On-Demand Macroscale Delivery System Based on a Macroporous

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Controlled Release and Delivery Systems

On-Demand Macroscale Delivery System Based on Macroporous Cryogel with High Drug Loading Capacity for Enhanced Cancer Therapy Pilseon Im, and Jaeyun Kim ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00911 • Publication Date (Web): 06 Sep 2018 Downloaded from http://pubs.acs.org on September 7, 2018

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ACS Biomaterials Science & Engineering

On-Demand Macroscale Delivery System Based on Macroporous Cryogel with High Drug Loading Capacity for Enhanced Cancer Therapy

Pilseon Im1 and Jaeyun Kim1,2,3*

1

School of Chemical Engineering, Sungkyunkwan University (SKKU), 2066 Seobu-ro,

Jangan-gu, Suwon 16419, Republic of Korea 2

Department of Health Sciences and Technology, Samsung Advanced Institute for Health

Science & Technology (SAIHST), Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea 3

Biomedical Institute for Convergence at SKKU (BICS), Sungkyunkwan University (SKKU),

2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea

*Corresponding author. E-mail: [email protected] Telephone: +82-31-290-7252. Fax: +82-31-290-7272

1

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Abstract The macroscale delivery system has been one of the practical platforms for controlled delivery system by acting as a local depot close to the target tissue. In this study, we fabricated macroporous alginate crygel incorporated with gold nanorods (GNRs) for ondemand release of chemotherapeutic drug from macroscale materials placed beside the target tumor. The macroporous crygel was prepared by ice-crystal templating of covalentlycorsslinked alginate hydrogel incorporated with GNRs. Mitoxantrone (MX), one of the potent anticancer drugs, with positive charge was strongly adsorbed on the negative alginate chains of the cryogels. This system enabled a high loading of MX and a successful on-demand release of strongly-bound MX from the GNR-loaded macroporous cryogels by near infrared (NIR) irradiation by dissociation of the interaction between alginate backbone and MX. Cell viability after NIR-irradiation of MX-loaded macroporous cryogel was significantly low compared to no stimuli condition. In vivo test showed repetitive NIR irradiations on MXloaded cryogel implanted near the tumor suppressed tumor volume 6 times more than that of control group. This simple approach to fabricate macroporous cryogel capable of on-demand release of bioactive cargos could be beneficial in various applications including cell, gene, and the other small molecule delivery system.

Keywords: on-demand delivery, cryogel, alginate, mitoxantrone, gold nanorods, cancer therapy

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Introduction Chemotherapy is one of the typical therapeutic methods to treat cancer. Although conventional systemic chemotherapy is the most reliable treatment for the cancer after surgical removal of tumors, it has been limited by undesired side effects to normal tissue, low targeting efficiency, and a requirement of high dosage of drugs to stimulate therapeutic effect. To circumvent these limitations, diverse drug delivery systems including nanocarriers,1-11 hydrogels,12-15 and scaffolds16-20 have been intensively researched. Macroscale delivery systems based on large-sized materials over a few millimeters, such as polymer hydrogel or three-dimensional macroporous scaffold, are one of the feasible material platforms for controlled drug delivery. Once implanted or injected near target sites, macroscale delivery systems start to act as a depot of pre-loaded drugs and release drugs in the local region close to the target sites. Macroscale delivery platform can circumvent limitations associated with nanoparticle-based systemic delivery system including unwanted distribution in non-targeted tissues, unidentified clearance from body, potential toxicity, and relatively low targeting efficiency.21 Locally placed multiscale delivery platform release drugs by diffusion and the drugs are more efficiently delivered to the target sites, which can lead to the use of lower dose and less side effect. For controlled release of drugs, timely and adequate dosage of drugs will be a key to guarantee therapeutic effects, reducing side effects in chemotherapy. Although the diffusionmediated drug release from macroscale delivery platform is valuable due to the sustained release profile, there are high needs for on-demand drug release system, in which the desired amount of drug is released from the depot at exactly wanted time point. Recently smart drug release systems based on their responsiveness to pH, temperature, and enzyme, have been intensively studied.22-26 However, there are several limitations related to controlling dosage of 3



