Fe3+-induced synchronous formation of composite hydrogels for

Subscriber access provided by The Libraries of the | University of North Dakota

Biological and Medical Applications of Materials and Interfaces

Fe3+-induced synchronous formation of composite hydrogels for effective synergistic tumor therapy in NIR-I/II biowindows Jiulong Zhao, Chunhua Zhou, Chenyao Wu, Huan Wu, Chunping Zhu, Changqing Ye, Shige Wang, and Duowu Zou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14649 • Publication Date (Web): 15 Nov 2018 Downloaded from http://pubs.acs.org on November 15, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 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

ACS Applied Materials & Interfaces

Fe3+-induced synchronous formation of composite hydrogels for effective synergistic tumor therapy in NIR-I/II biowindows

Jiulong Zhao1,†, Chunhua Zhou1,†, Chenyao Wu2,†, Huan Wu2, Chunping Zhu1, Changqing Ye 2, Shige Wang2,* and Duowu Zou1,* 1

Department of Gastroenterology, Changhai Hospital, Second Military Medical

University, No. 168 Changhai Road, Shanghai 200433, China 2

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

Jungong Road, Shanghai 200093, China

*To whom correspondence should be addressed email: [email protected] (Dr. Shige Wang) and [email protected] (Prof. Duowu Zou)

1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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

Page 2 of 31

ABSTRACT: Alginate-Ca2+ hydrogel has been used to immobilize photothermal materials as well as chemotherapy drugs at lesion sites to prevent their entry into the bloodstream. However, the alginate-Ca2+ gelation mechanism may result in hardening of the blood vessels due to Ca2+ migration to the lesion site. In this study, a unique and facile one-pot formation of chemotherapeutic (doxorubicin, DOX) and polypyrrole (PPy)-containing alginate hydrogel was designed by introducing Fe3+, which can synchronously induce the polymerization of pyrrole and gelatinization of alginate, into the DOX/pyrrole/alginate solution. The formed composite hydrogel was endowed with superior photothermal conversion properties in both the NIR-I (650–950 nm) and NIR-II (1000–1700 nm) biowindows and light-to-heat conversion efficiency higher than 50%, which enabled effective tumor hyperthermia treatment. Besides, NIR irradiation could be used as a remote controller to trigger the DOX-release due to the heat generation, thus achieving continuous and on-demand tumor chemotherapy. The composite

polymer

hydrogels

exhibited

favorable

hemo-,

cyto-,

and

histocompatibility, as well as simple and cost-effective preparation and good clinical prospects.

KEYWORDS: hydrogel; synchronous formation; polypyrrole; NIR-I/II; photothermal

therapy 2


Page 3 of 31 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


1. INTRODUCTION Photothermal therapy (PTT) has been recognized as a minimally invasive tumor ablation technique in which photothermal conversion material localized in tumors absorbs near-infrared light (NIR) and converts the light to heat to kill cancer cells 1-2. In the NIR spectrum, there are two biologically transparent windows characterized by low scattering and energy absorption, leading to maximal depth of light penetration and high light absorbance by the photothermal agents (PTAs)

3.

The NIR-I

biowindow comprises wavelengths 650–950 nm, and the NIR-II region comprises wavelengths 1000–1700 nm. To date, most studies that concentrated on tumor PTT focused on the NIR-I biowindow, which conforms to the excitation wavelength of PTAs

4-7.

Laser in the NIR-II biowindow has unique advantages, such as stronger

tissue penetration and higher allowed power density (according to US standards, the skin can be safely exposed to 1 W/cm2 at 1064 nm, but only 0.33 W/cm2 at 808 nm) 8. Unfortunately, despite these features, NR-II window has not been widely used owing to the lack of responsive PTAs that can generate acceptable heat levels when stimulated by NIR-II lasers. In addition, with the development of biological agents, the functions of treatment platforms are not merely limited to PTT, but also combined with other tumor therapy approaches, especially chemotherapy

9-10.

This is because

traditional chemotherapy lacks a degree of specificity, where drugs tend to be spontaneously released to normal tissues and insufficiently released to tumors 11. To date, various NIR-absorbing biomaterials have been widely studied; however, although effective PTT ability has been demonstrated, widespread concerns of these 3



Page 4 of 31

PTAs have been raised over their lack of biodegradability and potential long-term toxicity

12-15.

As a promising alternative, conjugated polymers such as polypyrrole

(PPy), has sparked tremendous interest not only due to their inherent outstanding NIR photothermal conversion ability, but also their favorable biocompatibility 16-17. PPy is a dark pigment produced via the electrochemical coupling reaction through metal organics or chemical pyrrole (Py) monomer polymerization in the presence of oxidizing agents such as Fe3+ or (NH4)2S2O8 18-19. Unique photothermal therapies have been reported by varying the sizes of PPy nanoparticle, when the therapeutic agent was I.T. or I.V., injected to the target 20-21. However, due to the enhanced permeability and retention (EPR) effect of blood vessels in tumor, the PPy nanoparticles tend to enter the bloodstream, resulting in their low levels at the tumor site and causing latent toxicity to normal tissues. Polymer hydrogel, on the condition that it possesses good biocompatibility and biodegradability, has found tremendous application prospects in biomedicine

22-26.

Firm binding among biodegradable, non-toxic polymer hydrogels, PTAs, and drugs has been exploited to avoid premature drug release and permit in situ delivery of PTA to lesion sites

27-29.

Moreover, most kinds of hydrogel carriers can release

chemotherapeutic agents on-demand upon external stimuli such as photothermal treatment and UV light irradiation

30-31.

The formation of polymer hydrogel delivery

systems can be initiated by changing the pH or temperature, contact with certain ions, and exposure under light

24-25.

As a water-soluble linear polysaccharide, alginate has

abundant carboxyl groups in its chain, which enable the sodium ion exchange with 4


Page 5 of 31 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


divalent calcium ions (Ca2+) to form a cross-linked and reinforced alginate-Ca2+ hydrogel

32.

