heparin Nanoparticles for Chemo- Photodynamic

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Pluronic/heparin Nanoparticles for Chemo-Photodynamic Combination Cancer Therapy through Photo-induced Caspase-3 Activation Nisar Ul Khaliq, Dal Yong Park, Hye Jin Lee, Keun Sang Oh, Jae Hong Seo, Sang Yoon Kim, Chang Soon Hwang, Tae-Hong Lim, and Soon Hong Yuk ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00572 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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ACS Applied Nano Materials

Pluronic/heparin Photodynamic

Nanoparticles Combination

for

Chemo-

Cancer

Therapy

through Photo-induced Caspase-3 Activation Nisar Ul Khaliq, †, # Dal Yong Park, †, # Hye Jin Lee,† Keun Sang Oh, † Jae Hong Seo, ‡ Sang Yoon Kim,§ Chang Soon Hwang,ǁǁ Tae-Hong Lim,Ѻ and Soon Hong Yuk†, ‡, * †

College of Pharmacy, Korea University, 2511 Sejongro, Sejong 30019, Republic of Korea

‡

Biomedical Research Center, Korea University Guro Hospital, Guro-gu, Seoul 08308, Republic of Korea §

Department of Otolaryngology, Asan Medical Center, University of Ulsan, College of Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Republic of Korea ǁǁ

TGel Bio Co., Ltd., 8 Samsungro, 86 gil,Gangnam-gu, Seoul 06185, Republic of Korea

Ѻ

Department of Biomedical Engineering, The University of Iowa, Iowa City, Iowa 52242, USA

*

Corresponding author: Soon Hong Yuk, Ph.D. (Telephone: 82-44-860-1612, Fax: 82-44-860-

1606, E-mail: [email protected]) #

These authors contributed equally to this paper

KEYWORDS:

apoptosis,

photodynamic

therapy,

prodrug,

combination

therapy,

Pluronic/heparin nanoparticles

ACS Paragon Plus Environment

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ABSTRACT: The Pluronic/heparin nanoparticles (NPs) were prepared for chemo-photodynamic combination cancer therapy through photo-induced caspase-3 activation. Doxorubicin (DOX) prodrug and methylene blue (MB) were assembled into a single structure by electrostatic interaction with heparin and further loaded into the NPs. MB is a photosensitizer, which can be used for photodynamic therapy, which can elicit cell death by producing reactive oxygen species (ROS). The DOX prodrug was DOX conjugated to a labile tetrapeptide (DEVD) by caspase-3 upon apoptosis. ROS was measured to verify ROS-mediated apoptosis. Membrane integrity test and flow cytometry analysis were performed to examine induced apoptosis. The tumor targeting and the in vivo antitumor efficacy were measured to evaluate chemo-photodynamic combination cancer therapy in tumor-bearing mice.


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INTRODUCTION A combination cancer therapy refers to a strategy which is based on the utility of several approaches to destroy cancer cells more efficiently through different modes of action, such as targeting various signal transduction pathways or overcoming cellular heterogeneity. Moreover, it provides synergistic effects with reducted doses of toxic components as well as a significant lessening of nonspecific toxicity.1-4 Photodynamic therapy (PDT) is a cancer therapeutic modality, which eradicates specific tumor area with antitumor vasculature effects as well as by apoptosis or necrosis.5-18 Cationic sensitizer such as methylene blue (MB) has been found to be effective in PDT (high light absorption at 665 nm) against several diseases.19-21 Furthermore, several reports have suggested multifunctional characteristics of MB, such as, low toxicity,22-24 in vivo staining of tumors,25-26 the deactivation of nucleic acid,27 and the generation of singlet oxygen.28 Photo-irradiation with MB could induce apoptosis with caspase-3 expression, critically contributing to the anticancer effect.29 Doxorubicin (DOX) is considered to be extremely effective as a cancer therapy agent. However, drug resistance and cardiotoxicity restrict its potential use as an effective chemotherapeutic agent.30-32 To overcome these limitations, DOX Prodrug was prepared and characterized as an apoptosis-activatable prodrug in our previous works.33-35 DEVD-S-DOX maintains an inactive state with tetrapeptide consisted of Aspartic acid, Glutamic acid, Valine and Aspartic acid (DEVD). The conversion of DOX Prodrug into its active form (DOX) has been demonstrated by radiation, the small amount of DOX, and PDT in our previous reports.33-35 Nanoparticles (NPs) are a type of delivery system that can deliver various modalities, such as, chemotherapeutic drugs, photosensitizers, or diagnostic agents.36-37 Owing to their structural versatility, NPs have been used to deliver multiple therapeutic cargoes simultaneously to target


