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Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Bio-compatibility, drug release, antimicrobial activity, and biofilm model
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Journal of Microbiology, Immunology and Infection xxx (xxxx) xxx
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Original Article
Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Bio-compatibility, drug release, antimicrobial activity, and biofilm model Chun-Hsing Liao a,b, Chiang Sang Chen c,d, Yu-Chun Chen d,e, Ni-En Jiang d, Chui Jia Farn f, Yi-Shan Shen d,g, Ming-Lun Hsu h,**, Chih-Hung Chang d,i,* a
Department of Medicine, Yang-Ming University, Taiwan Division of Infectious Disease, Far Eastern Memorial Hospital, New Taipei City, Taiwan c Department of Material and Fiber, Oriental Institute of Technology, New Taipei City, Taiwan d Department of Orthopaedic Surgery, Far Eastern Memorial Hospital, New Taipei City, Taiwan e College of General Studies, Yuan Ze University, Taoyuan City, Taiwan f Department of Orthopaedic Surgery, National Taiwan University Hospital, Taiwan g Department of Biomedical Engineering, National Taiwan University, Taipei, Taiwan h School of Dentistry, National Yang-Ming University, Taipei, Taiwan i Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan, Taiwan b
Received 2 July 2019; received in revised form 8 August 2019; accepted 13 August 2019
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KEYWORDS Antibiotic hydrogel; Vancomycin; Biofilm
Abstract Background: Prosthesis infection is a difficult-to-treat situation. Hydrogel is a novel biomaterial, which can be applied by simply spraying or by coating on implants before surgery and can be easily mixed with antibiotics. Methods: In order to evaluate the potential use of antibiotic-loaded hydrogel, we incorporated vancomycin into oxidized hyaluronic acid (HA) and adipic acid dihydrazide and evaluated the drug release and antimicrobial activity against methicillin-resistant Staphylococcus aureus (ATCC 29213). Results: The average release percentage of vancomycin on day 3 was about 86%. The antibiotic-loaded gel was biocompatible with mesenchymal stem cell, MC3T3, and L929 cell lines. The in vitro inhibition zones of vancomycin-loaded hydrogel [500X minimal inhibition concentration (MIC), 50X MIC, 10X MIC, and blank hydrogel] were 21, 13, 9, and 5 mm,
* Corresponding author. Department of Orthopaedics, Far Eastern Memorial Hospital, No.21, Sec. 2, Nanya S. Rd., Banciao Dist., New Taipei City, 220, Taiwan. ** Corresponding author. School of Dentistry, National Yang-Ming University, No.155, Sec. 2, Linong St., Taipei City, 112, Taiwan. E-mail addresses: [email protected] (M.-L. Hsu), [email protected] (C.-H. Chang). https://doi.org/10.1016/j.jmii.2019.08.008 1684-1182/Copyright ª 2019, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article as: Liao C-H et al., Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Biocompatibility, drug release, antimicrobial activity, and biofilm model, Journal of Microbiology, Immunology and Infection, https:// doi.org/10.1016/j.jmii.2019.08.008
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C.-H. Liao et al. respectively. In the Ti6Al4V implant biofilm model, 0.01e1% vancomycin-loaded gel exhibited significant anti-biofilm activity, measured by the MTT assay. Conclusions: Vancomycin could be loaded onto oxidized HA and adipic acid dihydrazide, which exhibited excellent drug release and in vitro antimicrobial activity with minimal cell toxicity. Copyright ª 2019, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
Introduction Surgical site infection (SSI) is one of the most common healthcare-associated infection. The US Centers for Disease Control and Prevention estimates that 500,000 surgical site infections occur annually and account for 3% of surgical mortality, prolonged length of hospital stay, and increased medical costs.1 Previous studies have predicted that the cost increases from £814 to £6626 ($1341 10,922) per patient in England,2 and $25,546 per infection in the United States.3 When a prosthesis is implanted, "race to the surface" phenomenon is exhibited in the host cells and bacteria.4e6 Unlike immune cells, bacteria have a faster reproductive rate and extreme environmental adaptation ability, and thus, biofilm could form immediately after a few hours of implantation. Once a biofilm has formed, it is not easily penetrable by the innate immune system or any antibiotics. Prosthesis-related infection is also usually associated