the poor targetability of free Ce6 molecules, more than half (56.32% ± 2.9%) of the HT29 cells were alive after treatment even with laser illumination. To further confirm the PDT-induced cancer cell death, calcein AM/propidium iodide (PI) costaining was performed. Coincident with the results of the MTT assay, the HT29 cells that received the HANP/Ce6 complexes with laser irradiation were stained red, which was indicative of cell death, whereas only a few cells were found dead with laser irradiation in the absence of HANP/Ce6. Furthermore, only cancer cells subjected to laser irradiation underwent apoptosis, and those not exposed to the laser spot remained alive, thereby suggesting that PDT affects tumors locally and does not affect adjacent tissues (Figure 3c). Fluorescence Imaging of Tumor Targeted Delivery of HANP/Ce6 Complexes 14


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Following Systemic Administration. Optical imaging is a promising method for preclinical and clinical drug development because it is inexpensive, does not involve irradiation, and is easy to operate. Optical imaging allows the determination of whole body distribution and monitoring of the intratumoral accumulation of a labeled agent. To develop an effective tumor PDT, it is essential to determine the laser irradiation time window. Because Ce6 elicits strong fluorescent signals, optical imaging was performed to investigate the tumor accumulation profile of the HANP/Ce6 complexes and free Ce6 molecules in HT29 tumor-bearing mice. NIR fluorescence imaging was performed after intravenous injection of HANP/Ce6 complexes and Ce6 molecules (dose: 5 mg/kg). Because HANP will be readily degraded to low-molecular weight components by Hyal and release Ce6 after being taken up by cancer cells34, it is expected that the recovered Ce6 fluorescent signal will indicate the location of the tumor, which may then be used as guide in the subsequent PDT application. As Figure 4a shows the strong fluorescent signals in the tumor site after the systemic administration of HANP/Ce6 complexes via both active CD44-targeting and passive EPR effects, with the signals peaking at 4 h post-injection (p.i.). Tumor/muscle ratio increased from 1.49 ± 0.32 at 1 h p.i. to 3.47 ± 0.46 at 4 h p.i., and then subsequently decreased to 1.19 ± 0.26 at 24 h p.i. (Figure 4b). Weak fluorescent signals from free Ce6 molecules were observed, which were mainly due to their non-targetability. No significant changes in tumor/muscle ratio (1.10 ± 0.13, 1.10 ± 0.24, 1.13 ± 0.14, 1.11 ± 0.26, 1.05 ± 0.15, and 1.10 ± 0.25) in the mice that received free Ce6 molecules at various time points (1, 2, 4, 6, 12, and 24 h p.i.) after injection were observed. In order to verify the specific 15



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accumulation of HANP into tumor via CD44 targeting, we performed a blocking experiment. Free HA was intravenously administrated into tumor-bearing mouse to saturate CD44 before HANP/Ce6 was injected. As Figure S4 shown, tumor uptake of HANP was reduced in vivo and ex vivo, suggesting HANP accumulation is mediated by targeting of CD44. It has to note that there is still some optical signals in tumor even with free HA blocking, and this is because of the tumor EPR effect of HANP. A similar competitive study result was also reported elsewhere26. To further explore the biodistribution of the HANP/Ce6 complexes, an ex vivo experiment was performed by sacrificing the mice at 4 h p.i.. The tumor and major organs were harvested, imaged, and quantified by optical imaging (Figures 4c and d). The HANP/Ce6 complexes accumulated in the HT29 tumor compared to the Ce6-treated group, thereby suggesting that 4 h p.i. is the optimal time point for initiating PDT in the tumor area. A significant amount of HANP/Ce6 complexes and free Ce6 molecules also accumulated in the liver, which might be attributable to the non-specific uptake of agents by the Kupffer cells of the liver. Modification of the HANP/Ce6 complexes with PEG might also have contributed to the decrease in nonspecific uptake and increase in the accumulation in the tumor. Furthermore, because optical imaging is semi-quantitative, an average fluorescent signal (p/sec/cm2/sr) was used to represent the distribution of the HANP/Ce6 complexes in normal organs and tumors in our study. In such scenario, the signals in certain tissues with a large surface area such as the liver might therefore be lower than a tumor with a small surface area. Further experiments will be needed to improve the behavior and quantification of the HANP/Ce6 complexes in vivo. 16


