Polyisocyanopeptide Biomimetic

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Oligo (p-phenylene vinylene) /Polyisocyanopeptides Biomimetic Composite Hydrogel-Based Three-dimensional Cell Culture System for Anti-cancer and Antibacterial Therapeutics Jingqi Guo, Chengfen Xing, Hongbo Yuan, Ran Chai, and Yong Zhan ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00217 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 11, 2019

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ACS Applied Bio Materials

Oligo (p-phenylene vinylene) /Polyisocyanopeptides Biomimetic Composite Hydrogel-Based Threedimensional Cell Culture System for Anti-cancer and Antibacterial Therapeutics Jingqi Guo,† Chengfen Xing, *†,‡ Hongbo Yuan,† Ran Chai‡ and Yong Zhan† Key Laboratory of Hebei Province for Molecular Biophysics, Institute of Biophysics, Hebei

†

University of Technology, Tianjin 300401, P.R. China School of Materials Science and Engineering, Hebei University of Technology, Tianjin 300130,

‡

P.R. China ABSTRACT: Cells are normally cultured in 2D environment which is usually inconsistent with the real microenvironment in vivo and it is rarely reported that effective cancer cells killing process occurs in 3D network environment. Herein, a kind of new biomimetic composite hydrogel which can achieve 3D cell culture has been prepared and constructed by assembly of polyisocyanopeptide (PIC) with cationic oligo (p-phenylene vinylene) (OPV). The polymer chains of PIC can be bound and frizzled to form 3D network when the temperature rises above the gelation temperature, followed by encapsulating the cells into biomimetic composite hydrogel. Cells grow and proliferate well in 3D composite hydrogels with excellent cell viability.

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When the cells undergo cancerization or microbial infection during the 3D culture, the addition of the luminol luminescence system can cause strong bioluminescence resonance energy transfer (BRET) process to produce highly active reactive oxygen species (ROS) in 3D culture and kill the cancer cells and pathogenic microorganism effectively. Utilizing the BRET process in 3D composite biomimetic hydrogels provides an efficient antibacterial and anticancer approach in 3D culture to overcome the light-penetration limitation. KEYWORDS: biomimetic hydrogel, three-dimensional cell encapsulation, bioluminescence resonance energy transfer, anti-cancer, anti-microbial

INTRODUCTION Cells are the basic units of the growth and development of the entire living body. Teeny changes in the microenvironment of the cell culture seriously affect the physiological behavior of the cells. For the growing cells, the hardness of the substrate materials of cell culture has an extensive influence on the morphological characteristics, migration orientation, proliferation and differentiation of the cells.1 The mechanical signals transmitted by the extracellular matrix are closely related to many physiological and pathological processes. However, cells are generally cultured in 2D environment and the hardness of the substrate materials such as plastics and glass culture dishes used in cell culture is too stiff to mimic the extracellular matrix and control the real microenvironment of cell growth.2 Therefore, it is difficult to accurately study cell mechanically responsive behaviors3 and carcinogenesis process.4 In addition, it is not conducive to a correct understanding of the pathogenesis of the diseases and the adoption of effective treatments. Nowadays, cells are usually cultured on 2D planar hard plastics and glass surface, which compel the cells to adhere to the surface of artificial5 and hard materials that influence the


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cell metabolism6 and normal growth. The experimental results obtained in 2D planar culture are usually inconsistent with the real situation in vivo.7 In recent years, 3D cell culture has aroused great interest of scientists and has become the focus of scientists' research. 3D cell culture lays the foundation for the scientific research, including cellular responses with higher biological relevance,8 tumor formation,9,

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drug development11,

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and testing,13 which mimic the

extracellular matrix (ECM). Therefore, materials with good mechanical properties and biocompatibility will be selected as the suitable substrate for cell culture,14-16 which can achieve quantitative controllability of mechanical properties and be conducive to understand the pathogenesis of disease and adopt effective treatments. Polyisocyanopeptide (PIC) has excellent biocompatibility17 and biopolymer-likely stressstiffing effect18, thus the strength of the PIC increases as the external stress increases and can simulate the mechanical properties of microtubules, actin, intermediate filaments and collagen.1921