Alginate-Ca2+ hydrogel has been used to immobilize photothermal

materials and antibodies as well as chemotherapy drugs at lesion sites to prevent their entry into the bloodstream

22, 33.

However, the alginate-Ca2+ gel may lead to

angiosclerosis of the blood vessels due to Ca2+ migration at the lesion site

34.

Therefore, developing other ionic cross-linkers is of fundamental importance to broadening the biomedical application of alginate-based hydrogels. In this study, a unique one-pot formation of PPy-containing alginate (PA) hydrogel and its DOX loading formula (DPA hydrogel) were proposed via introducing Fe3+ into Py/alginate or DOX/Py/alginate solution, for combined tumor therapy in both the NIR-I (650–950 nm) and NIR-II (1000–1700 nm) NIR windows. As a difunctional agent, Fe3+ simultaneously acts as a cross-linker to induce the gelatinization of the alginate solution by chelating with the carboxylic acid groups along alginate backbones, and as an oxidant to simultaneously cause the chemical polymerization of Py in the alginate solution. The formed composite hydrogel was employed as a localized therapeutic implant for suppression of postoperative recurrence of tumor. An adventurous feature of the DPA hydrogel is that it can package DOX and PPy nanoparticles to prevent their leakage and thus enhance the therapeutic efficiency and in vivo biosafety. More importantly, PPy equipped the DPA hydrogel with an excellent photothermal ability. Consequently, NIR laser can act as a remote control to trigger the on-demand DOX-release. To the best of our knowledge, such a synchronous, one-pot synthesis of PPy-containing medicated 5



alginate hydrogels with a therapeutic feature in NIR-I and NIR-II biowindows using Fe3+ as a difunctional agent has not yet been reported.

2. MATERIALS AND METHODS 2.1 Materials Sodium alginate powder and LIVE/DEAD BacLight Kit were obtained from Sigma-Aldrich (St. Louis, MO, USA). Py was obtained from Adamas Co., Ltd. (Shanghai, China). FeCl3·6H2O was commercially obtained from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). All chemicals were directly used without any treatment. Mouse fibroblast (L929) cell line and human colon (HT29) cell line were commercially obtained from the Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences (Shanghai). Roswell Park Memorial Institute-1640 medium (RPMI-1640), penicillin-streptomycin, fetal bovine serum (FBS), cell counting kit-8 (CCK-8) and trypan blue were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Flasks and plates for cell culture were bought from Corning, Inc. (Corning, NY, USA). Experimental animals, including 4–6-week-old Nude Balb/c mice and Kuming (KM) mice were commercially obtained from Shanghai Slac Laboratory Animal Center (China). All animal experiments in this study were carried out in the animal center in Changhai Hospital, Second Military Medical University. These animal experiments were also performed in accordance with the policies of the National Ministry of Health. 2.2 Material synthesis and characterization 6


Page 6 of 31

Page 7 of 31 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


PPy-containing alginate (PA) hydrogel was synthesized by a simple and mild two-step process. In detail, Py (32 μL) was mixed with 1 mL sodium alginate solution (3 wt. %) by stirring. Then, FeCl3·6H2O aqueous solution (56 μL, 1 g/mL) was added thereinto to allow simultaneous gelatin synthesis and polymerization for 4 h; the obtained hydrogel was denoted as PA1. For the preparation of PA2 hydrogel, FeCl3·6H2O aqueous solution (56 μL, 1 g/mL) was mixed with 1 mL sodium alginate solution (3 wt. %) under stirring. Py (32 μL) was then added into the above mixed solution to allow simultaneous gelatin synthesis and polymerization for 4 h. For the preparation of PA3, FeCl3·6H2O aqueous solution (14 μL, 1 g/mL) was mixed with 1 mL sodium alginate solution (3 wt. %) under stirring. Py (8 μL) was then added into the above mixed solution to allow simultaneous crosslinking and polymerization for 4 h. For the preparation of pure PPy nanoparticles, 32 μL Py was added into 1 mL distilled water. Then, 56 μL FeCl3·6H2O (1 g/mL aqueous solution) was introduced to allow polymerization for 4 h. Pure alginate hydrogel was prepared by adding 56 μL FeCl3·6H2O (1 g/mL) into 1 mL sodium alginate solution (3 wt. %) under stirring for 4 h. The materials surface morphologies were observed by field-emission scanning transmission electron microscopy (FESEM). 2.3 Photothermal conversion performance NIR laser of different power densities were generated with a multimode pump laser machine (Shanghai Connet Fiber Optics Co., China). PA1 (50 mg) and PA2 (50 mg) hydrogels were irradiated with NIR-I and NIR-II laser (808 nm was used for NIR-I and 1064 nm for NIR-II) at varying densities, namely, 0.1 W/cm2, 0.2 W/cm2, 7



Page 8 of 31

and 0.3 W/cm2 for 5 min and 0.5 W/cm2 and 0.8 W/cm2 for 1 min. Distilled water was set as control. To simulate the in vivo photothermal performance of PA hydrogel, we investigated the heat generation of PA hydrogel via continuously stimulating the PA hydrogel that was submerging in saline with 808 and 1064 nm lasers for 5 min at different power densities (0.1 W/cm2, 0.2 W/cm2, 0.3 W/cm2, and 0.5 W/cm2). Temperature increases (△T) over time throughout the above treatment and their infrared thermal images were photographed using a infrared camera (FLIR E60, Wilsonville, OR, USA). The photothermal stability of the PA1 and PA2 hydrogel was further characterized under laser irradiation with both 808 and 1064 nm lasers at 0.3 W/cm2; 0.1 g hydrogel in 0.1 mL distilled water was subjected to 10 cycles of 5 min on/off laser irradiation, and the values of △T were monitored with the FLIR E60. In the evaluation of photothermal conversion efficiency, 808 and 1064 nm lasers were used to irradiate 0.1 g PA1 or PA2 hydrogel or 0.1 mL distilled water for 10 min. Then, the irradiated laser or water was allowed to cool for 10 min, and the formula (eq 1) was used to calculate the photothermal conversion efficiency (η): η=

hS(Tmax ― Tsurr) ― Qdis 𝐼(1 ― 10

―𝐴𝜆

)