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sites as a part of combination therapy for cancer treatment.38-39 For the safe journey of the NPs in the bloodstream, polyethylene oxide (PEO)-based NPs have been utilized extensively.40-41 Pluronics are composed of PEO and poly(propylene oxide (PPO). They have different molecular weights depending on PEO/PPO ratios with PEO-PPO-PEO structure. With PEO blocks internally, Pluronic-based NPs show prolonged circulation time in the bloodstream.42 In this study, the Pluronic/heparin NPs were constructed and characterized for a targeted chemo-photodynamic combination chemotherapy as presented in Scheme 1. This combination therapeutic approach was accomplished through the photosensitization of MB for PDT. Induced caspase-3 will cleave DOX prodrug to accomplish the combination therapy. Because the Pluronic/heparin NPs were coated by Pluronic F-68, enhanced permeation retention (EPR) effect may contribute to the elevated tumor accumulation.43 Antitumor efficacy was examined with the cardiotoxicity to support the proposed chemo-photodynamic combination cancer therapy.


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Scheme 1. Schematic description of chemo-photodynamic combination cancer therapy through photo-induced caspase-3 activation. EXPERIMENTAL METHODS Materials. DOX (anhydrous form) and heparin sodium (Molecular weight: 12500) were supplied by Sigma (St. Louis, MO, USA) and Celsus Laboratories (Cincinnati, OH, USA), respectively. BASF Corp. and Amersham Bioscience (Piscataway, USA) supplied Pluronic F-68 (Molecular weight: 8,350) and Cy5.5, respectively. ORIENT BIO Inc. (Daejeon, Korea) supplied Male C3H/HeN mice and Sprague-Dawley (SD) rats.


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Preparation of the NPs. The detailed preparation method of DOX prodrug (DEVD-S-DOX) and the Pluronic/heparin NPs was presented in Supporting Information and our previous reports.33-35 The NPs in this study were prepared as a single assembly carrying MB and DOX prodrug. To understand the behavior of the NPs in the bloodstream, Cy5.5 was loaded into the NPs (loading amount: 0.1 wt%). Characterization of the Pluronic/heparin NPs. Particle size analyzer and transmittance electron microscope were utilized to characterize the morphology of the Pluronic/heparin NPs. Detailed experimental condition was presented in our previous reports.34, 35 In Vitro Drug Release Characteristics. Dialysis bag system with the aqueous solution containing the NPs was utilized to observe the release profile of MB or DOX prodrug from the NPs. It was equilibrated with 20 mL of PBS in shaking water (shaking speed: 100 rpm, 37 °C). Periodical sampling was performed by withdrawing 2 mL aliquots from the dialysis bag. To measure the drug loading amount, the Pluronic/heparin NPs were equilibrated with distilleddeionized water (10 mg/mL) at 25oC for 24 hours and the aqueous solution of Pluronic/heparin NPs was centrifuged at 3500 rpm for 5 minutes. The supernatant of each aqueous solution (3 ml) was mixed with DMSO (3:1v/v) and passed through 0.22 µm filter for quantification. Detailed experimental condition was presented in our previous reports.34, 35 Singlet Oxygen Assay. The singlet oxygen species was quantitatively evaluated on the basis of the oxidation of 4-nitroso-N, N-dimethyl-aniline (RNO) measurements as described in earlier works.44,

45

To do this, the numbers of singlet oxygen species generated from the MB were

evaluated using p-nitroso-dimethylaniline. Detailed experimental condition was presented in our previous reports.35, 45