The cytoskeletal stiffening effect of PIC biomimetic hydrogel make it available for bio-like

applications due to the biomimetic mechanical properties,22 which mimics the extracellular matrix and the microenvironment of cell growth.23,

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The PIC can be dissolved in aqueous

solution at low temperature25 and its polymer chains can be bound and frizzled to form porous 3D network when the temperature rises above the gelation temperature.26 It is reported that fibroblasts, endothelial cells, adipose-derived stem cells and melanoma cells can survive and differentiate in 3D PIC hydrogels23 and PIC hydrogels can facilitate wound repair without causing foreign body reaction or excessive inflammation.27 Therefore, it is advisable for us to choose the PIC as the material of 3D cell culture. As pathogen infections increase in clinic, pathogen infection has become a public health concern around the global. Fungi and bacteria are the main causes of pathogen infections and


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have serious consequences, including expensive medical costs, life-threatening diseases and increasing patient mortality.28, 29 At present, there are more than 3.5 million new cancer cases and more than 2 millions of those die each year in China. Cancer has become one of the major diseases that seriously endanger human health.30 When the normal tissue was infected by microbial or cell carcinogenesis occurs, how to simulate the true microenvironment of tissue infection or cell carcinogenesis and take effective antibacterial and anticancer methods are of great significance.31, 32 Thus, it is urgent to explore effective treatment for microbial infections and cell carcinogenesis in 3D culture. Photodynamic therapy (PDT) utilizing the photosensitive drugs and laser activation has been widely used as a novel treatment for a variety of neoplastic diseases, ophthalmology and dermatology-related diseases, which is highly selective, minimally invasive and effective.33-35 Irradiation of the site with specific laser wavelength enables the activation of the photosensitizer selectively clustered in the tissue and the excited photosensitizer transfer energy to the surrounding oxygen to produce highly active reactive oxygen species (ROS) which can kill tumor cells or pathogenic bacteria effectively.36-42 Compared with traditional oncology therapy, PDT has the advantage of precise and effective treatments with minimal side effects.43 However, the requirement of external light source for photodynamic therapy limits its effective application to deeper diseased tissues for the reason that the light absorption and scattering effects of biological tissues hinder the external light to penetrate deeper tissues. Therefore, it is of great significance to develop a new PDT method that does not rely on external light sources. Bioluminescence resonance energy transfer (BRET) system is a new PDT, which has been reported to effectively kill cancer cells in vitro, inhibit tumor growth in vivo44 and efficiently kill pathogen.45 In BRET system, the photosensitizer is activated by chemical molecules, not an


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external light source. The donor is usually luminescent enzyme which glows when it is catalyzed or oxidized and the receptor is normally fluorescent protein or other fluorescent materials that absorb the energy of the donor and emit longer wavelength light.46 The BRET system overcomes the defects of traditional PDT system and eliminates the need of an external light source to excite the donor. Therefore, BRET system has potential applications in the regulation of lightdependent cellular signaling pathways and optogenetic control,47,

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providing guidelines for

designing a new model of photodynamic therapy system. Effective BRET requires the emission spectrum of the donor and the excitation spectrum of the acceptor overlaps. Here, we choose luminol as the donor and OPV as the receptor for that the absorption spectra of OPV in aqueous solution ranges from 350 to 550 nm and the bioluminescence of luminol exhibits a maximum emission peak at 425 nm under alkaline conditions in the presence of hydrogen peroxide and horseradish peroxidase (HRP),49 which meets the condition of the bioluminescence resonance energy transfer (BRET)50 as the donor−acceptor pair owing to the overlap of absorption spectra of OPV and luminescence spectra of luminol. OPV is cationic oligo (p-phenylene vinylene) with unique light-harvesting like traditional conjugated polymers51-54 and optical signal amplification capabilities.55-58 Under alkaline conditions, luminol is oxidized in the presence of hydrogen peroxide and HRP to form a negatively charged luminescent intermediate.47 When the luminescent intermediate returns to the ground state, it emits blue light and transfers the energy to the cationic OPV electrostatically bound to the luminescent intermediate. OPV is excited to sensitize oxygen in the surrounding to produce ROS, which can effectively kill electronegative cancer cells or pathogenic bacteria electrostatically bound to the cationic OPV. It is noted that this BRET system for photodynamic therapy exhibits excellent antitumor and antibacterial


