Design of Phase-Changeable and Injectable Alginate Hydrogel for

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Design of Phase-Changeable and Injectable Alginate Hydrogel for Imaging-Guided Tumor Hyperthermia and Chemotherapy Jiulong Zhao, Jialing Li, Chunping Zhu, Fei Hu, Hongyu Wu, Xiaohua Man, Zhaoshen Li, Changqing Ye, Duowu Zou, and Shige Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17608 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 9, 2018

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ACS Applied Materials & Interfaces

Design

of

Phase-Changeable

and

Injectable

Alginate

Hydrogel

for

Imaging-Guided Tumor Hyperthermia and Chemotherapy

Jiulong Zhao†, Jialing Li†, Chunping Zhu†, Fei Hu ‡, Hongyu Wu†, Xiaohua Man†, Zhaoshen Li†, Changqing Ye ‡, Duowu Zou*,† and Shige Wang*,‡

†

Department of Gastroenterology, Changhai Hospital, Second Military Medical University, No. 168

Changhai Road, Shanghai 200433, China ‡

College of Science, University of Shanghai for Science and Technology, No. 334 Jungong Road,

Shanghai 200093, China

1

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ABSTRACT: The objective of the present study was to construct an alginate (AG) phase-changeable and injectable hydrogel for imaging-guided tumor hyperthermia and chemotherapy. Based on the binding between the α-L-guluronic blocks of AG and calcium ions, the AG/MoS2/Bi2S3-PEG (MBP)/doxorubicin (DOX) solution formed a cross-linked hydrogel to simultaneously encapsulate MBP nanosheets and DOX within the hydrogel matrix. The in situ-formed hydrogel can act as a reservoir to control the release of entrapped drug molecules, and the doped MBP nanosheets and DOX can realize computed tomography/photoacoustic dual-modal imaging-guided in vivo tumor PTT and chemotherapy, respectively. The AMD hydrogel exhibited excellent photothermal conversion properties with mass extinction coefficient of 45.1 L.g−1.cm−1 and photothermal conversion efficiency of 42.7%. Besides, the heat from the photothermal transformation of MBP can promote drug diffusion from the hydrogel to realize on-demand drug release. Additionally, the hydrogel system can restrain MBP and DOX from entering into the blood stream during therapy, and therefore substantially decrease their side effects on normal organs. More importantly, the drug loading of the AG hydrogel was general and can be extended to the encapsulation of antibiotics, such as AMX, for the prevention of postoperative infections.

KEYWORDS: Phase-changeable, Injectable, Alginate, Hydrogel, Tumor Hyperthermia 2


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1. INTRODUCTION Photothermal therapy (PTT) has been intensively studied as a minimally invasive tumor ablation method. One of the necessary prerequisites for efficient and successful tumor PTT is a photothermal 1

transducing agent (PTA). Various nanomaterials, such as carbon7-10

metal-chalcogenide/dichalcogenidenanosheet-based nanomaterials

, metal-

2-6

, transition

11

, transition metal oxide-

, and black phosphorus

12-15

, have been extensively explored as active PTA candidates.

Moreover, intelligent agents that can simultaneously realize tumor imaging and hyperthermia within a single platform have been the latest trend in PTA design

13, 16-18

. During PTT, the employed

photo-absorbing agents can be specifically accumulated in tumors, and their temperatures are selectively and accurately elevated to burn tumor tissue 19. Therefore, PTT has distinct advantages as a highly efficient tumor therapy when compared with traditional physical therapy approaches, such as microwave 20, focused ultrasound 21, and radiofrequency thermotherapy 22. As for the in vitro and in vivo therapeutic performance of currently reported PTAs, although no obvious toxicity has been reported in labs, the PTAs have suffered from other disadvantages, such as limited accumulation in tumor sites and undesired drug loss

23-24

. For this reason, exploring a representative therapeutic

modality to increase the utilization of PTAs and drugs and decrease side effects while coordinately enhancing therapeutic efficiency may create more opportunities for highly efficient and on-demand tumor therapy. Biocompatible polymers can form hydrogels under the activation of certain stimuli such as temperature, pH, ions, and solvents, among others

25-29

. Hydrogels are three dimensional networks

that imbibe water or biological fluids through physically or chemically cross-linked hydrophilic polymers

30

. Due to their high water content, biodegradability, biocompatibility, and softness,

polymer gels have attracted much attention in regenerative medicine research and especially for in situ therapeutic delivery

3, 31-33

. These therapeutic-laden polymer hydrogels can be prepared directly 3



by dissolving polymer and therapeutic into a solvent. For example, by simply mixing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), hydrophobic superparamagnetic iron oxide nanoparticles, and thermosensitive biodegradable and thermosensitive poly(organophosphazene) polymers, Song et al. proposed a magnetic hyperthermia-mediated TRAIL release system for combined tumor therapy 34. Other kinds of polymers, such as poly(N-isopropylacrylamide) (NIPAm) 30

, chitosan-agarose 35, polypeptide

36-37

, glycerophosphate 38, hyaluronic acid 39, and silk 40, among

others, have also been extensively studied in the construction of composite hydrogels for different biomedical applications. Alginate (AG) is a water-soluble linear polysaccharide composed of alternating blocks of 1,4-linked α-L-guluronic and β-D-mannuronic acid residues. The α-L-guluronic blocks allow the AG polymer to selectively and cooperatively bind multivalent cations, such as calcium ions, to form Ca-AG hydrogels

25, 41

. Moreover, the multivalent cations cause a mild gelation process, and the

degradability of the formed hydrogel is adjustable. As a U. S. Food and Drug Administration (FDA)-approved biocompatible polymer, AG has been widely used in a variety of biomedical applications, such as cell and drug transport

42-45

and tissue engineering scaffolds 46, among others.

Therefore, it is rational to anticipate that the AG hydrogel also possesses an excellent biocompatibility. Li et. al prepared methylene blue (MB)- and laponite (LP)-containing AG hydrogels by injecting aqueous solutions of LP/AG/MB into a calcium chloride solution under magnetic stirring, and found that the formed hydrogel was a good candidate for the controlled delivery of cationic drugs under acidic conditions

41

. In another study, Mazutis et al. reported the

microfluidic production of AG hydrogel particles for extended antibody encapsulation and release 47. However, to our knowledge, sufficient study of an AG hydrogel-based platform for imaging-guided multifunctional tumor therapy is still lacking in literature.

