A Hydrogel-Based Hybrid Theranostic Contact ... - ACS Publications

May 31, 2016 - 510642, China. ‡. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre, Sun Yat-Sen University, Guangzhou 510064, China...
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A Hydrogel-Based Hybrid Theranostic Contact Lens for Fungal Keratitis Jian-Fei Huang,†,# Jing Zhong,‡,# Guo-Pu Chen,†,# Zuan-Tao Lin,§,# Yuqing Deng,‡ Yong-Lin Liu,† Piao-Yang Cao,† Bowen Wang,‡ Yantao Wei,‡ Tianfu Wu,§ Jin Yuan,*,‡ and Gang-Biao Jiang*,† †

Department of Pharmaceutical Engineering, College of Materials and Energy, South China Agricultural University, Guangzhou 510642, China ‡ State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre, Sun Yat-Sen University, Guangzhou 510064, China § Department of Biomedical Engineering, University of Houston, Houston, Texas 77204, United States S Supporting Information *

ABSTRACT: Fungal keratitis, a severe ocular disease, is one of the leading causes of ocular morbidity and blindness, yet it is often neglected, especially in developing countries. Therapeutic efficacy of traditional treatment such as eye drops is very limited due to poor bioavailability, whereas intraocular injection might cause serious side effects. Herein, we designed and fabricated a hybrid hydrogel-based contact lens which comprises quaternized chitosan (HTCC), silver nanoparticles, and graphene oxide (GO) with a combination of antibacterial and antifungal functions. The hydrogel is cross-linked through electrostatic interactions between GO and HTCC, resulting in strong mechanical properties. Voriconazole (Vor), an antifungal drug, can be loaded onto GO which retains the drug and promotes its sustained release from the hydrogelbased contact lenses. The contact lenses also exhibited good antimicrobial functions in view of glycidyltrimethylammonium chloride and silver nanoparticles. The results from in vitro and in vivo experiments demonstrate that contact lenses loaded with Vor have excellent efficacy in antifungal activity in vitro and could significantly enhance the therapeutic effects on a fungus-infected mouse model. The results indicate that this hydrogel contact lenses-based drug delivery system might be a promising therapeutic approach for a rapid and effective treatment of fungal keratitis. KEYWORDS: fungal keratitis, ophthalmology treatment, graphene oxide, nanosilver, antifungal

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often treated with the therapeutic keratoplasty (TKP) combined with medical therapy.13 However, apart from the overall difficulty and complexity of the surgical technique, another concern is that, in many cases, TKP leads to poorer vision. Today, the systemic treatment of fungal keratitis still remains a challenging, risky, and prolonged therapy.14 Voriconazole (Vor), a triazole antifungal agent, is a secondgeneration synthetic derivative of fluconazole, which is superior to the traditional antifungal agents and can be effective against yeast and filamentous fungi infection.15,16 Indeed, it has been widely used to treat serious, invasive fungal infections in clinical settings, yet only oral and intravenous formulations are available.17 Vor comes as a tablet or liquid suspension, and requires a long period of time to treat fungal infection via oral administration. For instance, patients should take Vor for at least 14 days to treat esophageal candidiasis.18 For Aspergillosis infection, patients need to take it for several months or even

ungal keratitis is a serious ocular disease that often causes ocular morbidity and blindness. The overall prevalence of fungal keratitis has a clearly increasing trend in recent years, accompanied by wide fungal variety in different geographical location, especially in developing countries.1 For example, out of all cases of infectious keratitis, fungal keratitis accounts for 6−20% in the United States,2 39% in North India3 and approximate 61.9% in North China.4 The disease is usually caused by saprophytic fungal pathogens including Fusarium and Aspergillus.5 The major pathogenic factors are associated with frequent and chronic use of topical corticosteroids, the rapid emergence of antimicrobial drug resistance, previous corneal injury, extended-wear contact lens and growing number of corneal surgeries.6−8 Since fungal keratitis results in a strong diffuse infection and is a leading cause of blindness, a rapid and effective treatment is critical once the diagnosis of fungal keratitis is confirmed. However, current therapies against fungal keratitis are mostly inefficient due to various factors, including the absence of an ideal form of sustained release and drug insensitivity and resistance.9,10 As an intractable disease,11,12 fungal keratitis is © 2016 American Chemical Society

Received: January 25, 2016 Accepted: May 31, 2016 Published: May 31, 2016 6464

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Figure 1. (a) Synthesis of HTCC/Ag/GO/Vor; (b) schematic illustration of drug loaded contact lenses and controlled drug release.

longer.19 Hence, it might cause many serious side effects, such as transient visual disturbances, fever, rash, vomiting, diarrhea, peripheral edema, and respiratory disorder; and the prolonged course of treatment could increase the risk of these occurrences.20 In particular, visual disturbances are unique to Vor compared to other azole antifungal agents.21,22 Thus, topical therapy using Vor has been employed, including subconjunctival, intrastromal/intraocular injection, and eye drops, to improve drug concentration in the disease site and to reduce side effects.23 However, eye injection has been associated with various drawbacks in many clinical cases, including the damage of liver function, second endophthalmitis, and visual disturbances.24 In clinical settings, the most commonly used method is antifungal eye drops, which is a noninvasive, cost-effective, and easy-to-use therapeutic approach.25 Nevertheless, quite often this method suffers from low bioavailability, due to poor solubility, dispersity, and intraocular permeability of the drug, the eye tissue tolerance to medicines, and the physiological barriers of the eyes.26 Particularly, the short acting time via eye drops results in only 1−7% of the dosage actually reaching the focus areas, while the rest is cleaned up by eye’s movement and the scouring nasolacrimal system. Besides, gutta and oculentum forms, which account for 60% of the formulation of current ophthalmic remedies, have poor bioavailability. Therefore, the eye drops must be administered frequently; for example, Sharma et al. instructed a group of patients to receive topical eye drops every 4 h.27 Unfortunately, frequent daily dosing might cause side effects and compromise patient obedience.