drugs; for example, in the clinical trial, required dosage or timing of drugs may not be precisely managed in target sites due to a various physiological characteristics in body.27 To get over these problems, real-time controlled drug delivery systems based on external stimuli have been proposed by actuating on/off-switchable phase at instant time. There are several key points to develop ideal on-demand drug delivery system based on mascroscale delivery platform.28 High loading of drugs in the matrix and almost no release of drugs from the matrix in the normal condition is required because longer duration is highly desired after single implantation of macroscale system in the body. In addition, the release of drugs from matrix should be precisely controlled by signals induced in the treatment, such as external stimuli. In this study, we proposed near infrared (NIR)-responsive macroscale delivery system based on macroporous alginate cryogel incorporated with gold nanorods (GNRs) for precisely controlled on-demand release of chemotherapeutic drug (Figure 1a). Alginate hydrogels, composed of α-L-guluronic (G) and β-D-mannuronic (M) acid sugar residues, have been used as macroscale delivery materials, because they have low toxicity, biocompatibility, and characteristic of structure from M and G residues to incorporate various drugs, cell, and small molecules.29-32 Strong electrostatic interactions between positivelycharged anticancer drugs and negatively-charged alginate matrix in macroporous scaffold prepared by ice-templating resulted in high loading of drugs and very slow release from scaffold without an external stimulus once loaded. High temperature induced by phothothermal effect of GNRs after NIR irradiation led to enhanced release rate of electrostatically loaded drugs. When the NIR laser was turned off, increased temperature immediately decreased and the drug release from alginate matrix was suspended. Thus, a small amount of drug was released at off-phase in NIR-responsive system unlike on-phase. 4


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Subcutaneous implantation of GNR/alginate cryogel near tumors in mice and subsequent repetitive NIR irradiation showed that tumor volume was significantly reduced compared to control groups (Figure 1b), representing the potential of the on-demand anticancer drug delivery system.

Experimental Section Materials. Alginic acid sodium salt from brown algae, mitoxantrone dihydrochloride (MX), doxorubicin

hydrochloride

(DOX),

methotrexate

(MTX),

calcium

sulfate,

hexadecyltrimethylammonium bromide (CTAB), gold(III) chloride hydrate (HAuCl4·3H2O), 2,6-dihydrobenzoic acid, thiazolyl blue tetrazolium bromide (MTT), and RPMI-1640 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Silver nitrate (AgNO3) and sodium borohydride (NaBH4) were purchased from Samchun Chemicals (Seoul, Korea). L-ascrobic acid was purchased from Tokyo Chemical Industry (Chou-ku, Tokyo, Japan). Methoxy polyethylene glycol sulfhydryl (mPEG-SH) was purchased from Sunbio (Anyang, Korea). All reagents were used as received without further purification. Synthesis of gold nanorods. Gold nanorods (GNRs) were synthesized by a modified seedmediated growth method.33, 34 First, the gold seed solution was prepared by progressively adding a HAuCl4·3H2O (0.01 M, 0.25 mL) and aqueous ice-cold NaBH4 solution (0.01 M, 0.6 mL) to a CTAB solution (0.1 M, 7.5 mL) under stirring for 2 min. Then, the color of the mixture solution was changed to light brown, representing the formation of gold seed nanoparticles. Next, growth solution was prepared by mixing HAuCl4·3H2O (0.01M, 10 mL), 2,4-dihydroxbenzoic acid (0.067M, 20 mL), AgNO3 (0.01 M, 2 mL), and L-ascorbic acid (0.1M, 1.6 mL) to CTAB solution (0.1 M, 240 mL) under vigorous stirring. To synthesize gold nanorods, 2 mL gold seed solution was added into 271.6 mL growth solution with mild 5