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Reactive Oxygen Species (ROS) Detection. ROS detection was carried out with cells (1 ×106/well) treated with free MB and MB in the NPs (MB concentration: 25 µg/mL). Murine squamous cell carcinoma (SCC-7) cancer cells were used as a cell system throughout the experiment. Photo-irradiation was performed using a Laser (690 nm, 0.01 W/cm2) for 10 minutes, providing a total amount of energy of 6 J. This condition was maintained throughout the experiment. Detailed experimental condition was presented in our previous reports.35 Membrane Integrity Tests Using Trypan Blue. The toxicity induced by photo-irradiation was assessed through membrane integrity test. The cells (5×105/well) were treated with the NPs containing MB or those containing MB and DOX prodrug at selected concentrations (equivalent concentration: 25 µg/ mL each). Trypan blue was used as a marker and detailed experimental condition was presented in our previous reports.35 MTT Assay. To observe the effect of photo-irradiation in the cellular level, the cytotoxicity of free DOX, free MB, and a mixture of MB and DOX prodrug were evaluated in a concentrationdependent manner with or without photo-irradiation. The cytotoxicity levels of the Pluronic/heparin NPs with various combinations (empty Pluronic/heparin NPs, the Pluronic/heparin NPs with MB, DOX prodrug, or MB/DOX prodrug combination) were then examined with or without photo-irradiation. Detailed experimental condition was presented in our previous reports.34, 35 Caspase-3 Activation Analysis. Caspase-3 expression by photo-irradiation was assessed in the cellular level (5×105/well) with the NPs containing MB or those containing MB and DOX prodrug at selected concentrations (equivalent concentration: 25 µg/ mL each). Detailed experimental condition was presented in our previous reports.34, 35


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Cellular Uptake Behavior. Cellular uptake behavior and cell apoptosis were examined in the cells (5×105/well) with the NPs containing MB or those containing MB and DOX prodrug at selected concentrations (equivalent concentration: 25 µg/ mL each). The correlation between them was determined using a flow cytometry analysis. Detailed experimental condition was presented in our previous reports.34 In Vivo Biodistribution of the NPs. To observe the tumor accumulation, tumor-xenografted C3H/HeN mice (20-25g) were injected with various active substances through the tail vein. The fluorescence images were taken at 0, 1, 3, 5, 7, 9 and 24 hours post-injection. Detailed experimental condition was presented in our previous reports.34 Pharmacokinetics studies were performed to support the tumor accumulation of the NPs and various substances were delivered through intravenous injection via the tail vein to SD rats (2025g). After periodical withdrawing (200 µL), a refrigerated centrifuge was then operated to collect plasma for 10 minutes at 1500xg. For fluorescence intensity measurement, the plasma samples and fresh plasma was examined by a microplate reader at 485/590 nm. In Vivo Antitumor Efficacy. The same experimental condition for in vivo biodistribution study was applied to the mice to observe the antitumor efficacy. At the tumor size of 50-100 mm3, several experimental groups of 5 mice were prepared and treated with various formulations. Every formulation used a dose of 2 mg/kg MB or DOX-equivalent. Photo-irradiation (180 J/cm2) was applied to the mice 1 hour after intravenous injection. Detailed experimental condition was presented in our previous reports.34 In vivo experiments were performed based on the regulations of Korea University, Republic of Korea. The visualization of DOX at the tumor tissues was also performed as presented in our previous reports.34.


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Cardiotoxicity Assay. Troponin-T Mouse ELISA kits (Kamiya Biomedical Company) was utilized to assess the cardiotoxicity. After withdrawing the blood sample (50 µL), collected blood samples were transferred to 50 µL of PBS containing sodium heparin (2-5 IU/tube). A microtiter plate was used to measure the cardiac Troponin-T (cTnT) level. Detailed experimental condition was presented in our previous reports.34

