Co-delivery of antiviral and antifungal therapeutics for the treatment of sexually transmitted infections using a moldable, supramolecular hydrogel.

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Co-Delivery of Antiviral and Antifungal Therapeutics for the Treatment of Sexually Transmitted Infections using a Moldable, Supramolecular Hydrogel Ashlynn L.Z. Lee, Victor W.L. Ng, Ghim Lee Poon, Xiyu Ke, James L. Hedrick, and Yi Yan Yang* total estimated direct medical costs, accounting for more than 81% of the total burden. This value underestimates the total damages incurred as it does not include indirect costs (e.g., loss of productivity) or intangible costs (e.g., pain and discomfort).[1] HIV infection is incurable and often fatal. The virus weakens the human immune system primarily by infecting CD4+ helper T cells. In the absence of antiviral treatment, HIV infection results in the gradual loss of CD4+ T cells and a progressive immune deficiency that cripples the body’s ability to fight opportunistic infections.[2] On the average, it takes approximately 11 years from HIV infection to the development of acquired immunodeficiency syndrome (AIDS) and eventual death. According to the World Health Organization (WHO), there were approximately 35.3 million people living with HIV globally (in 2012) and the virus has claimed more than 36 million lives to date.[3] The development of curative treatment against HIV has been met with numerous challenges due to the three key properties of the virus—the persistency of the latently infected CD4+ T cells, de novo infection of target host cells (ongoing viral replication) and the ability to breakdown the immune system and escape destruction by host immune cells.[4] Despite the inherent difficulties in achieving a cure for HIV infection, the use of antiretroviral drugs have significantly improved the well being and prolonged life expectancy of infected individuals. With global efforts in increasing the accessibility of antiretroviral treatment across the world, close-to-complete viral suppression can be maintained for decades in compliant patients who are infected with drug-susceptible viral strain.[5,6] However, despite sizable efforts in HIV research over the past few decades, there has been little or no successful clinical outcome. As such, the most effective method in curtailing the spread of HIV infection boils down to preventive measures and there is an urgent need to develop an effective prophylaxis against the disease. Previous report by the Centre for the AIDS Program of Research in South Africa (CAPRISA), 004 trial had demonstrated success in the prevention of HIV acquisition in women

In this investigation, a therapeutic co-delivery hydrogel system is developed to provide effective HIV prophylaxis, alongside the prevention and/or treatment of candidiasis. Two components—a HIV reverse transcriptase inhibitor, tenofovir, and a cationic macromolecular antifungal agent derived from a vitamin D-functionalized polycarbonate (VD/BnCl (1:30))—are formulated into biodegradable vitamin D-functionalized polycarbonate/PEG-based supramolecular hydrogels. The hydrogels exhibit thixotropic properties and can be easily spread across surfaces for efficient drug absorption. Sustained release of tenofovir from the hydrogel is observed, where approximately 85% tenofovir is released within 3 h. VD/BnCl (1:30) does not impede drug diffusion from the hydrogel as the drug release profiles are similar with and without the polycation. Antimicrobial efficacy studies indicate that the hydrogels kill C. albicans efficiently with a minimum bactericidal concentration (MBC) of 0.25–0.5 g L−1. These hydrogels also eradicate C. albicans biofilm effectively at 4× MBC. When human dermal fibroblasts (as model mammalian cells) are treated with these hydrogels, cell viability remains high at above 80%, demonstrating excellent biocompatibility. When applied topically, this dualfunctional hydrogel can potentially prevent HIV transmission and eliminate microbes that cause infections in the vulvovagina region.

1. Introduction Many diseases and infections can be transmitted from one individual to another during sexual contact. These diseases are caused by a myriad of pathogens such as bacteria, viruses, fungi, and parasites. Among the various sexually transmitted infections (STIs), viral infections account for the bulk of the economic burden. In a report published in 2013, approximately 95% of the total STI-associated medical cost in the United States is due to viral STIs.[1] Currently, human immunodeficiency virus (HIV) is by far the most costly STIs in terms of

Dr. A. L. Z. Lee, Dr. V. W. L. Ng, G. L. Poon, X. Ke, Prof. Y. Y. Yang Institute of Bioengineering and Nanotechnology 31 Biopolis Way, The Nanos, Singapore 138669, Singapore E-mail: [email protected] Dr. J. L. Hedrick IBM Almaden Research Center 650 Harry Road, San Jose, CA 95120, USA