Additionally, fungal keratitis infection often leads to the formation of a biofilm, which is particularly difficult to be cleaned and penetrated by antifungal agents especially via eye drops, because of its encasement in a protective and impermeable extracellular matrix.28−30 It has been shown that Vor has an eminent antifungal effect compared to natamycin in vitro experiments (original literature); however, the topical Vor solution was not superior to natamycin in terms of clinical outcomes in a randomized and double-blinded clinical trial of fungal keratitis.30 Therefore, it is urgent to explore a more effective drug delivery system for topical Vor over gutta and oculentum forms. Contact lenses have been successfully utilized for vision correction. Their comfort structure and capacity for drugloading make contact lenses suitable as an ocular drug delivery system. The last few decades have witnessed advances in hydrogel research and nanotechnology that enable soft contact lenses to carry and release drugs at sustained rates to achieve more efficient therapeutic effects with fewer side effects. In particular, considerable efforts have been made to design and fabricate hydrogels based on natural polymers by virtue of theirs biocompatibility and biodegradation.31 The most extensively used natural polymers are polysaccharides among which chitosan (CS) and its derivatives have drawn increasing attention due to their excellent properties such as the ease of chemical modifications.32,33 As antimicrobial and therapeutic agents can be easily incorporated into CS-based multifunctional membranes, antimicrobial drugs-loaded CS membranes have been widely employed in medical area.34 For example, Norowski et al. used genipin cross-linked CS membranes loaded with minocycline to prevent infection through the local 6465

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ACS Nano delivery of antibiotics during bone healing.35 However, because of the lack of softness and flexibility, membrane-based native CS is not a good choice to form contact lenses. Quaternized CS (HTCC) is positively charged and thus possesses antimicrobial capabilities, it also retains good biocompatibility.36 Zhou et al. demonstrated that quaternary functionalized CS fibers could be used as potential wound dressing for skin regeneration.37 Unfortunately, CS loses its membrane-forming capability after being quaternized. On the other hand, the drug loading capacity of hydrophilic CS derivatives such as HTCC is very low, and the drugs carried by HTCC based on hydrogel or membrane will still be released and cleaned quickly. Graphene, a single layer of carbon atoms, has exhibited good drug loading capability for hydrophobic drugs especial for drugs with an aromatic ring by hydrophobic interactions and π−π stacking interactions. Graphene oxide with partial carboxylic groups (GO) can also be used as a crosslinker for the HTCC to form a stable hydrogel. In this article, as Figure 1 shows, a drug delivery system based on hydrogel contact lenses with integrated antifungal functions is designed and fabricated for the treatment of fungal keratitis. The hydrogel is composed of HTCC, GO (with little carboxylation), silver nanoparticles, and Vor. Both HTCC and silver nanoparticles enable contact lenses to possess antimicrobial functions; while the GO serves as the drug carrier, it also provides the anionic group to create electrostatic cross-linking with the cationic group of HTCC. This sustained drug delivery system was designed as contact lenses with softness and flexibility; they are capable of adhering easily to the cornea and exhibit excellent cyto-compatibility and outstanding antimicrobial activity in vitro and in vivo. The platform showed excellent efficiency in antifungal functions and might be promising in the applications of therapeutic contact lenses, especially for the treatment of fungal keratitis.

the presence of GO in HTCC/GO.42 In the IR spectra of GO, it can be found that two peaks at 1720 and 1620 cm−1 belonged to the stretching vibration of CO and ketonic species of −CO.43 From the IR spectra of HTCC/GO (Figure S1d) and HTCC (Figure S1c), the absorption peaks at 1655 cm−1 and at 1590 cm−1 were attributed to the vibration of −CO of the amide group, while the peaks at 1720 and 1620 cm−1 disappeared in GO, demonstrating that GO was linked to HTCC. In addition, the absorbance of silver nanoparticles was at 403 nm through the ultraviolet and visible spectrum (UV− vis spectrum) (Figure S4), suggesting silver nanoparticles were obtained. The mechanics property of the theranostic contact lens matrix is of importance. The hydrogel membranes with excellent flexibility were prepared. To test the mechanical performance of the hydrogel membrane, the mechanical strength was evaluated. Since the HTCC could not form strong hydrogel membranes for mechanical strength test, 5% of CS was incorporated into HTCC solution to form a membrane for the test. Figure 2 presents the stress−strain curve of five

RESULTS AND DISCUSSION Characterization of Composites. To confirm the successful synthesis of HTCC, Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (NMR) analysis were performed. As shown in Supporting Information, Figure S1, in the IR spectrum of HTCC (Figure S1c), a new peak at 1438 cm−1 appeared, which was the methyl absorption peak of the trimethylamine group; however, the peak intensity at 1594 cm−1 which presents NH2 deformation was weakened dramatically compared to that in the spectrum of CS. This indicated that the amine groups of CS were partly grafted by glycidyltrimethylammonium chloride (GTMAC) via the ring opening of epoxy group.38 The evidence of quaternization of HTCC was also shown in the 1H NMR spectra (Figure S2). In contrast to the 1H NMR spectrum of CS (Figure S2a), the N,N,N-trimethyl protons were observed as a characteristic signal at 3.13 ppm in the 1H NMR spectrum of HTCC (Figure S2b). The 1H NMR spectrum of HTCC was in agreement with those in previous reports.39,40 These results confirmed that GTMAC was grafted to CS. The degree of quaternization (DQ) of HTCC was determined by using the potentiometric precipitation titration method,39 and the value of DQ calculated was 42% according to the reported equation.41 In the high resolution C 1s X-ray photoelectron spectroscopy (XPS) spectrum of HTCC/GO (Figure S3), the C 1s component can be attributed to C−C/ CC (284.7 eV, C in graphite), C−OH (285.5 eV), C−O (286.3 eV, C epoxy/ether), and CO (287.8 eV), indicating

Figure 2. Stress−strain behavior of membranes of CS, HTCC, HTCC/Ag, HTCC/GO, and HTCC/Ag/GO. HTCC and HTCC/ Ag were mixed with 5% CS solution in the process of casting of membranes, while HTCC/GO and HTCC/Ag/GO were not.

types of hydrogel membranes. As determined by the slope of the stress−strain curves at 1% strain, the value of elastic modulus of the engineered membrane of HTCC/GO, HTCC/ Ag/GO, CS, HTCC, and HTCC/Ag calculated were 11.75, 10.81, 6.62, 6.44, 0.67 MPa, respectively. Mechanical properties of the engineered membranes were significantly enhanced by the addition of GO. Compared with that of the HTCC and HTCC/Ag membranes, the tensile strength and elastic modulus of HTCC/GO and HTCC/Ag/GO membranes increased with the blending of GO. This behavior is mainly attributed to the strong H-bonding between the HTCC and GO.44 Without GO, HTCC could not form an intact membrane. In the case of the HTCC membrane, the introduction of quaternary ammonium into CS decreases the original crystal of CS, which results in the poor membrane formation of HTCC. However, with the blending of a small amount of GO, the HTCC can form fairly soft and flexible hydrogel membranes or contact lenses. Compared with those of the HTCC membrane, a higher tensile strength and elastic modulus of the CS membrane were observed, but the membrane based on native CS is too hard and stiff to adhere 6466

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Figure 3. (A) UV−vis spectra of HTCC/Ag/GO/Vor and Voriconazole in PBS: (a) HTCC/Ag/GO/Vor, (b) Voriconazole; (B) accumulated release curve of Vor in PBS (pH7.4).