4


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Based on the outstanding features of AG, we designed a Ca-AG-based multifunctional hydrogel consisting of AG, MoS2/Bi2S3-PEG (MBP), and doxorubicin (DOX) for tumor photothermal and chemotherapy. The photothermal performance, biocompatibility, and therapeutic effectiveness of the hydrogel were evaluated both in vitro and in vivo. Moreover, antibiotics (e.g., amoxicillin (AMX)) can also be encapsulated in the hydrogel to avoid potential wound infection during tumor therapy. This research provides the first demonstration that the in situ-formed AG hydrogel can realize highly synergistic tumor photothermal and chemotherapy and can exert anti-inflammatory effects simultaneously. Compared with the intratumoral injection of MBP nanosheets and DOX solution in which a substantial amount of MBP nanosheets and DOX were captured by reticuloendothelial system 14, the utilization of MBP nanosheets was improved since the in situ-formed AG hydrogel can act as a macro-vessel to restrict their access to body fluid circulation.

2. MATERIALS AND METHODS

2.1 Materials

(NH4)2MoS4 (ammonium tetrathiomolybdate) powder was obtained from J&K Chemical. Co., Ltd. (China). AG, Bi(NO3)3·5H2O, monoethanolamine, calcium chloride (CaCl2), and PEG (molecular weight = 400 Da) were provided by Sinopharm Chemical Reagent Co., Ltd., (China). AMX and DOX were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (China) and Beijing Huafeng Pharmaceutical Co., Ltd. (China), respectively. All chemicals were used without further purification. Mouse fibroblast (L929) and human colon (HT29) cell lines were obtained from the Institute of Biochemistry and Cell Biology (the Chinese Academy of Sciences, Shanghai, China). Cell culture reagents, including penicillin-streptomycin, Roswell Park Memorial Institute-1640 medium (RPMI-1640), fetal bovine serum (FBS), trypan blue, and Cell Counting Kit-8 (CCK-8), 5



were purchased from Gibco (Shanghai, China). Cell culture flasks and plates were from Corning Incorporated (Shanghai, China). Staphylococcus aureus (S. aureus) for the antibacterial test was purchased from Shanghai Fuzhong Biotechnology Development Co., Ltd. (Shanghai, China). Beef extract and peptone were purchased from Beijing Aoboxing Biotechnology Co., Ltd. (Beijing China). Balb/c nude mice and Kuming mice (KM, 4-6 weeks old) were obtained from Shanghai Slac Laboratory Animal Center (Shanghai, China). All animal experiments were performed under the guidance of Changhai Hospital, Second Military Medical University and in accordance with the policies of National Ministry of Health. Water used in this study with resistivity higher than 18.2 MΩ·cm was purified with the Pall Cascada Laboratory Water System (Pall Corporation, Port Washington, NY, USA).

2.2 Preparation and characterizations of MBP nanosheets and AMD hydrogels

To synthesis MBP nanosheets

14

, 150 mg (NH4)2MoS4 and 150 mg Bi(NO3)3·5H2O were

dissolved into 30 mL PEG-400 with magnetic stirring for 30 minutes. Afterwards, 30 mL H2O was added into the solution and magnetically stirred for another 30 minutes. The resultant solution was subsequently transferred into a 100-mL polyphenylene-lined stainless steel autoclave and heated at 220 °C in an oven for 12 h. The products were thoroughly washed with monoethanolamine solution (50%, in water, v/v) and water 3 times and dispersed in water for further use. UV-Vis-NIR spectra of MBP nanosheets was collected using a UV-3600 Shimadzu UV-Vis-NIR spectrometer. The MBP nanosheets concentration was fixed at 0.25 mg/mL and the blank quartz cell was filled with distilled water. The Mo and Bi concentrations were determined with an Agilent 700 Series ICP-OES system (Agilent Technologies, Santa Clara, CA, USA). To prepare the hydrogels, a homogeneous AG solution was first prepared by dissolving 3.0 g AG powder into 97 mL water (the mass ratio of AG was 3%). 1 mL of the resultant AG solution was 6


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directly mixed with 0.1 mL CaCl2 aqueous solution (0.1 M). For construction of AG/MBP/DOX (denoted as AMD) hydrogels, MBP and DOX were dissolved into the AG solution. The concentration of MBP and DOX was set as 2.5 mg/mL and 0.1 mg/mL, respectively. The formed solution was then directly mixed with CaCl2 aqueous solution (0.1 M, 0.1 mL). AG and AMD hydrogel appearances were recorded using a Nikon D5300 digital camera, and their microstructure and energy dispersive spectroscopy (EDS) mapping spectra and the microstructure of MBP nanosheets were analyzed by field-emission scanning transmission electron microscopy (FESEM, FEI Magellan 400, FEI Co., Hillsboro, OR, USA). For the microstructure study of AG and AMD hydrogel, the hydrogel was air-dried and sprayed with gold for 3 min using JS-1600 (Beijing, China) before observation. For the microstructure study of MBP nanosheets, 10 µL dispersion (500 ppm in ethanol) was dripped onto the sample holder, air-dried and sprayed with gold for 3 min using JS-1600 (Beijing, China). The crystalline structures of AG and AMD hydrogels were studied using a Rigaku D/max-2200 PC XRD system (Rigaku, Tokyo, Japan) at 40 kV and 40 mA (with Cu Kα radiation, λ = 1.54 Å, the scan was performed from 5o to 80o (2θ)). Before the analysis, the hydrogel with appropriate size was air-dried and fixed onto the sample holder. UV-Vis-NIR spectra of AMD hydrogel was also collected using a UV-3600 Shimadzu UV-Vis-NIR spectrometer. The MBP nanosheets concentration was fixed at 0.25 mg/mL and the blank quartz cell was filled with pure AG hydrogel. The mass extinction coefficient (κ) of AMD hydrogel at the wavelength of 800 nm was calculated according to Lambert-Beer's law: A = κ·C·L (A is the absorbance at 800 nm, C is the MBP nanosheets concentration (mg/mL) and L is the optical length (1 cm) of the quartz cell). Note that the concentration of MBP was set as 0.25 mg/mL, this is due to the absorbance of MBP nanosheets with higher concentration will exceed the measuring range of the spectrometer. The temperature in the photothermal conversion efficiency study was 22 oC.