with compromised soft tissue condition and poor antibiotic penetration due to venous thrombosis or vessel destruction. Secondary surgical removal is usually necessary to reduce the possibility of becoming another source of infection. Antibiotics delivered locally by a carrier could overcome this problem and may help in reaching a therapeutic level. For all these reasons, the development of an antibioticloaded hydrogel for coating implantable biomaterials is needed. It can prevent adhesion, colonization, and formation of biofilm.7,8 The most commonly used antibiotic carrier is poly-methyl methacrylate (PMMA), which is poorly absorbed, radio-opaque, and can interfere with radiographic interpretations. Thus, development of an absorbable or degradable antibiotic carrier is needed. Vancomycin is one of the major antimicrobial agents for SSI treatment due to the emergence of methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative Staphylococcus-related infections.9e12 However, vancomycin treatment failure is not uncommon, especially when there is foreign body-related biofilm formation. There are limited in vitro tests and models for the efficacy of vancomycin-loaded hydrogel and biofilm-related infection.13,14 Hyaluronic acid (HA) is made up of D-glucuronic acid and N-acetyl-D- glucosamine. It has been used as a hydrophilic polymer to coat on polyurethane catheters and can reduce the adherence of Staphylococcus epidermidis.15 Surfaces coated with sulfated HA have shown a marked reduction in bacterial growth as compared to uncoated surfaces.16 Furthermore, HA is biocompatible because its chemical
structure has been identified in all species including bacteria, animals, and human. In a previous study, we described an injectable hydrogel developed by oxidized HA and adipic acid dihydrazide (oxi-HA/ADH) for intervertebral disc regeneration.17 This kind of combination could be gelled within a few minutes at room temperature and forms a thin layer on the implant’s surface. In order to develop a hydrogel for coating the surfaces of orthopedic implants to reduce subsequent bacterial infection, we tested oxi-HA/ ADH hydrogel-loaded with vancomycin for its biocompatibility, drug release, in vitro antimicrobial activity, and its effectiveness in a biofilm model.
Material and methods Parameter optimization for the antibiotic hydrogel preparation The antibiotic-loaded hydrogel was prepared by mixing oxidized HA (oxi-HA), adipic acid dihydrazide (ADH), and antibiotics. HA (1% w/v) was dissolved in double-distilled water and then sodium periodate (2.67% w/v) was added with stirring. The oxidation reaction proceeded in a dark environment for 24 h at room temperature. The reaction was stopped by the addition of ethylene glycol (0.5 ml). A dialysis tube was used to separate the byproduct and oxiHA. Double-distilled water was used as a dialysis buffer solution, and water was changed three times a day for four days. Silver nitrate (1%) was used to check the amount of periodate in the outer dialysis buffer. Water was changed until there was no precipitate. The final oxidized HA product was obtained by freeze-drying. Before hydrogel preparation, oxi-HA and ADH were dissolved in normal saline individually. The hydrogel could be easily prepared from mixing oxi-HA, ADH, and antibiotics by gentle pipetting and was transformed from solution type to gel type within 3e5 min. Various concentrations of oxi-HA and ADH were mixed together to obtain the optimal parameters of antibiotic hydrogel preparation.
Degradation time of antibiotic-loaded hydrogel The liquid-state antibiotic-loaded hydrogel solution was introduced into the cylinder mold and allowed to set for 10 min to form a gel-like matrix. After transferring the cylinder of antibiotic-loaded hydrogel into a 24-well culture plate, 3 ml of PBS was added to each well. At Day 1, 3, 7, 14, 21 and 28, the hydrogel was removed, and
Please cite this article as: Liao C-H et al., Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Biocompatibility, drug release, antimicrobial activity, and biofilm model, Journal of Microbiology, Immunology and Infection, https:// doi.org/10.1016/j.jmii.2019.08.008
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Vancomycin-loaded HA and dihydrazide hydrogel lyophilization of the hydrogel was carried out using a freeze-drying method to obtain the dry weight (Wd). The degradation percentage was calculated using the formula (Wd - Wi)/Wi 100%, where Wi is the initial weight of hydrogel (Wi) on day 0. All experiments are performed in triplicate.
