Photosensitizer Loaded Multifunctional Chitosan Nanoparticles for

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Photosensitizer Loaded Multifunctional Chitosan Nanoparticles for Simultaneously in Situ Imaging, High Efficient Bacterial Biofilms Eradication and Tumor Ablation Lin Sun, Wenya Jiang, Hengrui Zhang, Yishun Guo, Wei chen, Yingying Jin, Hao Chen, Kanghui Du, Hangdong Dai, Jian Ji, and Bailiang Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19522 • Publication Date (Web): 24 Dec 2018 Downloaded from http://pubs.acs.org on December 24, 2018

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ACS Applied Materials & Interfaces

Photosensitizer Loaded Multifunctional Chitosan Nanoparticles for Simultaneously in Situ Imaging, High Efficient Bacterial Biofilms Eradication and Tumor Ablation Lin Suna,#, Wenya Jianga,#, Hengrui Zhanga, b, Yishun Guoa,Wei Chena, b, Yingying Jina, Hao Chena, b*, Kanghui Dua, Hangdong Daia, Jian Jic, and Bailiang Wanga, b* aSchool

of Ophthalmology & Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, 325027, China bWenzhou Institute of Biomaterials and Engineering, Chinese Academy of Sciences, Wenzhou, 32500, China cMOE Key Laboratory of Macromolecule Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China * Corresponding authors. Fax: +86 577 88017524. E-mail: [email protected] (H. Chen); [email protected] (B. L. Wang) # These authors contributed equally to this work and should be considered co-first authors. ABSTRACT In recent decades, bacterial, viral infections and chronic inflammatory response have emerged as important causes of cancer. Also, infections remain a significant cause of morbidity and mortality in cancer patients. In this work, carboxymethyl chitosan nanoparticles (CMC NPs) were synthesized in a facile and green way, and further combined with ammonium methylbenzene blue (MB) as crosslinking agent as well as fluorescent molecules and photosensitizer for self-imaging photodynamic therapy (PDT). The obtained CMC-MB NPs exhibited an apparent pH responsive release behavior of MB, which was released for a prolonged period in simulated physiological environment (pH 7.4) for more than 15 d and the time reduced to only 3.5 h in acidic conditions (pH 5.5). When irradiated by 650 nm laser at 202 mW/cm2 for 5 min, the CMC-MB NPs showed efficient bactericidal and biofilm eradication properties as well as suppression of the tumor cells growth in the similar acidified microenvironment. Furthermore, in in vivo rabbit wound bacterial infection model, the rapid sterilization of CMC-MB NPs played a crucial role in bacterial infections, inflammation inhibition and wound healing. As a PDT treatment against cancer, the CMC-MB NPs also exhibited an efficient anti-tumor therapeutic effect in subcutaneous tumor mice model. Keywords: photodynamic therapy; bacterial infections; biofilms; cancer; in vivo

1. Introduction Bacterial infections and cancer are both greatly life threatening owing to the increasing morbidity and mortality1-2. More importantly, the problem will be trickier and out of control once both of them are intertwined3. On one hand, long-term exposure to infections and chronic inflammatory response can easily lead to the incidence of cancer. 16.1% of newly diagnosed cancers are estimated to be attributable to infections4. For those suffering with liver, colon or stomach cancer,

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one of the highest risks that patients face is the infections and chronic inflammatory response. Generally caused by viral or bacterial infection, this kind of problem can cause otherwise healthy cells to turn cancerous, and make the disease more aggressive as cancer grows5. On the other hand, the development of infections put cancer patients at greater risk6. One of the most common and potentially life-threatening side effects of chemotherapy is neutropenia and the subsequent development of infections7. The Centers for Disease Control and Prevention (CDC) estimates that about 60,000 cancer patients are hospitalized with infections every year in the United States8-9. Fungal and mycobacterial infections after hematopoietic stem-cell transplantation, bleeding events or life-threatening infection manifesting as febrile neutropenia and sepsis are examples of cancer therapy-related infections. Moreover, to the needs for eradicating the infection is crucial before another cycle of chemotherapy, radiation therapy or any surgical procedures can be performed. Preventing or eradicating infection is vital to continue their cancer-targeted therapy10. As a result, the simultaneous treatment of both bacterial infections and cancer is of great advantage. Patients with late stage tumors and compromised immune function are susceptible to a wide variety of infections11. In addition, it is known that the combination of chemotherapeutic drugs and antibiotics greatly influences the efficacy of chemotherapy drugs and even produces toxic effects12. For example the antibiotic chloramphenicol inhibits o-phenylendiamine in the body. o-Phenylendiamine has the therapeutic effect of reducing reactive metabolites, therefore the anti-cancer effect is reduced13. These challenges make it essential to find a facile way to reduce the risk of infection during tumor treatment. Bacterial infections are the main cause of many diseases such as delayed wound healing, periodontitis, endophthalmitis, bacteremia and even cancer14-18. Drug-resistant bacteria and bacterial biofilms are in particular tricky problems in clinical therapy19-20. Especially, people with immune deficiency such as tumor patients during treatment are more prone to serious infections. The currently treatment for bacterial infection is antibiotics injection. However the overuse of antibiotics has led to the emergence of drug-resistant bacteria21-23. A 2014 report by the World Health Organization on the global surveillance of antibiotic resistance stated clearly that, “antibiotic resistance is no longer a prediction for the future, it is happening right now, across the world, and is putting at risk the ability to treat common infections in the community and hospitals”24-26. This emergence of drug-resistant bacteria makes the exploration and development of new antibiotics urgent27-28. However, the rate of drug-resistant bacteria development is much fast than the development of new antibiotics. Various open treatments in hospitals (urinary and central venous catheters) are common sources of bacterial infection29-30, with bacteria easily attaching to the surface of tubing and other implants leading to biofilm formation31. Biofilms have strong resistance to antibiotics, as antibiotics cannot penetrate the biofilm32-34. Currently, the main method of biofilm treatment is to remove the suture or graft from the body, which needs a secondary surgery and implantation. As a result, it is necessary to find other ways to efficiently prevent the formation or eliminate the already existing biofilms35-36. Our laboratory been focused on the study of smart drug delivery systems responsive to bacterial infections microenvironment34, 37, degradable anti-adhesive coatings and reversibly switchable antibacterial surface with bactericidal and antifouling properties38-39. Although these antibiotics delivery systems for targeted and local drug delivery showed delayed the development of drug-resistant bacteria and improved the therapeutic effect, it is not the wise way to fundamentally solve the problem at all. Researchers devoted great efforts in finding other efficient