7



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2.3 In vitro photothermal performance of AMD hydrogel

The in vitro photothermal performance of the AMD hydrogel was analyzed by continuously irradiating it with an NIR laser (wavelength = 808 nm, produced by a high power multimode pump laser, Shanghai Connet Fiber Optics Company, Shanghai, China). The laser power density was varied from 0.4-0.8 w/cm2, and the doped MBP concentration was set to 1.0, 2.5, or 5.0 mg/mL. AG hydrogels irradiated with a 0.8 w/cm2 NIR laser were selected as controls (blank). The irradiation time was 5 min, and the temperature increase and corresponding infrared thermal images of AMD hydrogels were recorded with a FLIRTM E60 infrared camera (FLIR Systems, Wilsonville, OR, USA). The temperature in the photothermal conversion efficiency study was 10 oC. The photothermal conversion efficiency (η) of AMD hydrogel (ALG/MBP hydrogel, due to the absorbance in the wavelength of 808 nm, DOX was not added when measuring the η) was measured by a modified method similarly to Korgel’s report

48

, where η can be calculated according to the

following equation (1):

η=

hS (Tmax − TSurr ) − Qin ,Surr I (1 − 10( − A ( λ )) )

(1)

In this equation, S is the surface area of the sample and the value of hS was determined by measuring the rate of temperature drop after removing the light source. Tmax is the maximum temperature of AMD hydrogel under NIR laser irradiation and Tsurr is ambient temperature. Qin,surr is the heat lost to the surroundings. I is the laser power (in units of mW) and A(λ) is the absorbance at the wavelength of 808 nm. More details about the calculation can be found in supporting information. The photothermal stability of the hydrogel was monitored by recording the temperature increase and decrease during 9 cycles laser on/off irradiation.

8


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2.4 In vitro bio- and hemocompatibility of AMD hydrogel

For the in vitro biocompatibility assay of the AMD hydrogel, 0.1 mL AG and AG/MBP (MBP concentration: 2.5 and 5.0 mg/mL) solution was mixed with 10 µL CaCl2 solution to allow hydrogel formation. The formed AG and AG/MBP hydrogels were transferred to another 96-well cell culture plate that was seeded with L929 cells (1 × 104 cells per well) and cultured for 1 day. Afterwards, the AG and AG/MBP hydrogels were discarded and cells were rinsed with phosphate-buffered saline (PBS) 3 times. The cell viability was quantitatively evaluated in a CCK-8 assay and qualitatively studied by microscopic observation (using inverted phase contrast microscopy and trypan blue staining). Hemolysis assay of mouse red blood cells (mRBCs, collected from KM mouse blood and dispersed in saline) was used to evaluate the in vitro hemocompatibility of the composite hydrogel. In brief, 1.0 mL mRBCs was treated with 4.0 mL water (positive control), PBS (negative control), and 4.0 mL saline containing 0.3 g AG/MBP hydrogel. After incubation at 37 °C for 2 h, the above mRBCs were centrifuged (10000 rpm, 1 min) to obtain the supernatant. The absorbance of the supernatants at 541 nm, a wavelength associated with hemoglobin concentration, of AG/MBP (Dt)-, PBS (Dnc)-, and water (Dpc)-treated mRBCs was monitored using a Lambda 25 UV-Vis spectrophotometer (Perkin Elmer, Inc., Waltham, MA, USA). Hemolytic percentage (HP) was calculated according to equation (2) 19. HP (%) =

( Dt − Dnc ) × 100% ( D pc − Dnc )

(2)

The kinetic clotting time method was used to evaluate the potent coagulant behavior of the AMD hydrogel according to procedures reported in literature

49-50

. Breifly, 0.1 g AMD hydrogel was put

into individual well of 12-well cell cultrue plate. Holes (n = 3) without hydrogel were used as control. Then, a 300 µL of fresh mice blood (stablized with heparin) was dropped onto the surface of 9



the hydrogel or directly added into the cell culture hole. After incubated at 37 oC for a predetermined period of time (10, 30, and 60 min, n = 3 at each time point; the control group was cultured for 60 min), 2.5 mL distilled water was put into each well and incubated at 37 oC for 5 min to dissolve the red blood cells. The absorbance at 541 nm of the above solution which in direct proportion to the hemoglobin concentration was determined using a Lambda 25 UV-Vis spectrophotometer (Perkin Elmer, Inc., Waltham, MA, USA).

2.5 In vitro and in vivo CT and PA imaging

Tumor models for in vivo tests were established by subcutaneously injecting 150 µL serum-free RPMI-1640 medium containing 1 × 107 HT29 cells into the backs of Balb/c nude mice. After 2 weeks of feeding, tumor nodules with diameters of ~0.5-1.0 cm were found and used for experiments. Before in vitro CT imaging, 1 mL of AG/MBP/DOX solution with an Mo concentration of either 0 (control), 0.05, 0.1, or 0.2 M was pipetted into an Eppendorf tube. For in vivo CT, HT29 tumor-bearing Balb/c nude mice were in situ injected with 50 µL AG/MBP/DOX solution (MBP concentration = 2.5 mg/mL, DOX concentration = 100 ppm) and then anesthetized to harvest tumors into 1.5-mL Eppendorf tubes. The in vitro and in vivo CT imaging capacity (indicated as CT contrast enhancements, Hounsfield units, HU) were recorded with a GE CT imaging system (operated at 100 kV, 200 mA and a slice thickness of 0.625 mm) (GE Healthcare, Chicago, IL, USA). The tubes and tumors used for CT scanning were then further analyzed to evaluate their in vitro and in vivo PA imaging capacity at 808 nm using the Vevo LAZR PA Imaging System (VisualSonics, Toronto, Canada).