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activity with distinct advantage of overcoming the light-penetration limitation without requiring the external light sources.59

Scheme 1. a) Schematic illustration of composite hydrogel encapsulating cells and BRET caused cell death in 3D hydrogel cube; b) chemical structure of OPV and PIC. As illustrated in Scheme 1, a new composite biomimetic hydrogel was designed and constructed by non-covalent assembly of cationic OPV and helical PIC. OPV and PIC can interact with each other via hydrophobic interactions and combine the advantages of them. Moreover, PIC can promote the absorption capacity of OPV and lead to an increase of the fluorescence emission intensity of OPV at 550 nm. The presence of PIC enhances its ability to produce ROS in the BRET system when luminol is chosen as the donor and OPV is chosen as the receptor. In addition, PIC produces little effect on the electrostatic binding of cationic OPV with cells and OPV does not affect the gelation temperature and the excellent thermo-mechanical


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properties of PIC as well. A 3D network was constructed to mimic the extracellular matrix, which commendably realizes 3D culture of cells in a 3D composite biomimetic hydrogel (OPVPIC) system and facilitates cell growth and proliferation. Under alkaline conditions with HPR and H2O2, utilizing the energy of bioluminescence within the biological system transferred to OPV to occur a strong BRET process and effective ROS generation, cancer cells in 3D culture environment and pathogenic microorganism were effectively killed or damaged. Furthermore, the killing effect of the BRET system with PIC is more effective than that without PIC. The achievement of BRET process occurring in 3D culture to cause the cancer cell death is rarely reported, which starts a new point for treatment of tumor and pathogen infections. RESULTS AND DISCUSSION OPV-PIC composites were prepared by physical mixing OPV with PIC. In order to prove the interaction between OPV and PIC, we performed dynamic light scattering (DLS) experiments to measure the changes of particle sizes of OPV in the presence of different concentrations of PIC in aqueous solution. As shown in Figure 1a, the average particle sizes of OPV-PIC composites increased gradually from 255 nm to 531 nm with the increase of PIC concentration. In addition, to further prove the interaction between OPV and PIC, the absorption and fluorescence spectra of OPV with different concentrations of PIC were measured. The absorption (Figure 1b) and fluorescence intensity (Figure S1b) of OPV also increased as the concentration of PIC increased and the increase of fluorescence intensity was illustrated obviously when the concentration of PIC reached to 1.0 mg mL−1, indicating that the presence of PIC can promote the absorption capacity of OPV and lead to an increase of the fluorescence emission intensity of OPV at 550 nm. Zeta potentials in Figure S1a indicated that the addition of PIC to OPV produces little influence


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on the zeta potential of OPV owing to the electroneutral property of PIC. These results indicate that OPV can interact with PIC via hydrophobic interactions. The thermo-mechanical properties of composite hydrogels were measured by rheological analysis. PIC has been proved to have temperature sensitivity and the PIC aqueous solution gradually changes from a solution state to a gel state as gelation temperature is over 18 °C.25 Figure 1c shows temperature sweeps of the storage loss Gʹ as a function of the temperature. The sharp transitions of PIC marked the formation of gels and no obvious changes were observed for the gelation temperature of the composite hydrogels with a stable value of 18 °C, which indicates that the presence of OPV does not affect the gelation temperature of PIC. PIC is stress-stiffening and nonlinearly stress responsive that is the strength of the PIC itself increases as the external stress increases.21,

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To accurately study the mechanical response behavior, the differential

modulus K' (K' = ∂σ/∂γ) were calculated. Figure 1d shows the differential modulus K' as a function of applied stress and no obvious changes of the differential modulus K' were obtained with the increase of OPV concentration. These results demonstrate that the addition of OPV does not influence the hardness of PIC in the linear region, the stress-stiffening properties of PIC in the nonlinear region and the gelation temperature of PIC. To study the influence on PIC structure after the addition of OPV (Figure S2). The PIC hydrogel and composite hydrogel (OPV-PIC) show 3D porous structure, which means OPV does not influence the structure of PIC hydrogel.