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antibacterial agents alternative to antibiotics to treat resistant bacteria and biofilms related infections40-41. Antimicrobial agents such as quaternary ammonium salts, metal ions in particular silver ions42-43, antimicrobial peptides and other novel treatment fashion such as photodynamic therapy (PDT) and photothermal therapy (PTT) etc have drawn more and more attentions44. However, the in vivo use of metal ions can lead to their accumulation in the body, which caused serious toxicity, such as hepatotoxicity and kidney toxicity. Quaternary ammonium salts constructed on biomaterial surfaces often lead to obvious cytotoxicity to mammalian cells45. Antimicrobial peptides have a broad-spectrum bactericidal effect and are able to work on multiple targets in the bacteria46. Owing to the limited natural sources of antimicrobial peptides, chemical synthesis and genetic engineering are the main routes to antimicrobial peptides synthesis, which are too expensive for widely application. In comparison, PDT and PTT methods exhibited superior treatment effect owing to the human controllability and inhibition of drug-resistant bacteria development47. Photosensitizers (PS) loaded in nanoparticles (NPs) are known as the third generation PS which is mainly used in photodynamic therapy (PDT). PS-loaded NPs can be designed with smart characteristics for controlled and responsive release features for precise treatment. PDT basically needs to include three components: photosensitizer, laser irradiation at the certain wavelength, and molecular oxygen. Under irradiation, PS can produce molecular oxygen and other reactive oxygen species (ROS)48, which can induce autophagy, apoptosis, and necrosis of both bacteria and tumor cells49-50. Compared with conventional antibiotics, ROS work through a multi-targeted mechanism, which makes it less possible to develop bacterial resistance. PDT has many advantages such as small trauma, less adverse reactions, lower toxicity, curative effect and no development of drug resistance. As the first generation PS, hematoporphyrin derivatives such as dihematoporphyrin persist in vivo for a long time so that light should be avoided for more than 4 weeks. The second generation PS with shorter light sensitive period, wider wavelength range, deeper effective penetration depth and more singlet oxygen partly overcome the drawbacks of the first generation51. However, highly concentrated local PS causes cytotoxicity, which gave rise to the third generation of PS NPs. Furthermore, bacterial infections and tumor site have the similar microenvironment, such as lower pH, highly concentrated ROS, hypoxia and high expression of specific enzymes34. 37. As a result, it is very convenient to delivery functional drug into the pathological sites for both bacterial infections and cancer treatment. In this work, carboxymethyl chitosan nanoparticles (CMC NPs) were synthesized in a facile and green way with or without genipin (GNP) as crosslinking agent to load ammonium methylbenzene blue (MB) as PS as well as crosslinking agent (Scheme 1). The release behavior of the loaded PS in CMC NPs will be explored both in simulated body fluid environment (pH 7.4) and in weakly acidic medium (pH 5.5). Antibacterial properties will be investigated against four types of bacteria Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and methicillin-resistant Staphylococcus aureus (MRSA). Both bacteria live/dead kit and scanning electron microscope (SEM) methods were used to verify the anti-biofilm functions of PDT. The cellular biocompatibility of CMC-MB NPs will be determined against mouse fibroblast cells (L-929) and adriamycin-resistant human breast cancer cells (MCF-7/ADR), respectively. Rabbit wound bacterial infection model and subcutaneous tumor mice model also will be used to verify the in vivo antibacterial and antitumor properties.



Scheme 1. Illustration of CMC-MB NPs synthesis for bacterial biofilms eradication and tumor ablation multifunction.

2 Results and discussions 2.1 Bactericidal and cellular biocompatibility of MB As a common second generation PS, MB was combined with nano-carrier into the third generation PS CMC-MB NPs for controlled release2. Firstly, the standard curve line of MB absorption against concentration was measured at 630 nm by UV-vis spectrophotometry (Figure S1). The absorbance increases gradually and linearly with increasing concentration. Cytocompatibility is a primary consideration for the application of biofunctional materials20. The cytotoxicity of free MB was determined through incubation different concentrations of MB with L929 cells (Figure 1A). It was found that MB showed good cell compatibility when the concentration was lower than 2 μg/mL under 202 mW/cm2 for 5 min. The cell compatibility greatly decreased as the increase of MB concentration. Therefore, the concentration of MB at 2 μg/mL was chosen for preliminary bactericidal activity test against S. aureus. The effects of laser intensity and laser irradiation time on bacteria killing efficacy were also explored using S. aureus as bacterial model, the most common pathogenic bacteria in implant-related infections, wound infections and bacteremia40-41, 52 (Figure 1B, C). As indicated in Figure 1B, neither laser irradiation nor MB alone displayed nearly any bacteria killing effect comparing with the control without any treatment. However, the PDT showed excellent bacterial eradication function laser irradiation intensity was higher than 202 mW/cm2 with the concentration of MB at 2 μg/mL and the laser irradiation time at 10 min. The influence of laser irradiation time on bacteria killing property was also tested at under laser irradiation intensity of 202 mW/cm2 and 2 μg/mL of MB against E. coli, S. aureus and MRSA (Figure 1C). A totally 100% percent of Gram positive S. aureus and MRSA was killed only after 5 min irradiation. As for Gram negative E. coli, almost 95% of the bacteria were eliminated after laser irradiation. The difference in bactericidal efficiency can be attributed to the difference of cell wall structure of two kinds of bacteria. Compared to Gram-positive S. aureus, Gram-negative E. coli have an additional lipopolysaccharide (LPS)-containing membrane as the outmost layer24, 52. This creates a secondary LPS barrier that further prevents the ROS from lysing E. coli44.