2.6 In vitro DOX release and tumor synergetic therapy

AMD hydrogel (1 g) was added to a vial containing 5 mL PBS solution and stored at 37 °C or 10


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55 °C water bath. At predetermined time points, 1 mL PBS was removed from the vial and replaced with 1 mL new PBS. A Lambda 25 UV-Vis spectrophotometer was used to determine the released DOX concentration in PBS according to the concentration-absorbance standard curve at 480 nm. For the in vitro tumor synergetic therapy, HT29 cells were seeded into a new 96-well cell culture plate at a density of 1 × 104 cells per well and cultured in the cell incubator for 12 hours. Solutions of 0.1 mL AG/DOX, AG/MBP, and AMD were added to the 96-well cell culture plate and mixed with 10 µL CaCl2 solution. The formed hydrogels were transferred to the cell-seeded plate. Cells treated with AG/MBP and AMD were irradiated with an NIR laser (0.8 w/cm2) for 5 minutes. After that, the hydrogels were discarded and cells were rinsed 3 times with PBS. Cell viability was quantitatively evaluated using the CCK-8 assay and qualitatively studied with microscopic observation (using inverted phase contrast microscopy and trypan blue staining).

2.7 In vitro and in vivo hemocompatibility

KM mice were subcutaneously injected with 50 µL AG/MBP/DOX solution and 10 µL CaCl2 solution (0.1 M), and mice in the control group were subcutaneously injected with 50 µL saline. The mice were anesthetized for heart puncturing to collect blood on days 1, 3, 7 after the injection (n = 3, in each group and each time point). Routine blood evaluation was carried out on a Sysmex XS-800i automated hematology analyzer (Sysmex Co., Kobe, Japan). The studied parameters included red blood cells (RBC), white blood cells (WBC), platelets (PLT), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), hemoglobin (HGB), and red cell distribution width (RDW).

2.8 In vitro Mo ion release and in vivo biodistribution and histocompatibility

KM mice were subcutaneously administered 50 µL AG/MBP/DOX and 10 µL CaCl2 solution. 11



At days 1, 3, 7, and 14 post materials injection, major organs (heart, liver, spleen, lung, and kidney) were harvested, weighted, and thoroughly digested by aqua regia solution. The total amounts of Mo ions in these organs were determined with an Agilent 700 Series ICP-OES. Three KM mice treated with AMD hydrogels and 3 KM mice treated with saline were fed for 28 days, and their body weights were recorded every two days. KM mice were subcutaneously injected with 50 µL Agar/MBP/DOX and euthanized after 14 days of feeding to harvest hearts, livers, spleens, lungs, and kidneys. Standard H&E staining was performed according to the manufacturer’s instructions with untreated KM mice as controls. The images were recorded using a Leica DM IL LED inverted phase contrast microscope (Leica Microsystems, Wetzlar, Germany).

2.9 In vivo DOX release study and combined in vivo tumor therapy

For the in vivo DOX release study, HT29 xenografted tumor-bearing mice with tumor diameters of 0.5-1.0 cm were randomly divided into two groups (n = 6 for each group). Then, mice in this two groups were administered 50 µL AG/MBP/DOX solution (DOX concentration = 500 ppm) and 10 µL 0.1 M CaCl2 solution, or 50 µL AG/MBP/DOX solution and 10 µL saline into tumors. At 12h 24h and 72 h after the treatment, 1 mL blood and organs including heart, liver, spleen, lung, and kidney were harvested. The blood was centrifuged to obtain the serum. Then, 0.3 g of each organ was weighted and grinded with 500 µL lysis solution using a tissue grinder. For the serum, 500 µL serum was directly mixed with 500 µL lysis solution. The above solution (200 µL) was mixed with 100 µL triton and then homogenized in 1 mL extraction solution (0.75 mM HCl in isopropanol) and incubated at 37 °C overnight. A Lambda 25 UV-Vis spectrophotometer was used to determine the absorbance of the solution at 480 nm which was in direct proportion to the released DOX concentration. 12


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For the combined in vivo tumor therapy study, 4 groups (group I-IV, n = 7 per group) of HT29 xenografted tumor-bearing mice with tumor diameters of 0.5-1.0 cm were employed for in vivo tumor therapy. Then, mice were in situ administered 50 µL AG and 10 µL 0.1 M CaCl2 solution (group I, control), 50 µL AG/MBP/DOX solution and 10 µL 0.1 M CaCl2 solution (groups II and IV), or 50 µL AG/MBP solution and 10 µL CaCl2 solution (group III) into tumors. After 1 h to allow thorough hydrogel formation, mice in group I, III, and IV were irradiated with an NIR laser for 5 minutes (0.8 W/cm2). During the 5 min irradiation, tumor temperature and thermal images in groups I and IV were recorded using the FLIR™ E60 camera. After PTT, tumors (n = 1 per group) were harvested and fixed with 10% neutral buffered formalin for immunohistochemical analysis (CD31, Ki67, and TUNEL staining) to comparatively assess the tumor therapy efficiency. A Leica DM IL LED inverted phase contrast microscope was used to record the immunohistochemical images. The tumor growth (indicated as relative tumor volume, V/V0, where V0 represents the initial tumor volume at day 0) and tumor appearance in the four groups was also recorded.

2.10 In vitro AMX release and antibacterial activity assay

AMX with concentrations of 0, 100, 200, and 400 ppm was directly dissolved into AG/MBP solution and mixed with CaCl2 solution to prepare AG/MBP/AMX hydrogels (MBP concentration = 2.5 mg/mL, volume of AG/MBP solution: volume of CaCl2 solution = 10:1). The AMX release profile of the AG/MBP/AMX hydrogels was monitored as was the DOX profile: by recording the absorbance at 230 nm using a Lambda 25 UV-Vis spectrophotometer. The released AMX concentration was calculated according to the concentration-absorbance standard curve. The antibacterial activity of AG/MBP/AMX hydrogels was evaluated in liquid and solid media 50-51

. Briefly, bacterial concentration in liquid medium was determined by recording the solution

absorbance at 625 nm using a Lambda 25 UV-Vis spectrophotometer. For this, a 15-mL glass tube 13



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with 5 mL bacterial solution (absorbance at 625 nm was ~ 0.2) was stored in an air-bath (37 oC). Then, 0.3 g AG/MBP/AMX hydrogels with AMX concentrations of 0 (control group, without sample) 100, 200, and 400 ppm was added into each tube and cultured with shaking at 37 oC for 24 h. Finally, the OD values in the liquid medium of the control (IC) and experimental groups (Is) at 625 nm were recorded to calculate the inhibition percentage according to equation (3): Bacterial inhibition (%) = (Ic − Is)/Ic × 100

(3)

For the antibacterial activity assay, 3.2 g agar was dissolved in 100 mL distilled water and autoclaved. Then, 15 mL agar solution was poured onto Petri dishes and air-dried. AG/MBP/AMX hydrogels (diameter: ~1 cm) with 30 µg AMX were attached to the middle of Petri dishes. Afterwards, 100 µL S. aureus suspension (absorbance: ~0.2 at 625 nm) was poured onto the solid agar medium plate. In a parallel test, the 100 µL S. aureus suspension was poured onto the solid agar medium 4 h after hydrogel attachment to allow AMX release. These above plates were incubated at 37 oC for certain time periods (1d, 2d, 4d, and 7d) to quantitatively evaluate the bacterial inhibition efficiency.