after HTCC was strengthened by GO. It was observed that the endothermic process of HTCC/GO was between 41.63 and 76.47 °C (endothermic peak at 45.72 °C), leading to 5.83% of weight loss, which corresponds to the vapor of water molecules physically absorbed. The water content of HTCC/GO was higher than that of HTCC because the blending of the GO thin layer could block the volatilization of water during the drying process. However, the endothermic peak of HTCC/GO was at 264.60 °C and shifted slowly toward higher temperature, which was increased by 20 °C, associated with a 37.83% of weight loss which was higher than the weight loss of 20.14% in the HTCC hydrogel contact lens. Probably there was a dehydration reaction between the amino groups of HTCC and carboxyl groups of GO. From Figure S6c, it can be observed that the endothermic process was shifted insignificantly, corresponding to the weight loss of 7.42% (water evaporation). However, the exothermic process was between 163.27 and 300 °C, indicating that there was a complex thermal decomposition of HTCC/Ag/GO, such as the disruption of hydrogen bond and the forces between groups containing nitrogen and silver nanoparticles. The weight loss of the exothermic process was 36.57%. The thermal stability of HTCC/Ag/GO was increased compared to HTCC, suggesting that the carboxyl groups of GO were linked to the naked amine groups of HTCC via electrostatic interactions, which is in agreement with the result of FT-IR. In Vitro Drug Release from HTCC/Ag/GO/Vor. The amount of Vor loading onto HTCC/Ag/GO was determined by UV spectrum at 255 nm (Figure 3A). PBS solution was used to simulate the surroundings of the cornea. The release profiles of Vor from the contact lenses exhibited a sustained rate at pH 7.4 (37 °C) (Figure 3B). The whole release process could be divided into three phases. The first phase corresponded to an initial burst release for the first 3 h owing to fast diffusion of the drug blending into the HTCC matrix of the contact lenses, and 35% of total loaded drugs were released at the this stage. Different from drug release used for treatment of other ocular diseases, this first burst release was unquestionably beneficial for the treatment of fungal keratitis. It is important to note that the Vor concentration could quickly reach an adequate effective antifungi concentration. However, when the PBS solutionsoaked HTCC/Ag/GO/Vor is placed onto the eyes, the liquid surrounding the contact lenses is limited, which will retard the drug diffusion, thus an initial burst phase is desired. In this

in the mold because of the crystal structure formed by hydrogen bonds.45,46 In summary, the increased mechanical property of the HTCC/Ag/GO membrane is essential for a theranostic contact lens for fungal keratitis. The morphology of the HTCC/Ag hydrogel contact lens was presented in Figure S5a. The distribution of Ag on HTCC was uniform. After the blending of GO, the hydrogel integrated with GO was rough (Figure S5b). With the blending of Ag, the HTCC/Ag/GO hydrogel contact lens was smooth, but some small granular particles appeared on the surface as presented in Figure S5c. The most noticeable change was that the blending of GO changed the material distribution and the surface topography markedly. Since the HTCC structures were bound together by GO, the HTCC/Ag/GO hydrogel contact lens can present a stronger mechanical performance and excellent flexibility. Another function of the blending of GO is to increase the drug-loading capacity and sustain drug release; as Vor is insoluble and contains a benzene ring that is apt to adhere to GO by π−π stacking interactions. Figure S5d shows that the rodlike Vor adheres extensively onto GO and is welldistributed, adhering onto the surface of the hydrogel contact lens. The support for the presence of Vor loaded in the hydrogel was confirmed by the XPS wide spectrum in Figure S5e. In the XPS spectrum of HTCC/Ag/GO/Vor, the signal of element fluorine (F) at 689.5 eV was observed, which exclusively results from the drug Vor. The content of F on the surface of the hydrogel was 14.6%, suggesting a significant component of Vor in the hydrogel (Figure S5f). The surface accumulation of Vor occurs because of the hydrophobic interactions and π−π stacking of GO.47 The thermal stability of CS and its derivatives was studied by differential thermal analysis (DTA)/thermogravimetry (TG), and the curves were shown in Figure S6. The DTA curve of HTCC shown in Figure S6a exhibited a prominent endothermic process around 42−77.60 °C (endothermic peak at 47.78 °C). This was attributed to the vapor of water molecules that HTCC physically absorbed, followed by an endothermic process between 199.40 and 251.27 °C (exothermic peak at 244.52 °C), indicating the depolymerization and decomposition of the polymeric units and dehydration of saccharide rings. The calculated HTCC losses were 4.52% and 20.14% of its weight in the endothermic process and exothermic process, respectively. As shown in Figure S6b, the thermal stability of HTCC/GO was enhanced dramatically 6467

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Figure 4. Diameter datum of zone of inhibition of CS and its derivatives composites against four kinds of microorganism: (a) against bacteria S. Aureus. and E. coli; (b) against fungi F.solani and A.fumigatus (all measured by filter paper method).

study, this fast initial release might benefit the rapid treatment of fungal keratitis in an animal model for about 1 week. The following release was a near zero-order release for the second period of 21 h. At the end of this phase the release rate reached 52% of total amount of drug loaded on the contact lenses. After 24 h, the drug release rate became slow; perhaps, as the solubility of Vor is poor, a large amount of Vor adhering onto GO can only be slowly released, which contributes to retention of the drug in the GO drug depot for sustained release. The in vitro release results indicated that HTCC/Ag/GO contact lenses could be used as a favorable carrier for Vor. In Vitro Antimicrobial Activity. Since the contact lenses are composed of chitosan and Ag, and they have good antimicrobial activity, the antimicrobial activity was investigated. The results are presented in Figure 4. The antifungal activity of HTCC was the same as that of CS, while the antibacterial ability of HTCC was slightly lower than that of CS. It was noticeable that the antimicrobial abilities of HTCC/ Ag and HTCC/Ag/GO were significantly enhanced. The diameter of the zones against and E. coli with HTCC/Ag and HTCC/Ag/GO were 90.37% and 94.56% higher than that with chitosan, while the size of zones against S. aureus with HTCC/ Ag and HTCC/Ag/GO were 50.08% and 63.64% higher than that with chitosan. For the antifungal activity, 36.67% and 21.11% higher inhibition zone diameter against F. solani and A. f umigatus with HTCC/Ag/GO were observed which indicated that HTCC/Ag/GO has antifungal activity although it is not as strong as its antibacterial activity. Therefore, the contact lenses matrix exhibits high antimicrobial activity. Additionally, the minimum inhibitory concentration (MIC) value of HTCC/Ag/GO/Vor and Vor hydrogel matrix was measured by serial dilution. The MIC of Vor to F. solani was 2.5 μg/mL, but the MIC to A. fumigatus was 2.5−5 μg/mL. In contrast, the MIC of HTCC/Ag/GO/Vor hydrogel matrix to both F. solani and A.fumigatus was 1.25 μg/mL. These results suggest that HTCC/Ag/GO hydrogel matrix is able to increase the antimicrobial activity of Vor, and the HTCC/Ag/GO/Vor hydrogel matrix has a better antimicrobial effect than antibiotics when used alone. Cell Cytotoxicity. Biocompatibility is one of the most important criteria for contact lenses. To investigate the effect of the contact lenses on the proliferation of normal cells, human corneal epithelial cells were used in in vitro experiments to confirm the safety of the various composites used for hydrogel contact lenses. The solutions of HTCC, HTCC/Ag, HTCC/ Ag/GO, and HTCC/Ag/GO/Vor were coincubated with corneal cells for 24 h, respectively. The control group is the group treated without the contact lenses. As shown in Figure 5, compared to the control group, the experimental groups HTCC and HTCC/Ag had a weak inhibitory effect on the