Drug release experiments Vancomycin (1%) was added into the hydrogel for antibiotic release test. Antibiotics-containing hydrogel (0.2 mL) was then immersed in a vial containing 6 mL of PBS and incubated at 37 C without shake. PBS was changed and collected at regular time intervals (1, 2, 3, 6, 24, 48, 72, and 168 h). The collected PBS samples were analyzed by high-performance liquid chromatography (HPLC) to estimate the concentration of the released antibiotics. For vancomycin, the mobile phase was phosphate buffer (pH, 4.2) and methanol (70:30). A C8 column was used with at a flow rate 1 mL/min. The detector was set to monitor the wavelength at 298 nm.
3 specimens were made into round shapes, the ZOI was represented as diameter in this study.
Antimicrobial activity in the biofilm model We prepared the biofilm by immersing Ti6Al4V implant having a diameter of 16 mm into the MRSA ATCC 33592 containing medium with an initial bacterial density of 12X106 CFU/ml for 24 h.18 After several washes, the biofilm containing Ti6Al4V implants were removed to the wells with various vancomycin-loaded hydrogel groups for anti-biofilm formation ability test. The concentration of vancomycin within the hydrogel was 0.01e1%. MTT assay was performed on day 1 and 3,19 and the absorbance was recorded at 570 nm for the evaluation of MRSA proliferation.
Statistical analysis All the experiments were performed in triplicate. Means and standard deviation were calculated and t-test was used for p value. Data were analyzed with EXCEL for Windows.
Cell-compatibility of antibiotic-loaded hydrogel
Results
Cell-compatibility was tested by the extraction medium method. Briefly, the extraction medium was prepared by incubating the antibiotic-loaded hydrogel with a standard culture medium for 72 h at 37 C. The extraction medium (200 mL) was transferred on a monolayer of cells in 96-well plates and cultured for 1 and 3 days at 37 C in the presence of 5% CO2 to evaluate the cell viability and cytotoxic effects on the L929, MC3T3 and mesenchymal stem cells. For cellcompatibility evaluation, water-soluble tetrazolium-8 (WST-8) assay was used to evaluate the cell viability. Briefly, 0.2 ml of water-soluble tetrazolium-8 (WST-8) working solution was transferred to each well. After 2 h incubation, the WST-8 working solution should show color change due to cleavage of the tetrazolium salt forming formazan by a cellular mitochondrial dehydrogenase. NP cell viability was quantitatively assessed by a spectrophotometer at 450 nm. The reference wavelength was 650 nm.
The release curve of vancomycin-containing HA hydrogels is shown in Fig. 1. As HA was gradually dissolved in the PBS environment, the release rate of vancomycin in the hydrogel was little faster than expected, nearly 4/5th of the antibiotic was released into the PBS on day 3. The average release percentage of vancomycin on day 3 was about 86%. The rate of HA gel degradation was about 20% on day 1, 30% on day 7, 40% on day 14, and complete degradation on day 21. For the biocompatibility assay, the cell viability was evaluated by WST-1 in the presence of the antibioticloaded hydrogel (Fig. 2AeC). HA gel hydrogel slightly affected mesenchymal stem cell (MSC) and MC3T3, but not L929. As for the effect of vancomycin, there was no significant difference between the HA hydrogel and HA hydrogel containing vancomycin groups. Even HA hydrogel containing 1% vancomycin carries little toxicity across three cell types. Most of the cells died in the negative control group. To test the antimicrobial activity of vancomycin-loaded hydrogel, we added 500X, 50X, and 10X minimum inhibitory concentration (MIC) of antibiotics inside, and called them HA hydrogel-Van1 (hydrogel with 500X MIC vancomycin, 625 mg/ml), HA hydrogel-Van2 (hydrogel with 50X MIC vancomycin, 62.5 mg/ml), HA hydrogel-Van3 (hydrogel with 10X MIC vancomycin, 12.5 mg/ml). Fig. 3 shows the results of the antimicrobial activity of vancomycin containing hydrogel. The average zone of inhibition of HA HydrogelVan1, -Van2, -Van3, and HA Hydrogel was about 21.4 0.7, 13.1 0.2, 9.4 0.7, and 5.7 0.6 mm after overnight culture, respectively. The zone diameter did not change on day 3 and day 7. In the MRSA contaminated Ti6Al4V implant model, HA hydrogel with different concentration of vancomycin (0.01e1%) showed excellent activity in reducing the number of MRSA cells as demonstrated by MTT assay (Fig. 4). The absorbance of all vancomycin containing hydrogel