DOI: 10.1002/adhm.201400340

Adv. Healthcare Mater. 2014, DOI: 10.1002/adhm.201400340

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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who were administered with a vaginal gel containing tenofovir, an HIV reverse transcriptase inhibitor.[7] These hydrogels could be topically applied over the vulvovaginal area to achieve a high bioavailability at the targeted tissues and potentially reduce the side effects associated in most oral formulations. In view of their importance, we have specifically designed an antiviral/ antifungal hydrogel system that contains vitamin D-functionalized antifungal polycarbonates and tenofovir. The hydrogels were prepared using an “ABA”-type triblock copolymer, that is, a hydrophilic PEG middle block flanked on both ends by hydrophobic vitamin D-functionalized polycarbonate blocks. Vitamin D is a group of lipid-soluble secosteroids that is recognized for its role in bone mineralization and calcium homeostasis. It is an FDA-approved supplement and is associated with the ability to regulate innate and adaptive immunity.[8,9] Several studies have demonstrated the antibacterial effects of vitamin D, especially towards mycobacterium tuberculosis (TB). Martineau et al. has reported a reciprocal relationship between vitamin D levels and TB occurrence, which is directly linked to the increase of HIV incidence due to compromised immunity.[10] The incorporation of vitamin D not only adjusts the amphiphilicity of the polycarbonate blocks for micellization and subsequent gel formation, but may also play a complementary role to enhancing patients’ immunity. The polymeric networks were formed based on intermolecular hydrophobic interactions between vitamin D-functionalized polycarbonate blocks. The incorporation of tenofovir and cationic vitamin D-containing polycarbonate into the hydrogel conferred antiviral and antifungal properties, respectively. The presence of cationic polycarbonate in the hydrogels would exert antifungal effects through direct membrane lysis of microbes, and this combination treatment may prevent the emergence of drug resistant subpopulation. This will consequently facilitate the prevention and/or elimination of fungal infections that are potentially transmitted through sexual contact. Overall, the topically delivered formulation will provide excellent convenience for frequent usage and can serve as an effective prophylaxis against HIV transmission. Our primary target is vulvovaginal candidiasis, which is an infection of the vagina’s mucous membrane caused by Candida supp. It has been reported that approximately 70%–75% of all women develop the infection at least once in their lifetime and 5%–8% experience recurrent vulvovaginal candidiasis.[11] Having new sexual partners or more frequent and intensive sexual intercourse could upset the symbiotic balance of the human microbiota and result in breaching of C. albicans.[12] In immunocompromised patients, they can cause mucosal, skin, or systemic infections. Vulvovaginal candidiasis is conventionally treated with antifungal agents such as topically or orally applied azole drugs. However, prolong usage of such antifungal agents such as fluconazole can eventually lead to reduced susceptibility and clinical response.[13] In particular, many human immunodeficiency virus (HIV)-infected patients who received long-term, low-level azole antifungal therapy showed the emergence of azole-resistant isolates of C. albicans.[14] The goal of this work is to develop topically applied antiviral/ antifungal hydrogel that can potentially enhance HIV prevention and simultaneously circumvent STIs, such as vulvovaginal candidiasis, by encouraging users’ compliance through ease of

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application. Furthermore, drug resistance of the fungal population can possibly be mitigated via the incorporation of macromolecular antimicrobial polycarbonates. The hydrogels were assessed for their mechanical properties and tenofovir release profiles. The antifungal efficacy of the hydrogel was evaluated by measuring minimum biocidal concentration (MBC), and its ability in eradicating C. albicans biofilms was assessed through metabolism quantification, biomass removal, and scanning electron microscopy. In addition, the cytotoxicity of the cationic polymer-formulated hydrogels was also investigated.

2. Results and Discussion 2.1. Monomer and Polymer Synthesis The 5-methyl-5-(ergocalciferyl)carboxyl-1,3-dioxan-2-one (MTCVD) monomer was synthesized via a direct coupling between MTC-Cl and ergocalciferol (vitamin D2) in the presence of triethylamine (Scheme 1a). The reaction was carried out in the dark to prevent premature decomposition of the light-sensitive vitamin D reactant. The final product was obtained using flash chromatography (gradient ethyl acetate/n-hexanes) followed by recrystallization from methanol as white solids in approximately 50% yield. The inclusion of ergocalciferol resulted in the creation of a hydrophobic cyclic carbonate. Organocatalytic ring-opening polymerization (ROP) was employed to synthesize the desired “ABA”-triblock hydrogel and antimicrobial polycarbonate precursors using DBU and thiourea derivative as catalysts (Scheme 1b). The ABA triblock hydrogel precursor was obtained by polymerizing MTC-VD from a difunctional PEG macroinitiator; the conversion of polymerization was 40%–50% as analyzed by 1H NMR. The modest conversion likely resulted from ring-chain equilibrium issues associated with the homopolymerization of the cyclic carbonate containing the bulky-appended vitamin D. The excess MTC-VD monomer and other reagents were removed easily by repeated precipitation with diethyl ether. The actual degree of polymerization (DPm) of MTC-VD was 0.98 according to 1H NMR analysis, that is, (MTC-VD)0.98-PEG(20k)-(MTC-VD)0.98. The vitamin D-containing cationic polycarbonates were obtained by copolymerization of MTC-VD and 5-methyl-5-(4chloromethyl)benzylcarboxyl-1,3-dioxan-2-one (MTC-BnCl) in the presence of DBU/TU co-catalysts using benzyl alcohol as the initiator (Scheme 1b). The polymerization reactions went to completion as judged by quantitative conversion of monomer to polymer as evidenced by 1H NMR analysis. Subsequent quaternization using trimethylamine resulted in total conversion to the desired cationic polymers. The polycarbonates are coded based on the feed ratios of MTC-VD to MTC-BnCl, respectively, for example VD/BnCl (1:30). The 1H NMR spectra of VD/ BnCl (1:30), before and after quaternization, are presented in Figure 1. All the major peaks could be assigned, and the ratios of MTC-VD to MTC-BnCl were referenced to the six methyl protons of MTC-VD (peak f) compared to the benzyl protons of MTC-BnCl (peak c). In addition, the degree of quaternization was ascertained by comparing the trimethylammonium-CH3 protons (peak h) to the benzyl protons of MTC-BnCl (peak c). Complete quaternization was observed for all polymers.




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Scheme 1. Synthesis of MTC-VD (a), “ABA”-triblock hydrogel precursor and cationic antimicrobial polycarbonates (b).