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Antitumor Efficacy of the HANP/Ce6 Complexes In Vivo by PDT. Based on the observed superior tumor recognition and accumulation, the effect of the HANP/Ce6 complexes on tumor ablation was evaluated. Figures 5a and b show that HT29 tumor growth was not affected in the saline treatment group, and tumor growth was not inhibited in the free Ce6 and HANP/Ce6 treatment groups in the absence of laser application to the tumor. The rate of tumor growth slightly decreased in the free Ce6 treatment group with laser irradiation, possibly due to poor Ce6 accumulation in the tumor. On the other hand, tumor growth was terminated with the application of HANP/Ce6 complexes and laser irradiation after only two continuous PDTs. Compared to the control group, 81.73 ± 3.23% of the tumor was inhibited by HANP/Ce6-mediated PDT. Although the tumor did not diminish by this approach, the application of PDT effectively controlled or inhibited tumor growth in some non-surgical tumors or patients with poor physical conditions too weak to undergo surgery. The combination of PDT with chemotherapy may also increase the anti-tumor effect, thus decrease the use of chemotherapeutic drugs, which in turn reduced the risk for side effects. The body weight of the mice used in this study did not significantly change (Figure 5c), thereby indicating that HANP/Ce6-mediated PDT des not induce systemic toxicity. PET is a noninvasive molecular imaging modality that involves the use of short-lived, highly sensitive positron emitting bioprobes. This technique has been used for in vivo biochemical investigations in several medical fields such as oncology, cardiology, and neurology. One of the widely used PET agents is ([18F]FDG). [18F]FDG that is injected into patients accumulates in tumor cells as glucose analogs due to the upregulation of 17



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hexokinase, which, among other mechanisms, induces high glucose cellular uptake. This study is the first to utilize [18F]FDG PET to monitor of PDT responses to HANP/Ce6 complexes in a mouse tumor model. [18F]FDG PET images were obtained before (day 0) and 1, 7, and 14 days after PDT. PET images scanned at day 0 before PDT was set as baseline, and the PDT response to HANP/Ce6 complexes was monitored at the time points of 1, 7, 14 days after light irradiation. Figure 6 shows a prominent increase in [18F]FDG uptake in the control group tumors of saline, free Ce6, and HANP/Ce6 without laser irradiation. In the saline group, tumor [18F]FDG uptake increased from 2.62 ± 0.11% ID/g before PDT to 4.93 ± 0.46% ID/g, 5.52 ± 0.21%,ID/g, and 5.15 ± 0.53% ID/g on days 1, 7, and 14 (Figure 6), which coincides with the observed growing tumors in these groups. Similarly, [18F]FDG uptake in free Ce6 and HANP/Ce6 without laser treatment also shown an upward tendency from day 1 to day 14. On the other hand, [18F]FDG tumor uptake did not increase in mice that were treated with HANP/Ce6 complexes and laser irradiation as early as 1 day after PDT.

No

obvious changes of [18F]FDG uptake was observed in HANP/Ce6 with laser treated group for 14 days (2.41 ± 0.23% ID/g, 2.33 ± 0.28 % ID/g, 2.5 ± 0.23% ID/g, and 2.26 ±0.27% ID/g on days 0, 1, 7, and 14 after PDT), which coincided with the observed inhibited tumor growth in the PDT-treated group (Figures 5a and 6). In addition to the tumors, [18F] FDG was also observed in the brain, heart, and bladder, which are sites with high metabolic rates, even in the absence of tumors. These results suggest that PDT-induced cell death occurs shortly after the initiation of treatment, and the 18F[FGD] PET imaging technique effectively predicts PDT tumor responses before changes in 18