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Figure 1 A: The CCK-8 viability test of L929 cells cultured with the different concentrations of MB measured by microplate reader at 450 nm compared with TCPS at 100%; B: The effect of laser irradiation intensity on S. aureus eradication through counting the number of colonies after coating on agar plate; C: Effect of laser irradiation time on antibacterial against S. aureus (106 CFU/mL, laser: 202 mW/cm2, MB: 2 μg/mL) through plate counting method.

2.2 Synthesis and characterization of CMC-MB NPs In this work, spherical CMC nanoparticles were synthesized through reverse phase microemulsion method with TritonX-100 as surfactant, n-octanol as co-surfactant, cyclohexane as oil phase and water as aqueous phase. Two kinds of CMC-MB NPs were synthesized with MB or MB/GNP as the crosslinking agents. It is usually known that crosslinking agents such as glutaraldehyde or GNP should be used in the formation of chitosan NPs. In our work, it was surprising to find that CMC-MB NPs formed without adding any crosslinking agents. It was found that CMC and MB reacted immediately after mixing and formed into dark blue solid that was poorly soluble in water. Therefore, we explored the effect of MB concentration on the synthesis of CMC-MB NPs. It was found that with increasing MB concentration, the color of the products deepened and the quality of NPs gradually increased (Figure S2). The obtained NPs were characterized using transmission electron microscope (TEM) and SEM and the results showed the formation of uniform NPs which were distributed between 40 and 60 nm (Figure 2B, B’ and Figure S3). Based on the low solubility of MB, a concentration of 10 mg/mL was chosen to form CMC-MB NPs (Figure 2E). The crosslinking property of MB in this system might be attributed to the electrostatic interactions, hydrogen bonding and hydrophobic interaction between CMC and MB1, 53. The structure of the NPs was stable at the pH 7.4. However, a small amount of white precipitate in the solution at pH 6.0, which indicated the change in charge stability which led to the NPs aggregation (Figure 2D). When the feeding concentration of MB increased from 1 mg/mL to 5 mg/mL and 10 mg/mL, the MB encapsulation efficiency changed from 98.6±0.4% to 92.2±0.5% and 87.9±0.8% respectively.



The loading capacity of MB into CMC-MB NPs was 0.35, 1.75 and 3.5 wt%, respectively. Then, the release behavior of MB from the CMC-MB NPs without GNP crosslinking showed fast release in 24 h in PBS (Figure 3B). In order to control the MB release in a sustained way, extra 1 mmol/mL GNP as crosslinking agent was added during CMC-MB NPs synthesis. The particle size and surface charge property were also examined through dynamic light scattering (DLS) and TEM. As showed in Figure 2A, A’, C, after adding GNP the particle size increased much bigger than that of CMC-MB NPs without GNP. As examined by DLS, the diameter was approximately 200 nm which was greatly bigger than that measured by TEM. The obvious size difference of NPs tested by TEM and DLS could be due to the dry and wet test environments of TEM and DLS, respectively. It also could be observed that the CMC-MB NPs have high swelling performance in aqueous environment as the diameter increased approximately four times. Both DLS and TEM characterizations proved that the uniform NPs successfully formed through the facile reverse phase microemulsion method.

Figure 2 A, A’: TEM measurement of the particle size and distribution of CMC-MB NPs with additional GNP as crosslinking agent; B, B’: TEM measurement of the particle size and distribution of CMC-MB NPs without additional GNP; C: DLS measurement of the particle size and distribution of CMC-MB NPs with and without additional GNP as crosslinking agent; D: White precipitation of CMC-MB NPs without additional GNP in the solution at pH 6.0; E: The photograph of CMC-MB NPs formation without additional GNP as the increase of MB concentration.

2.3 The release of MB from CMC-MB NPs The microenvironment of bacterial infections and tumor shared some important similarity such as weak acidification, high expression of various enzymes and the host's own serious immune response54-55. PH is an important stimulus for antibacterial coatings construction, since many bacteria metabolically acidify their local environment. In particular, different bacteria produce different acidic substances such as lactic or acetic acid. The abnormal microenvironments provide us ideas to design and construct microenvironment responsive drug delivery systems for targeted and local disease treatment56-57. In this way, the similar microenvironment of cancer and infections provided us opportunity to solve the problems through “one stone for two birds”. To the best of our knowledge, seldom works have been reported about the serious problem of mutual


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reinforcement or coordination between cancer and infections. The simultaneous treatment options may provide a new perspective and solution to the problem. The first issue need to be dealt with is the controlled release of the drug from the NPs. Chitosan is commonly used in drug delivery systems due to the pH responsiveness38. As shown in Figure 3A, B, the CMC-MB NPs crosslinked by GNP showed very slow release of MB from the NPs at pH 7.4, which indicated the stable interactions between MB and CMC. MB sustained for 15 d due to the improved stability and was suitable for long term treatment. However, at pH 5.5, the release of MB greatly increased from the NPs within 3 h. There are reasons contributing to the obvious pH responsive MB release behavior. The first reason should be owing to the presence of amino and carboxyl groups in CMC molecules, which are the common and well known pH responsive groups. The transition between protonation and deprotonation of amino and carboxyl groups greatly influenced hydration behavior and the (interand intra-) molecular interactions of CMC in the matrices58. Amino and carboxyl groups protonated at lower pH and thus had an extended and opened structure, which caused the collapse of the polymeric structure due to instantaneous deprotonation of CMC chains, resulting in expulsion of drug molecules59. As showed in Figure 3A, B, it was found that at pH 5.5 the particle size increased to approximately twice the original size at pH 7.4, and the particle size could not be measured at pH 4 indicating the total dissolution of NPs. As a result, the changed permeability of CMC-MB NPs enhanced the MB diffusion from the matrices. Another important reason should be the high swelling ability of the NPs as indicated in the size difference measured by TEM and DLS (Figure 2). The high water content in the NPs provided sufficient diffusion channels for MB60. The CMC molecular chain was in a diastolic state at such low pH, which was the reason for high water content. The third reason should be attributed to the weakening of interactions between CMC and MB, which led to the disassembly of the CMC-MB NPs. At pH 6, a small amount of white precipitate began to form in the solution and the amount of white precipitate gradually increased as the gradual decrease of pH (Figure 2D). Therefore, the NPs began the release of the loaded MB through disassembly in the gradual increased acidified environment. In addition, the surface characteristics of the NPs were studied by zeta potential measurements (Figure 3D). The zeta potential of the spherical CMC-MB NPs was close to zero at pH 6.0 which had more negative charges as pH increased. As the main component of CMC-MB NPs was CMC, the pKa of the NPs was also close to that of CMC. We also tested the stability of CMC-MB NPs at different pH by tracking the intensity changes in DLS. As showed in Figure S4, the CMC-MB NPs with GNP crosslinking mainly maintained the size after release of MB as showed in Figure 3A and 3B in the first 8 h at both pH 5.5 and 7.5. However, the CMC-MB NPs without GNP crosslinking at pH 5.5 greatly decomposed in the first 2 h and totally degraded after 8 h. As for the CMC-MB NPs without GNP crosslinking at pH 7.5, the size well maintained indicating the highly pH responsiveness of the CMC-MB NPs.