Figure 5. Cytotoxicity of four materials in human corneal epithelial cells.

growth of cornea cells as demonstrated by the protein contents in the cells. In the groups treated with HTCC/Ag/GO or HTCC/Ag/GO/Vor, there was not obvious inhibition on corneal cells compared to the control group. Indeed, since the HTCC and HTCC/Ag contact lenses have more positive charges and adhered to the corneal cell membrane easily, the normal function of the corneal cell had changed, thereby the growth of corneal cells were restrained to some extent, yet not significantly. Therefore, the antimicrobial material HTCC/Ag/ GO/Vor is considered safe and nontoxic to the cornea demonstrating good biocompatibility. This could be further verified in animal studies. The cytotoxicity of samples to HCECs is exhibited in Figure 5. The viability and the protein contents of the cells treated with HTCC, HTCC/Ag, HTCC/Ag/GO, or HTCC/Ag/GO/ Vor contact lenses were comparable to those of the untreated cells. The HTCC and HTCC/Ag contact lenses clearly showed inhibition to some extent, while the one treated with HTCC/ Ag/GO contact lenses did not have any inhibition. This could be on an account of cationic toxicity of HTCC; whereas the formulations containing silver nanoparticles and GO were nontoxic. In Vivo Antimicrobial Activity. To further explore the function of these antifungal materials, contact lenses of suitable size were made and placed onto the left eye of mice with fungal keratitis, and the therapeutic effects were observed under a clinical slit lamp for seven consecutive days. Clinical scores (Figure 6A) were evaluated upon disease progression on days 1, 3, 5, and 7. As shown in Table 1, the scores from all three criteria were tallied daily according to disease severity for each eye to yield a total score of 0 to 12. A total score of 5 or less 6468

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Figure 6. Severity of keratomycosis in mice carried with antifungal materials. C57BL/6(B6) mice were infected with 108 CFU/mL of Aspergillus fumigates routinely and visually evaluated for their corneal involvement for 7 days. The control group was fungal keratitis mouse model and untreated while the others were stuck with antifungal materials. A possible score of 0 to 4 was assigned to each of the following three criteria: area of opacity, density of opacity, and surface regularity. The scores from all three categories were tallied daily for each eye to yield a possible total score of 0 to 12. Through observation during seven consecutive days, clinical score (A) and representative pictures (B) are shown. Photographs indicated the disease progression at 1, 3, 5, and 7 days. Magnification ×16. ((∗) p < 0.05; (∗∗) p < 0.01; (∗∗∗) p < 0.001 by Student’s t test; NS, not significant).

Table 1. Visual Scoring System for Murine Fungal Keratitis grade 1 area of corneal opacity density of corneal opacity surface regularity

grade 2

grade 3

1%−25%

26%−50%

51%−75%

slight cloudiness, outline of iris and pupil discernible slight surface irregularity

cloudy, but outline of iris and pupil cloudy, opacity not uniform remain visible rough surface, some swelling significant swelling, crater or serious descemetocele formation

was categorized “mild”, a total score of 6 to 9 was considered “moderate”, and a total score more than 9 was “severe”.48 As shown in Figure 6A, compared with the control group, all four groups treated with various contact lenses have significant therapeutic effect on keratomycosis which were induced by

grade 4 76%−100% uniform opacity perforation or descemetocele

A.f umigates infection. On Day 1, eyes treated with HTCC, HTCC/Ag, and HTCC/Ag/GO contact lenses showed lower clinical scores compared to the untreated group (p < 0.05). Particularly, the HTCC/Ag/GO/Vor contact lenses presented more significant difference compared to other groups (p < 6469

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silver nanoparticle to Ag+ was retarded. Along with the release of Vor, the space on GO was increased, thereby the release speed of Ag+ was accelerated. Hence, silver nanoparticles enhanced the antifungi activity, yet the antifungi activity was inhibited by GO at the initial stage. During the course of 7 days, the antifungi effect mostly resulted from Vor from Day 1 through Day 3 or Day 5, though silver nanoparticles clearly revealed antifungal effects at a later stage. Histological Analysis. To examine the effect of HTCC/ Ag/GO/Vor contact lenses on cornea healing, histological evaluation of the mouse cornea was performed on Day 7 after treatment. The corneas from both groups were paraffinembedded and stained with hematoxylin and eosin (H&E). The pathological differences in cornea between experimental groups and the control group were observed under microscope (Figure 8). The results clearly indicated that the cornea

0.001). In addition, corneal involvement of contact lenses (HTCC/Ag/GO) with or without drug loading was obviously distinct on Day 1 (p < 0.05). Furthermore, the cornea treated with drug-loaded contact lenses (HTCC/Ag/GO/Vor) were scored markedly lower than the ones without drug loading on Day 7 (p < 0.001). From Day 1 through Day 7, the clinical scores were lower in the HTCC/Ag/GO/Vor treated group compared to all the other groups. In the untreated control group, the corneal disease developed and deteriorated over time course; however, the mice treated with antifungal contact lenses on Day 7 exhibited a lower average clinical score than the untreated group on Day 5. During the whole process, corneal diseases showed the mildest inflammatory responses in the antifungal contact lenses group. Representative findings from Day 1 and Day 7 were shown in Figure 6B. In addition, based on our experimental observation, no significant biodegradation of the HTCC/Ag/GO/Vor contact lenses was noticed over 7 days. Variety of the activity of fungus in the cornea clinical scoring and slit-lamp examination demonstrated that the mice treated with HTCC/Ag/GO/Vor contact lenses exhibited significantly reduced keratitis compared to other groups (Figure 6A and 6B). The fungal plate count method (Figure 7) was used to

Figure 7. Variety of the activity of fungus in the cornea.