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tumor size are observed. To further confirm the changes in the histological features of the tumor, the mice that received different treatments were sacrificed at 14 days after PDT. The normal organs and tumors were collected and subjected to H&E staining (Figure 7). Histological assessment showed that the tumor tissues were destroyed, and the number of tumor cells significantly decreased after HANP/Ce6 treatment and laser irradiation (Figure 7a), whereas tumors in the HANP/Ce6-treated group without laser illumination showed no detectable histological changes, thereby suggesting that PDT effectively inhibits tumor growth. In addition, tumor tissues exhibited no histological changes in the free Ce6 molecule-treated group regardless of laser irradiation, which was similar to that in the saline-treated group. Figure 7b shows that the no histological changes were observed in the normal organs from all treatment groups, thereby indicating that localized PDT effectively inhibits tumor growth without affecting nearby normal organs. CONCLUSIONS We successfully developed a simple and robust polymeric nanoparticle photodynamic agent by encapsulating Ce6 molecules into HANPs, which we hereby named as HANP/Ce6 complexes. These nanocomplexes are capable of binding to CD44 ligands, which are overexpressed in cancer cells and predominantly accumulate in tumors at 4 h p.i. via passive EPR effect and active CD44 targeting as observed by fluorescent imaging. After low power near infrared laser illumination, tumor growth in HANP/Ce6treated mice was significantly inhibited compared to that in the control groups 19



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regardless of laser irradiations, and no detectable side effects were observed. To evaluate the HANP/Ce6-mediated tumor PDT, [18F]FDG PET was utilized before and after PDT, which coincided with changes in tumor size upon [18F]FDG uptake. Our in vitro and in vivo findings indicate that our PDT targeting agent is effective, shows excellent biocompatibility, tumor targetability, and tumor suppression capacity. The HANP-based drug delivery system can also be used in other PS delivery, especially for those that are unfit for chemical modifications. This novel PDT agent and strategy for determining PDT responses may be potentially used in the clinic. ASSOCIATED CONTENT Supporting Information Standard curve and drug release profile are presented in this section. This material is available free of charge via the Internet at http://pubs.acs.org. Author Contributions All authors contributed to the writing of this manuscript. All authors have given their approval to the final version of the manuscript. Notes The authors declare no competing financial interests. Acknowledgments The National Science Foundation of China (Grant Nos. 81571708, 81771869, 81501506, 51373144, and 81201129), National High Technology Research and Development Program of China (863 Program) (Grant No. 2014AA020708), the Fundamental Research Funds for the Central Universities (Grant No. 20720150064), 20


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the Research Fund of Science and Technology Department of Jilin Province (Grant No. 20160101001JC), the Department of Education of Jilin Province for Thirteen-Five Scientific Technique Research (Grant No. [2016]460), and the Norman Bethune Program of Jilin University (Grant No. 2015219) supported this study. 21



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Figure legends: Scheme 1. Schematic illustration of the preparation of the HANP/Ce6 complexes as photodynamic therapy agents. (a) The preparation of the HANP/Ce6 complexes. (b) Molecular mechanism of the HANP/Ce6 complexes for photodynamic therapy (PDT). HANP/Ce6 complexes reach the tumor site by EPR effect and binding of HA to CD44, then the drug is released via enzymatic hydrolysis of hyaluronidase for PDT. Figure 1. Characterization of HANP/Ce6 complexes. (a) Stability test for the HANP/Ce6 complexes and free Ce6 molecules in FBS, cell medium, PBS, and water for 7 days. No visible precipitation was observed after incubation. (b) NIR-UV-vis absorbance spectra of the HANP/Ce6 complexes and free Ce6 molecules. (c) Fluorescence spectra of the HANP/Ce6 complexes and free Ce6 molecules. (d) Size distribution of HANP and HANP/Ce6 complexes by dynamic light scattering (DLS). Figure 2. Cellular uptake of HANP/Ce6 complexes by HT29 and NIH3Ts cell lines. The HT29 and NIH3T3 cells were incubated with Ce6 and HANP/Ce6 complexes at the same equivalent Ce6 concentration (40 µg/mL) for 4 h and then imaged by confocal laser scanning microscopy (CLSM). The red color represents the Ce6 molecules, whereas the blue color indicates DAPI staining. The scale bar is 20 μm. The strong red color in the HT29 cells indicates that the HANP/Ce6 complexes gained entry into the cells via CD44 binding. Figure 3. In vitro PDT of cancer cells. (a) Generation of ROS in the HANP/Ce6 complex- and free Ce6 molecule-treated HT29 cells. ROS was detected with a commercialized ROS probe (DCFH-DA). Higher fluorescent intensities indicate 22