Figure 3 A: Release curves of MB from CMC-MB NPs with and without additional GNP as crosslinking agent at pH 5.5 and 7.4 for 360 h; B: Release curves of MB from CMC-MB NPs with and without additional GNP as crosslinking agent at pH 5.5 and 7.4 for 24 h; C: Particle size changes measured by DLS at various pH values; D: Zeta potential changes of CMC-MB NPs measured by DLS at various pH values.

2.4 In situ imaging of the MB The in situ imaging of bacterial infections was examined as the special self-fluorescence of MB. A commonly nanoscaled functional material for in situ imaging of disease needs additional grafted or loaded imaging probes. The cumbersome preparation process, toxicity, metabolism of fluorescent molecules and other problems are difficult to avoid61. As a result, it has great advantages for biomaterials with simultaneous function of in situ imaging as well as enhanced therapeutic efficiency. As showed in Figure S5, as the increase of MB concentration, red fluorescence was observed under the fluorescence microscope. When the concentration was lower than 1 μg/mL, only some of the cells could be observed. However, as the MB concentration increased into 2 μg/mL, almost all of the bacterial cells (MRSA, E. coli and P. aeruginosa) and MCF-7/ADR tumor cells could be excited with clear red fluorescence which indicated the good in situ imaging property of MB. As showed in Figure 4, all of the three kinds of living cells including L929 cells, S. aureus and MCF-7/ADR tumor cells were stained with remarkable red fluorescence without any extra dye after incubation with CMC-MB NPs. PDT has been used to diagnose and to treat disease through PS to produce reactive oxygen species (ROS) especially singlet oxygen under appropriate irradiation. These methods can be used to treat infectious diseases caused by multidrug-resistant bacteria and to delay the development of bacterial biofilms44. Absorbing photons of light, the PS molecule becomes activated from the ground state to a short-lived excited singlet state (1PS*)2. The excited PS can emit fluorescence and return to the ground state, which can be used for clinical imaging. As the number of MB molecules got into cells increased, the cytotoxic and antibacterial


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effect would increase. As a basic dye, the cations of quinone imine have a staining effect in combination with the acidic substance of tissue cells, which can be excited out fluorescence for in situ imaging62.

Figure 4 In situ imaging of L929 cells (A and D), MCF-7/ADR tumor cells (B and E) and S. aureus (C and F) stained with or without 10 mg/mL CMC-MB NPs for fluorescent images at 630 nm.

2.5 Antibacterial properties of the CMC-MB NPs 2.5.1 Bactericidal properties The bactericidal and biofilms eradication properties of CMC-MB NPs were explored in four strains of popular bacteria including Gram-positive S. aureus and MRSA and Gram-positive E. coli and P. aeruginosa. The antibacterial efficiency was characterized by plate counting, bacteria live/dead staining and scanning electron microscope (SEM). Bacteria solutions without any treatment, treated by 2 μg/mL CMC-MB NPs or laser irradiation alone were as parallel controls to examine the PDT effects. As indicated in Figure 5, 100% of S. aureus and MRSA were killed through PDT treatment which should the excellent bactericidal function of the CMC-MB NPs. What’s more, about 99% of the E. coli and 95% of the P. aeruginosa were sterilized suggesting that the two strains are with slightly higher resistance to MB PDT. However, no bacterial killing property was observed for the three control groups showing no bacterial killing property of MB or laser irradiation treatment alone. It is also important to know whether the efficient bactericidal properties could be due to the photodynamic effects of the encapsulated MB in CMC-MB NPs. We centrifuged and re-dispersed bacteria in different buffer at pH 7.4 PBS and pH 5.5 MES. The bacteria killing results showed almost the same efficiency against both S. aureus and E. coli even at a much lower CMC-MB NPs concentration (0.5 μg/mL). As a result, the bactericidal properties could be mainly attributed to the production of ROS from the NPs. However, fast release of MB from the NPs in the lower pH environment was also beneficial to the absorption of light and ROS production.



Figure 5: Antibacterial properties of CMC-MB NPs against S. aureus, E. coli, MRSA and P. aeruginosa as bacterial model through incubation and plate counting method (A: Bacterial colonies and B: Number of the baceria ). (bacterial concentration: 104/mL; Laser: 202 mW/cm2, 5 min); C: Measurement of intracellular levels of ROS with MRSA through Cellular Reactive Oxygen Species Detection Assay Kit (DCFH-DA). To explore the mechanism of efficient bactericidal property of CMC-MB NPs, Cellular Reactive Oxygen Species Detection Assay Kit (2',7'-Dichlorodihydrofluorescein diacetate, DCFH-DA) was used to measure the intracellular levels of ROS in MRSA. As shown in Figure 5C, only the bacteria treated by CMC-MB NPs combined with laser irradiation showed bright green fluorescent indicating the production of plenty of ROS which should accounted for the main reason for bacteria killing. Compared with conventional antibiotics, ROS work through a multi-targeted mechanism, which makes it less possible to develop bacterial resistance63-64. PDT has many advantages such as small trauma, less adverse reactions, lower toxicity, curative effect and no development of drug resistance. Besides activation from the ground state to a short-lived excited singlet state (1PS*) for imaging of the cells, PS can also be converted into a triplet excited state by intersystem crossing (3PS*). The PS in the triplet excited state can interact directly with cell membranes and generate superoxide radicals, hydroxyl radicals, and peroxides (type I reaction), through proton or electron transfer. Alternatively, excited state PS molecules can transfer energy to nearby oxygen molecules to produce singlet oxygen (type II reaction). Reactive oxygen species (ROS) obtained from both the type I and type II reactions can induce autophagy, apoptosis, and necrosis in the cells. It is generally believed that the type II reaction that produces singlet oxygen plays the major role in these processes of bacterial killing65.