RESULTS AND DISCUSSION The Pluronic/heparin NPs for Combination Therapy. As described in Scheme 1, the Pluronic/heparin NPs were formed in an aqueous medium via complex formation between positive MB or DOX prodrug and negative heparin with subsequent stabilization using Pluronic F-68. As demonstrated in Figure 1a, the complexation of MB, DOX prodrug, and heparin led to precipitation in the aqueous solution indicating hydrophobic characteristics. Through stabilization using freeze-drying, the complexed mixtures were encapsulated into the hydrophobic core of Pluronic F-68 to form the Pluronic/heparin NPs as shown in Figure 1b (Pluronic F-68 formed polymeric micelle in the aqueous medium with a central hydrophobic core (PPO) and PEO shell).42 Several Pluronic/heparin NPs were prepared at various polymer to drug ratios to confirm the stable Pluronic/heparin NPs, as described in Table S1 (Supporting Information). Among them, 1 mg of MB, 1 mg of DOX prodrug, and 10 mg of heparin with 200 mg of Pluronic F-68 exhibited the best stability. The size distribution and TEM pictures revealed that the Pluronic/heparin NPs had a diameter of 202.9+12.0 nm with a core/shell structure as shown in Figure 1c (PDI: 0.317). To verify stable Pluronic/heparin NPs in the blood circulation, the particle size analysis was performed with the NPs in the serum. The diameter was 204.3+ 2.6nm (PDI : 0.348 ).


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The drug loading (%) of the Pluronic/heparin NPs is described in Table S2.

Figure 1. (a) MB and DOX prodrug in the aqueous medium, (b) MB/DOX prodrug combinationloaded NPs in the aqueous medium, and (c) TEM picture of MB/DOX prodrug combinationloaded NPs with their size distribution.


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In Vitro Release Kinetics. The release patterns of active agents were studied from the respective micellar structures, which were correspondingly as shown in Figures 2a and 2b. Cationic MB/DOX prodrug combination and anionic heparin were transformed into ionic complex, which was subsequently stabilized to form the Pluronic/heparin NPs. Retarded release during the systemic circulation was accomplished with Pluronic/heparin NPs as shown in Figure 2. The rapid release patterns of free MB and DOX prodrug with released amounts of 47% and 67% were observed for 1 hour and more than 80 % of free agents were released within 6 hours. In contrast, the Pluronic/heparin NPs showed the retardation of the release rate because of the stable formation of ionic complex and their surface coating with Pluronic-F68. On the whole, the sustainability was accomplished with the Pluronic/heparin NPs.

Figure 2. Release profile of (a) MB and (b) DOX prodrug from the Pluronic/heparin NPs.

Photo-Induced Apoptosis. To evaluate the degree of photo-induced apoptosis, photoirradiation-mediated ROS was quantified and a membrane integrity was examined. Figure 3a showed the concentration change of RNO (a singlet oxygen sensor) 46, 38 as a function of time at


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various concentrations of MB, suggesting that the RNO concentration decreased with an increased time of laser exposure. This was due to the generation of singlet oxygen with photoirradiation. We observed that photo-irradiation of MB at concentrations of 25 µg/mL and 50 µg/mL led to a considerable decrease in the RNO concentration because of generated singlet oxygen. Based on this observation, we used MB of 25 µg/mL throughout the experiment. The quantification of ROS was also performed with the NPs containing MB. Compared to free MB, a further increase was observed in the ROS level with the cells incubated with the Pluronic/heparin NPs (Figure 3b). This may have been due to the high uptake of the NPs containing MB and to the resistance of the NPs to the photo-bleaching of the MB during photoirradiation process.46

Figure 3. (a) Time-dependent singlet oxygen generation from free MB with photo-irradiation power (0.01 W/cm2) at various concentrations of MB and (b) ROS activation in the cell system during 4 hours (n =3).


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Previous reports suggested that the generation of ROS by photo-irradiation possibly might be disruptive to the integrity of the cell membrane and this led to the entrance of trypan blue into the cytoplasm.47, 48 To assess this, the morphological changes of the cells caused by ROS generation were evaluated after a treatment with the Pluronic/heparin NPs as shown in Figure 4. The Pluronic/heparin NPs without photo-irradiation showed minor morphological changes in the cells. As a result, slight trypan blue coloration was observed on the surfaces of cells minimally stained with trypan blue dye. In contrast, photo-irradiation on the cells treated with the Pluronic/heparin NPs demonstrated a significant change in the cell morphology with intense trypan blue dye staining. These observations revealed that photo-irradiated cells treated with the Pluronic/heparin NPs could efficiently produce ROS and disturb the plasma membrane to trigger apoptosis. This led to the caspase-3 expression in the cells with further cleavage of DOX prodrug into an active form of DOX. The reason to maintain blue background was only to show the obvious mechanistic difference between the treatment groups. The experiments were performed several times and we found that too much excessive washing with PBS could not retain the cells in the culture plate. Furthermore, the experiment was a multistep procedure of combination therapy and further washing could not guarantee the cells confluence. The control image provided in the study was the best possible picture of SCC-7 cancer cells which maintained their structural integrity after several washing. The difference in the control background was due to several washing. Membrane integrity test provided us the qualitative results. Trypan blue was used in the study for membrane integrity test to figure out whether the ROS production after photoirradiation could induce loss to the membrane integrity of the cells. The ruptured membrane easily allowed trypan blue to stain the cells.