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Figure 1. DLS analysis (a) and the absorption spectra (b) of OPV in the presence of different concentrations of PIC. [OPV] = 20.0 μM, [PIC] = 0-1.0 mg mL−1; Storage modulus Gʹ as a function of the temperature (c) and differential modulus Kʹ (Kʹ = ∂σ/∂γ) as a function of stress σ (d) for composite hydrogels. [PIC] = 2.0 mg mL−1, [OPV] = 0-10.0 μM. Wang et al. have reported that the positively charged OPV can bind to negatively charged cell membrane and there is no significant change in the zeta potential measurement of HeLa cells before and after the addition of OPV for the reason that high charge density and linearity of OPV might lead to OPV insert into the phospholipid bilayer of the cell membrane.59 To verify that the presence of PIC does not affect the interaction of OPV with cells (HeLa cells and NIH-3T3 cells), Zeta potential characterization was performed. As shown in Figure S1a, the addition of PIC did not cause significant changes on the zeta potential of cells because of the electrical neutrality of PIC. It can be concluded that the presence of PIC does not affect the interaction of OPV with cells. To study the growth of cells in biomimetic composite hydrogels (OPV-PIC), we


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encapsulated NIH-3T3 cells into 3D composite hydrogel constructs for 36 h of culture. To directly observe the growing state of NIH-3T3 cells in 3D culture, confocal laser scanning microscopy (CLSM) was used. NIH-3T3 cells remained viable, typical fibroblasts-likely spindle morphology and stretched (Figure 2a). The nucleus of NIH-3T3 cells shows no significant changes in morphology and keeps oval (Figure 2b), indicating that NIH-3T3 cells can grow well in the three-dimensional environment of the composite hydrogels. In order to directly visualize that OPV can still bind to cells in 3D composite hydrogel constructs, NIH-3T3 cells were cultured in 3D composite hydrogels. The bright field and the fluorescence field images were captured by CLSM. As shown in Figure 2c-d and Figure S3, the surface of the cell membrane exhibits green fluorescence revealing that NIH-3T3 cells were bound to OPV in 3D composite hydrogel constructs. As for the three-dimensional image of the Z-axis scanning, the NIH-3T3 cells are evenly dispersed in 3D composite hydrogels. As control, NIH-3T3 cells treated with OPV were cultured in 2D planar culture (Figure S3a-d). As shown in Figure S3d, the threedimensional image of 2D planar culture demonstrates that all the NIH-3T3 cells grow on the same plane.


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Figure 2. a-d) CLSM images of NIH-3T3 cells after culturing in 3D composite hydrogel constructs for 36 h. [OPV] = 5.0 μM, [PIC] = 1.5 mg mL−1; e) Proliferation rate of NIH-3T3 cells in 3D hydrogel constructs. [OPV] = 5.0 μM, [PIC] = 1.0 mg mL−1. On the basis of 3D culture conditions, cell proliferation in 3D composite hydrogel was determined utilizing the CCK-8 kit. The absorbance at 450 nm was measured and the obtained values were normalized to 0 day. As the culture time prolonged, when the NIH-3T3 cells were cultured for 96 h in the presence of OPV-PIC composite hydrogel, the cells proliferated 2.3 fold relative to 0 h under 3D conditions (Figure 2e and Figure S4), indicating that the 3D biomimetic polymer network constructed by water-soluble cationic OPV with biocompatible PIC is conducive to cell growth and proliferation. The 3D biomimetic network simulated and controlled the real microenvironment of cell growth, providing the basis for studying the mechanical response behavior of cells, revealing the processes of carcinogenesis and exploring efficient antibacterial and anti-tumor methods. Cell viability was directly visualized by confocal laser scanning microscopy (Figure 3) using the CellTrace calcein red-orange AM to staining live cells with red-orange. It is noted that OPV can bind to the cell membrane and exhibits green fluorescence using a 488 nm laser. After encapsulation NIH-3T3 cells into 3D composite biomimetic hydrogel system for 36 h, OPV-bound cells almost coincided with the cells stained with living cell fluorescent dye indicating that NIH-3T3 cells exhibit excellent cell viability in the 3D composite hydrogels.