2.5.2 Biofilms eradication For the biofilms eradication testing, substrates were firstly cultivated with bacterial solutions in Luria-Bertani (LB) broth medium for 7 d. After bacteria live/dead staining, it showed that a thick layer of biofilms evenly distributed on the substrates under fluorescence microscopy. As showed in Figure 6, the MB together with laser irradiation could remove more than 99% of the three kinds of bacteria S. aureus, MRSA and P. aeruginosa leaving only some individual and sporadic


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bacteria on the surface. However, the biofilms removal property against E. coli was not very ideal with only 70% removal efficacy. It is believed that longer laser irradiation time or higher intensity will improve the biofilms treatment efficiency. The resistance of E. coli has been shown in other works as well, which could be attributed to difference of cell wall structure from S. aureus1, 24. Biofilms treated with MB or laser irradiation showed nearly no removal functions.

Figure 6 ： Anti-biofilm properties of CMC-MB NPs against S. aureus, E. coli, MRSA and P. aeruginosa as bacterial model determined using a live/dead bacterial staining kit. (CMC-MB NPs: 10 mg/mL, laser: 202 mW/cm2, 5 min). SEM observation also confirmed the excellent biofilms eradication property of CMC-MB NPs against MRSA and P. aeruginosa at different magnifications. Before treatment, the biofilms formed on the silicon wafer through a process including bacterial adhesion, bacterial growth and extracellular matrix secretion into the densely packed biofilms. In general, the biofilm matrix primarily contains four components: extracellular polysaccharides, extracellular DNA (eDNA), enzymes and other proteins, which all have various special functions. Extracellular polysaccharides account for the major component of the biofilm matrix, which crosslink with eDNA to stabilize the framework of biofilms66. As showed in Figure 7, the attached biofilms could be removed by PDT treatment with only few single bacteria or extracellular polysaccharides residue remaining on the substrates. There some reasons contributed to the rapid removal of the biofilms. On one hand, the activated ROS could high efficient eliminate the living bacteria which has been showed in Figure 1. The nutrient and waste drainage channels were also fit for small molecular MB diffusion into the biofilms. The PDT and release of ROS also enhanced the degradation of extracellular matrix through damage to biofilms components. On the other hand, the pH responsive and rapid MB release at lower pH was very beneficial to the PDT. It should be emphasized that the fast responsive PS release and short time therapy time were fit for reducing the toxicity to mammalian cells and tissues.



Figure 7 Anti-biofilm properties of CMC-MB NPs against S. aureus and P. aeruginosa as bacterial model determined using SEM with magnification at ×2.2 k or ×8 k. (CMC-MB NPs :10 mg/mL, laser: 202 mW/cm2, 5 min).

2.5.3 In vivo antibacterial property test Based on the in vitro antibacterial tests and the wavelength of the laser irradiation (650 nm), subcutaneous rabbit wound bacterial infection model was carried out to explore the in vivo antibacterial property of CMC-MB NPs. Different treatments were applied after subcutaneous injection of bacterial solutions for 1 d, which showed obvious inflammatory response caused by bacterial infections with redness and bulge (Figure 8). As showed in Figure 8, at day 7, the appearance of the infected position showed great difference between CMC-MB NPs/laser irradiation treatment and the three control groups. The redness and inflammation in the skins of CMC-MB NPs/laser irradiation treated groups almost disappeared comparing with the other three control groups. In addition, the redness and area of the three control groups gradually increased indicating the development of inflammation and infections. Especially for the infected skins treated by laser irradiation, the appearance of many herpes was the precursor of decay. The infected skins were also cut and opened to explore the internal inflammatory response appearance and physiological inflammatory response. As indicated in Figure 8, there was small amount of necrotic tissue and white pus in the infected wounds of three control groups. However, the wound of CMC-MB NPs/laser irradiation treated group showed hardly any abnormal appearance. The quantitative characterization of bacterial infections development in the infected tissues was done through measuring the number of bacteria by agar plate counting method. The result showed that 324 ± 48, 236 ± 29 and 310 ± 53 CFU/mL of the tissue extract of control, CMC-MB NPs and laser treated groups. However, the number greatly decreased into 12 ± 4 CFU/mL for the CMC-MB NPs/laser irradiation groups. The H&E staining results also visually represented physiological inflammatory response through distinguishing and counting the


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inflammatory cells around the wounds. The presence of inflammatory cells in the tissues of control groups illustrated the delayed inflammatory response state of the wounds. On the contrary, there almost no inflammatory cells existing in the wounds treated by CMC-MB NPs/laser irradiation. This phenomenon proved that the wound had inhibited the inflammation by controlling infections development. The inflammation biomarkers including TNF-α and IL-6 in the tissue fluid were also analyzed with a commercially available ELISA kit according to the manufacturer’s protocol. As showed in Figure S6, only the group of CMC-MB NPs/laser showed highly reduction of both TNF-α and IL-6, which might be due to the high efficient bacterial infections inhibition activity of CMC-MB NPs under laser irradiation. We also have tried to get the clear images of in vivo imaging as showed in Figure S7. It showed clear imaging under animal living imaging device and the fluorescence continued 8 h. The enrichment of fluorescence around the bacterial infections position indicated the obvious pH responsive release of MB from CMC-MB NPs into this area.

Figure 8 In vivo antibacterial property test through taking photographs at day 1 for different treatments, at day 3 for the appearance observation, day 7 for the opened pockets observation and images of hematoxylin and eosin-stained sections of the surrounding connective tissues treated with CMC-MB NPs/laser irradiation, CMC-MB NPs, laser or no treatment.