(1)

where h stand for the heat transfer coefficient, S stands for the surface area of the irradiated sample, Tmax refers to the maximum temperature at equilibrium of PA hydrogels under NIR-I and NIR-II laser irradiation, the corresponding ambient temperature is denoted as Tsurr, Qdis stands for the lost energy from a blank cell, I refer to the power of laser (in W), and Aλ stands for the PA hydrogels absorbance at 808 or 8


Page 9 of 31 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


1064 nm. The detailed calculation process can be found in the corresponding supporting information. 2.4 In vitro biocompatibility of PA hydrogel L929 cells or HT29 cells (with density of 8 × 103 per well) were seeded into 96-well culture plates in triple (group I-III). Groups I and II received 0.05 g PA1 or PA2 along with 0.1 mL fresh 1640 cell culture medium. Group III (control) received 0.1 mL 1640 cell culture medium (cell viability set to 100%) only. After 24 h of culture, the hydrogels were discarded, and the treated cells were washed thrice with phosphate-buffered saline (PBS), and then their viability was evaluated by CCK-8 assay and LIVE/DEAD BacLight Kit. Cells stained with LIVE/DEAD BacLight Kit were imaged with inverted phase-contrast microscopy. 2.5 In vivo biocompatibility of PA hydrogel The hemocompatibility of the PA hydrogel was evaluated using KM mice blood as a model. In brief, healthy KM mice were subcutaneously implanted with 0.05 g PA1 hydrogel or 0.05 g PA2 hydrogel. Mice without any treatment were set as control. These grouped mice (n = 3 in each group) were normally fed for one day, one week, or two weeks. At these predetermined time points, mice blood was collected after anesthetization by cardiac puncture for the serum biochemistry parameter test and blood routine examination. The serum biochemistry parameter test was performed on Beckman Coulter Unicel DxC 800 automatic biochemical analyzer, and the blood routine examination was carried out with Sysmex XS-800i automated hematology analyzer. In addition, the body weight of the above KM mice was also recorded. 9



For the in vivo histocompatibility evaluation, healthy KM mice were subcutaneously implanted with 0.05 g PA1 hydrogel or 0.05 g PA2 hydrogel or 0.05 g DPA2 hydrogel. Mice without any treatment were set as control. These grouped mice (n = 3 per group) were normally fed for one day, one week, or two weeks and anesthetized to collect the heart, liver, spleen, lung, and kidney for H&E staining. The morphologies of the stained organ slices were observed by Leica DM IL LED inverted phase contrast microscopy (Wetzlar, Germany). 2.6 In vitro DOX loading and release The preparation of DOX-loaded PA hydrogel was similar to that of PA, but the used alginate solution contained DOX (1 mg/mL). The formed PA1- and PA2-mediated hydrogels (Figure S1) were denoted as DPA1 and DPA2 hydrogels, respectively. The impact of temperature on the drug release and the difference in DOX release by the two types of DPA hydrogels were monitored at 480 nm using a Lambda 25 UV-Vis spectroscopy system (Perkin Elmer, Waltham, MA, USA). In brief, the drug-loaded DPAs (DPA1 and DPA2) were wrapped in dialysis bags and separately placed in bottles of 5 mL PBS (pH = 7.4), which were then placed in 37 (DPA1 and DPA2) or 50 °C (DPA2) water baths. After culturing for predetermined time durations, 1 mL PBS was took out to measure the DOX concentration, and 1 mL equal new solution was added. 2.7 In vitro PTT For the in vitro tumor therapy study, 1 × 104 HT29 cells in 100 μL 1640 cell culture medium per well were seeded in a 96-well cell culture plate. After a 12-h 10


Page 10 of 31

Page 11 of 31 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


culture, these cells were divided into 4 groups (6 wells per group) that received different treatments (I: added with 50 mg PA2 hydrogel per well; II: added with 50 mg DPA2 hydrogel per well; III: 50 μL 1 mg/mL DOX solution; IV: untreated). Cells in all groups were continually irradiated for 5 min at 808 or 1064 nm (0.5 W/cm2). After the irradiation, cells were incubated for another 24 h, following which their viability was quantitatively monitored by the CCK-8 assay and qualitatively evaluated using the LIVE/DEAD BacLight Kit along with inverted phase-contrast microscopy. 2.8 In vivo PTT The therapeutic efficiencies of the PA and DPA hydrogels were analyzed by evaluating the anti-postoperative recurrence of tumors. The HT29 xenografted tumor was constructed by subcutaneous injection of 5 × 106 HT29 cells (in serum free RPMI-1640 culture medium, 150 μL) into the back of mouse. HT29 xenografted tumors with nodular diameters of approximately 0.5 cm were excised from Balb/c nude mice. Note that to better study the anti-postoperative recurrence of tumor, tumor tissue with size of 1 mm × 2 mm was left in the back of mouse. After the surgery, the mice were randomly divided into 5 groups. Groups I, II, and III were implanted with 50 mg DPA2 hydrogel directly in the tumor location, group IV was implanted with 50 mg PA2 hydrogel directly in the tumor location, while group V was untreated. The above tumor sites were finally sutured using the catgut. The former tumor cites in groups I, II and IV were exposed to an 808 nm or 1064 nm laser (5 min, 0.5 W/cm2). After the treatment, mice were continuously fed for 28 days to monitor the tumor volume, and tumor morphology was observed by imaging to prove the therapeutic 11



effect of the hydrogels. 2.9 Statistical analysis The experimental data significance was analyzed with one-way ANOVA using Origin 75. The significance level was select as p < 0.05, and data indicated with (*) stands for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001.

3. RESULTS AND DISCUSSION 3.1 Preparation and characterization of hydrogels In the presence of Ca2+, alginate can transform into an alginate-Ca2+ hydrogel to immobilize photothermal materials and chemotherapy drugs at lesion site and prevent their entry into the bloodstream

35.