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Figure 4. The observation of cell membrane integrity with photo-irradiation (0.01 W/cm2 for 10 minutes or 6 J). To study caspase-3 activation by ROS in photo-irradiated apoptotic cells, caspase-3 expression was quantified in photo-irradiated cell system incubated with the Pluronic/heparin NPs (Figure 5). Time-dependent caspase-3 expression was observed with significant increase after the incubation with the Pluronic/heparin NPs for 4 hours. These findings revealed that the ROSinduced caspase-3 expression cleaved DOX prodrug into active DOX and this provoked caspase3 expression with the amplification of this cycle.


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160 Caspase-3 Activity (RFU 485/535 nm)

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140

the Pluronic/heparin NPs with MB + PDT 6J the Pluronic /heparin NPs with MB and DEVD-S-DOX+ PDT 6J

**

120 100 80 60 40 20 0 0.5 h

1h

2h

4h

Time (hours)

Figure 5. Caspase-3 expression in the cell system during 4 hours (n =3). MTT Assay. The cytotoxicity levels of free agents and the Pluronic/heparin NPs were measured in a concentration-dependent manner followed by photo-irradiation for 24 hours as described in Figure S1. Free agents such as MB and DOX prodrug showed the highest level of cell viability in the absence of photo-irradiation (see Figure S1a). This revealed that the MB was nontoxic with a free form and that DOX prodrug maintained its nontoxic nature. Free MB reduced the cell viability significantly in the presence of photo-irradiation. This occurred because ROS was generated after photo-irradiation. Likewise, MB combined with DOX prodrug reduced cell viability significantly, as ROS production after photo-irradiation of MB induced apoptosis with caspase-3 expression. 29 This led to the cleavage of DOX prodrug into DOX. The highest cell mortality and the least cell viability were observed in DOX-treated cells. Additionally, the concentration-dependent cytotoxicity was examined with various Pluronic/heparin NPs (see Figure S2). The cell viability did not change significantly with the Pluronic/heparin NPs during 24 hours without photo-irradiation. In contrast, photo-irradiation


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increased the cytotoxicity with the Pluronic/heparin NP. A more significant cytotoxicity was exhibited with the combined delivery of MB and DOX prodrug using the Pluronic/heparin NPs. Similar to the free DOX, DOX in the NPs also exhibited the lowest degree of cell viability. Overall, the cell viability associated with the Pluronic/heparin NPs was higher than that of the free agents because of controlled release of MB and DOX prodrug from the Pluronic/heparin NPs. The IC50 values of every substance are presented in Table S3. Cellular Uptake Behavior. Time-dependent behaviors of various substances in the cell system were presented in Figure 6. At any time points, free DOX was observed in the nucleus as reported previously (Figure 6a).49,

50

In contrast, the DOX in the Pluronic/heparin NPs was

observed in the cytoplasm up to 30 minutes. At 1 hour, DOX was observed in the nucleus up to 4 hours as shown in Figure 6b. This suggested that DOX was translocated into the nuclear region after endocytosis of the Pluronic/heparin NPs. However, DOX prodrug from the Pluronic/heparin NPs was found in the cytoplasmic region without photo-irradiation at all time points (Figure 6c). After endocytosis, DOX prodrug from the Pluronic/heparin NPs maintained its form as a prodrug and was restricted with regard to entry into the nucleus. DOX prodrug was activated by caspase-3 expression upon apoptosis.33 Due to such property, the entry of DOX prodrug into the nucleus was restricted and DOX prodrug stayed in the cytoplasm as a prodrug. Once ROS was generated after photo-irradiation of MB and induced apoptosis with caspase-3 expression, DOX prodrug was cleaved into free DOX. Free DOX was a small molecule, and they could permeate into the nucleus with cell death.