2.6 Anti-tumor properties of CMC-MB NPs 2.6.1 In vitro anti-tumor efficacy Under laser irradiation, PS could be excited for further ROS generation, which has a toxic effect on tumor cells as well as normal mammalian cells. A CCK-8 kit was used to investigate the cytotoxicity after incubation with either L929 fibroblast cells or MCF-7/ADR cancer cells for 24 h. As shown in Figure 9A, B, the PDT process exhibited totally different cytotoxicity against two cells. Almost no cytotoxicity was observed against L-929 cells, which maintained more than 80% the cell viability of the control without any treatment. Neither MB nor laser irradiation alone showed any cytotoxicity against L-929 cells as well. As a result, the generation of ROS during PDT was the main cause of cytotoxicity. However, as for the cell viability of MCF-7/ADR tumor



cells (Figure 9A), approximately 70% of the cancer cells were killed after CMC-MB NPs/laser irradiation treatment. It was not surprisingly to find that neither MB nor laser irradiation alone showed any cytotoxicity against the cancer cells. The great cytotoxicity difference between L929 fibroblast cells or MCF-7/ADR cancer cells could be ascribed to the different of growth and physiological state of two types of cells. The rapid proliferation of cancer cells and tumor tissues, including abnormal fast neovascularization and the extraction of nutrients promoted the diffusion and interactions with tumor cells. It should be emphasized that the optimized laser irradiation (202 mW/cm2; 5 min) not only showed acceptable cytotoxicity against mammalian cells, but also ensured high efficiency in bacterial biofilms eradication and tumor ablation. We further explored the mechanism of anti-tumor properties of CMC-MB NPs through measuring the intracellular levels of ROS in L-929 or MCF-7/ADR cells using DCFH-DA method. As showed in Figure 9C, for both of L-929 and MCF-7/ADR cells, most of the cells without any treatment could be distinguished but without green fluorescent, which meant the absent of ROS production. Also, for the cells in the control groups treated either MB or laser irradiation alone showed nearly no green fluorescent under the microscope. However, for the CMC-MB NPs under laser irradiation groups, two kinds of cells indicated the accumulation of ROS which would result in significant damage to cellular structures. As a result, the production of ROS could be the main reason for the excellent bacterial biofilms eradication and tumor ablation functions.

Figure 9 The cell viability assay through CCK8 method of A: MCF-7/ADR cancer cells and B: L929 cells; C: Measurement of ROS production in L929 cells and MCF-7/ADR cancer cells through DCFH-DA method.

2.6.2 In vivo anti-tumor efficacy Anti-tumor experiments were carried out in a subcutaneous tumor model in nude mice. Based on the previous in vitro anti-tumor result, free MB or MB equivalence CMC-MB NPs were injected into A549 tumor bearing mice to evaluate the tumor ablation property. After treatment, the tumor sizes were measured with calipers at weeks 1, 2, and 3. At week 3 the tumor were harvested, and pathological tissue slices were taken. The changes of the body weight, tumor volume, and survival rate were recorded during three weeks after the drug injection and laser irradiation. As shown in Figure 10B, the weight of mice did not significantly change during the experimental period


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indicating the normal living state. As for the tumor volume ratio changing, the tumor volume in PBS treated groups grew very fast with more than 10 times increase in just three weeks. However, the CMC-MB NPs combined laser irradiation treatment led to the absolute suppression of the tumor growth (Figure 10A, C, E). As for the MB combined laser irradiation treated group, the tumor showed moderate growth rate. With regard to the survival rate of the rice, the control group showed a much lower survival rate which could be due the decreased immunity of the immune system caused by rapid growth of the tumors67. Also the injection of free MB might lead to the death of the mice due to the high cytotoxicity of the PS under laser irradiation.

Figure 10 The in vivo anti-tumor efficacy of CMC-MB NPs. A: the representative photograph of the tumor; B: Changes of body weight; C: Volume ratio (VT: volume of the tumor at different time, V0: tumor volume before treatment); D: survival rate; E: the representative photograph of the tumor after treatment for 3 weeks; F: Histological images using H&E staining of the tumor tissues (50× magnification). The excellent therapeutic efficacy of the CMC-MB NPs against solid tumor could be due to the pH responsive MB release and rapid aggregation at the tumor site. As for the survival rate of mice, the free MB group showed a much higher death proportion than CMC-MB NPs treated group, which could be attributed to the systemic toxicity of MB as showed in Figure 1. Paraffin sections of the collected tumor tissues were subjected to histologic and apoptosis evaluation using H&E staining method. As indicated in Figure 10F, the PBS treated group showed an obvious hyper-cellularity and a remarkable nuclear polymorphism exhibiting a vigorous growth of the tumor tissue. In contrast, the tumor tissue treated with MB or CMC-MB NPs under laser irradiation groups showed more cell necrosis as well as relatively loose cell density. The results proved that CMC-MB NPs combined with laser irradiation showed significant anti-tumor activity.

Conclusions A novel and facile multifunctional nanomaterial composite of CMC and PS was constructed through reverse phase microemulsion method with self-assembly, in situ imaging, high efficient bacterial biofilms eradication and tumor ablation properties. The CMC-MB NPs could be



synthesized in a green way using MB as crosslinking agent with or without further adding of GNP as crosslinking agent. The MB release from NPs showed remarkable pH responsiveness because of the high swelling ability and pH sensitivity of NPs components which was very beneficial to bacterial infections and tumor treatment. The MB released from the CMC-MB NPs could be excited and detected with red fluorescence under the fluorescence microscope for in situ imaging. Antibacterial experiments both in vitro and in vivo carried out on E. coli, S. aureus, MRSA, and P. aeruginosa proved the excellent bactericidal and biofilms eradication properties. Also, the anti-tumor property tests both in vitro against drug resistant MCF-7/ADR tumor cells and in vivo using nude mice subcutaneous tumor model showed the high efficient tumor ablation activities. The results showed that remarkable green fluorescence could only be observed in the combined MB-NPS and laser irradiation group. In summary, the obtained CMC-MB NPs with self-imaging, biofilms eradiation and tumor ablation multifunction has great potential in tumor therapy at the risk of bacterial infections and biofilms development.