2.11. In vivo antibacterial activity

A subcutaneous abscess mouse model was constructed as follows: the backs of KM mice (n = 3 per group, two groups) were anesthetized, shaved, and disinfected with betadine and 70% ethanol 52. Then, 0.2 mL of sterile dextran beads (Cytodex-1; Sigma-Aldritch Co., St. Louis, MO, USA) containing 4×105 S. aureus was subcutaneously injected into the backs of the KM mice. Mice in the experimental group were subcutaneously injected with 0.1 mL AG/MBP/AMX solution and 10 µL CaCl2 solution, and mice in the control group were subcutaneously injected with 10 µL CaCl2 solution. After 1 and 7 d, mice were sacrificed and blood serum was harvested by centrifuging for IL-6 and TNF-α analysis using a mouse IL-6/TNF-α specific enzyme-linked immunosorbent assay 14


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(ELISA) kit (Anogen, Mississauga, Ontario, Canada) according to the manufacturer's instructions.

2.12 Statistical analysis

The significance of the experimental data was evaluated by one-way ANOVA. p < 0.05 was selected as the significance level, and data with probability less than 0.05 were indicated with * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. Unless specified, sample sizes were 3 (n = 3).

3. RESULTS AND DISCUSSION

3.1 Preparation and characterization of AMD hydrogels

Calcium ions will selectively and cooperatively bind with the α-L-guluronic blocks of the AG polymer to form CaCl2 crosslinked AG hydrogels. During the crosslinking, the introduced MBP nanosheets (with Bi2S3-decorated on the surface, lateral size of about 300 nm, figure 1b) and DOX were automatically encapsulated within the matrix to form AMD hydrogel. Under the NIR laser irradiation, the photothermal conversion of MBP nanosheets and chemotherapeutic effect caused a synergistic tumor killing effect (figure 1a). After dissolving MBP nanosheets and DOX into the AG solution, the formation of Ca cross-linked hydrogels was not affected. It was found that the homogeneous AG/MBP/DOX solution possess excellent syringeability and can be smoothly drawn into a standard 1 mL 21-gauge syringe and pumped out through the needle. The AG/MBP/DOX solution possessed a good syringeability and can be readily filled into and smoothly pumped out through the needle pinpoint (figure S-1). This is utmost important for its potential clinical application since it needed to be injected within tumor using the conventional puncture needle. AG/DOX, AG/MBP, and AG/MBP/DOX solutions exhibited gelling behavior similar to that of AG and formed hydrogel upon contacting Ca2+ ions (figure 1f). The AG/MBP/DOX hydrogels (denoted as AMD) had a conspicuously rough structure (figure 1c) like that of AG (figure 1d), indicating that 15



Page 16 of 37

the introduction of MBP nanosheets and DOX did not influence the hydrogel surface morphology. EDS analysis (figure S-2) and elemental mapping indicated a homogeneous distribution of Mo within the hydrogel (figure 1e); however, there were no other elements except Na, C, O, Ca, and Cl (figure S-3), implying that MBP nanosheets were thoroughly dispersed within the hydrogel after magnetic stirring. Additionally, the PEG chains coated onto the MBP nanosheet surfaces contributed to nanosheet dispersal in the hydrogel solution. Further structural analysis using XRD readily detected the existence of MBP nanosheets (MoS2: JCPDS card No. 17-0744; Bi2S3: JCPDS card No. 17-0320, figure 1f). The designed AMD hydrogel with rough surface morphology was anticipated to facilitate the diffusion of encapsulated drugs from the hydrogel matrix, and the homogenous MBP distribution was expected to confer an excellent photothermal capacity on the hydrogel.

3.2 In vitro photothermal performance

To assess the photothermal performance of the prepared AMD hydrogel, the light absorption of MBP nanosheets was studied in advance. It was found that both of the MBP nanosheets and AMD hydrogel can significantly absorb light in NIR region (750 - 850 nm, Figure 2a). The mass extinction coefficient (κ) of MBP nanosheets and AMD hydrogel was calculated to be 55.4 and 45.1 L.g−1.cm−1 at 800 nm. Although the κ value was decreased due to the the hydrogel encapsulation, it is still significantly higher than the previous reported agar hydrogel (7.2 L.g−1.cm−1)

29

, and the

photothermal conversion efficiency and time constant for heat transfer (τs) was calculated as

was

determined as 47.2% and 284.3s, respectively (figure 2a-b). After physically encapsulating the MBP nanosheets, the AMD hydrogels exhibited typical MBP concentration-, irradiation time-, and power density-related photothermal transformation capacity. AG hydrogels without MBP doping showed no detectable temperature increase (with a temperature increase (△T) of 0.36 oC) even under the NIR power density of 1.0 W/cm2. Under the NIR power density of 1.0 W/cm2, the △T of hydrogels with 16


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doped MBP increased dramatically, with △T of 29.78, and 66.14 oC when the doped MBP concentrations were 2.5, and 5.0 mg/mL (figure 2c), respectively. When the doped MBP concentration was 5.0 mg/mL, the △T was 26.45, 40.27, and 63.45 oC when the NIR power density was 0.4, 0.6, and 0.8 W/cm2, respectively. The corresponding infrared thermal images (figure 2 d and f) were consistent with the MBP concentration-, irradiation time-, and power density-dependent photothermal performance of AMD hydrogels. In addition to the excellent photothermal performance, AMD hydrogel also possess a favorable photothermal stability, which showed with no distinguishable variation in △T during 9 cycles laser irradiation (Figure S-4). Based on the in vitro photothermal study, optimized AMD hydrogels with MBP doping concentration of 2.5 mg/mL and laser power density of 0.8 W/cm2 were used for further in vivo experiments.