2.2. Antimicrobial and Hemolytic Activities of Cationic Polymers The antimicrobial efficacy of the vitamin D-containing cationic polycarbonates was evaluated against various bacterial and fungal strains, including S. aureus (Gram positive), E. coli (Gram negative), C. albicans (fungi), and C. neoformans (fungi). The antimicrobial (minimum inhibitory concentrations, MICs), hemolysis (HC50), and selectivity (HC50/MIC) results were summarized and presented in Table 1. In general, the polycarbonates with varying cationic components were effective and exhibited similar activity against all pathogens tested. The toxicity of the polymers was evaluated by hemolysis assay using rat red blood cells (RBCs) and we observed that hemolysis was low for all three polymers with HC50 values ≥ 1000 mg L−1. Hence, these vitamin D-functionalized polycarbonates are potentially useful broad-spectrum antimicrobial agents. Among the various polycations tested, VD/BnCl (1:30) gave the best results with regards to selectivity as it was the least toxic to the RBCs.This


observation can be rationalized by the fact that VD/BnCl (1:30) is the least hydrophobic and possibly exhibits the best amphiphilicity balance among the three cationic polymers. Therefore, we selected VD/BnCl (1:30) as our lead antimicrobial candidate, and investigated its co-delivery with an antiviral drug, tenofovir, in the subsequent study against the clinically-relevant microbe, C. albicans, in a two-prong antiviral/antifungal strategy.

2.3. Mechanical Properties of Hydrogel A minimum concentration of 4 wt% of (MTC-VD)0.98PEG(20k)-(MTC-VD)0.98 was found to be required for the formation of hydrogels (i.e., G′>G″).[15] As the concentration of polymer increased, the solid-like behavior (G′>>G″) of the hydrogels became more dominant (Table S1, Supporting Information), and the physical spreading of the matrix over surfaces would become less smooth. Hence, the 4 wt%



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Figure 1. 1H NMR spectra of VD/BnCl (1:30) (bottom) and its precursor (top).

hydrogel was selected for further studies. As shown in Table 2, the mean storage modulus G′ values for the hydrogels ranged between 402 to 486 Pa at 25 and 37 °C. The addition of the cationic polycarbonate VD/BnCl (1:30) to the hydrogels at both low (0.25 wt%) and high concentrations (2.5 wt%) did not result in significant differences to the stiffness at both temperatures. The ratio of G′/G′′ indicates whether the hydrogel was displaying a more liquid-like or solid-like behavior, which is in relation to the connectivity of the polymeric network.[16] A higher G′/G′′ ratio represents a predominantly solid-like or elastic response of the hydrogel. Interestingly, at the lower temperature, the loading of tenofovir (TEN) into the hydrogel resulted in the highest G′/G′′ (Figure 2A) corresponding to a more solid-like gel. The incorporation of tenofovir reinforced the elastic property of the hydrogel and this can be ascribed

to the presence of tenofovir within the hydrogel matrix.[17] On the other hand, lower G′/G′′ values were observed with the loading of VD/BnCl (1:30). Interactions between the vitamin D groups on (MTC-VD)0.98-PEG(20k)-(MTC-VD)0.98 and VD/ BnCl (1:30) might have weakened the intermolecular interactions between (MTC-VD)0.98-PEG(20k)-(MTC-VD)0.98, leading to a lesser elastic dominance of the hydrogels. Furthermore, the ratios of G′/G′′ were also lower at 37 °C as compared to 25 °C for the blank hydrogels, hydrogels with tenofovir or both tenofovir and VD/BnCl (1:30) (Figure 2A), thereby showing that viscous property was more dominant at higher temperatures. Flow sweep experiment was performed to simulate the spreading action of hydrogel onto the mucosal surface and this was done by monitoring the shear-dependent change in

Table 1. Antimicrobial activity and selectivity (HC50/MIC) of vitamin D-containing cationic polycarbonates. Polymer

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MIC [mg L−1], (selectivity)

Hemolysis HC50 [mg L−1]

S. aureus

E. coli

C. albicans

VD/BnCl (1:10)

31 (32)

63 (16)

125 (8)

31 (32)

≈1000

VD/BnCl (1:20)

31 (>32)

63 (>16)

125 (>8)

31 (>32)

>1000

VD/BnCl (1:30)

31 (>32)

63 (>16)

125 (>8)

16 (>63)

>1000



C. neoformans


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VitD0.98-(PEG20k)-VitE0.98 Tenofovir [wt%] [g L−1]

VD/BnCl (1:30) [g L−1]

G′ [Pa] at 25 °C

G′ [Pa] at 37 °C

4

0

0

439 ± 29

431 ± 78

4

1

0

445 ± 35

486 ± 72

4

0

0.25

452 ± 90

441 ± 111

4

0

2.5

477 ± 69

448 ± 104

4

1

0.25

421 ± 35

402 ± 73

4

1

2.5

423 ± 33

418 ± 65

120 100 80 60 TEN (1g/L) Gel

40

TEN (1g/L) + VD/BnCl(1:30) (2.5g/L) Gel TEN (1g/L) Sol

20

TEN (1g/L) + VD/BnCl(1:30) (2.5g/L) Sol

0 0

viscosity of the hydrogels (Figure 2B). As the hydrogels were subjected to increasing shear rate, the viscosity of the gels fell drastically, and became a thin liquid eventually. The shearthinning profiles of the hydrogels were due to the disruption of physical cross-links between the polymer chains under shear stress. This thixotropic property indicates that a thin layer of hydrogel can be topically applied onto the mucosal surface to provide more rapid drug absorption.

2.4. In Vitro Tenofovir Release In vitro drug release study was performed to assess the difference in tenofovir release kinetics between hydrogel and solution formulation. From Figure 3, tenofovir was released much more gradually from the hydrogels as compared to the solution formulations, thereby showing that the formulations can significantly affect the drug release profiles. Approximately 80% of the drug was released from the hydrogel by 3 h while a burst release of tenofovir was observed from the solutions where 100% drug release was achieved within the first 0.5 h. There was also no significant difference in tenofovir release profiles between the formulations loaded with tenofovir alone or those with the combination of tenofovir and VD/BnCl (1:30). The gradual release of tenofovir from the hydrogel formulations will enable a sustained delivery of the drug to the targeted tissues for HIV prevention.