Experimental Section Materials: Ammonium methylbenzene blue, iso-propyl alcohol, octanol, cyclohexane and ethanol traton-100 were purchased from Sigma-Aldrich. Carboxymethyl chitosan was purchased from Hang Seng Technology Co., Ltd. Genipin was purchased from Yuan Zhi Biological Technology Co., Ltd. DCFH-DA, Mouse IL-6 ELISA kit (ab100713), and Mouse TNF-alpha ELISA kit (ab100747) were purchased from Sigma-Aldrich (St Louis, MO). S. aureus (ATCC 6538), MRSA (ATCC 43300) and Escherichia coli (E. coli, ATCC 8739) was kindly provided by Prof. Jian Ji (Zhejiang University, Hangzhou, China); P. aeruginosa (AS12378) was purchased from China General Microbiological Culture Collection Center. The water used in all experiments was of Millipore Milli-Q grade. Determination of the MB standard curve: The absorbance was determined by UV-vis spectrometry. MB has a strong absorption peak at 630 nm. Double distilled water was used to prepare different concentrations of MB solution (0.2, 0.3, 0.5, 1.0, 2.0and 3.0 μg/mL). A calibration curve could be obtained through measuring the absorption values of different concentrations. Synthesis and characterization of CMC-MB NPs Synthesis of CMC-MB NPs without and with additional crosslinking agent: Cyclohexane (18.15 mL), n-octanol (4.41 mL), and Traton-100 (4.75 g) were added to a 50 mL centrifuge tube and mixed well with a homogenizer for 7 min. Then 1 mL 3 % CMC solution dissolved in DI water was then added and mixed uniformity. After that, 0.1 mL MB (10 mg/mL) was added into the mixture followed by overnight reaction kept away from sunlight at room temperature. Isopropyl alcohol twice and three times of ethanol were used to clean the resulting product through centrifugation. The product was then stored in alcohol or freeze dried. The CMC-MB NPs were synthesized with the additional 0.1mL genipin (1mmol/L) into the mixed solution. The rest of the steps were the same as the above process. Particle size and Zeta potential of CMC-MB NPs:The CMC-MB NPs were suspended in buffer


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solutions at the corresponding pH (4, 5, 6, 7, and 8) that was adjusted with sodium hydroxide or hydrochloric acid. Particle size was then measured by Zetasizer Nano S90. The CMC-MB NPs were suspended in buffer solution at the corresponding PH and the pH of the buffer solution was adjusted with sodium hydroxide or hydrochloric acid (pH=10, 9, 8, 7, 6 and 5). Zeta potential was then measured by Zetasizer Nano S90. Bactericidal activity of MB and biofilms eradication effects of CMC-MB NPs Primary bacteria culture: 12.5 g of soluble LB broth powder was added to a conical flask with 500 mL of deionized water, and shaken well until completely dissolved. The solution was then sterilized with high temperature and high pressure, and cooled to room temperature. A drop of bacteria was added and the mixture was incubated at 37 °C on a shaking table for 24 h. The mixture was then centrifuged (2000–5000 rad/min, 15min) to precipitate the bacteria. After removing the supernatant, the bacteria were washed twice with sterile PBS and were kept in sterile PBS at 4 °C. Effect of laser irradiation intensity and time on bactericidal activity: The irradiation intensity and irradiation time on bacterial activities were explored after incubating with the bacteria. At first, the irradiation intensity was studied under a fixed irradiation time of 10 min and MB 2.0 μg/mL. Bacteria (100μL, 104 CFU/mL) were added to 1.5 mL centrifuge tubes and irradiated with different intensities of laser at 202, 420 or 624 mW/cm2. The changes of bacteria concentration were observed through plate counting method. The laser irradiation time on bactericidal activity was studied fixing the irradiation intensity at 202 mW/cm2. After adding MB and followed 1, 3, 5 or 7 min of laser irradiation, the number changes of bacteria was observed through plate counting method. Antibacterial efficiency of CMC-MB NPs. Bacteria was added to a 1.5 mL centrifuge tube (1 mL, 104 CFU/mL) followed by different treatments (no treatment, CMC-MB NPs only, laser irradiation only, CMC-MB NPs combined with laser irradiation). Then, changes in bacteria numbers were observed through plate counting method. Biofilms eradication effects of CMC-MB NPs. As for the biofilms eradication effect, bacterial live/dead staining and SEM methods were used to measure the morphological and quantitative changes after PDT treatment of CMC-MB NPs. To prepare the bacterial biofilm in a 24-well plate, 900 μL of LB broth and 100 μL of bacteria (106 CFU/mL) were added to each well. Fresh LB broth was change every day and biofilm formed after one week. The biofilms were cleaned gently with sterile PBS followed with different treatment (no treatment, 0.1 mL 10 mg/mL CMC-MB NPs only, laser irradiation only, CMC-MB NPs combined with laser irradiation for 5 min, 202 mW/cm2). Then CMC MB NPs solutions were removed and samples were cleaned gently with the PBS. 300 μL of live/dead staining kit was then added to each well and left to stain for 15 min without light. Samples were then observed using a fluorescence microscope. As the SEM observation of biofilm, silicon wafer cut and put in a 24-well cell culture plate after cleaning with ethanol and ultrapure water. After being irradiated with UV for 30 min, 900 μL of LB broth and 100 μL of bacteria (106 CFU/mL) was then added to each well for bacterial biofilms growth in the same process as before. The biofilms were cleaned gently with sterile PBS followed with different treatment (no treatment, 0.1 mL 10 mg/ml CMC-MB NPs only, laser irradiation only, 10 mg/mL CMC-MB NPs combined with laser irradiation for 5 min, 202 mW/cm2). Then CMC MB NPs solutions were removed and samples were cleaned gently with the