3.3 In vitro bio- and hemocompatibility

A platform for potential biomedical applications should be bio- and hemocompatible; therefore, the bio- and hemocompatibility of AMD hydrogels was systematically studied. Due to the possible harmful effects of DOX to L929 cells and the absorbance interference to hemoglobin, AG/MBP hydrogels were chose as an alternative for the in vitro bio- and hemocompatibility assessments. After incubation with AG/MBP hydrogels for 24 hours, the metabolic activity of L929 cells was not affected (with the cellular viability higher than 95%, figure 3a), even under the high MBP doping concentration of 5.0 mg/mL. Consistently with the qualitative CCK-8 assay results, the phase contrast images of the L929 cells indicated no morphology difference between AG/MBP hydrogeland PBS-treated L929 cells (figure 3b, c). Moreover, trypan blue staining suggested that no AG/MBP hydrogel-treated cells were stained (figure 3d), implying that AG/MBP hydrogels were biocompatibile in vitro at the experimental dosages. The in vitro bio-compatibility assay results illustrated that the used Ca2+ has no apparent influence on the viability of L929, although the 17



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concentration of the used CaCl2 solution was very high (0.1 M). This is because that a large amount of Ca2+ has exchanged with the Na+ of alginate, and the produced NaCl was encapsulated within the hydrogel matrix. The hemocompatibility probe of AG/MBP hydrogels (figure 3e) manifested that the hemolytic ratio (about 0.63%) of AG/MBP was maintained at the same level as that of PBS (the hemolytic ratio was set as zero) and was negligible when compared with mRBCs that were exposed to water (the hemolytic ratio was set as 100%). The blood clotting behavior of AMD hydrogel was also studied (figure S-5) since the high concentrated Ca2+ can promote the coagulation of blood. The coagulated blood cells will not dissolved by water and do not release hemoglobin. Therefore, a higher OD value represents that the released hemoglobin concentration is high and thereby the clotting behavior is less obvious. It was found that the OD values of untreated and AMD treated blood cell after dissolving into water are on almost the same level, implying that although the concentration of Ca2+ is as high as 0.1 M, it will not cause the blood coagulation, further proving that these Ca2+ has linked with α-L-guluronic blocks and the amount of the unbonded Ca2+ is limited. These results clearly proving the excellent hemocompatibility of the Ca2+ cross-linked AG hydrogel.

3.4 In vitro and in vivo imaging capacity

Due to its MoS2 skeleton and high atomic Bi concentration, MBP nanosheets possess strong photothermal performance and high X-Ray attenuation capacity

14

. We thus expected the MBP

nanosheet-containing AMD hydrogels to have PA/CT tumor dual-modal imaging capacity. As shown in figure 4a, the brightness in CT imaging was detectable even under a lower Bi concentration of 0.05M, and it gradually increased with the Bi concentration. Additionally, a linear correlation between the X-Ray attenuation coefficient and Bi concentration was found (figure 4a). The CT imaging contrast enhancement of the tumors can also be clearly visualized after injection with AG/MBP/DOX solution, and the X-Ray attenuation coefficient increased significantly from 9.1 ± 18


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3.5 (control) to 132.7 ± 3.5 (***p < 0.001, figure 4b). Apart from the CT imaging, MBP nanosheets also gave the AMD hydrogels excellent PA imaging capacity; the PA contrast in tumors (red signal) was clearly enhanced after AG/MBP/DOX solution injection (figure 4c-d). Apparently, the CT/PA dual-modal imaging capacity of MBP nanosheets was well- retained after gelling in vivo. It is noteworthy that tumors for the CT/PA imaging was only injected with AG/MBP/DOX solution (without CaCl2). Therefore, the AMD hydrogel can be uniformly implanted within tumor since the precise location of AG/MBP/DOX solution within tumor can be exactly monitored and CaCl2 solution can be injected after the confirmation of the sufficient and thorough diffusion of AG/MBP/DOX solution within tumor.

3.5 Synergetic in vitro tumor therapy

The drug loading of AG hydrogels was based on simple physical blending; therefore, the loaded drug could be from many categories, and the loading amount can reach a high level provided that the drug is water soluble. In addition, the cross-linked structure of the Ca-AG hydrogel can act as a reservoir to control the release of entrapped drug molecules. As shown in figure 5a, DOX from the AMD hydrogels is released in a sustained pattern, regardless of pH and temperature; however, the diffuse rate of DOX can be accelerated by enhancing the temperature of the surrounding solution. About 9.8% release of DOX was achieved after 24 h at 37 oC, while about 25.5% release was attained at 55 oC at the same time point, which may be ascribed to the accelerated thermal motion of DOX molecules. This demonstrated that heat from the photothermal transformation of MBP can promote drug diffusion out from the hydrogel to realize on-demand drug release. Owing to the excellent photothermal effect of MBP and chemotherapy effect of DOX, the prepared AMD hydrogel possessed a combined tumor photothermal and chemotherapy capacity. The viability of HT29 cells decreased to 44.7 ± 1.3 % and 40.7 ± 2.1% after incubation with AG/DOX 19



and AG/MBP + NIR, respectively (***p < 0.001 versus control group, figure 5b). AMD hydrogels doped with MBP and DOX showed highly synergistic tumor chemotherapy and PTT effectiveness, with the viability decreased to 27% after NIR irradiation (*p < 0.05, versus AG/MBP + NIR, and AG/DOX treated group, figure 5b). This synergetic in vitro tumor therapy was further qualitatively proven using trypan blue staining. Consistent with the CCK-8 results in figure 5b, the portion of blue stained cells followed the order of control < AG/DOX < AG/MBP+NIR < AMD+NIR (figure 5c-f), further confirming the tumor therapy efficiency of DOX, MBP, and their synergism after being encapsulated in Ca-AG hydrogels.