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greater rate of ROS generation. (b) Cell viability of the HT29 cells that were incubated with various concentrations of free Ce6 molecules or HANP/Ce6 complexes after irradiation with a 630-nm laser (150 mW/cm2, 3 min) and without irradiation. The concentrations of the free Ce6 molecules and the HANP/Ce6 complexes are 2.5 µg/mL, 5 µg/mL, 10 µg/mL, 20 µg/mL, and 40 µg/mL, respectively. (c) Calcein AM/PI costaining by fluorescence microscopy. HT29 cells were incubated with HANP/Ce6 (20 µg/mL) and treated with 630-nm laser (150 mW/cm2 for 3 min). (1) HT29 cells without irradiation, (2) HT29 cells with irradiation, and (3) on the boundary of laser spot. Figure 4. (a) In vivo fluorescence images of HT29 tumor-bearing mice treated with HANP/Ce6 complexes and free Ce6 molecules. Circles indicate the tumor site. (b) Tumor/muscle (T/M) ratio of HT29 tumor-bearing mouse treated with HANP/Ce6 complexes or free Ce6 molecules. The color bar indicates radiant efficiency (Min: 7.09×107, Max: 1.38×108 scaled p/sec/cm2/sr). (c) Ex vivo fluorescence images of organs and tumors in HT29 tumor-bearing mice after 4 h post-injection of HANP/Ce6 complexes and free Ce6 molecules. The color bar indicates radiant efficiency (Min: 5.04×107, Max: 2.38×108 scaled p/sec/cm2/sr). (d) Fluorescence intensities of the tumor and organs in (c). Figure 5. In vivo PDT effect of the HANP/Ce6 complexes by intravenous administration into a HT29 tumor mouse model. (a) Representative photographs of SCC7 tumor-bearing mice at different days after treatment with HANP/Ce6 complexes, Ce6 molecules, or saline. (b) Tumor growth curves of different groups of HT29 tumorbearing mice. Error bars represent the standard deviations of 5 mice per group. *P < 23



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0.05. (c) The body weight of the mice was measured during the 14-day evaluation period under different conditions. Figure 6. [18F] FDG PET monitoring of HANP/Ce6 complex-mediated PDT. Quantitative small-animal PET region-of-interest analysis of tumor uptake of [18F] FDG in each group. *P < 0.05. Figure 7. H&E staining of tumor (a) and major organs (b). Tumor tissue was significantly destroyed after PDT, whereas no abnormalities in the heart, liver, spleen, lungs, or kidneys were observed. Scale bar: 20 µm.

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Scheme 1.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Figure 6.

8.0 7.0 6.0

% ID/g

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HANP/Ce6 w/ laser HANP/Ce6 w/o laser Ce6 w/ laser Ce6 w/o laser Saline

*

*

*

5.0 4.0 3.0 2.0 1.0

D0

D1

D7

D14

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Figure 7.

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Table 1. Ce6 loading efficiency at different conditions Ce6 to HANP ratio (wt%)

Loading content (wt%)

Yield (%)

1:9 1:4 2:3

7.39 19.56 25

73.9 97.83 62.5

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TOC figure

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Construction and Evaluation of a Targeted Hyaluronic Acid

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