Antimicrobial activity of vancomycin-loaded hydrogel The antimicrobial activity of the hydrogel was tested against methicillin-resistant S. aureus (MRSA), ATCC 33592 (vancomycin MIC, 0.5e2 mg/L). The bacterial suspension was cultured overnight in an adequate amount of broth to yield a slightly cloudy solution, and the turbidity was adjusted to McFarland 0.5e1.0 before use. Bacteria were inoculated on the dried surface of Mueller-Hinton agar plates by streaking with a swab, which allowed the bacterial suspension to be absorbed over the entire sterile agar surface. Five disks from each experimental group were gently placed on the aforementioned microbial plates. The plates were incubated at 37 C in a fully humidified atmosphere. After each 24-h period, the zone of inhibition (ZOI) was photographed and measured, and the disks were moved to fresh plates that were prepared in the same fashion. The plates were observed for 72 h. As the
Please cite this article as: Liao C-H et al., Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Biocompatibility, drug release, antimicrobial activity, and biofilm model, Journal of Microbiology, Immunology and Infection, https:// doi.org/10.1016/j.jmii.2019.08.008
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Figure 2. AeC. The cell viability evaluated by WST-1 in the presence of the different concentrations of vancomycin-loaded hydrogel. A: Mesenchymal stem cell (MSC), B: MC3T3, C: L929. HA Hydrogel 0% vancomycin, HA Hydrogel e Van1 0.01% vancomycin, HA Hydrogel e Van2 0.1% vancomycin, HA Hydrogel e Van3 0.5% vancomycin, HA Hydrogel e Van4 1% vancomycin. * indicate p < 0.05 compared with positive control group; # indicate p < 0.05 compared with HA hydrogel group.
Please cite this article as: Liao C-H et al., Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Biocompatibility, drug release, antimicrobial activity, and biofilm model, Journal of Microbiology, Immunology and Infection, https:// doi.org/10.1016/j.jmii.2019.08.008
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Figure 4. MRSA contaminated Ti6Al4V implant model, HA hydrogel with different concentration of vancomycin (0.01e1%), evaluated by MTT assay. * indicate p < 0.05 compared with Hydrogel group; # indicate p < 0.05 compared with No hydrogel group.
groups at 570 nm was lower than that of 0% vancomycin hydrogel group (p < 0.05). The McFarland value of 0% vancomycin hydrogel was similar to that of the group without containing hydrogel.
Discussion In this study, we demonstrated that vancomycin could be loaded into oxidized HA and adipic acid dihydrazide. The vancomycin-containing HA hydrogels exhibited excellent drug release and in vitro antimicrobial activity with minimal cell toxicity. Hydrogel is a novel material that can be mixed with antibiotics and applied easily to either wound or
prosthesis surface.13 The compositions of different types of hydrogel vary and each component has its own characteristic feature. For example, dextran-PEG hydrogel had been mixed with polymyxin B and vancomycin designed for wound dressing, PEG-based monomer coupling PEG with vancomycin, and chitosan/glycerophosphate thermosensitive hydrogel with microparticles containing vancomycin for local antibiotics delivery use.20e22 The price of HA is higher than that of dextran, PEG, and chitosan, but HA is a natural biomolecule, which is distributed widely in the connective, epithelial, and neural tissues. It can be degraded by hyaluronidases, and hence, it is biocompatible and biodegradable and more suitable material for antibiotic hydrogel preparation.