determine the number of the living fungi in the cornea and to verify that the alleviation of inflammation was in the wake of the decreased load of fungi in the cornea. The results demonstrated that the quantity of the fungus decreased gradually after infection in two groups, while the experimental group treated with HTCC/Ag/GO/Vor contact lenses was significantly decreased after the infection on days 1, 3, 5, and 7 compared with the untreated control group (p < 0.001). From the observation under clinical slit lamp for 7 days, all four types of contact lenses had effect on fungal keratitis. HTCC contact lenses, however, was found to inhibit slightly the growth of A. f umigatus in vivo. After the blending of silver nanoparticles, the HTCC/Ag contact lenses exhibited increased efficacy against keratitis. The mice treated with HTCC/Ag or HTCC/Ag/GO contact lenses exhibited similar disease severity to the mice treated with HTCC contact lenses from Day 1 through Day 5, however, from Day 5 through Day 7, the differences of severity became significant. This indicates that the contact lenses with silver nanoparticles had a delayed effect on keratitis. Silver nanoparticles have antimicrobial activity by virtue of the Ag+ released from Ag.49 This might because the release of Ag+ was delayed by HTCC polymeric matrix. The reducibility of polyamines such as chitosan derivative HTCC can resist the oxidation of silver nanoparticles. After the crosslinking with GO in HTCC/Ag/GO hydrogel, the oxidation of

Figure 8. H&E staining of cornea treated without or with HTCC/ Ag/GO/Vor on 1, 3, 5, and 7 d.

pathology was significantly ameliorated in the experimental groups compared to the control group after infection on Day 1, 3, 5, and 7. The dramatic reduction of infiltrated inflammatory cells and inflammatory responses in the cornea of the experimental group treated with HTCC/Ag/GO/Vor contact lenses suggests that this might represent a promising therapeutic strategy for fungal keratitis.

CONCLUSIONS Fungal keratitis is an intractable ophthalmic disease; however, traditional treatment such as eye injection or eye drops suffer from various shortcomings and it is difficult to achieve a satisfactory therapeutic outcome. In the present study, we have designed a therapeutic contact lens by incorporating the following components: (1) HTCC, which has a good hydrophilicity and could serves as an excellent hydrogel matrix; (2) GO, which could increase the drug loading capacity and prolong drug release time due to the hydrophobic property of Vor and the physical structure of GO; (3) silver nanoparticles, which are capable of increasing the antifungi activity and expanding antimicrobial spectrum; and (4) Vor, a potent 6470

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CD3CO2D (acetic acid-d4)/D2O and D2O as solvent using a 600 MHz DMX-600 spectrometer (Bruker, Germany), respectively. Fourier transform infrared (FT-IR) spectra of CS and its derivatives were recorded on Nicolet 6700 Infrared Detector (Thermo Fisher Scientific, USA). The chemical bonding states of GO and the contents of F in the HTCC/Ag/GO/Vor were analyzed by X-ray photoelectron spectroscopy (XPS) (ESCALAB 250, Thermo Scientific, USA) with Al Kα radiation as the exciting source. The UV−vis spectra (Shanghai Precision Scientific Instrument Cop., China) were used to detect the absorption peak of the particular substance, which was dissolved in double distilled water. The differential thermal analysis/thermogravimetry (DTA/TG) (Shimazu, Japan) was performed to examine the change of internal structure. The surface topography can be observed by field emission scanning electron microscope (FESEM) (Ultra 55, Zeiss, Germany). The mechanical behavior was tested by universal tensile testing machine under the conditions 10 mm/min (Shenzhen Suns Technology Cop., China). The values of elastic modulus of engineered membranes calculated were from the slope of the stress− strain curve at 1% strain. Antifungal Activity Studies. E. coli strain DH5α and S. aureus were inoculated into Luria−Bertani (LB) medium and cultivated in an incubator at 37 °C, while F.solani and A.fumigatus were cultured in Sabouraud’s medium. After the appearance of many colonies, they were eluted with saline for liquid culture. The final fungal concentration was adjusted to 1 × 105 CFU/mL before inoculation in a plate. Circular specimens (diameter =6 mm) of membranes were placed in different places of one inoculated Petri dish. After 24 h of incubation, the inhibition zone was measured with a Vernier caliper. The minimal inhibitory concentration (MIC) value of HTCC/Ag/ GO/Vor and voriconazole were measured by double dilution. The concentration of fungal concentration was diluted to 1 × 105 CFU/mL in each well of ELISA plate. After the addition of composite or Vor, the plate was incubated at 37 °C. After 12 h of incubation, the visible growth of the bacteria was assessed by measuring the optical density value at 600 nm (OD600) using UV−vis spectroscopy. The MIC was the lowest concentration of adding samples or Vor which inhibited the fungal growth. Each assay was carried out in triplicate. In Vitro Drug Release Studies. To determine the stability and release rate of Vor loaded on the HTCC/Ag/GO, the HTCC/Ag/ GO/Vor contact lenses (made by 30 mL HTCC/Ag/GO/Vor solution) was placed in dialysis bags (MWCO = 1000 Da). Each dialysis bag was completely immersed in 100 mL of PBS (pH = 7.4) in Erlenmeyer flasks which were constantly shaken in a shaker at 37 °C.51 At predetermined time intervals, the drug release medium was completely replaced with 4 mL of fresh PBS pre-equilibrated to 37 °C. The released Vor was evaluated by measuring the UV−vis absorbance at 255 nm. Cytotoxicity Assay. To test cytotoxicity of the drug formulations, human corneal epithelial cells (HCECs, CRL-11135; ATCC) were plated in a 96-well plate at a seeding density of 5000 cells/well in serum containing medium. After 24 h, the medium was replaced with 200 μL of serum free medium. The remaining cells were either untreated or incubated with 30 μL of drug-loaded composites for the next 24 h. Next, the medium in each well was replaced with 200 μL of fresh serum-free medium. To each well, 20 μL of 5 mg/mL MTT solution in PBS (pH 7.4) was added. Following incubation for 3 h at 37 °C, the medium was aspirated out and the formazan crystals formed in the well were dissolved in 200 μL of dimethyl sulfoxide (DMSO). The absorbance of the dissolved formazan crystals in DMSO was recorded at 570 nm and used to estimate cell viability relative to control cells. Protein contents of the cells lysates were quantified using the Micro BCA Protein Assay Kit (Thermo Scientific). In brief, a Micro BCA working reagent was prepared by mixing Micro BCA reagent A and B at a ratio of 50:1. Then 25 μL of the solution from the MTT assay was mixed with 200 μL of the Micro BCA working reagent and kept at 37 °C for 30 min. The absorbance of the mixture solution was measured using photospectrometry at 562 nm for protein concentrations. In this assay, bovine serum albumin was used as a standard.

antifungal drug. These components enable effective antifungal capabilities, sustained drug release, and good biocompatibility in the contact lenses. Importantly, fungal keratitis could be significantly improved using this contact lens in 7 days, indicating that this drug delivery system has great potential for an effective and rapid treatment of fungal keratitis.