inversion for 30 sec. The mixture solution was kept under static condition for 2 h to grow up gold nanorods from gold seed nanoparticles. Then, color of mixture solution was changed to deep purple. Next, to reduce cytotoxicity of CTAB for in vitro and in vivo experiment, CTAB on gold nanorods was substituted by PEG-SH.35 GNRs (1 mL) was centrifuged at 15,000 rpm for 35 min to remove excess CTAB two times and resuspended in PEG5000-SH solution (0.1 mM, 1 mL). The mixture solution was rotated for 24 h at 25 °C, centrifuged at 18,000 rpm for 10 min two times, and finally redispersed in water (30 mL). Fabrication of macroporous alginate/GNRs cryogel. Alginate solution (2.5 wt%, 2 mL), GNRs-PEG solution (200 µL), water (200 µL), and CaSO4 slurry (2 wt%, 80 µL) were mixed by Luer-lock syringe. Then the mixture in syringe was transferred between glasses (spacer height: 3 mm) and aged for 24 h at RT to allow complete calcium crosslinking of alginate. The resulting alginate hydrogel was washed two times by DPBS to remove excess calcium ions and cut into cylinder shape by using biopsy punch (diameter: 8 mm). The disc-shaped alginate/GNRs hydrogels were frozen at -20 °C for at least 6 h to form ice crystals which play a role as porogen. Finally, the frozen alginate hydrogels were lyophilized for three days to generate macroporosity in Alg/GNR cryogel. Drug loading. The macroporous Alg/GNR cryogels were immersed in 1 mL of different chemotherapeutic drugs, MTX, DOX, or MX, with desired concentration (e.g., 500 µg/mL) for 48 h in static condition and washed three times with excess DPBS to remove unbound drugs prior to the subsequent in vitro and in vivo experiments. The loading efficiency was calculated by measuring absorbance of supernatants collected washing steps at 305, 480, and 610 nm for MTX, DOX, and MX, respectively, using UV/VIS spectrometer (Multiscan GO, Thermos, Massachusetts, US). On-demand NIR-responsive drug release. The drug (MX)-loaded macroporous Alg/GNR 6


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cryogel disks were placed in 1 mL DPBS. Every 30 min, cryogel was subjected to NIR irradiation (1.0 W/cm2) for 1 min under continuous mode with a beam distance of 2 cm (Picasso, AMD laser, Indianapolis, USA), while no NIR irradiation was applied to control cryogel. To check amount of released MX, an aliquot of medium was collected and absorbance of MX in DPBS at a wavelength of 610 nm was measured. The same volume of fresh medium was refilled into the well for further release study. Temperature transition of the cryogel with irradiating NIR laser was recorded using IR camera (FLIR-349001, FLIR, Wilson, Oregon, USA). Cell viability. Mouse mammary tumor cells (4T1, ATCC CRL-2539) were cultured at 37 °C in RPMI medium supplied with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Lonza). Then 4T1 cells were retrieved and seeded at a concentration of 5.0 × 103 cells/well in 96 well plate and cultured until complete adherence of cells onto the bottom of plate. To investigate the cytotoxicity of free MX, MX was added to the cells with different concentrations (2, 4, 6, 10, 20, 40, and 80 µg/mL) and the cells were cultured for designated time period. Then the cells were washed with complete RPMI medium three times and incubated in complete medium for 24 h. Finally, cell viability was measured by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT)

assay

following

the

manufacturer’s protocol. To determine cell viability after NIR-irradiation of MX-loaded macroporous alginate/GNR cryogels, 4T1 cells at a concentration of 1.0 × 105 cells/5 mL in RPMI was seeded at 12 well plate for 24 h. Then macroporous cryogels were placed in each well and was subjected to NIR irradiation (1.0 W/cm2, continuous mode, beam distance of 2 cm) for 1 min at every 30 min for 2 h. After NIR irradiation, cryogels were removed and cells were incubated at 37°C for 3 h and the medium was replaced with fresh RPMI complete medium for further incubation of cells at 37 °C for 24 h. Cell viability was measured by MTT 7



assay and Live/Dead assay (Eugene, OR, USA) according to the manufacturer`s instruction. Tumor suppression study. To prepare tumor bearing mouse model, 100 µL of 4T1 cells dispersed in PBS at a concentration of 1.0 × 106 cells/mL was subcutaneously injected to Balb/c mouse (Orient Bio, Seongnam, Republic of Korea). When the nodule of tumors was formed at around 5 days after injection, Alg/GNR cryogels loaded with MX were implanted next to the tumor with a distance of 3 cm. Since Day 2 post-implantation, the implanted cryogel was irradiated with NIR (1.0 W/cm2, continuous mode, beam distance of 2 cm) for 5 min every 2 days, which resulted in the increase of temperature of tumor region to around 50 °C. The tumor area was measured with calipers during the experiments. After 30 days, mice were euthanized, and tumors were isolated to identify the effects of drug release. Statistical analysis. All values in this study were presented as mean ± standard deviation (as error bar). Statistical analysis was performed with F-test and student's t-test (two-tailed). A pvalue of less than 0.05 was considered a significant difference.