Please cite this article as: Liao C-H et al., Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Biocompatibility, drug release, antimicrobial activity, and biofilm model, Journal of Microbiology, Immunology and Infection, https:// doi.org/10.1016/j.jmii.2019.08.008
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6 The advantage of oxi-HA/ADH gel is that it could be gelled within a few minutes at room temperature, and forms a thin layer on the surface of the implant. In this study, the release of vancomycin from the gel was rapid and the gel degraded completely in 3 weeks under in vitro condition, and this would be much faster in the human body. The target rate of drug release depends on the purpose of the gel. If prevention of immediate infection is the purpose, early and rapid release might be the best design. Furthermore, osteointegration into prosthesis will not be affected since its degradation happens within weeks. On the contrary, for treatment of established infection, such as osteomyelitis, a prolonged release might be more appropriate. Currently, the release rate of the drug from the different gels is manageable by adding microparticles,22 charged copolymers,14 or adjusting of pH of the gel.23 Moreover, Lakes et al. showed that vancomycin release and material degradation were tunable via hydrophobic/hydrophilic content of the hydrogel matrix.21 There is no standard test to assess the efficacy of antibiotic-loaded gels. In this study, we used two models to examine the in vitro efficacy of the gel. Due to the plasticity of the gel, we first apply traditional disc diffusion method by molding vancomycin-loaded oxi-HA/ADH gel as the size of the standard disc and test antimicrobial activity of the disc.24 After overnight culture, the zone of inhibition of the hydrogel was easily observable. There was a dosedependent effect of vancomycin load gel as 500X MIC vancomycin hydrogel had an inhibition zone of 21 mm. Even a blank gel had an inhibition zone of 5 mm. HA is reported to have some anti-bacterial and fungal activities.25 The standard vancomycin paper disc (30 mg) used in the clinical microbiology lab could have an inhibition zone over 15 mm against MRSA, while our hydrogel with a 50X MIC of vancomycin (62.5 mg/ml) had an inhibition zone of 13 mm, which is quite comparable. This methodology could be easily applied to other kinds of hydrogels or drugs adding into hydrogels. The second in vitro test was a biofilm model modified from the study of Drago et al.18 In our MRSA-contaminated Ti6Al4V implant model, HA hydrogel with different concentration of vancomycin (0.01e1%) showed excellent activity in reducing the number of MRSA cells as measured by MTT assay. The results suggest that rapid releasing vancomycin-loaded oxi-HA/ADH gel has good effects on the surface-contaminated biofilm model even with a low concentration of vancomycin. The real challenge lies with deep and complicated infection, such as nails used in open contaminated wound. In an animal study, Giavaresi et al. showed that vancomycin-loaded hydrogel coating can reduce bacterial load from 72% to 99% on the prosthesis.26 Adding antibiotic into cement is a common practice nowadays to reduce subsequent prosthesis infection.24 Drug release from cement is generally slower than from hydrogel. A study with cement and calcium polyphosphate hydrogel combination showed that adding 10% CPP gel to cement led to a much lower burst release of vancomycin and extended duration of drug release.27 However, the degradation nature of HA gel makes it an unsuitable candidate for this type of design. On the other hand, some surgeons use antimicrobial powder directly while performing orthopedic surgery. It is not feasible to evaluate the
C.-H. Liao et al. pharmacodynamics and toxicity of drugs by this kind of approach. To sum up, oxi-HA/ADH hydrogel-loaded with vancomycin has an excellent bio-compatibility, rapid and complete drug release, and measurable in vitro antimicrobial activity and is quite effective in a biofilm model to prevent immediate bacterial colonization. With its bio-compatibility, 1% vancomycin-loaded gel can be used since high vancomycin concentration carries higher inhibition in the in vitro model.
Declaration of Competing Interest None.
Acknowledgements We would like to thank the Ministry of Science and Technology, Taiwan (MOST-105-2221-E-418-001, MOST-106-2221E-418-001), Far Eastern Memorial Hospital, Taiwan (FEMH2016-C-005, 105FN14) and AOTRAUMA (AOTAP16-08) for financial support. The Far Eastern Memorial Hospital Core Laboratories I & II for providing facilities and instruments.