However, Ca2+ migration may lead to

angiosclerosis of the blood vessels at the lesion site. As an alternative, we found that Fe3+ can induce the gelatinization of alginate due to its chelation with carboxylic acid groups along alginate backbones (Figure 1a). PA hydrogels were synthesized utilizing Fe3+ to enable not only Py polymerization, but also sodium alginate gel synthesis via Fe3+ crosslinking (Figure 1b). Upon introducing the crosslinker Fe3+ into the alginate solution, alginate-Fe3+ hydrogel with a relatively smooth surface morphology was facially synthesized (Figure 1c and Figure S1), while pure PPy characterized by a nanoparticle structure was obtained by directly mixing the Py monomer with Fe3+ (Figure 1d). The preparation of composite PPy-containing hydrogel is facile and can be carried out either by first mixing the Py with alginate solution and subsequently 12


Page 12 of 31

Page 13 of 31 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


initiating the synchronous gelatinization and polymerization using FeCl3 (this hydrogel is denoted as PA1), or by first mixing FeCl3 with alginate solution to form the alginate gel, and then introducing the Py to interact with FeCl3 (the formed hydrogel is denoted as PA2). In both cases, a dark color was developed by the chemical reaction between Py and Fe3+, implying the rapid polymerization of PPy (Figure S1). Additionally, the chemotherapeutic agent (DOX) dissolved in the alginate solution beforehand was readily entrapped to form drug delivery systems in both methods (the formed hydrogels are denoted as DPA1 and DPA2). SEM observation indicated a morphology consistent with those of PA1 and PA2 (Figure 1e, 1f), which showed an irregular rough surface. After introducing DOX, the formed DPA1 and DPA2 hydrogels retained their rough surface (Figure S2), indicating that DOX exerts no significant influence on the structure of PA hydrogel. Such a rough surface of DPA1 and DPA2 hydrogels is expect to promote the diffusion as well as release of doped DOX molecules. 3.2 Photothermal conversion performance The light absorbance of the hydrogel was studied before testing its photothermal conversion performance. PA hydrogels were irradiated with 808 and 1064 nm lasers to study the photothermal conversion characteristics in the two NIR biowindows. It was found that the remarkable photothermal conversion performance of PPy nanoparticles was well preserved after introducing them into the alginate hydrogel. The △T of the hydrogels gradually increased with time and laser power density (Figure 2). Notably, under irradiation of 808 nm laser for 1 min (Figure 2a), the △T of 13



PA1 immediately increased to 39.52 °C (0.5 W/cm2) and 47.1 °C (0.8 W/cm2). Under irradiation of 1064 nm laser for 1 min (Figure 2c), the △T of PA1 immediately increased to 36.36 °C (0.5 W/cm2) and 48.06 °C (0.8 W/cm2). Such a swift temperature increase can dry off within 5 min irradiation; therefore, the temperature of PA hydrogels under higher power density (0.5 W/cm2 and 0.8 W/cm2) was recorded only in the first 60 seconds. When the hydrogel was irradiated at the lower power density (0.1, 0.2, and 0.3 W/cm2), the maximum value of △T decreased in both biowindows, suggesting the typical power density dependent photothermal conversion performance of PA hydrogels (Figure 2a, c). Moreover, the △T curves of PA1 and PA2 exhibited obvious overlap when stimulated with lasers in both biowindows using the same power density (0.2 W/cm2) ( Figure 2a, c), implying the comparable in vitro photothermal conversion performance of PA1 and PA2 hydrogels. The IR thermal images further demonstrated that the PA hydrogels can rapidly convert light to heat in both biowindows (Figure 2b, d). To simulate in vivo photothermal conversion performance, the heat generation of PA hydrogel (PA2 hydrogel was selected as a representative) immersed in physiological saline was investigated (Figure 3). The maximal △T value after 808 nm irradiation for 5 min at power densities of 0.1 W/cm2, 0.3 W/cm2, and 0.5 W/cm2 was 6.63 °C, 21.29 °C, and 33.23 °C, respectively (Figure 3a). Under the stimulation of 1064 nm laser at the same power densities, the △T value was determined as 7.64 °C, 19.98 °C and 30.81 °C (Figure 3c), respectively. Obviously, the differences between △T of the hydrogel in saline stimulation with the 808 and 1064 nm lasers were not 14


Page 14 of 31

Page 15 of 31 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


significant, further proving the comparable in vitro photothermal conversion performance of PA1 and PA2 hydrogels. The IR thermal images also confirmed the swift temperature change of PA hydrogel in the saline over time (Figure 3b, d), further clearly indicating the excellent photothermal conversion efficiency of PA hydrogels. This prominent photothermal conversion of PA hydrogels in saline suggested that PA hydrogels possess good heat conductivity and can timely transfer the generated heat to its surroundings. Photothermal stability and conversion efficiency assays of PA hydrogel in saline were further performed to illustrate their splendid photothermal conversion performance. Recycling △T of two types of PA hydrogels under exposure to 808 and 1064 nm lasers were recorded. It was found that the maximal temperature and temperature variations of the two types of PA hydrogels in each laser on/off cycle showed no successive deterioration (Figure 3e, S3a), clearly implying the photothermal durability of PA hydrogel. Furthermore, photothermal conversion efficiencies were measured at 54.62% for 808 nm (Figure 3f) and 59.57% for 1064 nm (Figure 3g) of PA2, while the values of PA1 were 52.66% for 808 nm (Figure S3b) and 51.83% for 1064 nm (Figure S3c). This excellent photothermal conversion and good heat conductivity of PA hydrogel will guarantee a highly efficient tumor hyperthermia application of PA hydrogel. 3.3 In vitro cytocompatibility assay Giving that the PA hydrogels possessed an excellent photothermal conversion and good heat conductivity, the in vitro and in vivo compatibility of the hydrogels 15