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Figure 6. Cellular uptake behaviors of (a) free DOX, (b) DOX in the Pluronic/heparin NPs, (c) the Pluronic/heparin NPs with MB/DOX prodrug combination (without photo-irradiation), and (d) the Pluronic/heparin NPs with MB/DOX prodrug combination (with photo-irradiation) (x 40). Conversely, the co-delivery of MB and DOX prodrug using the Pluronic/heparin NPs showed different cellular uptake behavior with photo-irradiation (Figure 6d). Translocation of DOX was observed in the nuclear region and a slight morphological change was observed at 1 hour, representing the cleavage of DOX prodrug into free DOX. With Photo-irradiation, ROS was generated with ROS-mediated caspase-3 expression. The maximal morphological change (rounded cells) with a significant presence of DOX in the nuclear region was observed for 4 hours. Figure 7 showed the cellular uptake behavior by a flow cytometry. The treatment of free DOX or the Pluronic/heparin NPs containing MB with photo-irradiation was performed in the cell system as a positive control. Incubation with free DOX resulted in more than 80 % apoptotic cells and 10% necrotic cells for 4 hours. In contrast, the cells treated with Pluronic/heparin NPs containing MB followed by photo-irradiation showed a different apoptotic behavior with an early apoptotic phase (22.7%) and a late apoptotic phase (64%). This outcome indicated that photoirradiated Pluronic/heparin NPs with MB generated ROS, which induced apoptosis. Likewise, the application of the Pluronic/heparin NPs with MB/DOX prodrug combination exhibited 11.2 % early apoptotic cells and 79.6% late apoptotic cells in the presence of photo-irradiation because of the generation of DOX from DOX prodrug.33-35


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(a)

100

the pluronic/heparin NPs with

Free DOX

MB and DEVD-S-DOX+PDT the pluronic/heparin NPs

80

Necrosis (%)

Late apoptosis (%)

Early apoptosis (%)

Late apoptosis (%)

Necrosis (%)

20

Early apoptosis (%)

40

Necrosis (%)

60

Late apoptosis (%)

with MB +PDT

Early apoptosis (%)

Apoptotic cells vs Necrotic cells (%)

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

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0 Time (4 hours)

(b)

Figure 7. (a) Flow cytometry analysis of the cell system with various treatments with photoirradiation and (b) its statistical analysis.


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Tumor Accumulation of the NPs. Tumor accumulation behavior of the Pluronic/heparin NPs containing Cy5.5 was examined in tumor-xenografted mice (Figure 8). The Pluronic/heparin NPs revealed higher fluorescence intensity level in the vicinity of the tumor as compared to free Cy5.5 (a control). It was noted that the Pluronic/heparin NPs had accumulated in the tumor vicinity 1 hour after the injection. As positive controls, the Pluronic/heparin NPs with MB and DOX were utilized. As described in the preparation method and in the TEM imagery (Figure 1c), the surfaces of the Pluronic/heparin NPs were coated with Pluronic F-68, and this feature extended half-life in the blood stream.42 Pharmacokinetic studies also supported the longer systemic circulation of Pluronic/heparin NPs as shown in Figure S3. After injecting free DOX, DOX prodrug, and the NPs, the time-dependent plasma concentration was measured and the Pluronic/heparin NPs exhibited the highest value. Because of these interesting features, the majority of the NPs accumulated at the tumor instead of liver through the EPR effect.43, 49 The fluorescence intensities from dissected organs also supported the tumor targetability of the Pluronic/heparin NPs as shown in Figure 8b. The Pluronic/heparin NPs with MB/DOX prodrug combination had accumulated in greater amounts in the tumor tissues as compared to the Pluronic/heparin NPs with MB/DOX combination or free Cy5.5. Fluorescence at starting point was measured after the injection of sample through the tail vein of mice. 5 mice were used for one sample and injection time point for each mouse was different (the maximum difference of injection time point was more than 15 minutes). Therefore, fluorescence at starting point could be changed depending the order of injection time. Although the fluorescence at starting point from the Pluronic/heparin NPs with MB/DOX prodrug was higher comparing to other groups, this might be due to the stable systemic circulation of the Pluronic/heparin NPs containing Cy5.5.