MATERIALS AND METHODS Materials. Chitosan (CS, Mw = 190 kDa, 95% deacetylated) was obtained from Saland (Los Angeles CA, USA). Glycidyltrimethylammonium chloride (GTMAC) was purchased from Shanghai D B Chemicals Technology Co. Ltd. (Shanghai, China). All reagents were analytical grade and used without further purification. Graphene oxide (GO) was obtained from Guangzhou Nano Biotechnology Limited Company (Guangzhou, China). The content of carboxyl in the watersoluble graphene was 5%. F.solani, A.f umigatus (AS 3.772) and voriconazole (Vor) were kindly provided by Zhongshan ophthalmic center at Sun Yat-Sen University. Synthesis of N-[(2-Hydroxy-3-trimethylammonium) Propyl] Chitosan Chloride (HTCC). The synthesis of HTCC is shown in Figure 1 as described by Lim et al.40 with modifications. Briefly, CS (2 g, 10.7 mmol) was dispersed in isopropyl alcohol at 80 °C. GTMAC (20 mL, 32.1 mmol) was added dropwise into the CS solution in 2 h. After 10 h of reaction, the yellowish reaction solution was poured into cold acetone (100 mL) with stirring, and kept at 4 °C overnight. Acetone was decanted and the precipitated product was dissolved in MeOH (50 mL). The solution was precipitated in 4:1 acetone− ethanol (100 mL). The white product was obtained by filtration and further purified by washing with hot EtOH. The final product was dried at 60 °C overnight. Preparation of Sliver Nanoparticles and HTCC/Ag. Silver nanoparticles were obtained via the reduction of AgNO3 with NaBH4.50 The detailed process of synthesis displayed in Figure 1 was described as below: NaBH4 (550 mL, 2 mM) was cooled down for 20 min, then AgNO3 solution was added in slowly. Subsequently, the mixture was stirred for another 3 h. The resulting color of the solution was changed to brown gradually. So the silver nanoparticles were obtained and then stored at 4 °C. The silver nanoparticles were added into HTCC solution with stirring at 60 °C, and HTCC/Ag was obtained. Therein, the amount of silver nanoparticles in HTCC/Ag solution was 5%. Preparation of HTCC/Ag/GO. GO was dispersed through ultrasonication and dropped into HTCC/Ag solution. After being stirred for 3 h, a composite which combined the GTMAC with GO was obtained. In this compound, the weight ratio of GO to HTCC/Ag was 1:20. Vor Loading by GO. Vor was dissolved in the mixture of propylene glycol and ethanol, followed by dilution with sterilized saline. Next, Vor mixture was added dropwise into HTCC/Ag/GO composite solution and stirred overnight. Excessive Vor was removed by dialysis. Preparation of Hydrogel Membranes and Contact Lenses. The HTCC powder (0.2 g) was dissolved in 40 mL of deionized water to obtain the HTCC solution. For mechanical properties measurement and in vitro experiments, after defoaming by sonication, 5% CS was added into the HTCC solution and placed into the plate (diameter = 7 cm). The membranes of HTCC were obtained by drying under vacuum at 60 °C, and the thickness was 0.1 mm. The resulting HTCC/Ag and HTCC/Ag/GO suspensions were diluted by water and dropped onto a glass plate after defoaming by sonication. The HTCC/Ag/GO/Vor composite solution was flowed in a glass plate to cast membrane with 0.1 mm thickness of HTCC/Ag/GO/Vor at 37 °C. The hydrogel contact lenses were prepared in a contact lens mold. The HTCC solution was injected into a contact lens mold to cast the hydrogel contact lenses. Characterizations of Composites. To verify that CS was grafted quaternary ammonium, the 1H nuclear magnetic resonance spectroscopy (NMR) analysis of CS and HTCC were conducted in 2% (w/w) 6471

DOI: 10.1021/acsnano.6b00601 ACS Nano 2016, 10, 6464−6473

Article

ACS Nano Establishment of Fungal Keratitis Model and in Vivo Antifungal Activity Studies. In vivo antifungal activity was evaluated using C57BL/6 female mice aged 6−8 weeks, raised in specific pathogen-free (SPF) environment furnished by the experimental animal center of Ophthalmology Center at Sun Yat-Sen University. All the protocols for animal experiments were carried out according to the guidelines of the Council for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Public Health, China. The mice were individually housed in cages on a 12 h light/dark cycle under constant temperature with ad libitum access to food and water. The animals were anesthetized using 4.3% chloral hydrate (0.01 mL/g) via an abdominal cavity injection. After that, the models of fungal keratitis were established via injection in corneal stroma with F. solani suspension. Briefly, the tunnel of corneal which had reached stroma deeply was prepared through a 30-gauge needle in the peripheral cornea. Subsequently, 2 μL of F. solani suspension (108 CFU/mL) was injected with a 33-gauge needle head along the prepared tunnel. After inoculation for 12 h, the HTCC/Ag/GO/Vor contact lens was cut into fragments by a trephine with a diameter of 2.5 mm2, and a fragment was attached onto the left eye of the mice, whereas the right eyes were untreated. In each group, pathology observations were carried out on mice that were sacrificed 7 days after the treatment. Tissue samples were taken from the cornea to prepare pathological slides and were observed under a Nikon Eclipse TE2000U microscope. The corneas were collected after 1, 3, and 5 days of inoculation treated with or without HTCC/Ag/GO/Vor contact lenses, respectively. Then the corneas were broken and the fungus were released after grinding in a sterile solution containing 0.85% (w/v) NaCl and 0.25% BSA. Supernatant was collected by centrifugation and was diluted 10 times. A series of dilutions of samples were spread on Sabouraud medium which were incubated at 37 °C overnight, and the colonies were counted the next day.