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Table 2. Values of G′(storage modulus) of (MTC-VD)0.98-PEG(20k)(MTC-VD)0.98 (4 wt%) hydrogels at 25 °C and 37 °C.

Cumulative release (%)


1

2

3

4

5

Time (hours) Figure 3. In vitro tenofovir release from hydrogel and solution formulations.

2.5. Killing Efficiency VD/BnCl (1:30) was tested for its antimicrobial activity against C. albicans by treating the planktonic fungal cells with hydrogels containing various concentrations of the cationic polymer. VD/ BnCl (1:30) exerts its antimicrobial effects through the perturbation of membrane integrity of the fungal cells, which would lead to cytoplasmic leakage and eventual cell death.[18] Figure 4 shows that the MBC of the hydrogel that was loaded with VD/BnCl (1:30) alone was 0.25 g L−1. When 1.0 g L−1 of tenofovir was simultaneously incorporated with the cationic polymer, the antifungal efficacy of the VD/BnCl (1:30) remained similar (MBC = 0.5 g L−1) to that when it was delivered alone in the hydrogel. 2.6. Biofilm Eradication Biofilms are a major concern in the biomedical field as it had been estimated that pathogenic biofilms are responsible for 80% of human infectious disease[19] and are resistant to doses up to 1000 times that of planktonic microbes.[20] Furthermore, drug resistance can easily develop within biofilms and this can

B 50 25oC 25oC 37oC 37oC

G'/G''

40 30 20 10 0 TEN (g/L) VD/BnCl (g/L)

1

0 0

2 1.0 0

03 0.25

04 2.5

5 1.0 0.25

6 1.0 2.5

Figure 2. A) Ratio of G′/G′′ of hydrogels at 25 °C (gray bars) and 37 °C (black bars). B) Viscosity of hydrogels as a function of shear rate at 25 °C.




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100.0

Killing Efficiency (%)

100.00

99.85

100.00 100.00 100.00 100.00

98.23

98.0 96.0 94.0 92.0 90.0 0

0.125

0.25

0.5

1

Conc. of VD/BnCl(1:30)

VD/BnCl(1:30)

VD/BnCl(1:30) + TEN (1g/L)

Figure 4. Killing efficiency of (MTC-VD)0.98-PEG(20k)-(MTC-VD)0.98 (4 wt%) hydrogels loaded with VD/BnCl (1:30) in the presence or absence of tenofovir.

be attributed mainly to two reasons. First, biofilms possess a layer of extracellular matrix, which acts as a diffusion barrier against the diffusion of antimicrobial agents to the encased cells. Second, the biofilm provides an environment with close proximity for the exchange of resistant plasmids, and this can result in the generation of resistant microorganisms.[21] C. albicans could form biofilms in the vagina during extended vulvovaginal candidiasis infection.[22] As such, the eradication of C. albicans biofilms is critical in ensuring that the hydrogels are effective in preventing and treating candidiasis infection due to biofilm formation. To evaluate the effectiveness of the

hydrogels, antifungal cationic polymers were loaded at multiples of MBC concentrations (1, 2, and 4× MBC) for treatment on the biofilms. After 24 h of treatment, crystal violet and metabolic assays were performed to quantify the biomass and cell viability of the remaining biofilm, respectively. The blank hydrogels or the hydrogels containing tenofovir resulted in slight reduction of the biofilms as the cell viability was ≈90% compared to the untreated group (Ctrl) (Figure 5). This could be due to the retardation of nutrient and metabolite diffusion as a result of the hydrogels being present on the cell surface. Hydrogels loaded with 1× and 2× MBC of VD/BnCl (1:30) (0.25 and 0.50 g L−1 in the absence and presence of tenofovir) were effective at reducing the cell viability by 44% and 46%, respectively, but there was no significant reduction in the biomass after treatment with hydrogels. This indicates that VD/BnCl (1:30) at 1× and 2× MBC is only sufficient in reducing the number of viable cells within the biofilm, but it is inadequate in removing the biofilm. The greatest extent of biomass reduction was seen with the hydrogels loaded with 4× MBC of VD/BnCl (1:30) where the biomass was reduced by 58% and 50%, in the absence and presence of tenofovir, respectively. At this concentration, the cell viability of the remaining biofilm was also significantly lower than biofilms treated with hydrogels loaded with 1× and 2× MBC of VD/BnCl (1:30). The requirement of fourfold MBC for substantial biomass removal demonstrated that the cells encased in biofilms displayed significantly higher levels of antimicrobial resistance compared to planktonic cells. The extent of biofilm clearance was also observed through scanning electron microscopy (SEM) imaging (Figure 6).

A

B

*

*

80

*

Biomass (%)

Cell Viability (%)

80 60 40

60 40 20

20

0

0 Ctrl

Blank Gel

VD/BnCl 0.25g/L Gel

VD/BnCl 0.5g/L Gel

Ctrl

VD/BnCl 1.0g/L Gel

C

Blank Gel

VD/BnCl VD/BnCl 0.25g/L Gel 0.5g/L Gel

VD/BnCl 1.0g/L Gel

D *

100

100

*

80

*

60 40

*

80 Biomass(%)

Cell Viability(%)

*

100

100

60 40 20

20

0

0 Ctrl

Blank Gel TEN 1.0g/L TEN 1.0g/L TEN 1.0g/L TEN 1.0g/L + VD/BnCl + VD/BnCl + VD/BnCl 1.0g/L 2.0g/L 0.5g/L

Ctrl

Blank Gel TEN 1.0g/L TEN 1.0g/L TEN 1.0g/L TEN 1.0g/L + VD/BnCl + VD/BnCl + VD/BnCl 0.5g/L 1.0g/L 2.0g/L

Figure 5. A,C) Cell viability and B,D) biomass of C. albicans biofilms after 24 h treatment with (MTC-VD)0.98-PEG(20k)-(MTC-VD)0.98 (4 wt%) hydrogels loaded with VD/BnCl (1:30) in the presence or absence of tenofovir (1.0 g L−1). Statistical significance in difference was analyzed using Student’s t-test. P ≤ 0.05 was considered statistically significant.