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Figure 3. a-f) CLSM images of NIH-3T3 cells after encapsulation into 3D composite hydrogel constructs for 36 h of culture. Fluorescence images under the excitation of 488 nm (a, d), 543 nm (b, e) and overlap images (c, f). [OPV] = 5.0 μM, [PIC] = 1.5 mg mL−1. It is reported that the excited OPV can sensitize oxygen molecule in the surroundings to generate reactive oxygen species (ROS).55 To further verify that the effect of PIC on the ROS production by OPV sensitization, 2, 7-dichlorofluorescein diacetate (DCFH-DA) was utilized. The activated DCFH can be oxidized to 2, 7-dichlorofluorescein (DCF) in the presence of ROS and DCF has a distinct characteristic fluorescence emission peak at 525 nm.60 After irradiation of OPV with DCFH under white light for 0-4 min, emission peak at 525 nm can be detected in all samples with excitation wavelength of 488 nm. As shown in Figure 4a and Figure S5, the fluorescence intensity at 525 nm increased as the irradiation time of white light goes on, while the control groups without OPV all show very weak fluorescence emission. Surprisingly, the fluorescence intensities at 525 nm have a slight enhancement with the increase of PIC concentration (Figure 4b), which indicates that the presence of PIC can enhance the ability of OPV to sensitize oxygen molecule to produce ROS.


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It is noted that the absorption spectra of OPV in aqueous solution ranges from 350 to 550 nm and the bioluminescence of luminol exhibits a maximum emission peak at 425 nm under alkaline conditions in the presence of hydrogen peroxide and horseradish peroxidase (HRP) (Figure S6a), which meets the condition of the bioluminescence resonance energy transfer (BRET) as the donor−acceptor pair owing to the overlap of absorption spectra of OPV and luminescence spectra of luminol. Furthermore, PIC has no absorption peak from 350 nm to 550 nm so that the presence of PIC does not affect the BRET process. It is reported that the luminol emits blue light in the presence of hydrogen peroxide and horseradish peroxidase (HRP) under alkaline conditions when the negatively charged luminescent intermediate returns to the ground state and transfers the energy to the cationic intermediate OPV. The BRET process occurs and OPV is excited to sensitize oxygen in the surrounding to produce ROS.59 To investigate the effect of PIC

Figure 4. The fluorescence spectra (a) and the fluorescence intensity at 525 nm (b) of DCFH under white light irradiation. [OPV] = 1.0 μM, [DCFH] = 40.0 μM, [PIC] = 0-1.0 mg mL−1; The


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luminescence spectra of luminol (c) and BRET ratio (I550nm/I425nm) (d) with successive addition of OPV. [OPV] = 0-50.0 μM, [HRP] = 0.003 mg mL−1, [luminol] = 0.2 mM, [4-iodophenol] = 0.5 mM, [H2O2] = 0.5 mM, [PIC] = 1.0 mg mL−1. in the BRET process, we measured the luminescence of luminol under alkaline condition with the enzyme and substrate in the presence of different concentrations of OPV before and after the addition of PIC (Figure 4c and Figure S6b). As expected, effective BRET processes can be detected before and after the addition of PIC. As the concentration of OPV increases (0−50.0 μM), the luminescence intensity of luminol at 425 nm gradually decreases, while the fluorescence intensity of OPV at 550 nm gradually increases. To further directly investigate the effect of PIC in the BRET process, we calculated the ratio of fluorescence emission intensities at 550 nm versus 425 nm (I550nm/I425nm). As shown in Figure 4d, upon the addition of PIC, the ratio increased dramatically with the increase of OPV concentration. With the concentration of PIC increases, more obvious enhancement of the ratio (I550nm/I425nm) was observed, which suggests the addition of PIC can slightly promote the BRET process. These results consist with that of the ability of PIC to promote ROS production. These results of ROS and BRET measurements confirm the outstanding advantages of the composite biomimetic hydrogel system. To demonstrate that anti-cancer process can be achieved in a three-dimensional environment of composite hydrogels, the BRET process caused cells apoptosis was visualized using confocal laser scanning microscopy by Z-axis scanning with the scan height of 300 µm. BRET process from luminol to OPV caused the ROS generation, leading to cell death. HeLa cells were encapsulated into 3D composite hydrogel constructs for 24 h of culture. Upon the addition of luminol luminescence system (enzyme and substrate, abbreviated to E+S) and further culture for 0-24 h, the late apoptotic or dead cells were stained by EB using a 543 nm laser. As illustrated in