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Vancomycin-loaded HA and dihydrazide hydrogel 11. Stevens DL. The role of vancomycin in the treatment paradigm. Clin Infect Dis 2006;1:S51e7. 12. Darouiche RO. Treatment of infections associated with surgical implants. N Engl J Med 2004;350:1422e9. 13. Pitarresi G, Palumbo FS, Calascibetta F, Fiorica C, Di Stefano M, Giammona G. Medicated hydrogels of hyaluronic acid derivatives for use in orthopedic field. Int J Pharm 2013;449:84e94. 14. Gustafson CT, Boakye-Agyeman F, Brinkman CL, Reid JM, Patel R, Bajzer Z, et al. Controlled delivery of vancomycin via charged hydrogels. PLoS One 2016;11:e0146401. 15. Pavesio A, Renier D, Cassinelli C, Morra M. Anti-adhesive surfaces through hyaluronan coatings. Med Device Technol 1997; 8:20e1. 16. Hoekstra D. Hyaluronan modified surfaces for medical devices. Med Device Diagn Ind 1999:48e58. 17. Su WY, Chen YC, Lin FH. Injectable oxidized hyaluronic acid/adipic acid dihydrazide hydrogel for nucleus pulposus regeneration. Acta Biomater 2010;6:3044e55. 18. Drago L, Boot W, Dimas K, Malizos K, Ha ¨nsch GM, Stuyck J, et al. Does implant coating with antibacterial-loaded hydrogel reduce bacterial colonization and biofilm formation in vitro? Clin Orthop Relat Res 2014;472:3311e23. 19. Grela E, Kozłowska J, Grabowiecka A. Current methodology of MTT assay in bacteria e a review. Acta Histochem 2018;120:303e11. 20. Wang L, Li X, Sun T, Tsou YH, Chen H, Xu X. Dual-functional Dextran-PEG hydrogel as an antimicrobial biomedical material. Macromol Biosci 2018;18. https://doi.org/10.1002/mabi. 201700325.
7 21. Lakes AL, Peyyala R, Ebersole JL, Puleo DA, Hilt JZ, Dziubla TD. Synthesis and characterization of an antibacterial hydrogel containing covalently bound vancomycin. Biomacromolecules 2014;15:3009e18. 22. Mahmoudian M, Ganji F. Vancomycin-loaded HPMC microparticles embedded within injectable thermosensitive chitosan hydrogels. Prog Biomater 2017;6:49e56. 23. Hoque J, Bhattacharjee B, Prakash RG, Paramanandham K, Haldar J. Dual function injectable hydrogel for controlled release of antibiotic and local antibacterial therapy. Biomacromolecules 2018;19:267e78. 24. Hsu YM, Liao CH, Wei YH, Fang HW, Hou HH, Chen CC, et al. Daptomycin-loaded polymethylmethacrylate bone cement for joint arthroplasty surgery. Artif Organs 2014;38:484e92. 25. Ardizzoni A, Neglia RG, Baschieri MC, Cermelli C, Caratozzolo M, Righi E, et al. Influence of hyaluronic acid on bacterial and fungal species, including clinically relevant opportunistic pathogens. J Mater Sci Mater Med 2011;22: 2329e38. 26. Giavaresi G, Meani E, Sartori M, Ferrari A, Bellini D, Sacchetta AC, et al. Efficacy of antibacterial-loaded coating in an in vivo model of acutely highly contaminated implant. Int Orthop 2014;38:1505e12. 27. Zhou Z, Seta J, Markel DC, Song W, Yurgelevic SM, Yu XW, et al. Release of vancomycin and tobramycin from polymethylmethacrylate cements impregnated with calcium polyphosphate hydrogel. J Biomed Mater Res B Appl Biomater 2018;106:2827e40.