Results and Discussion Ideal on-demand drug delivery system requires both high loading of drugs in the matrix and slow release of drugs from the matrix in the normal condition without external stimuli. To choose optimal anti-cancer drug for on-demand delivery system based on macroporous alginate cryogels (Figure 2a), we first tested the adsorption of three representative anti-cancer drugs on alginate cryogels; methotrexate (MTX), doxorubicin (DOX), and mitoxantrone (MX). The charge of each drug is different; -2 for MTX, +1 for DOX, +2 for MX in the physiological solution (Figure 2b).36 As alginate chain has strong negative charges due to carboxylic groups, higher adsorption of more positively charged drug was expected. Figure 8


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1c shows the photographic images of control alginate cryogel in PBS and alginate cryogels incubated with 500 µg/mL of MX, DOX, and MTX. The colors of cryogels turned into pale yellow, deep red, and deep blue upon incubating with MTX, DOX, MX, respectively (Figure 2c). In addition, the size of DOX- and MX-loaded cryogels became smaller than that of control and MTX-loaded cryogels. These data represent that positively charged DOX and MX were adsorbed more efficiently and strongly on the negatively charged alginate cryogel than neutral MTX. We further investigated the adsorption kinetics of three drugs on alginate cryogels (Figure 2d). MTX showed lowest adsorption rate and the final adsorption amount was around 30 µg per cryogel. In contrast, DOX and MX showed significantly higher adsorption rate compared to MTX. Furthermore, final adsorption amounts of DOX and MX reached to 400 µg and 500 µg per cryogel, respectively. Of note, almost 100% of added MX was adsorbed on alginate cryogel. Based on these data, MX with highest loading efficiency was selected as a model anti-cancer drug for on-demand release based on alginate cryogels. To further evaluate the maximum loading capacity of MX per alginate cryogel, different concentrations of MX (0.1, 0.5, 1, 2 mg/mL) were incubated with alginate cryogels (5 mg at lyophilized form) for 72 h (Figure 2e). Almost all MX was adsorbed on alginate cryogels in 0.1, 0.5, and 1 mg/mL solution, while 92.5% MX was loaded on cryogel in 2 mg/mL solution, representing the maximum MX loading capacity of alginate cryogel was around 370 µg/mg cryogel. The photograph images of MX-loaded macroporous alginate cryogel disks after incubation of MX solution with different concentrations shows that higher concentration of MX resulted in the formation of darker alginate disk with smaller dimension (Figure 2f). This dimension change of cryogels is probably due to higher adsorption of MX on the alginate chains via strong electrostatic interactions and subsequent shrinkage of the hydrogel matrix. 9



As MX molecules are strongly adsorbed on alginate chains, we next investigated whether applying an external stimulus can induce enhanced release of MX from alginate cryogels. As enhanced diffusion and relaxation of electrostatic interactions could be induced at higher temperature, we measured release kinetics of MX from cryogels at varying temperature-induced from GNRs concentration (Figure 3a). MX-loaded cryogel disks containing 500 µg MX were incubated in 24 well plates filled with 1 mL DPBS at RT, 37 °C, and 65 °C for 20 h, showing that faster and higher MX release was resulted from higher temperature. Release profile at RT was very slow; only 1% for 72 h. The release amount at 37 °C was 3.5-fold higher than that at RT. In case of 65 °C, release rate at 20 h was 2- to 5fold higher, respectively than that of 37 °C and RT. Based on these data, we hypothesized that applying heat to cryogels or generating heat in cryogels could be the external signal to induce MX release from alginate cryogels at desired time point. Based on the thermally controlled release profile of strongly-bound MX from alginate cryogel, we investigated the potential of Alg/GNR cryogels for NIR-mediated controlled release of MX on-demand. As the transduction of NIR light energy to heat at local region could cause a rapid increase of temperature in local area near target tumor, which could induce on-demand release of drug. GNRs were prepared via a seed-mediated growth method in the presence of cetyltrimetylammonium bromide (CTAB) and the surface of GNRs were modified with polyethylene glycol (PEG) using PEG-thiol. TEM image shows that PEGmodified GNRs were well-dispersed in water (Figure 3b). The longitudinal absorption peak of GNRs was around 808 nm that can maximize the photothermal effect of GNRs using 808 nm NIR laser that we used (Figure 3c). The Alg/GNR cryogels with a different concentration of GNRs (0.95, 1.9, 3.8 µg/L) were fabricated and their NIR-responsiveness was evaluated by checking the temperature profile with an irradiation of NIR laser (1.0 W/cm2) on surface of 10