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Figure 5a, the quantity of ROS caused dead or late apoptotic cells were gradually increased with the increase of the further culture time after the addition of E+S and almost all the cells were dead after 24 h of further culture. The results indicated that the BRET in 3D composite hydrogel constructs occurred effectively and ROS production in the BRET significantly damaged and killed cancer cells in a three-dimensional environment. To quantitatively investigate the anti-cancer ability in 3D culture, MTT assay was carried out. As shown in Figure 5b, the OPV and OPV-PIC composite hydrogels alone show extremely low toxicity to HeLa cells while the BRET system in the presence of PIC displays enormous cytotoxicity and the cell viability decreases as the concentration of OPV increases. Moreover, the addition of PIC with the BRET system shows more obvious cell killing rate than that without PIC. Among the experimental samples, the BRET system in 3D composite hydrogel constructs shows the strongest cytotoxicity with the cell viability under 10%, while PIC alone exhibits excellent biocompatibility.24 These results consist with the PIC promoting effect in the ROS generation and BRET ability, for which we suppose that PIC hydrogels keep the ROS fixed in the hydrogels. However, unfortunately, this system is not selective for tumor cells and normal cells due to the lack of groups that specifically target the cancer cells. The BRET system in 3D composite hydrogel constructs can also kill the NIH-3T3 cells effectively (Figure S7). To the best of our knowledge, the BRET caused cell death in 3D culture is extremely rare.


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Figure 5. a) Three-dimensional view of CLSM images of HeLa cells apoptosis. [OPV] = 5.0 μM, [HRP] = 0.01 mg mL−1, [luminol] = 0.5 mM, [4-iodophenol] = 1.25 mM, [H2O2] = 0.4 mM, [PIC] = 1.5 mg mL−1; b) Cell viability of HeLa cells. [OPV] = 0-8.0 μM, [HRP] = 0.01 mg mL−1, [luminol] = 0.5 mM, [4-iodophenol] = 1.25 mM, [H2O2] = 0.4 mM, [PIC] = 1.0 mg mL−1. To show that the BRET system in the composite hydrogels can apply to the pathogenic microorganism as well, inhibition zone experiments were performed. The pathogenic microorganism includes the therapeutic fungi and bacteria, which is one of the important causes of the spread of infectious diseases. Fungi have been identified as major pathogens associated with critically ill patients for fatal infections and pathogenic bacteria are one of the important reasons for the increase of mortality and morbidity among patients with low immune function. We selected the representative gram-negative bacteria E. coli, the gram-positive bacteria B. subtilis and pathogenic fungi C. albicans as the experimental samples.61 As shown in Figure 6,


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the BRET system in the composite hydrogels (PIC+OPV+E+S) exhibits excellent antimicrobial activities with the extremely low concentration of OPV under 1.0 μM and the corresponding areas of inhibition zone continue to increase as the concentration of OPV in the composite hydrogel system (PIC+OPV+E+S) increases. As expected, comparing the BRET system (OPV+E+S) with the BRET system in the composite hydrogels (PIC+OPV+E+S), the presence of PIC in the BRET system displays more obvious antimicrobial activity for that the areas of inhibition zone exhibit slightly larger than that of BRET system (OPV+E+S) which is in accordance with the results that the presence of PIC promotes the BRET process and enhances the ability of ROS producing. It is reported that Fungi have stronger antimicrobial resistance than bacteria62 and the results we obtained coincide with that. As control group, all samples on the spread plate of the pathogenic microorganism show inconspicuous antimicrobial activities (Figure S8). These results indicate that the luminol luminescence system in the composite hydrogels can produce an effective BRET process causing OPV to sensitize the oxygen molecules in the surrounding environment to produce ROS, which has obvious antifungal and bacterial activities.