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Figure 8. (a) NIRF images and (b) fluorescence images from the major organs (n = 3). Antitumor Efficacy. The antitumor efficacy of the chemo-photodynamic combination therapy was studied with five groups of tumor-bearing mice as shown in Figure 9. For controls, PBS, free DOX (2 mg/kg), free MB (2mg/kg) and MB/DOX prodrug combination were given to the mice with photo-irradiation of 180 J/cm2. Upon photo-irradiation, the mice treated with free MB showed better antitumor efficacy in comparison to the PBS-treated mice during the experiment. Shrinkage of tumor was due to the effect of ROS generated by photo-irradiation. Slightly enhanced antitumor efficacy was observed in the DOX-treated group. Free DOX can permeate easily into the cell during MTT assay, however, it can be easily eliminated from the systemic


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circulation during animal experiment for antitumor efficacy.

As expected, an intravenous

injection of MB/DOX prodrug combination with photo-irradiation enhanced the antitumor efficacy compared to the PBS, MB and DOX outcomes. Although the difference in antitumor efficacy was observed between experimental groups treated with free agents, statistical difference was not significant. The most significant tumor shrinkage was found with the Pluronic/heparin NPs containing MB/DOX prodrug combination or DOX prodrug only. After the first administration of Pluronic/heparin NPs with MB/DOX prodrug combination with photoirradiation, MB induced ROS-mediated apoptosis with caspase-3 expression.29 Tumor regression levels were significantly enhanced through the photodynamic therapy with MB and chemotherapy with DOX stemming from DOX prodrug. This maintained the level of caspase-3 expression. The state of tumor suppression was maintained by additional administrations of the Pluronic/heparin NPs which contained DOX prodrug only. The MB was used to ignite the caspase-3 expression only happened at early-stage. Figure S4 presented the loss in the body weights of mice caused by the treatment-induced toxicity during the treatment. Apparent losses in body weights were observed except for the group treated with DOX.


23


PBS free MB free DOX the mixture of MB and DEVD-S-DOX the Pluronic/heparin NPs with MB and DEVD-S-DOX

3500 3000 3 Tumor Size (mm )

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2500

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N S N S N S

2000

*

1500 1000 500 0 0

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16

Time (Days)

Figure 9. Therapeutic efficacy with photo-irradiation (180 J/cm2) (n =3). After the measurement of antitumor efficacy, the remaining DOX in the tumor tissue was examined as shown in Figure 10. The visualization of free DOX was low at the tumor tissues because of low targetability and retention as well as lower plasma concentration (Figure S3 (Pharmacokinetic studies)). However, fluorescence intensity increased slightly in the groups treated with MB/DOX prodrug combination following photo-irradiation. The Pluronic/heparin NPs containing MB/DOX prodrug combination with photo-irradiation showed the highest fluorescence intensity. This outcome also verified that the highest amount of DOX in the tumor tissues was retained due to the four additional administrations of the Pluronic/heparin NPs which contained DOX prodrug only. These observations demonstrate the effectiveness of the codelivery of MB/DOX prodrug combination using single carrier.


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Figure 10. DOX visualization in the tumor tissue treated with (a) free DOX, (b) MB/DOX prodrug combination, and (c) the Pluronic/heparin NPs with MB/DOX prodrug combination. Considering the release profile of DOX prodrug from the NPs, the sustained release was attained. However, an appreciable amount of DOX prodrug from the NPs was appeared in the blood stream prior to their arrival at the tumor tissue. The released DOX prodrug may cause DOX-induced cardiac injuries. Therefore, cTnT level was measured as shown in Figure 11. Minimal elevation was observed until the third administration, however, the fifth administration of free DOX exhibited enhanced cTnT indicating the initiation of myocardial injury. However, a lower level of cTnT was observed with MB/DOX prodrug combination-treated group comparing to DOX-treated group. The lowest level of cTnT was found after one administration of the Pluronic/heparin NPs with MB/DOX prodrug combination and additional administrations of the


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Pluronic/heparin NPs with DOX prodrug. These results supported that the chemo-photodynamic combination therapy in this study can minimize the cardiotoxicity of DOX without reducing therapeutic efficacy using the Pluronic/heparin NPs supported by PDT. Slight increase in the control groups observed, however, it was not statistically significant. The only statistical significance (P-value < 0.05, *) was shown by the free DOX after fifth injection.