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cryogel for 1 min at every 10 min (Figure 3d). At lowest concentration of GNRs (0.95 µg/L), temperature raised up to 42 °C. The cryogels embedded with GNRs of higher concentration (1.9, 3.8 µg/L) resulted in similar higher temperature (54 - 58 °C) under same NIR irradiation. Based on this result, we selected Alg/GNR cryogels loaded with 1.9 µg/L GNR for further in vitro and in vivo studies. Next, temperature profile of Alg/GNR cryogel loaded with 1.9 µg/L was measured at a different power of NIR laser (0.2, 1, and 1.4 W/cm2) from 32 °C (Figure 3e). The temperature increase by 0.2 W/cm2 was not significant and the resulting temperature was similar to 37 °C, the physiological temperature, suggesting that an enhanced release of MX might not be significant. The temperature induced by NIR irradiation at 1.4 W/cm2 was very high up to 65 °C, which could have a possibility to damage tissue surrounding the target sites. The resulting temperature from NIR irradiation at 1.0 W/cm2 reached approximately 50 – 53 °C. Based on this data, we used 5W for further in vitro and in vivo study. We additionally tested a release profile of MX from Alg/GNR cryogels loaded with 1.9 µg/L GNR at a different duration times (0.5, 1, and 2 min) under 1.0 W/cm2 NIR laser (Figure S1, Supporting Information). The temperature was immediately increased by irradiation of NIR laser and there was no significant difference of resulting temperature although longer duration of high temperature was observed for longer irradiation. Based on the conditions that we have selected for the further experiments, we measured release properties of MX from alginate/GNR cryogels with or without applying NIR irradiation (1.0 W/cm2) for 30 sec at every 2.5 min (total 4 irradiations), while no NIR irradiation was applied to the control Alg/GNR cryogels (Figure S2, Supporting Information), showing that NIR irradiation 2-fold higher MX release compared to the control group. Next, to investigate on-demand release of MX by NIR irradiation, Alg/GNR cryogels 11



containing 500 µg of MX were placed with 5 mL DPBS in 12 well plate. NIR-responsive groups were subjected to NIR laser (1.0 W/cm2) for 1 min every 30 min, while control group was incubated without NIR irradiation. These experiments were conducted at 37 °C to mimic physiological condition of MX release in in vivo study. The cumulative release profile clearly showed that a stepwise increase of MX release upon NIR irradiation (Figure 3f). The total amount of released MX from NIR-responsive group was 1.5-fold higher than that of control group over 3 h. We further studied whether the high temperature caused by the output of NIR can influence the release profile of MX. Release amount of MX was increased by higher NIR power as this led to high temperature (Figure S3, Supporting information). Based on the NIR-responsive drug release capability of Alg/GNR cryogels, we further investigated the in vitro and in vivo anti-cancer drug delivery in the concept of on-demand delivery. First, in vitro anticancer activity of MX-loaded macroporous alg/GNR hydrogel to using 4T1 breast cancer cells was investigated (Figure 4). Experimental groups were divided into 4 groups; cancer cells treated by (1) PBS, (2) blank Alg/GNR cryogel with NIR irradiation on cyrogel, (3) MX-loaded Alg/GNR cryogel without NIR irradiation, and (4) MX-loaded Alg/GNR cryogel with NIR irradiation. In NIR-irradiation groups (group 2 and 4), NIR laser (1.0 W/cm2) was irradiated on surface of the cryogels for 1 min repeated 4 times at every 30 min. The cell viability of each group was accessed with MTT assay (Figure 4a). Cell viability of both (1) control groups and (2) blank Alg/GNR cryogel with NIR irradiation was almost same about 100 %. The temperature increase in blank Alg/GNR cryogel triggered by NIR irradiation did not affect to the cell viability, which is presumably because the macroporous cryogel was floating in cell culture media and thus did not have influence on cell seeded in bottom of the plate. On the contrary, cell viability of MX-loaded macroporous Alg/GNR cryogel without NIR irradiation was about 50 %. representing that a certain level of 12