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Figure 6. Antimicrobial activities of PIC hydrogels and OPV with different concentrations on spread plate. [OPV] = 0.1-1.0 μM, [HRP] = 0.004 mg mL−1, [luminol] = 0.2 mM, [4-iodophenol] = 0.5 mM, [H2O2] = 0.5 mM, [PIC] = 0-1.0 mg mL−1. CONCLUSION In summary, a kind of new biomimetic composite hydrogel has been prepared and constructed by assembling PIC with OPV. OPV interacts with PIC via hydrophobic interactions and produces little effect on the gelation temperature and the excellent thermo-mechanical properties of PIC. Moreover, PIC promotes the ability of OPV to sensitize oxygen molecule to produce ROS and enhance BRET efficiency, while PIC does not influence the electrostatic binding of cationic OPV with both NIH-3T3 cells and HeLa cells. The polymer chains of PIC are bound and frizzled to form hydrogels with porous 3D network when the temperature rises above the gelation temperature, mimicing the extracellular matrix and the real microenvironment of cell growth. NIH-3T3 cells were electrostatically bound to OPV and evenly dispersed in 3D composite hydrogels. Furthermore, encapsulation of HeLa cells into the 3D porous composite


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hydrogels to mimic the real situation of cell canceration, the addition of the luminol luminescence system can cause the strong BRET process to kill the cancer cells (HeLa cells) with the killing rate above 90%. Notably, the BRET system to the composite hydrogel achieves more efficient antibacterial effect than that without PIC. This application takes advantages of the efficient BRET efficiency of OPV and the stress-stiffness effect of PIC, providing the starting point to utilize the BRET strat in 3D composite biomimetic hydrogels for exploring efficient antimicrobial and anti-cancer strategies. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Additional information was noted in the supporting information, including details of materials, instruments, experimental procedures. The followings are also presented in the supporting information: zeta potentials data of HeLa cells and NIH-3T3 cells with and without OPV and PIC; fluorescence curve of OPV under different concentrations PIC; SEM images of PIC hydrogel and OPV-PIC hydrogel; CLSM images of NIH-3T3 cells with OPV in 2D planar and 3D culture; curves of proliferation rate in 2D planar culture and cell viability of NIH-3T3 cells; fluorescence curves of DCFH under white light irradiation with different concentrations of PIC; normalized absorption spectra of OPV, PIC and luminescence spectra of luminol; luminescence spectra of luminol with successive addition of OPV; antimicrobial activities of control groups. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (C. X.).


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Author Contributions C.X. designed experiments and organized the manuscript; J.Q. performed experimental part and analyzed data; H.Y. and R.C. performed experimental part; Y.Z. analyzed data. Funding Sources National Natural Science Foundation of China (No. 21574037, No. 21773054 and No. 51803046), the “100 Talents” Program of Hebei Province, China (No. E2014100004), the Natural Science Foundation of Hebei Province (No. B2017202051), the Program for Top 100 Innovative Talents in Colleges and Universities of Hebei Province (No. SLRC2017028). Notes The authors declare no conflict of interest. ACKNOWLEDGMENT The authors are gratefully acknowledged for Prof. L. B. Liu from Key Laboratory of Organic Solids, Institute of Chemistry Chinese Academy of Sciences (Beijing, China) and Dr. H. X. Yuan from school of science, Beijing Technology and Business University. The authors are also grateful for the financial support of the National Natural Science Foundation of China (No. 21574037, No. 21773054 and No. 51803046), the “100 Talents” Program of Hebei Province, China (No. E2014100004), the Natural Science Foundation of Hebei Province (No. B2017202051), the Program for Top 100 Innovative Talents in Colleges and Universities of Hebei Province (No. SLRC2017028). REFERENCES


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