was studied to check whether they meet the criteria for a safe in vivo application. The CCK-8 assay and FDA/PI staining were performed to determine the potential toxicity of PA hydrogels to L929 cells. After co-incubation with PA1 and PA2 hydrogels for 24 h (Figure 4a), the viabilities of L929 cells were measured as high as 99.8 ± 4.2 % (PA1) and 96.7 ± 3.7% (PA2), and no significant difference was detected between control group and PA1 hydrogel treated cells, and between control and PA2 hydrogel treated cells. In live/dead staining, the green region representing live cells showed obvious dominance over the red region representing dead cells, morphologically reflecting the integrated cell morphology of L929 cells and the biocompatible nature of PA1 and PA2 hydrogels (Figure 4b-d). The CCK-8 assay and FDA/PI staining clearly implied their excellent in vitro biocompatibility. 3.4 In vivo biocompatibility examination of PA hydrogel The hemo- and histocompatibility of PA hydrogels was further investigated after the in vitro cytocompatibility assay to confirm their translation potential in vivo. As indicated in Figure 5a and Figure S4a, the body weights of PA1, PA2 and DPA2 hydrogels-treated mice showed no significant deviation from control. In addition, the serum biochemistry index (Figure 5b-c, S4b-c and S5) and routine blood tests (Figure 6, S6, and S7) were assayed to determine the vivo hemocompatibility of the hydrogels, which proved that there was no meaningful fluctuation or abnormality between the experimental group and control at the predetermined time points. The superior biocompatibility of PA hydrogels was further evaluated by observing corresponding tissue lesions in heart, liver, spleen, lung, and kidneys of KM mice that 16


Page 16 of 31

Page 17 of 31 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


were subcutaneously implanted with PA and DPA hydrogels. H&E staining of organ slices from KM mice fed for 1, 7 and 14 d (Figure 5d, S4d, and S8),

showed no

obvious pathological tissue damage or toxicity in comparison to those in the control group, further implying that PA hydrogels exhibited long-term biosafety and moreover, it can decrease the toxicity of DOX. 3.5 In vitro PTT in the NIR-I and NIR-II biowindows The DOX loading of PA hydrogel was analyzed by directly dissolving DOX in alginate solution. These DOX molecules were simultaneously incorporated in the obtained medicated DPA hydrogel. It is worth to note that the drug loading capacity can be very high provided the DOX is soluble in alginate solution. Besides, the ferric ions will be slowly released out from the hydrogel under acidic condition, which affects the concentration determination of DOX, therefore, we only paid attention to the DOX release at the pH of 7.4. Owing to the promoted molecular motion of DOX, and thermal expansion-induced polymer chain distance increase, the DPA hydrogel showed a temperature-responsive DOX release manner, which is beneficial for the continuous and on-demand tumor chemotherapy. After 24 h, the accumulative release of DOX tended to balance with the final release amount of 54% at 50 oC (Figure 7a). More importantly, the accumulative release curves of DPA1 and DPA2 overlapped at 37 °C (the final release amounts were about 41%), highlighting the drug release similarity of the two types of DOX-loaded DPAs. Because there was no significant difference in the photothermal conversion performance and the in vitro and in vivo biosafety of PA1 and PA2 hydrogels, PA2 17



hydrogel, as a representative, was selected for the synergetic tumor therapy through a combination of PTT and chemotherapy. The tumor hyperthermia and chemotherapy outcome of DPA hydrogels were firstly evaluated in vitro. HT29 cells were incubated with PA2, or DPA2, and irradiated with 808 or 1064 nm lasers. It was determined that the viability of PA2 hydrogel-treated cells significantly declined upon NIR laser irradiation (NIR I, 26.53%, p < 0.01. NIR II, 32.32%, p < 0.01). The therapeutic effect of the DPA hydrogel was more pronounced than PA2 hydrogel (in both biowindows, p < 0.05) and pure DOX (p < 0.05, Figure 7b), clearly indicating the combined tumor photothermal and chemotherapy outcome. It is worth to note that the PTT induced cell death was a result of hyperthermia caused cell apoptosis.36-37 To check whether the PA hydrogel would induce cytotoxicity to cancer cells, HT 29 cells were cultured with PA1 and PA2 hydrogels for 24 hours. As shown in figure S9, the viabilities of HT29 cells were measured as high as 95% after treated with PA1 and PA2 hydrogels, and no significant difference between control group and PA1 hydrogel treated cells, and between control and PA2 hydrogel treated cells was found. Live/dead staining further morphologically reflecting the integrated cell morphology of HT29 cells (Figure S9b-d), clearly implying the alginate hydrogel has no contribution to the HT29 cell killing. The effectiveness of in vitro combined tumor therapy was further confirmed by calcein AM staining. In accord with the CCK-8 assay, the proportion of red stained cells follows the orders of control < PA2 < PA2 + DOX in both biowindows (Figure 7c). After treated with pure DOX, the portion of red stained HT29 cells also corresponded with the CCK-8 results (figure S10). These results 18


Page 18 of 31

Page 19 of 31 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


suggest that the synergistic in vitro tumor PTT and chemotherapy were achieved via incorporating DOX, clearly showing the superiority of the medicated hydrogel as a promising system for highly efficient tumor therapy. 3.6 In vivo PTT in the NIR-I and NIR-II biowindows The synergetic therapeutic effects of DPA hydrogel were further evaluated in vivo in the NIR-I (808 nm laser) and NIR-II (1064 nm laser) biowindows. When tumor sites in mice were irradiated using the 808 or 1064 nm laser, temperature changes at the lesion sites varied according to treatments. Compared with the control groups which experienced no obvious temperature increase (△T was 8.45 °C at 808 nm, 4.14 °C at 1064 nm) when irradiated for 5 min, PA2 hydrogel-treated mice exhibited significant △T values of 31.80 °C at NIR-I (808 nm) and 23.7 °C at NIR-II (1064 nm, Figure 8a-b), laying a foundation for effective tumor photothermal therapy in the NIR I and NIR II biowindows. In particular, the in vivo △T induced by 808 nm irradiation was markedly larger than that induced by 1064 nm under the same conditions, demonstrating the superiority of the in vivo photothermal performance of PA hydrogels in NIR I biowindow. The laser-treated mice were continually fed for 28 days for tumor volume recording. Regardless of the irradiated laser wavelength (808 or 1064 nm), the tumor recurrence of mice receiving synergistic treatment was completely eradicated during the feeding period, whereas the relative tumor volume in the control group expanded 22.53-fold after 28 days of feeding, and that in the DPA hydrogel-treated group and PA2 + 808 nm laser-treated group expanded 12.96-fold and 5.22-fold, respectively 19