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FULL PAPER Figure 6. SEM images of C. albicans biofilm after 24 h treatment with (MTC-VD)0.98-PEG(20k)-(MTC-VD)0.98 (4 wt%) hydrogels loaded with VD/BnCl (1:30) in the presence or absence of tenofovir (1.0 g L−1). Scale bar represents 2 µm.

Biofilms that were treated with VD/BnCl (1:30)-loaded hydrogels displayed a similar trend to the cell viability and biomass quantification assays where the clearance of biofilms was dependent on the concentration of VD/BnCl (1:30) used. Cells that were treated with the cationic hydrogels showed uneven and indented surface morphology. At 4× MBC, extensive cell destruction and biofilm eradication were observed.

2.7. Cytotoxicity Tests The effects of the newly formulated hydrogels with mammalian cells were evaluated by treating human dermal fibroblasts with the hydrogels loaded with 1, 2, and 4× MBCs of VD/BnCl (1:30). When treated with blank hydrogels, the cell viability remained ≈100% after 24 h, showing negligible toxicity (Figure 7) towards the cells. Furthermore, the presence of tenofovir within the hydrogels also did not induce cytotoxic effects on the cells. Hydrogels that were loaded with VD/BnCl (1:30) resulted in a slight decrease of cell viability but this was tolerated well within 20% of the untreated group.


4. Conclusions Biodegradable polycarbonate-based hydrogels capable of delivering both antiviral and antifungal therapeutics for effective HIV prevention and fungal infection have been developed. These hydrogels were fabricated from the “ABA”-type triblock copolymer, (MTC-VD)0.98-PEG(20k)-(MTC-VD)0.98 through physical cross-linking. An antireteroviral drug tenofovir and an antifungal polycation VD/BnCl (1:30) were loaded into the hydrogels during the gel formation process. These hydrogels exhibited thixotrophy, which is ideal for topical applications. The entrapment of tenofovir within hydrogels enabled an appreciably sustained release compared to solution formulations. No significant difference in tenofovir release profiles was observed between the formulations loaded with tenofovir alone or those in combination with VD/BnCl (1:30). Importantly, more than 99.9% killing efficiency of C. albicans was achieved for hydrogels loaded with the drug combination. Hydrogels loaded with 4× MBC of the polycation were able to effectively eradicate the biomass and reduce the viability of microbes residing as C. albicans biofilms. At the same concentrations, the hydrogels showed low cytotoxicity towards mammalian cells. These antiviral/antifungal



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Figure 7. Viability of human dermal fibroblasts after 24 h of treatment with (MTC-VD)0.98-PEG(20k)-(MTC-VD)0.98 (4 wt%) hydrogels loaded with VD/BnCl (1:30) at different concentrations in the presence or absence of tenofovir (1.0 g L−1).

hydrogels have potential for use as an HIV pre-exposure prophylaxis, as well as a treatment formulation for eliminating infections caused by both planktonic microbes and their biofilms.

4. Experimental Section Materials: Vitamin D-functionalized carbonate monomer, MTC-VD, MTCBnCl, and N-(3,5-trifluoromethyl)phenyl-N′-cyclohexylthiourea (TU) were synthesized by adapting a protocol previously reported (See Supporting Information for full experimental and characterization details).[23,24] All reagents were bought from Sigma–Aldrich and used as received unless otherwise mentioned. 1,8-Diazabicyclo[5,4,0]undec-7-ene (DBU) and benzyl alcohol were dried over calcium hydride and vacuum distilled twice before being transferred to the glovebox. Ergocalciferol (Vitamin D) was purchased from Tokyo Chemical Industry (TCI, Japan) while methanol and ethanol (ACS grade) were purchased from Tee Hai (Singapore). Glacial acetic acid was obtained from VWR (USA). Ultra pure (HPLC grade) water was obtained from J.T. Baker (USA). Tryptic soy broth (TSB) powder and yeast mold broth (YMB) powder were purchased from BD Diagnostics (Singapore) and used to prepare the microbial growth media according to the manufacturer’s instructions. Staphylococcus aureus (ATCC No. 29737), Escherichia coli (ATCC No. 25922), Candida albicans (ATCC No. 10231), and C. neoformans (ATCC No. 90112) were obtained from ATCC (USA), and reconstituted according to the suggested protocols. Tenofovir was obtained from Shanghai Uchem Inc. (China) and formalin solution, crystal violet, menadione, and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) were purchased from Sigma, USA. Nuclear Magnetic Resonance (NMR) Spectroscopy: The 1H NMR spectra of monomers and polymers were recorded using a Bruker Avance 400 spectrometer, and operated at 400 MHz, with the solvent proton signal as the internal reference standard. Molecular Weight Determination by Size-Exclusion Chromatography: SEC was conducted using THF as the eluent for monitoring the polymer conversion and also for the determination of polystyrene equivalent molecular weights of the polymers.[24] THF–SEC was recorded on a Waters 2695D (Waters Corporation, USA) with a separation module equipped with an Optilab rEX differential refractometer (Wyatt Technology Corporation, USA) and Waters HR-4E as well as HR 1 columns (Waters Corporation, USA). The system was equilibrated at 30 °C in THF, which served as the polymer solvent and eluent with a flow rate of 1.0 mL min−1. Polymer solutions were prepared at a known