PBS. Samples were fixed with 2.5% glutaraldehyde overnight, then dehydrated using an ethanol gradient dehydration method (50 wt%, 75wt%, 85wt%, 95wt%, 100wt%) for 15 min each step, and then dried in air. The resulting samples were observed under SEM. Cellular compatibility of MB and CMC-MB NPs Dark toxicity of MB against L-929 cells. The cytotoxicity was evaluated using a Cell Counting Kit-8 assay (CCK-8, Dojindo, Japan). Cells were cultured in DMEM/F12 medium supplemented with 10% FBS, and maintained at 37 °C under a humidified atmosphere of 5% CO2 and 90% relative humidity. After incubation, cells were seeded at a density of 5000 cells per well in 96-well plates. After 4 h, once cells were found to be adherent under microscope observation, MB solution with different concentrations was added (2, 5, 8, and 10 μg/mL). The cells were kept in carbon dioxide cell culture incubator for 24 h without light. Then wells were cleaned gently with serum-free culture medium twice to remove excess MB. 100μL of 10% of CCK-8 solution was added to each well and the plates were kept in a carbon dioxide cell incubator. After 2-4 hours, the above formazan solution were taken from each sample and added to one well of a 96-well plate, six parallel replicates were prepared. The absorbance at 450 nm (calibrated wave) was determined using a microplate reader (Multiskan MK33, Thermo electron corporation, China). Tissue culture plates (TCPS) without any sample were used as a control. Cellular compatibility of CMC-MB NPs against L929 cells and drug resistant MCF-7/ADR tumor cells. Cell viability of the CMC-MB NPs PDT treatment against L929 cells and drug resistant MCF-7/ADR tumor cells was quantitatively evaluated through CCK-8 (Beyotime, China) assay. After different treatment (no treatment, 0.1 mL 10 mg/ml CMC-MB NPs only, laser irradiation only, 10 mg/mL CMC-MB NPs combined with laser irradiation for 5 min, 202 mW/cm2), the L929 cells or MCF-7/ADR tumor cells solutions were replaced by 100 μL of new medium containing 10 μL of CCK-8. After incubation for 2 h at 37 °C, water-soluble formazan formed in the solution. The formazan solution (100 μL) was aspirated and added to the well of a new 96 well plate. A microplate reader (Multiskan MK33, Thermo Electron Corporation, China) was used to determine the absorbance at 450 nm. TCPS without any surface modification were used as control. Measurement of molecular oxygen A Cellular Reactive Oxygen Species Detection Assay Kit (2',7'-Dichlorodihydrofluorescein diacetate, DCFH-DA) was used to measure the intracellularly produced molecular oxygen. Firstly, 3.5mg DCFH-DA was dissolved in 721μL ethanol and diluted 10 times with DMEM/F12 used as stock solution at -20 °C. When used, the stock solution was dissolved at room temperature and diluted 100 times with DMEM/F12 into working solutions. After the cells and biofilm were prepared as in the previous method, the cells or biofilms were treated with MB of CMC-MB NPs combined with laser irradiation. For DCFH-DA staining, the samples were incubated with working solutions in dark for 30 minutes and then cleaned gently with serum-free culture medium to remove excess working solutions. And samples were observed under a fluorescence microscope for green fluorescence. In vivo antibacterial and anti-tumor tests In vivo antibacterial tests. In vivo antibacterial property of the CMC-MB NPs was tested through


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a Japanese big ear rabbit subcutaneous infection model. The subcutaneous infection in rabbits was established through a subcutaneous injection of 0.5 mL 107 CFU/mL S. aureus for 24 h. Different treatments were applied at day 1 and continues observation for one week before cut open of the pockets. Care was taken to ensure broad physical separation between the implants to eliminate cross-contamination risks. Rabbits were housed individually and given ad libitum access to food and water for 1 week, after which they were returned to the operating room and prepared as above. Each pocket was opened via a small incision distinct from the prior wound, through which two sterile cotton swabs were inserted, sequentially. Animals were then euthanized, and the incision extended. After obtaining culture samples, each pocket was excised en bloc, and transmural sections from representative areas taken. Specimens were fixed in 10% formalin and embedded in paraffin blocks. Tissue sections, 5 mm thick, were mounted onto slides, which were stained using Hematoxylin and Eosin (H&E). The levels of IL-6, and TNF-α were determined in tissue fluid around the wound. The tissue fluid was analyzed with a commercially available ELISA kit according to the manufacturer’s protocol. Analysis of optical density or fluorescence was performed in a plate reader. The in situ imaging of the CMC-MB NPs was observed using a Maestro 2 in vivo imaging system at the exciting wavelength of 550 nm. The fluorescence emission intensity at 630 nm was recorded in a time-correlated manner. Anti-tumor experiments in animals. Tumors were induced in nude mice for 4-8 weeks by subcutaneous administration of 0.1 mL, 107/mL MCF-7/ADR cells in the thigh. After tumor grows to 0.8-1cm3 (V0), mice were divided into three groups: injection of PBS, MB injection combined with laser irradiation, and CMC-MB NPs combined with laser. Free MB or CMC-MB NPs was injected into the tail vein at a MB concentration of 2.5 mg per kg body weight. PBS instead of MB or CMC-MB NPs solution was used to create the control group. After 4 hours, laser irradiation was applied at 202mw/cm3 for 5min. Two subsequent treatments were performed after week 1 and 2 after the first treatment. The survival rates and changes of body weight were recorded. Vernier calipers were used to measure the size of the tumors at 1, 2, and 3 weeks. The tumor volume (mm3) was calculated using the following equation: VT =a × b2/2 where “a” and “b” stand for the longest and shortest diameter, respectively. The animals were euthanized at week 3, histological changes and apoptotic cells in tumor tissues were evaluated using H&E staining using the kit (Beyotime® Biotechnology Co. Ltd, Jiangsu, China), according to the manufacturers’ instructions. Statistical analysis All data were obtained from at least three independent experiments with six parallel samples and expressed as mean ± standard deviation (SD) of typical images. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:. The standard curve of MB, color changes during the process of CMA-MB NPs precipitation and separation, scanning electron microscope image, and the results of phase diagram, and In situ imaging of the bacterial cells (PDF) Acknowledgment This work was financially supported by the National Natural Science Foundation of China



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