(Figure 8c-d). Different from many other reported PTAs that are only responsive to single NIR laser, the PA hydrogel system exhibited excellent in vivo photothermal efficiency in not merely NIR I biowindow, but also in NIR II laser, with a significant enhanced penetration depth, thus is expected to have a more promising translational potential in clinical tumor therapy. 4. CONCLUSION In this study, we developed a facile way to produce PPy and DOX-loaded composite polymer hydrogel for in vitro and in vivo synergistic tumor PTT and chemotherapy. The concept of design was realized by introducing Fe3+, which can synchronously induce the polymerization of pyrrole and gelatinization of alginate, into the pyrrole/alginate solution. In addition, DOX was completely encapsulated into the Fe3+-crosslinked hydrogel during gel formation to form a medicated DPA hydrogel. The PA2 hydrogel showed rapid increases in temperature and a photothermal conversion efficiency of 54.62 % and 59.57% when stimulated with the 808 and 1064 nm laser, respectively. While for PA1 hydrogel, this value changed to 52.66 % and 51.83% when stimulated with the 808 and 1064 nm laser, respectively. In addition, the hydrogel possessed good heat conductivity and can timely transfer the generated heat to its surroundings. More importantly, the generated heat permitted on-demand DOX-release in which NIR irradiation could be used as a remote control to realize the on-demand drug release. The composite hydrogel showed a distinct in vitro and in vivo synergistic tumor therapeutic effect in both the NIR-I and NIR-II biowindows. With favorable hemo-, cyto-, and histocompatibility, as well as simple 20


Page 20 of 31

Page 21 of 31 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


and cost-effective preparation, the designed composite hydrogel is anticipated to find diverse applications in tumor treatment, with high efficiency.

ASSOCIATED CONTENT Supporting Information Photothermal conversion efficiency calculation, characterization results: digital images, FESEM, temperature and heating curves, serum biochemistry index, routine blood test results, and H&E staining.

AUTHOR INFORMATION Corresponding Author *[email protected] (Dr. Shige Wang) and [email protected] (Prof. Duowu Zou) Author Contributions † These authors contributed equally to this work. Notes The authors declare no competing financial interest.

Acknowledgments Authors thank the financial support from China National Natural Science Foundation of China (51702214, 81670485). The study was also supported by Chenguang Program supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (15CG52), as well as Shanghai Sailing Program (17YF1412600) supported by the Shanghai Committee of Science and Technology, and the Young Medical Talents Program and Natural Science Research Project of 21



Universities in Jiangsu Province (CH Zhou, QNRC2016863, 18KJB320021).

Figures

Figure 1. (a) Schematic illustration of chemical molecular structure of alginate-Fe hydrogel; (b) schematic illustration of the Fe3+ induced one-pot formation of PAD hydrogel; (c-f) FESEM of (c) alginate-Fe hydrogel, (d) PPy nanoparticles; (e) PA1 hydrogel, and (f) PA2 hydrogel.

22


Page 22 of 31

Page 23 of 31 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


Figure 2. (a, c) NIR laser irradiation ((a) 808 nm, (c) 1064 nm) induced temperature changes of PA hydrogels at with different power density; (b, d) photothermal images corresponded to (a) and (c), respectively.

23



Figure 3. Temperature changes of PA2 hydrogel in saline at varied power densities under 808 nm (a) and 1064 nm (c) lasers and the responding IR thermal image ((b) 808 nm, (d) 1064 nm). (e) Temperature curves of PA2 hydrogel in water over 10 laser on/off cycles under the irradiation of 808 nm and 1064 nm laser; (b-c) heating curves (violet) and time constant (τs, blue) of PA2 hydrogel under the irradiation of (f) 808 nm and (g) 1064 nm laser.

24


Page 24 of 31

Page 25 of 31 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


Figure 4. (a) L929 cell viabilities and (b-d) LIVE/DEAD staining imaging of L929 cells (b) without any treatment, and treated with (c) PA1 and (d) PA2 hydrogel.

Figure 5. In vivo toxicity evaluation. (a) The relative body weight of two types PA hydrogels treated KM mice; (b-c) serum biochemistry index of KM mice under PA2 treatment; (d) H&E staining images of major organs of PA1 and PA2 treated KM 25



mice anesthetized on day 14.

Figure 6. Routine blood test of KM mice fed with different days which treated with PA2 hydrogel. (a) hemoglobin (HB); (b) hematocrit (HCT); (c) mean corpuscular hemoglobin content (MCH); (d) concentration of mean corpuscular hemoglobin (MCHC); (e) mean corpuscular volume (MCV); (f) platelet (PLT); (g) red blood cell count (RBC); (h) red cell distribution width (RDW); (i) white blood cell count (WBC).

26


Page 26 of 31

Page 27 of 31 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


Figure 7. (a) DOX release profile of DPA2 hydrogel; (b) HT29 cells viability under after varied treatments; (c) LIVE/DEAD staining of HT29 cells co-incubated different hydrogels and NIR laser as noted.

Figure 8. In vivo PTT. (a) Tumor temperature of control and mice injected with 27



DPA2 hydrogel and irradiated with NIR-I (808 nm) and NIR-II (1064 nm) lasers; (b) the corresponding thermal images; (c) the time-dependent mice tumor growth and (d) photographs of HT29 tumor-bearing mice on different days after different treatments.