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concentration (ca. 3 mg mL−1) and an injection volume of 100 µL was used. Data collection and analysis were performed using the Astra software (Wyatt Technology Corporation, USA; version 5.3.4.14). The columns were calibrated with series of polystyrene standards ranging from Mp = 360 Da to Mp = 778 kDa (Polymer Standard Service, USA). Polymer Synthesis and Characterization: Triblock copolymers of vitamin D-functionalized polycarbonate and poly(ethylene glycol), and vitamin D-containing cationic polycarbonates were synthesized for preparation of antiviral/antifungal hydrogels. Triblock Copolymer ((MTC-VD)0.98-PEG(20k)-(MTC-VD)0.98): In a 20-mL vial containing a magnetic stir bar in a glove box, MTC-VD (67.3 mg, 125 µmol, 5.0 equiv.), HO-PEG-OH (20 kDa, 500 mg, 25 µmol, 1.0 equiv.) and TU (18.5 mg, 50 µmol, 2.0 equiv.) were dissolved in dichloromethane (4 mL). To this solution, DBU (7.5 µL, 50 µmol, 2.0 equiv.) was added to initiate polymerization.[23] The reaction mixture was allowed to stir at room temperature and aliquots of samples were taken to monitor the monomer conversion and evolution of molecular weight by 1H NMR spectroscopy and SEC. After 3 h, the reaction mixture was quenched by the addition of excess (≈20 mg) of benzoic acid and was precipitated into ice-cold diethyl ether (2 × 50 mL). The resultant polymer was dried in a Falcon tube for about 1–2 d until a constant sample mass was obtained, as white powder. Selected 1H NMR (400 MHz, CDCl3): δ3.40–4.00 (s, 1815H, OCH2CH2 PEG), 1.10–3.00 (overlapping peaks, VD), 1.00 (d, 5.9H, J = 6.4 Hz, VD-CH3), 0.90 (d, 5.9H, J = 6.4 Hz, VD-CH3), 0.82 (t, 11.7H, J = 6.4 Hz, VD-CH3). PDI from GPC: 1.07. Vitamin D-Containing Cationic Polycarbonates (VD/BnCl (1:30)): In a 20-mL vial containing a magnetic stir bar in the glove box, MTC-BnCl (500 mg, 1.67 mmol, 30 equiv.), MTC-VD (30.0 mg, 56 µmol, 1.0 equiv.), and TU (20.6 mg, 56 µmol, 1.0 equiv.) were dissolved in dichloromethane (3 mL). To this solution, BnOH (5.8 µL, 56 µmol, 1.0 equiv.) followed by DBU (8.3 µL, 56 µmol, 1.0 equiv.) were added to initiate polymerization. The reaction mixture was allowed to stir at room temperature for 20 min and quenched by the addition of excess (≈30 mg) of benzoic acid. The mixture was then precipitated into ice-cold methanol (50 mL) and centrifuged at –5 °C for 30 min. The resultant semi-transparent oil was dried under vacuo until a foamy white solid was obtained. GPC analysis of the intermediate was carried out and the polymer was used without further purification. The polymer was subsequently dissolved in acetonitrile, transferred to a Teflon-plug sealable tube, and chilled to 0 °C. Trimethylamine was added to start the quarternization process. The reaction mixture was stirred at room temperature for 18 h in the sealed tube. Precipitation of an oily material was observed during the course of reaction. The mixture was evacuated to dryness under vacuo and freeze-dried to finally yield a white crisp-foamy solid. The desired polymer was characterized by 1H NMR (integral values are approximated to the nearest DP). VD/BnCl (1:30): 1H NMR (400 MHz, CDCl3) δ 7.20–7.70 (m, 117H, overlapping peaks of initiator Ph and Bn), 6.16 (d, 2H, C C on VD), 5.92 (d, 2H, C C on VD), 5.00–5.40 (m, 58H, overlapping peaks of initiator CH2Ph and Bn), 4.67 (m, 56H, CH2(N(CH3))), 4.30 (m, 112H, MTC-CH2), 3.07 (br s, 252H, N(CH3)), 1.00–2.00 (overlapping peaks, VD), 0.81 (t, 6H, CH3 on VD); PDI of intermediate (GPC): 1.21; Actual DP of VD/BnCl (1:30) (from 1H NMR) ≈1:28. VD/BnCl (1:10) and VD/BnCl (1:20) were obtained in a similar manner using the respective feed ratio of monomer. VD/BnCl (1:10): 1H NMR (400 MHz, CDCl3) δ 7.20–7.70 (m, 41H, overlapping peaks of initiator Ph and Bn), 6.16 (d, 2H, C C on VD), 5.92 (d, 2H, C C on VD), 5.00–5.40 (m, 20H, overlapping peaks of initiator CH2Ph and Bn), 4.67 (m, 18H, CH2(N(CH3))), 4.30 (m, 36H, MTC-CH2), 3.07 (br s, 81H, N(CH3)), 1.00–2.00 (overlapping peaks, VD), 0.81 (t, 6H, CH3 on VD); PDI of intermediate (GPC): 1.18; Actual DP of VD/BnCl(1:10) (from 1H NMR) ≈1:9. VD/BnCl(1:20): 1H NMR (400 MHz, CDCl3) δ 7.20–7.70 (m, 81H, overlapping peaks of initiator Ph and Bn), 6.16 (d, 2H, C C on VD), 5.92 (d, 2H, C C on VD), 5.00–5.40 (m, 40H, overlapping peaks of initiator CH2Ph and Bn), 4.67 (m, 38H, CH2(N(CH3))), 4.30 (m, 76H, MTC-CH2), 3.07 (br s, 171H, N(CH3)), 1.00–2.00 (overlapping peaks, VD), 0.81 (t, 6H, CH3 on VD); PDI of intermediate (GPC): 1.19; Actual DP of VD/BnCl(1:20) (from 1H NMR) ≈1:19.