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Original Article
Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Bio-compatibility, drug release, antimicrobial activity, and biofilm model Chun-Hsing Liao a,b, Chiang Sang Chen c,d, Yu-Chun Chen d,e, Ni-En Jiang d, Chui Jia Farn f, Yi-Shan Shen d,g, Ming-Lun Hsu h,**, Chih-Hung Chang d,i,* a
Department of Medicine, Yang-Ming University, Taiwan Division of Infectious Disease, Far Eastern Memorial Hospital, New Taipei City, Taiwan c Department of Material and Fiber, Oriental Institute of Technology, New Taipei City, Taiwan d Department of Orthopaedic Surgery, Far Eastern Memorial Hospital, New Taipei City, Taiwan e College of General Studies, Yuan Ze University, Taoyuan City, Taiwan f Department of Orthopaedic Surgery, National Taiwan University Hospital, Taiwan g Department of Biomedical Engineering, National Taiwan University, Taipei, Taiwan h School of Dentistry, National Yang-Ming University, Taipei, Taiwan i Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan, Taiwan b
Received 2 July 2019; received in revised form 8 August 2019; accepted 13 August 2019
Available online - - -
KEYWORDS Antibiotic hydrogel; Vancomycin; Biofilm
Abstract Background: Prosthesis infection is a difficult-to-treat situation. Hydrogel is a novel biomaterial, which can be applied by simply spraying or by coating on implants before surgery and can be easily mixed with antibiotics. Methods: In order to evaluate the potential use of antibiotic-loaded hydrogel, we incorporated vancomycin into oxidized hyaluronic acid (HA) and adipic acid dihydrazide and evaluated the drug release and antimicrobial activity against methicillin-resistant Staphylococcus aureus (ATCC 29213). Results: The average release percentage of vancomycin on day 3 was about 86%. The antibiotic-loaded gel was biocompatible with mesenchymal stem cell, MC3T3, and L929 cell lines. The in vitro inhibition zones of vancomycin-loaded hydrogel [500X minimal inhibition concentration (MIC), 50X MIC, 10X MIC, and blank hydrogel] were 21, 13, 9, and 5 mm,
* Corresponding author. Department of Orthopaedics, Far Eastern Memorial Hospital, No.21, Sec. 2, Nanya S. Rd., Banciao Dist., New Taipei City, 220, Taiwan. ** Corresponding author. School of Dentistry, National Yang-Ming University, No.155, Sec. 2, Linong St., Taipei City, 112, Taiwan. E-mail addresses: [email protected] (M.-L. Hsu), [email protected] (C.-H. Chang). https://doi.org/10.1016/j.jmii.2019.08.008 1684-1182/Copyright ª 2019, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article as: Liao C-H et al., Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Biocompatibility, drug release, antimicrobial activity, and biofilm model, Journal of Microbiology, Immunology and Infection, https:// doi.org/10.1016/j.jmii.2019.08.008
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C.-H. Liao et al. respectively. In the Ti6Al4V implant biofilm model, 0.01e1% vancomycin-loaded gel exhibited significant anti-biofilm activity, measured by the MTT assay. Conclusions: Vancomycin could be loaded onto oxidized HA and adipic acid dihydrazide, which exhibited excellent drug release and in vitro antimicrobial activity with minimal cell toxicity. Copyright ª 2019, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
Introduction Surgical site infection (SSI) is one of the most common healthcare-associated infection. The US Centers for Disease Control and Prevention estimates that 500,000 surgical site infections occur annually and account for 3% of surgical mortality, prolonged length of hospital stay, and increased medical costs.1 Previous studies have predicted that the cost increases from £814 to £6626 ($1341 10,922) per patient in England,2 and $25,546 per infection in the United States.3 When a prosthesis is implanted, "race to the surface" phenomenon is exhibited in the host cells and bacteria.4e6 Unlike immune cells, bacteria have a faster reproductive rate and extreme environmental adaptation ability, and thus, biofilm could form immediately after a few hours of implantation. Once a biofilm has formed, it is not easily penetrable by the innate immune system or any antibiotics. Prosthesis-related infection is also usually associated with compromised soft tissue condition and poor antibiotic penetration due to venous thrombosis or vessel destruction. Secondary surgical removal is usually necessary to reduce the possibility of becoming another source of infection. Antibiotics delivered locally by a carrier could overcome this problem and may help in reaching a therapeutic level. For all these reasons, the development of an antibioticloaded hydrogel for coating implantable biomaterials is needed. It can prevent adhesion, colonization, and formation of biofilm.7,8 The most commonly used antibiotic carrier is poly-methyl methacrylate (PMMA), which is poorly absorbed, radio-opaque, and can interfere with radiographic interpretations. Thus, development of an absorbable or degradable antibiotic carrier is needed. Vancomycin is one of the major antimicrobial agents for SSI treatment due to the emergence of methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative Staphylococcus-related infections.9e12 However, vancomycin treatment failure is not uncommon, especially when there is foreign body-related biofilm formation. There are limited in vitro tests and models for the efficacy of vancomycin-loaded hydrogel and biofilm-related infection.13,14 Hyaluronic acid (HA) is made up of D-glucuronic acid and N-acetyl-D- glucosamine. It has been used as a hydrophilic polymer to coat on polyurethane catheters and can reduce the adherence of Staphylococcus epidermidis.15 Surfaces coated with sulfated HA have shown a marked reduction in bacterial growth as compared to uncoated surfaces.16 Furthermore, HA is biocompatible because its chemical
structure has been identified in all species including bacteria, animals, and human. In a previous study, we described an injectable hydrogel developed by oxidized HA and adipic acid dihydrazide (oxi-HA/ADH) for intervertebral disc regeneration.17 This kind of combination could be gelled within a few minutes at room temperature and forms a thin layer on the implant’s surface. In order to develop a hydrogel for coating the surfaces of orthopedic implants to reduce subsequent bacterial infection, we tested oxi-HA/ ADH hydrogel-loaded with vancomycin for its biocompatibility, drug release, in vitro antimicrobial activity, and its effectiveness in a biofilm model.