References (1) Wang, S.; Zhao, J.; Yang, H.; Wu, C.; Hu, F.; Chang, H.; Li, G.; Ma, D.; Zou, D.; Huang, M. Bottom-up Synthesis of WS2 Nanosheets with Synchronous Surface Modification for Imaging Guided Tumor Regression. Acta Biomater. 2017, 58, 442-454. (2) Yang, H.; Zhao, J.; Wu, C.; Ye, C.; Zou, D.; Wang, S. Facile Synthesis of Colloidal Stable MoS2 Nanoparticles for Combined Tumor Therapy. Chem. Eng. J. 2018, 351, 548-558. (3) Lin, H.; Gao, S. S.; Dai, C.; Chen, Y.; Shi, J. L. A Two-Dimensional Biodegradable Niobium Carbide (MXene) for Photothermal Tumor Eradication in NIR-I and NIR-II Biowindows. J. Am. Chem. Soc. 2017, 139, 16235-16247. (4) Liu, H.; Li, W.; Cao, Y.; Guo, Y.; Kang, Y. Theranostic Nanoplatform Based on Polypyrrole Nanoparticles for Photoacoustic Imaging and Photothermal Therapy. J. Nanopart. Res. 2018, 20, 57. (5) Zhao, J.; Xie, P.; Ye, C.; Wu, C.; Han, W.; Huang, M.; Wang, S.; Chen, H. Outside-in Synthesis of Mesoporous Silica/molybdenum Disulfide Nanoparticles for Antitumor Application. Chem. Eng. J. 2018, 351, 157-168. (6) Cai, X.; Gao, W.; Ma, M.; Wu, M.; Zhang, L.; Zheng, Y.; Chen, H.; Shi, J. A Prussian Blue-Based Core-Shell Hollow-Structured Mesoporous Nanoparticle as a Smart Theranostic Agent with Ultrahigh pH-Responsive Longitudinal Relaxivity. Adv. Mater. 2015, 27, 6382-6389. (7) Cai, X.; Gao, W.; Zhang, L.; Ma, M.; Liu, T.; Du, W.; Zheng, Y.; Chen, H.; Shi, J. Enabling Prussian Blue with Tunable Localized Surface Plasmon Resonances: Simultaneously Enhanced Dual-Mode Imaging and Tumor Photothermal Therapy. ACS Nano 2016, 10, 11115-11126. (8) Ke, K.; Yang, W.; Xie, X.; Liu, R.; Wang, L.-L.; Lin, W.-W.; Huang, G.; Lu, C.-H.; Yang, H.-H. Copper Manganese Sulfide Nanoplates: A New Two-Dimensional Theranostic Nanoplatform for MRI/MSOT Dual-Modal Imaging-Guided Photothermal Therapy in the Second Near-Infrared Window. Theranostics 2017, 7, 4763-4776. (9) Liu, H.; Chen, D.; Li, L.; Liu, T.; Tan, L.; Wu, X.; Tang, F. Multifunctional Gold Nanoshells on Silica Nanorattles: a Platform for the Combination of Photothermal Therapy and Chemotherapy with Low Systemic Toxicity. Angew. Chem. 2011, 123, 921-925. (10) Liu, T.; Wang, C.; Gu, X.; Gong, H.; Cheng, L.; Shi, X.; Feng, L.; Sun, B.; Liu, Z. Drug Delivery with PEGylated MoS2 Nano-sheets for Combined Photothermal and Chemotherapy of Cancer. Adv. Mater. 2014, 26, 3433-3440. (11) Wu, C.; Zhao, J.; Hu, F.; Zheng, Y.; Yang, H.; Pan, S.; Shi, S.; Chen, X.; Wang, S. Design of Injectable Agar-based Composite Hydrogel for Multi-mode Tumor Therapy. Carbohydr. Polym. 2018, 180, 112-121. (12) Zhang, Z.; Wang, J.; Chen, C. Near Infrared Light Mediated Nanoplatforms for Cancer Thermo-Chemotherapy and Optical Imaging. Adv. Mater. 2013, 25, 3869-3880. (13) Zha, Z.; Yue, X.; Ren, Q.; Dai, Z. Uniform Polypyrrole Nanoparticles with High Photothermal 28