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Rheological Characterization of Hydrogels: Blank hydrogel and antimicrobial polymer-loaded hydrogels were prepared by first dissolving vitamin D-containing cationic polycarbonate in DI water at 25 °C to the desired concentrations by ultrasonication for 1 min. The resultant solution was then transferred to the triblock copolymer (MTC-VD)0.98PEG(20k)-(MTC-VD)0.98 (4 wt%), and the mixture was stirred using a pipette tip. After that, the mixture was quickly spun-down using a minicentrifuge and left to stand at room temperature for 4 h for gelation to occur. Rheological properties of hydrogels were characterized on ARES-G2 rheometer (TA instruments, USA) equipped with a plate–plate geometry of 8 mm diameter.[25] The hydrogels were equilibrated to either 25 °C or 37 °C and measurements were taken between an upper plate fixture of 8 mm diameter and a Peltier plate of 1.0 mm gap. The data were collected under a controlled strain of 0.5% with a frequency scan of 1.0 to 100 rad s−1. Viscoelastic properties of the polymer solutions was monitored by measuring the shear storage modulus (G′), and the loss modulus (G′′), at each point. Thixotropic properties of the hydrogels were determined by measuring the changes in viscosity of the hydrogels as a function of shear rate from 0.1 to 10 s−1. In VitroRelease of Tenofovir: The release of tenofovir from the (MTC-VD)0.98-PEG(20k)-(MTC-VD)0.98 hydrogels was studied by placing 0.5 mL of hydrogel containing 1.0 mg L−1 of tenofovir in a dialysis membrane tube with MWCO of 1 kDa (Spectrum Laboratories, USA). This was then immersed in 40 mL of the release medium phosphatebuffered saline (PBS, pH 7.4). To investigate the effect of the antimicrobial polycarbonate on drug release, 2.5 mg L−1 of VD/BnCl (1:30) was added to the hydrogel prior to the gelation process. In order to compare the differences in release rates, tenofovir samples were also prepared in solution formulations. The samples were kept shaking on an orbital shaker at 100 rpm at 37 °C. At designated time intervals, 0.5 mL of the release medium was removed and replaced with fresh PBS. The collected medium was analyzed for its drug content via HPLC. The samples were analyzed under the following conditions: Column temperature 28 °C, sample temperature 15 °C, mobile phase: A – 50 mM KH2PO4, B – acetonitrile at 30%A/70%B; detection: 270 nm; flow rate: 0.8 mL min−1. Hemolysis Assay: Fresh mouse blood was obtained and diluted with PBS buffer to reach a concentration of approximately 4 vol% of the blood cells. 300 µL of PBS solution containing a polymer at various concentrations (15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 mg/L) was placed in a 1.5 mL microfuge tube, followed by the addition of an equal volume (300 µL) of red blood cell suspension. The mixture was incubated at 37 °C for 1 h to allow for the hemolysis to occur. At the end of the incubation, intact red blood cells were separated by centrifugation at 1000 rpm for 5 min. Aliquots (100 µL) of the supernatant were transferred to a 96-well plate, and the release of hemoglobin was measured by UV-absorbance at 576 nm using a microplate reader (TECAN, Switzerland). Two controls were provided in this assay: an untreated red blood cell suspension in PBS solution was used as the negative control; a solution containing red blood cells lysed with 1% Triton-X was used as the positive control. Each condition was performed in four replicates, and the data were expressed as means and standard deviations of the four replicates. Percentage of hemolysis was calculated using the following formula: Hemolysis (%) = [(O.D. 576 nm of the treated sample-O.D. 576 nm of the negative control)/(O.D. 576 nm of positive control- O.D. 576 nm of negative control)] × 100%.[24] Minimal Inhibitory Concentration (MIC) Measurements: Bacteria (S. aureus, E. coli) and fungi (C. albicans and C. neoformans) obtained from ATCC were reconstituted from their lyophilized form according to the manufacturer’s protocol. Bacterial samples were cultured in tryptic soy broth (TSB) at 37 °C, and fungi were cultured in yeast mannitol broth (YMB) at 25 °C under constant shaking of 100 rpm. The MICs of the polymers were measured using the broth microdilution method.[25] Briefly, 100 µL of the respect broth medium containing a polymer at various concentrations (15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 mg L−1) was placed into each well of a 96-well tissue culture plate. An equal volume of microbial suspension (3 × 105 CFU mL−1) was added into each well. Prior to mixing, the microbial sample was first inoculated overnight to enter its log growth phase. The concentration of microbial