REFERENCES (1) Chang, D. C.; Grant, G. B.; O’Donnell, K.; Wannemuehler, K. A.; Noble-Wang, J.; Rao, C. Y.; Jacobson, L. M.; Crowell, C. S.; Sneed, R. S.; Lewis, F. M.; Schaffzin, J. K.; Kainer, M. A.; Genese, C. A.; Alfonso, E. C.; Jones, D. B.; Srinivasan, A.; Fridkin, S. K.; Park, B. J. Multistate Outbreak of Fusarium Keratitis Associated with Use of a Contact Lens Solution. JAMA, J. Am. Med. Assoc. 2006, 296, 953−963. (2) Jurkunas, U.; Behlau, I.; Colby, K. Fungal Keratitis: Changing Pathogens and Risk Factors. Cornea 2009, 28, 638−643. (3) Chowdhary, A.; Singh, K. Spectrum of Fungal Keratitis in North India. Cornea 2005, 24, 8−15. (4) Xie, L. X.; Zhong, W. X.; Shi, W. Y.; Sun, S. Y. Spectrum of Fungal Keratitis in North China. Ophthalmology 2006, 113, 1943− 1948. (5) Ahn, M.; Yoon, K.-C.; Ryu, S.-K.; Cho, N.-C.; You, I.-C. Clinical Aspects and Prognosis of Mixed Microbial (Bacterial and Fungal) Keratitis. Cornea 2011, 30, 409−413. (6) Rosa, R., Jr; Miller, D.; Alfonso, E. C. The Changing Spectrum of Fungal Keratitis in South Florida. Ophthalmology 1994, 101, 1005− 1013. (7) Tanure, M. A. G.; Cohen, E. J.; Sudesh, S.; Rapuano, C. J.; Laibson, P. R. Spectrum of Fungal Keratitis at Wills Eye Hospital, Philadelphia, Pennsylvania. Cornea 2000, 19, 307−312. (8) Panda, A.; Sharma, N.; Das, G.; Kumar, N.; Satpathy, G. Mycotic Keratitis in Children: Epidemiologic and Microbiologic Evaluation. Cornea 1997, 16, 295−299. (9) Liu, L.; Wu, H.; Riduan, S. N.; Ying, J. Y.; Zhang, Y. Short Imidazolium Chains Effectively Clear Fungal Biofilm in Keratitis Treatment. Biomaterials 2013, 34, 1018−1023. (10) Frei, R.; Breitbach, A. S.; Blackwell, H. E. 2-Aminobenzimidazole Derivatives Strongly Inhibit and Disperse Pseudomonas Aeruginosa Biofilms. Angew. Chem., Int. Ed. 2012, 51, 5226−5229. (11) Shukla, P.; Kumar, M.; Keshava, G. Mycotic Keratitis: an Overview of Diagnosis and Therapy. Mycoses 2008, 51, 183−199. (12) Gopinathan, U.; Garg, P.; Fernandes, M.; Sharma, S.; Athmanathan, S.; Rao, G. N. The Epidemiological Features and Laboratory Results of Fungal Keratitis: a 10-Year Review at a Referral Eye Care Center in South India. Cornea 2002, 21, 555−559. (13) Rogers, G. M.; Goins, K. M.; Sutphin, J. E.; Kitzmann, A. S.; Wagoner, M. D. Outcomes of Treatment of Fungal Keratitis at the University of Iowa Hospitals and Clinics: a 10-Year Retrospective Analysis. Cornea 2013, 32, 1131−1136. (14) Klont, R. R.; Eggink, C. A.; Rijs, A. J.; Wesseling, P.; Verweij, P. E. Successful Treatment of Fusarium Keratitis with Cornea Transplantation and Topical and Systemic Voriconazole. Clin. Infect. Dis. 2005, 40, e110−e112. (15) Mehta, H.; Mehta, H. B.; Garg, P.; Kodial, H. Voriconazole for the Treatment of Refractory Aspergillus Fumigatus Keratitis. Indian J. Ophthalmol. 2008, 56, 243−245. (16) Herbrecht, R. Voriconazole: Therapeutic Review of a New Azole Antifungal. Expert Rev. Anti-Infect. Ther. 2004, 2, 485−497. (17) Levêque, D.; Nivoix, Y.; Jehl, F.; Herbrecht, R. Clinical Pharmacokinetics of Voriconazole. Int. J. Antimicrob. Agents 2006, 27, 274−284. (18) Pappas, P. G.; Rex, J. H.; Sobel, J. D.; Filler, S. G.; Dismukes, W. E.; Walsh, T. J.; Edwards, J. E. Guidelines for Treatment of Candidiasis. Clin. Infect. Dis. 2004, 38, 161−189. (19) Herbrecht, R.; Denning, D. W.; Patterson, T. F.; Bennett, J. E.; Greene, R. E.; Oestmann, J.-W.; Kern, W. V.; Marr, K. A.; Ribaud, P.; Lortholary, O.; et al. Voriconazole Versus Amphotericin B for Primary Therapy of Invasive Aspergillosis. N. Engl. J. Med. 2002, 347, 408−415. (20) Purkins, L.; Wood, N.; Ghahramani, P.; Greenhalgh, K.; Allen, M.; Kleinermans, D. Pharmacokinetics and Safety of Voriconazole Following Intravenous-To Oral-Dose Escalation Regimens. Antimicrob. Agents Chemother. 2002, 46, 2546−2553. (21) Lazarus, H. M.; Blumer, J. L.; Yanovich, S.; Schlamm, H.; Romero, A. Safety and Pharmacokinetics of Oral Voriconazole in Patients at Risk of Fungal Infection: a Dose Escalation Study. J. Clin. Pharmacol. 2002, 42, 395−402.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b00601. FT-IR spectra of components, 600 MHz 1H NMR spectra of CS and HTCC, C 1s XPS spectrum of HTCC/GO, UV−vis spectra of components, FESEM images of hydrogel contact lenses, XPS wide spectrum of HTCC/Ag/GO/Vor and its relative percentage of elements, and the DTA/TG study of hydrogel contact lenses (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *Email: [email protected]. Author Contributions

# Jian-Fei Huang, Jing Zhong, Guo-Pu Chen and Zuan-Tao Lin contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This investigation was supported by grants from the Natural Science Foundation of Guangdong Province, China (2016A010103026, 2014B090901037), the National Natural Science Foundation of China (81560031, 81270972), and the Natural Science Research Team Foundation of Guangdong Province, China (S2011030003134). T.W. and Z.L. were partly supported by the Lupus Research Institute. 6472