Page 28 of 31

Page 29 of 31 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


Conversion Efficiency for Photothermal Ablation of Cancer Cells. Adv. Mater. 2013, 25, 777-782. (14) Yin, W.; Yan, L.; Yu, J.; Tian, G.; Zhou, L.; Zheng, X.; Zhang, X.; Yong, Y.; Li, J.; Gu, Z. High-throughput Synthesis of Single-layer MoS2 Nanosheets as a Near-infrared Photothermal-triggered Drug Delivery for Effective Cancer Therapy. ACS Nano 2014, 8, 6922-6933. (15) Song, X.; Gong, H.; Yin, S.; Cheng, L.; Wang, C.; Li, Z.; Li, Y.; Wang, X.; Liu, G.; Liu, Z. UltraSmall Iron Oxide Doped Polypyrrole Nanoparticles for In Vivo Multimodal Imaging Guided Photothermal Therapy. Adv. Funct. Mater. 2014, 24, 1194-1201. (16) Wang, C.; Xu, H.; Liang, C.; Liu, Y.; Li, Z.; Yang, G.; Cheng, L.; Li, Y.; Liu, Z. Iron Oxide @ Polypyrrole Nanoparticles as a Multifunctional Drug Carrier for Remotely Controlled Cancer Therapy with Synergistic Antitumor Effect. ACS Nano 2013, 7, 6782-6795. (17) Yang, K.; Xu, H.; Cheng, L.; Sun, C.; Wang, J.; Liu, Z. In Vitro and In Vivo Near-Infrared Photothermal Therapy of Cancer Using Polypyrrole Organic Nanoparticles. Adv. Mater. 2012, 24, 5586-5592. (18) Chen, X.; Zhang, M.; Li, S.; Li, L.; Zhang, L.; Wang, T.; Yu, M.; Mou, Z.; Wang, C. Facile Synthesis of Polypyrrole@metal-organic Framework Core-shell Nanocomposites for Dual-mode Imaging and Synergistic Chemo-photothermal Therapy of Cancer Cells. J. Mater. Chem. B 2017, 5, 1772-1778. (19) Wang, Q.; Wang, J.; Lv, G.; Wang, F.; Zhou, X.; Hu, J.; Wang, Q. Facile Synthesis of Hydrophilic Polypyrrole Nanoparticles for Photothermal Cancer Therapy. J. Mater. Sci. 2014, 49, 3484-3490. (20) Tian, Y.; Zhang, J.; Tang, S.; Zhou, L.; Yang, W. Polypyrrole Composite Nanoparticles with Morphology-dependent Photothermal Effect and Immunological Responses. Small 2016, 12, 721-726. (21) Woo, H. Y.; Jung, W. G.; Ihm, D. W.; Kim, J. Y. Synthesis and Dispersion of Polypyrrole Nanoparticles in Polyvinylpyrrolidone Emulsion. Synth. Met. 2010, 160, 588-591. (22) Chao, Y.; Xu, L.; Liang, C.; Feng, L.; Xu, J.; Dong, Z.; Tian, L.; Yi, X.; Yang, K.; Liu, Z. Combined Local Immunostimulatory Radioisotope Therapy and Systemic Immune Checkpoint Blockade Imparts Potent Antitumour Responses. Nature Biomedical Engineering 2018, 2, 611-621. (23) Li, Y.; Rodrigues, J.; Tomas, H. Injectable and Biodegradable Hydrogels: Gelation, Biodegradation and Biomedical Applications. Chem. Soc. Rev. 2012, 41, 2193-2221. (24) Li, Y.; Maciel, D.; Rodrigues, J.; Shi, X.; Tomas, H. Biodegradable Polymer Nanogels for Drug/Nucleic Acid Delivery. Chem. Rev. 2015, 115, 8564-8608. (25) Li, Y.; Maciel, D.; Tomas, H.; Rodrigues, J.; Ma, H.; Shi, X. pH Sensitive Laponite/alginate Hybrid Hydrogels: Swelling Behaviour and Release Mechanism. Soft Matter 2011, 7, 6231-6238. (26) Wang, C.; Wang, J. Q.; Zhang, X. D.; Yu, S. J.; Wen, D.; Hu, Q. Y.; Ye, Y. Q.; Bomba, H.; Hu, X. L.; Liu, Z.; Dotti, G.; Gu, Z. In Situ Formed Reactive Oxygen Species-responsive Scaffold with Gemcitabine and Checkpoint Inhibitor for Combination Therapy. Sci. Transl. Med. 2018, 10, eaan3682. (27) Jin, H.; Wan, C.; Zou, Z.; Zhao, G.; Zhang, L.; Geng, Y.; Chen, T.; Huang, A.; Jiang, F.; Feng, J.-P.; Lovell, J. F.; Chen, J.; Wu, G.; Yang, K. Tumor Ablation and Therapeutic Immunity Induction by an Injectable Peptide Hydrogel. ACS Nano 2018, 12, 3295-3310. (28) Wang, J.; Wang, D.; Yan, H.; Tao, L.; Wei, Y.; Li, Y.; Wang, X.; Zhao, W.; Zhang, Y.; Zhao, L.; Sun, X. An Injectable Ionic Hydrogel Inducing High Temperature Hyperthermia for Microwave Tumor Ablation. J. Mater. Chem. B 2017, 5, 4110-4120. (29) Yang, L.; Li, Y.; Gou, Y.; Wang, X.; Zhao, X.; Tao, L. Improving Tumor Chemotherapy Effect Using an Injectable Self-healing Hydrogel as Drug Carrier. Polym. Chem. 2017, 8, 3071-3076. 29



(30) Qiu, Y.; Park, K. Environment-sensitive Hydrogels for Drug Delivery. Adv. Drug Del. Rev. 2001, 53, 321-339. (31) Qiu, Y.; Park, K. Environment-sensitive Hydrogels for Drug Delivery. Adv. Drug Del. Rev. 2012, 64, 49-60. (32) Fajardo, A. R.; Silva, M. B.; Lopes, L. C.; Piai, J. F.; Rubira, A. F.; Muniz, E. C. Hydrogel Based on an Alginate-Ca2+/chondroitin Sulfate Matrix as a Potential Colon-specific Drug Delivery System. Rsc Advances 2012, 2, 11095-11103. (33) Komatsu, M.; Konagaya, S.; Egawa, E. Y.; Iwata, H. Maturation of Human iPS Cell-derived Dopamine Neuron Precursors in Alginate-Ca2+ Hydrogel. Biochim Biophys Acta 2015, 1850, 1669-1675. (34) Guohua, D.; Tong, Z.; Huijuan, L. Discussion on the TCM Treatment of Hypertension from Pathogenetic Viewpoints of Modern Medicine. Chin. J. Integr. Med. 2004, 10, 304-307. (35) Zhao, J. L.; Li, J. L.; Zhu, C. P.; Hu, F.; Wu, H. Y.; Man, X. H.; Li, Z. S.; Ye, C. Q.; Zou, D. W.; Wang, S. G. Design of Phase-Changeable and Injectable Alginate Hydrogel for Imaging-Guided Tumor Hyperthermia and Chemotherapy. ACS Appl. Mater. Interfaces 2018, 10, 3392-3404. (36) Ali, M. R.; Ali, H. R.; Rankin, C. R.;

El-Sayed, M. A. Targeting Heat Shock Protein 70 Using

Gold Nanorods Enhances Cancer Cell Apoptosis in Low Dose Plasmonic Photothermal Therapy. Biomaterials 2016,102, 1-8. (37) Pérez-Hernández, M.; Del, P. P.; Mitchell, S. G.; Moros, M.; Stepien, G.; Pelaz, B.; Parak, W. J.; Gálvez, E. M.; Pardo, J.; Jm, D. L. F. Dissecting the Molecular Mechanism of Apoptosis During Photothermal Therapy Using Gold Nanoprisms. ACS Nano 2015, 9 (1), 52-61.

30


Page 30 of 31

Page 31 of 31 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


TOC 150x61mm (300 x 300 DPI)


Fe3+-induced synchronous formation of composite hydrogels for

Recommend Documents