3.6 In vitro and in vivo hemo- and histocompatibility of AMD hydrogels

The potential influence of AMD hydrogels on the function of blood and organs was evaluated so as to determine the long-term safety and highlight the translational prospects of the hydrogels. The potential interactions between mRBCs and hydrogels were monitored via an in vivo routine blood test. There existed no statistical difference between any of the various routine blood parameters in the control and AMD hydrogel-treated KM mice (figure S-6), implying the excellent in vivo hemocompatibility of AMD hydrogels. The employed MBP nanosheets are confined within tumors during AG hydrogel treatment and rarely enter body fluid circulation. As a consequence, the Mo uptake in major organs was insignificant during the pre-determined time intervals (lower than 4 µg/g organ, figure 6a). Apparently, although the injected MBP concentration in the hydrogel was higher than that in MBP solution14 (50 µL, 2.5 mg/mL versus 100µL 100 ppm, note that the molar ratio of Mo to Bi content in MBP nanosheet was determined to be ~ 0.95 by ICP, therefore the total concentration of MBP was 300-350 ppm in reference14), the Mo accumulated amount in major organs was obviously lower than mice that were I. T. injected with MBP (up to 115µg/g organ in reference

14

). During the therapy, the MBP nanosheets were mainly trapped within tumor site and 20


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may finally fall off from the mice with the burned tumor scar. Additionally, the DOX diffusion into serum and main organs of mice injected with AMD hydrogel was obviously lower than mice injected with AG/MBP/DOX solution (serum: p < 0.01 and p < 0.05, after 12h and 24h, respectively; liver: p < 0.01, p < 0.01, and p < 0.05 after 12, 24 and 48h, respectively; lung: p < 0.05 after 24 h; kidney: p < 0.05 after 12 h; figure 6b), clearly indicating that AMD hydrogel are able to restrict the entrance of DOX into circulatory system than AG/MBP/DOX solution and thereby enhancing the utilization ratio of DOX. In addition, the tested KM mice maintained a normal growth rate compared with that of mice in the control group (figure 6c), confirming that AMD hydrogels imposed few adverse effects on the health of the mice. On account of the low levels MBP nanosheets and DOX allowed into body fluid circulation and organs, AMD hydrogels exerted no detectable damage to or influence on the structure and physiological function of KM mice compared with normal mice (figure 6d). In general, the in vivo DOX and Mo distribution data clearly illustrate the advantages of using AMD hydrogels over MBP nanosheets/DOX solution.

3.7 Combined in vivo tumor therapy

We next evaluated the in vivo synergistic antitumor effects of the MBP and DOX co-entrapped Ca-AG hydrogel. Apparently, after being encapsulated into the hydrogel, the excellent photothermal efficiency of MBP nanosheets was not compromised. A significant temperature increase (△T) of 25.6 oC was obtained after 30-s laser irradiation (0.8 w/cm2), and the maximum △T was as high as 37 oC during 5-min exposure to the laser (figure 7a). In contrast, the temperature increase in tumors treated with AG alone was not significant (△T ＝ 4.78 oC after irradiation with an NIR laser for 5 minutes, figure 7a). The corresponding infrared thermal images (figure 7b-c) further confirmed the photothermal performance of AMD and AG hydrogels. This excellent photothermal performance 21



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together with the chemotherapy efficiency of DOX in situ gave AMD hydrogels an outstanding synergistic tumor therapy capacity. Tumor growth in AMD hydrogel-injected mice was totally inhibited after NIR irradiation and 15 days of feeding (figure 7d-f). Due to hyperthermia, the growth of tumors treated with AG/MBP and NIR was inhibited to some extent (the tumor volume expanded 3.07 times after 28 days of feeding, figure 7d-f). Similarly, the chemotherapy efficiency of the released DOX also partially inhibited the malignant proliferation of tumors, allowing volume to expand 2.01 times after 28 days of feeding (figure 7d-f). Tumor growth in the control group was uncontrolled, and volume increased about 4.96 times after 28 day of feeding (figure 7d-f). The tumor volume results clearly suggested that photothermal therapy or chemotherapy alone were minimally efficient in inhibiting tumor proliferation. Immunohistochemical staining was then performed to further verify the in vivo synergistic antitumor performance of AMD hydrogels (figure S-7). Mice treated with AMD hydrogels and an NIR laser showed the lowest CD31 expression levels, implying the highest tumor cell proliferation suppression. Additionally, Ki-67 antibody and TUNEL staining clearly indicated that AMD hydrogels more efficiently inhibit the malignant proliferation of tumors than do either PTT or chemotherapy alone (figure S-7).

3.8 In vitro and in vivo anti-inflammatory efficiency of AG/MBP/AMX hydrogels

Patients with solid malignancy are especially vulnerable to nosocomial infections because of treatment- and disease-related changes in their immune systems, however, postoperative infections in patients with cancer is another issue that should not be ignored in clinic

43

. In this research, the

drug loading of Ca-AG hydrogels was extended to the encapsulation of antibiotics, such as AMX. As in the case of DOX, the cross-linked structure of AG hydrogels restricts the release of AMX, which also followed a sustained pattern. Approximately 22.6% and 25.8% AMX was released within the first 2 and 4 h, respectively, and sustained release was maintained as long as 48 h (figure 8a). The 22


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low-rate release of AMX contributed positively to the long-term antibacterial activity of the AMX-loaded hydrogels. We qualitatively and quantitatively evaluated the in vitro antibacterial activity of AMX-loaded hydrogels in liquid medium. Total bacterial inhibition was obtained under the experimental AMX dosages (100, 200, and 400 ppm, figure 8b-c); however, pure AG hydrogels exhibited no detectable anti-bacterial activity, implying that bacteria were killed solely by the released AMX. Bacterial growth was significantly inhibited on solid medium, and the inhibition zones were legible after 1 d, 2 d, and 7 d of culture, regardless of whether the hydrogels were removed after drug release (figure 8d). The prepared AG/MBP/AMX hydrogels also possess excellent in vivo anti-inflammatory effects. As shown in figure 8e-f, KM mice showed significantly decreased TNF-α and IL-6 levels at day 1 and 7 (p < 0.05), clearly indicating the potent in vivo anti-inflammatory effects of the hydrogels. With the sustained release profile and long-term anti-inflammatory effects, the antibiotic-laden Ca-AG hydrogel are expected to have promising clinical translation potential in pharmaceutical science such as efficient anti-inflammatory treatment and localized tumor therapy.