DOI: 10.1021/acsnano.6b00601 ACS Nano 2016, 10, 6464−6473

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ACS Nano (22) Denning, D. W.; Ribaud, P.; Milpied, N.; Caillot, D.; Herbrecht, R.; Thiel, E.; Haas, A.; Ruhnke, M.; Lode, H. Efficacy and Safety of Voriconazole in the Treatment of Acute Invasive Aspergillosis. Clin. Infect. Dis. 2002, 34, 563−571. (23) Hariprasad, S.; Mieler, W.; Lin, T.; Sponsel, W.; Graybill, J. Voriconazole in the Treatment of Fungal Eye Infections: a Review of Current Literature. Br. J. Ophthalmol. 2008, 92, 871−878. (24) Kadikoy, H.; Barkmeier, A.; Peck, B.; Carvounis, P. E. Persistent Photopsia Following Course of Oral Voriconazole. J. Ocul. Pharmacol. Ther. 2010, 26, 387−388. (25) Yuan, X.; Marcano, D. C.; Shin, C. S.; Hua, X.; Isenhart, L. C.; Pflugfelder, S. C.; Acharya, G. Ocular Drug Delivery Nanowafer with Enhanced Therapeutic Efficacy. ACS Nano 2015, 9, 1749−1758. (26) Sharma, N.; Chacko, J.; Velpandian, T.; Titiyal, J. S.; Sinha, R.; Satpathy, G.; Tandon, R.; Vajpayee, R. B. Comparative Evaluation of Topical Versus Intrastromal Voriconazole As an Adjunct to Natamycin in Recalcitrant Fungal Keratitis. Ophthalmology 2013, 120, 677−681. (27) Prajna, N. V.; Mascarenhas, J.; Krishnan, T.; Reddy, P. R.; Prajna, L.; Srinivasan, M.; Vaitilingam, C.; Hong, K. C.; Lee, S. M.; McLeod, S. D. Comparison of Natamycin and Voriconazole for the Treatment of Fungal Keratitis. Arch. Ophthalmol. 2010, 128, 672−678. (28) Sengupta, J.; Saha, S.; Khetan, A.; Sarkar, S. K.; Mandal, S. M. Effects of Lactoferricin B against Keratitis-Associated Fungal Biofilms. J. Infect. Chemother. 2012, 18, 698−703. (29) Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Bacterial Biofilms: from the Natural Environment to Infectious Diseases. Nat. Rev. Microbiol. 2004, 2, 95−108. (30) Flemming, H.-C.; Wingender, J. The Biofilm Matrix. Nat. Rev. Microbiol. 2010, 8, 623−633. (31) Zelikin, A. N. Drug Releasing Polymer Thin Films: New Era of Surface-Mediated Drug Delivery. ACS Nano 2010, 4, 2494−2509. (32) Khor, E.; Lim, L. Y. Implantable Applications of Chitin and Chitosan. Biomaterials 2003, 24, 2339−2349. (33) Kim, H.-J.; Zhang, K.; Moore, L.; Ho, D. Diamond NanogelEmbedded Contact Lenses Mediate Lysozyme-Dependent Therapeutic Release. ACS Nano 2014, 8, 2998−3005. (34) Rao, W.; Wang, H.; Han, J.; Zhao, S.; Dumbleton, J.; Agarwal, P.; Zhang, W.; Zhao, G.; Yu, J.; Zynger, D. L.; Lu, X.; He, X. ChitosanDecorated Doxorubicin-Encapsulated Nanoparticle Targets and Eliminates Tumor Reinitiating Cancer Stem-Like Cells. ACS Nano 2015, 9, 5725−5740. (35) Norowski, P. A.; Babu, J.; Adatrow, P. C.; Garcia-Godoy, F.; Haggard, W. O.; Bumgardner, J. D. Antimicrobial Activity of Minocycline-Loaded Genipin-Crosslinked Nano-Fibrous Chitosan Mats for Guided Tissue Regeneration. J. Biomater. Nanobiotechnol. 2012, 3, 528−532. (36) Qin, C.; Xiao, Q.; Li, H.; Fang, M.; Liu, Y.; Chen, X.; Li, Q. Calorimetric Studies of the Action of Chitosan- N-2-Hydroxypropyl Trimethyl Ammonium Chloride on the Growth of Microorganisms. Int. J. Biol. Macromol. 2004, 34, 121−126. (37) Zhou, Y.; Yang, H.; Liu, X.; Mao, J.; Gu, S.; Xu, W. Potential of Quaternization-Functionalized Chitosan Fiber for Wound Dressing. Int. J. Biol. Macromol. 2013, 52, 327−332. (38) Huang, R.; Chen, G.; Sun, M.; Hu, Y.; Gao, C. Studies on Nanofiltration Membrane Formed by Diisocyanate Cross-Linking of Quaternized Chitosan on Poly (acrylonitrile) (PAN) Support. J. Membr. Sci. 2006, 286, 237−244. (39) Ji, Q. X.; Zhong, D. Y.; Lü, R.; Zhang, W. Q.; Deng, J.; Chen, X. G. In Vitro Evaluation of the Biomedical Properties of Chitosan and Quaternized Chitosan for Dental Applications. Carbohydr. Res. 2009, 344, 1297−1302. (40) Lim, S.-H.; Hudson, S. M. Synthesis and Antimicrobial Activity of a Water-Soluble Chitosan Derivative with a Fiber-Reactive Group. Carbohydr. Res. 2004, 339, 313−319. (41) Wu, J.; Su, Z.-G.; Ma, G.-H. A Thermo- and PH-Sensitive Hydrogel Composed of Quaternized Chitosan/Glycerophosphate. Int. J. Pharm. 2006, 315, 1−11.

(42) Yang, Y.; Wang, J.; Zhang, J.; Liu, J.; Yang, X.; Zhao, H. Exfoliated Graphite Oxide Decorated by PDMAEMA Chains and Polymer Particles. Langmuir 2009, 25, 11808−11814. (43) Acik, M.; Lee, G.; Mattevi, C.; Chhowalla, M.; Cho, K.; Chabal, Y. J. Unusual Infrared-Absorption Mechanism in Thermally Reduced Graphene Oxide. Nat. Mater. 2010, 9, 840−845. (44) Han, D.; Yan, L.; Chen, W.; Li, W. Preparation of Chitosan/ Graphene Oxide Composite Film with Enhanced Mechanical Strength in the Wet State. Carbohydr. Polym. 2011, 83, 653−658. (45) Okuyama, K.; Noguchi, K.; Miyazawa, T.; Yui, T.; Ogawa, K. Molecular and Crystal Structure of Hydrated Chitosan. Macromolecules 1997, 30, 5849−5855. (46) Francis Suh, J.-K.; Matthew, H. W. Application of ChitosanBased Polysaccharide Biomaterials in Cartilage Tissue Engineering: a Review. Biomaterials 2000, 21, 2589−2598. (47) Bao, H.; Pan, Y.; Ping, Y.; Sahoo, N. G.; Wu, T.; Li, L.; Li, J.; Gan, L. H. Chitosan-Functionalized Graphene Oxide as a Nanocarrier for Drug and Gene Delivery. Small 2011, 7, 1569−1578. (48) Wu, T. G.; Wilhelmus, K. R.; Mitchell, B. M. Experimental Keratomycosis in a Mouse Model. Invest. Ophthalmol. Visual Sci. 2003, 44, 210−216. (49) Kittler, S.; Greulich, C.; Diendorf, J.; Koller, M.; Epple, M. Toxicity of Silver Nanoparticles Increases during Storage Because of Slow Dissolution under Release of Silver Ions. Chem. Mater. 2010, 22, 4548−4554. (50) Cha, K.; Hong, H.-W.; Choi, Y.-G.; Lee, M. J.; Park, J. H.; Chae, H.-K.; Ryu, G.; Myung, H. Comparison of Acute Responses of Mice Livers to Short-Term Exposure to Nano-Sized or Micro-Sized Silver Particles. Biotechnol. Lett. 2008, 30, 1893−1899. (51) Sahoo, N. G.; Bao, H.; Pan, Y.; Pal, M.; Kakran, M.; Cheng, H. K. F.; Li, L.; Tan, L. P. Functionalized Carbon Nanomaterials As Nanocarriers for Loading and Delivery of a Poorly Water-Soluble Anticancer Drug: a Comparative Study. Chem. Commun. 2011, 47, 5235−5237.

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DOI: 10.1021/acsnano.6b00601 ACS Nano 2016, 10, 6464−6473