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MX was released from the cryogel at 37 oC as observed in Figure 3f and affected to cells. In contrast, the cell viability of MX-loaded Alg/GNR cryogel with NIR irradiation was decreased down to 20%, which was a half of MX-loaded Alg/GNR cryogel withour NIR. These results indicate that the on-demand NIR-mediated MX release could lead to most efficient cancer cell death in vitro. Interestingly, although release rate of MX in on-demand (NIR irradiation) was 1.5-fold higher than the sustained (no NIR irradiation) group (Figure 3f), the cytotoxic effect with MX in on-demand group was more significant to kill cancer cells than that of sustained group. We further investigated cell viability via fluorescent images of live/dead assay (Figure 4b). According to the fluorescent images, control and blank Alg/GNR cryogel groups showed high cell viability, approximately 100 % without dead cells. In MX-loaded Alg/GNR cryogel without NIR irradiation, although dead cells were rarely detected, we could observe significantly less proliferation of cells compared to control group. In contrast, cytotoxic effect by on-demand drug release fron MX-loaded Alg/GNR cryogel via NIR irradiation was significantly higher with dead cells and shrunk cell morphology of even live cells, representing on-demand MX release using the developed cryogel was efficient to kill the cancer cells, which was consistent to the MTT assay result. To study the therapeutic effect of on-demand drug release system with MX-loaded Alg/GNR cryogel combined with NIR irradiation in vivo system, the mice bearing tumor were divided into 4 groups: control group treated by PBS, blank Alg/GNR cryogel combined with NIR irradiation on cryogel, MX-loaded Alg/GNR cryogel without NIR irradiation, and MX-loaded Alg/GNR cryogel combined with NIR irradiation on cryogel. The amount of MX loaded in the cryogels implanted in the animal was 500 µg per cryogel. At day 5 after subcutaneous inoculation of 4T1 cells, the cryogel disks were implanted near tumor site (Figure 1b). Then the cryogels were irradiated with NIR laser (1.0 W/cm2, continuous mode, 13



beam distance of 2 cm) for 5 min at every 2 days and the size of tumor was monitored over time (Figure 5a). There was no severe skin irradiation on the irradiation site in all animals. Surprisingly, despite such a high dose of mitoxantrone (25 mg/kg) compared to the LD50 for mouse with i.v. injection (~10 mg/kg), all mice received the MX-loaded cryogel survived after implantation. This result supports one of the advantages of our cryogel system; a high drug loading with a high affinity to enable very slow release of drug without an external stimulus even after implantation in the body. Tumor volume of control groups treated by PBS injection was gradually increased over time (Figure 5b). The treatment with blank Alg/GNR cryogel with NIR irradiation showed tumor growth similar to the control group at early stage up to day 17, but slowed down the tumor growth afterwards. This might be because the heat generated from NIR irradiation played a role as photo-thermal effect and might affect to the tumors once tumor became bigger and thus closer to the cryogel. The treatment with MXloaded Alg/GNR cryogel significantly reduced the tumor growth compared to control and blank Alg/GNR scaffold with NIR irradiation, but at last the tumors became bigger up to a similar size observed in blank Alg/GNR cryogel with NIR irradiation. This indicates that spontaneous MX release from Alg/GNR cryogel under body temperature could slow down the tumor growth at certain extent but could not achieve significant therapeutic effect because therapeutic efficacy of MX gradually decreased in the end due to the deficient amount of release MX. On the other hand, MX-loaded Alg/GNR cryogel combined NIR irradiation showed remarkable effect in tumor suppressing down to 16 % in comparison with the control groups. It is meaningful that on-demand drug release using Alg/GNR cryogel could show a significant result at tumor suppression. At the final point of the study, tumors were excised, and their sizes were compared from 4 groups of mice (Figure 5c), showing that significantly small tumor mass was resulted in the on-demand MX release group. 14


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Taken together with in vitro cell cytotoxicity results, controlling the drug release profile at desired time point could be an important way to suppress the tumor growth. The developed macroporous cryogels consisting of Ca-crosslinked alginate matrix and NIR-absorbing GNRs have a potential for powerful material platform on the on-demand release of anti-cancer drugs with high affinity to alginate polymer chain.