4. CONCLUSION In this study, an FDA-approved biocompatible AG polymer-based multifunctional hydrogel was designed for imaging-guided tumor hyperthermia and chemotherapy. The binding between α-L-guluronic blocks of AG and calcium ions allows the AG/MBP/DOX solution to gelatinize in vitro and in vivo. The in situ-formed hydrogel can act as a reservoir to control the release of entrapped drug molecules (chemotherapeutics: DOX, and anti-inflammatory drug: AMX). In addition, the doped MBP nanosheets and DOX molecules conferred on the composite hydrogel the capacity for computed tomography/photoacoustic dual-modal imaging-guided in vivo tumor PTT and chemotherapy, respectively. The AMD hydrogel exhibited excellent photothermal conversion 23



properties with mass extinction coefficient of 45.1 L.g−1.cm−1 and photothermal conversion efficiency of 47.2%. Heat transformation upon NIR laser irradiation can not only cause significant tumor coagulation necrosis, but also can trigger DOX release from the hydrogel matrix and thereby enhance chemotherapeutic efficiency. After the combined photothermal therapy and chemotherapy, the tumors were completely erased without reoccurrence. Another feature of the hydrogel is that MBP nanosheets and anticancer agents will not diffuse into body fluid circulation; therefore, any potential long-term side effects of MBP nanosheets and DOX on normal issue and organs are significantly mitigated. With convenient handling and administrating procedures, low required dosages, and high in vitro and in vivo hemo-/histocompatibility, the AMD hydrogel is anticipated to have promising clinical translation potential in localized tumor therapy.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] and [email protected] Author Contributions J.Z and J.L. contributed equally to this work. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (Grant No. 51702214, No. 81370493, No. 81670485), the Shanghai Sailing Program (17YF1412600) supported by the Shanghai Committee of Science and Technology, the Chenguang Program supported by the Shanghai Education Development Foundation and Shanghai Municipal

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Education Commission (15CG52), and the Shanghai Young College Teachers Training Project (slg16054).

Supporting Information Calculation of the photothermal conversion efficiency, the supplementary characterization results: digital photo, EDS spectrum, UV-Vis-NIR spectra, photothermal stability analysis, coagulant behavior, In vivo routine blood test and Immunohistochemical staining.

25



Figures

Figure 1. (a) Schematic illustration of the preparation of CaCl2 crosslinked AG gel and synergistic tumor PTT and chemotherapy; (b-d) FESEM images of (b) MBP nanosheets, (c) AMD and (d) AG hydrogel; (e) distribution mapping of Mo Bi within AMD hydrogels; (f) XRD patterns of AG and AMD hydrogels (▲: MoS2; ◆: Bi2S3); insert of (f) is the digital photos of AG (left) and AMD hydrogels (right).

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Figure 2. In vitro PPT performance of AMD hydrogels. (a) Laser on induced temperature increase and laser off induced temperature decrease of ALG/MBP hydrogel; (b) time constant for heat transfer of ALG/MBP hydrogel (τs = 284.3 s); (c) NIR induced PTT of AMD hydrogels with different MBP concentrations; (d) infrared thermal images of AMD hydrogels corresponding to panel (c); (e) the relationship between power density and PTT performance of AMD hydrogels; (f) infrared thermal images of AMD hydrogels corresponding to panel (e).

27



Figure 3. (a) Cell viability of L929 cells, which were co-cultured with PBS or AMD hydrogels with different concentrations of MBP nanosheets; (b-c) Phase contrast image of L929 cells cultured with (b) PBS and (c) AMD hydrogels with MBP concentration of 0.2 mg/mL; (d) Trypan blue staining of L929 cells corresponding to panel (c); (e) Hemolytic ratio and (f) photograph of distilled water-, PBS-, and AG/MBP hydrogel-treated mRBCs.

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Figure 4. CT and PA imaging capacity of AMD hydrogels. (a) In vitro X-ray attenuation (HU) intensity of AMD hydrogels as a function of Bi concentration; the insert shows the corresponding CT images of different AMD hydrogels; (b) in vitro HU intensity of saline and AMD hydrogel-injected tumor; the insert shows the corresponding CT images tumor; (c-d) in vivo photoacoustic images of tumor injected with saline (c) and AMD hydrogels (d).

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Figure 5. (a) In vitro cumulative release of DOX from AMD hydrogels at 37 oC and 55 oC (mimic NIR induced photothermal transformation of AMD hydrogels); (b) viability of HT29 cells upon different treatments; (c-f) trypan blue staining of HT29 cells after treatment with (c) saline, (d) AG/DOX, (e) AG/MBP hydrogel and NIR, and (f) AMD hydrogel and NIR.

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Figure 6. (a) In vivo distribution of Mo ions in heart, liver, spleen, lung, and kidney at different time points after the hydrogel injection; (b) in vivo biodistribution of DOX after 12h, 24h and 48h treatment with AG/MBP/DOX solution and AMD hydrogel; (c) body weight changes of saline (blank) or AMD hydrogel-treated mice; (d) H&E staining of major organs (heart, liver, spleen, lung, and kidney), sections of saline (blank), or AMD hydrogel-treated mice.

Figure 7. (a) Temperature changes in tumors injected with saline (blank) or AMD hydrogels after irradiation with an NIR laser for 5 min; (b-c) in vivo thermal infrared images of mice I.T. injected with (b) saline and (c) AMD hydrogels; (d) volume-increasing curves of tumors after different treatments as indicated; (e-f) digital photos of HT29 tumor-bearing mice on day 0 and day 28 after 31



various treatments.

Figure 8. (a) In vitro cumulative release of AMX from AG/MBP/AMX hydrogels; quantitative (b) and qualitative (c) antibacterial effects of AG/MBP/AMX hydrogels with different AMX concentrations (from left to right: 0, 100, 200 and 400 ppm); (d) photograph of inhibition zones of AG/MBP/AMX hydrogels on agar plates after incubation at 37 oC for 1, 2, and 7 days; (e-f) quantitative determination of TNF-α (e) and IL-6 (f) of KM mice with artificial wound-infection that treated with either saline (control) or AG/MBP/AMX hydrogel.

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graphic for abstract 119x97mm (300 x 300 DPI)


Design of Phase-Changeable and Injectable Alginate Hydrogel for

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