solution was adjusted to give an initial optical density (O.D.) reading of approximately 0.07 at 600 nm wavelength on a microplate reader (TECAN, Switzerland), which corresponds to the concentration of Mc Farland 1 solution (3 × 108 CFU mL−1), the microbial solution was further diluted by 1000 times to achieve an initial loading of 3 × 105 CFU mL−1. The bacterial samples were kept in an incubator at 37 °C for 18 h, while the fungi samples were kept at room temperature for 42 h under constant shaking of 100 rpm. The MIC was taken as the concentration of the polymer at which no microbial growth was observed with unaided eyes and the microplate reader at the end of the respective incubation time. Broth containing microbial cells alone was used as negative control, and each test was carried out in six replicates. Killing Efficiency Tests: C. albicans were reconstituted from its lyophilized form according to the manufacturer’s protocol, and cultured in YMB at 25 °C under constant shaking of 55 rpm. Prior to treatment, the microbes were first inoculated overnight to enter into log growth phase. Cationic polycarbonate (VD/BnCl (1:30)-containing hydrogels were prepared using the triblock copolymer (MTC-VD)0.98-PEG(20k)(MTC-VD)0.98 (4 wt%) and varying concentrations of VD/BnCl (1:30). The concentration of microbe solution was adjusted to an equivalent of McFarland 1.0 solution (3 × 108 CFU mL−1) via the measurement of optical density (O.D.) reading at 600 nm wavelength on a microplate reader (TECAN, Switzerland), and then diluted by 1000 times. The microbe solution (50 µL) was placed in each well of a 96-well plate, to which 50 µL of the hydrogel was added. The culture plate was kept under constant shaking of 50 rpm for 24 h at room temperature. Subsequently, the microbial samples were subjected to a series of tenfold dilution, and plated onto agar plates. The plates were incubated at room temperature for 72 h. The number of colony-forming units (CFU) was then counted. The negative control was designated as the group of microbes treated with hydrogel without the cationic polycarbonates, and each test was carried out in three replicates. The minimum bioicidal concentration, MBC, was defined as the concentration of the cationic polycarbonate that gives a killing efficiency of >99.9%.[25] Biofilm Formation and Treatment: C. albicans was grown overnight in YMB at 25 °C and diluted to 3 × 105 CFU mL−1 prior to use. The cell suspension (100 µL) was then transferred into each well of 96-well plate and cultured for 7 d for biofilm formation.[25] The biofilms were washed with PBS once every day to remove the planktonic and loosely adhered cells before it was replaced with fresh medium. After that, the biofilm was incubated with 50 µL of hydrogel for 24 h and subsequently, biomass and XTT assays and SEM observation were performed. Biomass Assay: After treatment, the residual biomass was analyzed using crystal violet (CV) staining assay.[25] The spent medium and hydrogel was gently removed and the biofilm was washed with PBS to remove the planktonic cells. Fixation was carried out by treating the biofilms with 100 µL of methanol for 15 min. Crystal violet staining (0.1 w/v%, 100 µL) was added to the fixed biofilm and incubated for 10 min. Excess crystal violet was washed off thoroughly using water of HPLC grade. The remaining crystal violet bound to the biofilm was extracted using 200 µL of 33% glacial acetic acid. An aliquot of 150 µL was then taken from each well and transferred to a fresh 96-well plate. The absorbance was then measured at 570 nm using a microplate reader (Tecan, Switzerland) and the biomass of the remaining biofilm was expressed as a percentage of the control. Statistical analysis was carried out using the student’s t-test. P ≤ 0.05 was considered statistically significant. XTT Reduction Assay: XTT assay was used to quantify the cell viability of the biofilms after treatment.[25] The assay was carried out by measuring the mitochondrial enzyme activity of the cells based on the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]2H-tetrazolium hydroxide (XTT) to a water-soluble formazan in the metabolically active microbial cells. Briefly, XTT solution (1 mg mL−1) and menadione solution (0.4 mM) were dissolved separately using deionized (DI) water. Right before the assay, the two components were mixed together at a volume ratio of 5:1 (XTT:menadione). The spent medium was first removed and the biofilm was gently washed with PBS to remove the planktonic and loosely adhered cells. PBS (120 µL) containing XTTmenadione (14.4 µL) was then added to each well and incubated at



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www.MaterialsViews.com room temperature for 3 h. An aliquot of 100 µL from each well was then transferred to a fresh 96-well plate. The absorbance was then measured at 490 nm using the microplate reader (Tecan, Switzerland) and cell viability of the remaining biofilm was expressed as a percentage of the control group. Statistical analysis was carried out using the student’s ttest and P ≤ 0.05 was considered statistically significant. Field Emission-Scanning Electron Microscopy (FE-SEM): After treatment with the hydrogels, the biofilm was gently washed with PBS. Fixation as carried out by incubation with 4% formaldehyde for 30 min. The fixed biofilm was then washed with DI water to remove the formaldehyde and a series of ethanol washes (35%, 50%, 75%, 90%, 95%, and 100%) was carried out for dehydration of the samples. After the samples were dried, they were mounted onto copper tape and coated with platinum for SEM analysis under a field emission-scanning electron microscope (JEOL JSM-7400F, Japan).[25] Cytotoxicity Test: Human dermal fibroblasts (HDF) were seeded at a density of 20 × 103 cells per well onto a 96-well plate and incubated overnight at 37 °C. On the day of treatment, the medium was removed and 50 µL of colorless DMEM was added to each well.[25] Following this, 50 µL of the hydrogels containing different concentrations of cationic polycarbonates and/or tenofovir was added to the cells. The plate was then incubated for another 24 h at 37 °C. CellTitre-blue (Promega, USA) and DMEM were then mixed at a volume ratio of 2:5 and 100 µL of this mixture was then added to each well and the cells were left to incubate in the dark at 37 °C for 4 h. Untreated cells were used as the control. Subsequently, the absorbance at 570 nm was measured and the readings were expressed as a percentage of the cell viability of the control.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements A. L. Z. L. and V. W. L. N. contributed to the study equally. This work was supported by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore). Received: June 19, 2014 Revised: August 21, 2014 Published online:

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