Conclusion For NIR-responsive on-demand system, macroporous Alg/GNR cryogels were fabricated and MX with positive charge was strongly adsorbed on the negative alginate chains of the cryogels. This system enabled a successful on-demand release of strongly-bound MX from the GNR-loaded macroporous cryogels by NIR irradiation. Heat triggered from GNRs could break interaction between alginate backbone and MX compared no stimuli groups. Cell viability after NIR irradiation of MX-loaded cryogel was significantly lower than no stimuli condition. Moreover, in vivo tumor suppressing effect of MX-loaded hydrogel with NIR irradiation was remarkable compared to the other groups. This study demonstrates that this simple approach to fabricate macroporous cryogel capable of on-demand could be beneficial in various applications including cell, gene, and the other small molecule delivery system.

Supporting Information. Release profiles of MX from Alg/GNR cryogels under different irradiation conditions including irradiation time, repetition, and power of NIR laser. Acknowledgements

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This work was supported by grants funded by the National Research Foundation under the Ministry of Science and ICT, Republic of Korea (Grants 2015R1A2A2A01005548, 20100027955, and 2014M3A9B8023471).

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Figures

Figure 1. Schematic illustration of (a) preparation of macroporous Alg/GNR cryogel and (b) its application to on-demand chemical drug release for cancer therapy in subcutaneous tumor model by implanting mitoxantrone-loaded Alg/GNR cryogels beside tumor and the subsequent NIR irradiation.

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Figure 2. (a) Cross-sectional SEM image of macroporous alginate cryogel. (b) Molecular structures of methotrexate (MTX), doxorubicin (DOX), and mitoxantrone (MX). (c) Representative images of control macroporous alginate cryogels and alginate cryogels loaded with MTX, DOX, and MX. (d) Adsorption kinetics at a same concentration of 500 µg/mL of drugs; MX, DOX, and MTX. (e) Adsorption kinetics of MX with different concentrations incubated with macroporous alginate cryogels. (f) Representative images of MX-loaded cryogels obtained after the adsorption experiments shown in (e).

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Figure 3. (a) Cumulative release of MX from alginate (Alg) cryogels loaded with MX at different temperature. (b) Representative TEM image and (c) absorption spectrum of gold nanorods (GNRs). (d) Temperature profile after NIR irradiation of Alg/GNR cryogels loaded with 0.95, 1.9, and 3.8 µg/L GNR. (e) Temperature profile of Alg/GNR cryogels loaded with 1.9 µg/L GNR per cryogel after irradiation with 0.2, 1.0, and 1.4 W/cm2 NIR laser. (f) Cumulative release profiles of MX from MX-loaded Alg/GNR cryogels incubated in 37 oC applied with repetitive irradiation with NIR laser (1.0 W/cm2, 1 min) at every 30 min or without applying NIR stimuli.

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Figure 4. (a) Cell viability of 4T1 cells access with MTT assay and (b) the representative fluorescent images after live-dead assay after incubation of cells with PBS, blank Alg/GNR cryogel with NIR irradiation, MX-loaded Alg/GNR cryogel without applying NIR, and MXloaded Alg/GNR cryogel with NIR irradiation. NIR laser (1.0 W/cm2) was irradiated on surface of the cryogels for 1 min repeated 4 times at every 30 min. Scale bar 100 µm.

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Figure 5. (a) Timeline for in vivo on-demand drug delivery for anti-cancer therapy. (b) Tumor volume changes of animals received with PBS (control), blank Alg/GNR cryogels with NIR irradiation, MX-loaded Alg/GNR cryogel without applying NIR, and MX-loaded Alg/GNR cryogel with applying NIR irradiation. The cryogels were irradiated with NIR laser (1.0 W/cm2) for 5 min every 2 days. (c) Representative images of tumor isolated from the mouse at the end of the study shown in (b).

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TABLE OF CONTENTS (TOC) GRAPHIC

On-Demand Macroscale Delivery System Based on Macroporous Cryogel with High Drug Loading Capacity for Enhanced Cancer Therapy Pilseon Im and Jaeyun Kim

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On-Demand Macroscale Delivery System Based on a Macroporous

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