0.06

Plasma cTnT level (ng/ml)

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

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0.04

Control free DOX mixture of MB and DEVD-S-DOX the Pluronic NPs with MB and DEVD-S-DOX

0.02

0.00

0

3

5

Number of injection

Figure 11. The level of Cardiac Troponin-T (cTnT) with various formulations.


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CONCLUSIONS A chemo-photodynamic combination therapy was accomplished through a single assembly of the Pluronic/heparin NPs with MB/DOX prodrug combination in the tumor-bearing mice. The Pluronic/heparin NPs, composed of an ionic complex core with active substances and a Pluronic shell, brought MB and DOX prodrug together to tumor area proficiently based on the EPR effect. The photodynamic therapy was accomplished efficiently through photo-irradiation in the tumor region with ROS-mediated caspase-3 expression. This led to effective chemotherapy in the tumor region through free DOX from DOX prodrug via caspase-3 expression. Although a considerable amount of DOX prodrug was released before its arrival at the tumor tissue, the inactive form of DOX prodrug did not exhibit DOX-associated side effects. The strategy introduced in this study can be considered as a combination therapy to overcome the side effects from free DOX with maintenance of antitumor efficacy.


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AUTHOR INFORMATION Corresponding Author *Soon Hong Yuk, Ph.D. (Telephone: 82-44-860-1612, Fax: 82-44-860-1606, E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. # Nisar Ul Khaliq, # Dal Yong Park contributed equally to this paper. Notes The authors declare that they have no conflict of interest. ACKNOWLEDGMENTS This work was supported by the Technology Innovation Program (10077501) by the Ministry of Trade, Industry & Energy (MOTIE), the Technology development Program (S2561925) funded by the Ministry of SMEs and Startups (MSS), and National R&D Program for Cancer Control (1420390), Ministry of Health and Welfare, Republic of Korea. Supporting Information. See supporting information for the synthesis of DOX prodrug (DEVD-S-DOX), cells cytotoxicity and pharmacokinetic study of Pluronic/heparin NPs. This material is available free of charge via the Internet at http://pubs.acs.org.


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FIGURE CAPTIONS Scheme 1. Schematic description of chemo-photodynamic combination cancer therapy through photo-induced caspase-3 activation. Figure 1. (a) MB and DOX prodrug in the aqueous medium, (b) MB/DOX prodrug combinationloaded NPs in the aqueous medium, and (c) TEM picture of MB/DOX prodrug combinationloaded NPs with their size distribution. Figure 2. Release profile of (a) MB and (b) DOX prodrug from the Pluronic/heparin NPs. Figure 3. (a) Time-dependent singlet oxygen generation from free MB with photo-irradiation power (0.01 W/cm2) at various concentrations of MB and (b) ROS activation in the cell system during 4 hours (n =3). Figure 4. The observation of cell membrane integrity with photo-irradiation (0.01 W/cm2 for 10 minutes or 6 J). Figure 5. Caspase-3 expression in the cell system during 4 hours (n =3). Figure 6. Cellular uptake behaviors of (a) free DOX, (b) DOX in the Pluronic/heparin NPs, (c) the Pluronic/heparin NPs with MB/DOX prodrug combination (without photo-irradiation), and


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(d) the Pluronic/heparin NPs with MB/DOX prodrug combination (with photo-irradiation) (x 40). Figure 7. (a) Flow cytometry analysis of the cell system with various treatment with photoirradiation and (b) its statistical analysis. Figure 8. (a) NIRF images and (b) fluorescence images from the major organs (n = 3). Figure 9. Therapeutic efficacy with photo-irradiation (180 J/cm2) (n =3). Figure 10. DOX visualization in the tumor tissue treated with (a) free DOX, (b) MB/DOX prodrug combination, and (c) the Pluronic/heparin NPs with MB/DOX prodrug combination. Figure 11. The level of Cardiac Troponin-T (cTnT) with various formulations.

Graphical abstract


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heparin Nanoparticles for Chemo- Photodynamic

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