Material and methods Parameter optimization for the antibiotic hydrogel preparation The antibiotic-loaded hydrogel was prepared by mixing oxidized HA (oxi-HA), adipic acid dihydrazide (ADH), and antibiotics. HA (1% w/v) was dissolved in double-distilled water and then sodium periodate (2.67% w/v) was added with stirring. The oxidation reaction proceeded in a dark environment for 24 h at room temperature. The reaction was stopped by the addition of ethylene glycol (0.5 ml). A dialysis tube was used to separate the byproduct and oxiHA. Double-distilled water was used as a dialysis buffer solution, and water was changed three times a day for four days. Silver nitrate (1%) was used to check the amount of periodate in the outer dialysis buffer. Water was changed until there was no precipitate. The final oxidized HA product was obtained by freeze-drying. Before hydrogel preparation, oxi-HA and ADH were dissolved in normal saline individually. The hydrogel could be easily prepared from mixing oxi-HA, ADH, and antibiotics by gentle pipetting and was transformed from solution type to gel type within 3e5 min. Various concentrations of oxi-HA and ADH were mixed together to obtain the optimal parameters of antibiotic hydrogel preparation.
Degradation time of antibiotic-loaded hydrogel The liquid-state antibiotic-loaded hydrogel solution was introduced into the cylinder mold and allowed to set for 10 min to form a gel-like matrix. After transferring the cylinder of antibiotic-loaded hydrogel into a 24-well culture plate, 3 ml of PBS was added to each well. At Day 1, 3, 7, 14, 21 and 28, the hydrogel was removed, and
Please cite this article as: Liao C-H et al., Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Biocompatibility, drug release, antimicrobial activity, and biofilm model, Journal of Microbiology, Immunology and Infection, https:// doi.org/10.1016/j.jmii.2019.08.008
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Vancomycin-loaded HA and dihydrazide hydrogel lyophilization of the hydrogel was carried out using a freeze-drying method to obtain the dry weight (Wd). The degradation percentage was calculated using the formula (Wd - Wi)/Wi 100%, where Wi is the initial weight of hydrogel (Wi) on day 0. All experiments are performed in triplicate.
Drug release experiments Vancomycin (1%) was added into the hydrogel for antibiotic release test. Antibiotics-containing hydrogel (0.2 mL) was then immersed in a vial containing 6 mL of PBS and incubated at 37 C without shake. PBS was changed and collected at regular time intervals (1, 2, 3, 6, 24, 48, 72, and 168 h). The collected PBS samples were analyzed by high-performance liquid chromatography (HPLC) to estimate the concentration of the released antibiotics. For vancomycin, the mobile phase was phosphate buffer (pH, 4.2) and methanol (70:30). A C8 column was used with at a flow rate 1 mL/min. The detector was set to monitor the wavelength at 298 nm.
3 specimens were made into round shapes, the ZOI was represented as diameter in this study.
Antimicrobial activity in the biofilm model We prepared the biofilm by immersing Ti6Al4V implant having a diameter of 16 mm into the MRSA ATCC 33592 containing medium with an initial bacterial density of 12X106 CFU/ml for 24 h.18 After several washes, the biofilm containing Ti6Al4V implants were removed to the wells with various vancomycin-loaded hydrogel groups for anti-biofilm formation ability test. The concentration of vancomycin within the hydrogel was 0.01e1%. MTT assay was performed on day 1 and 3,19 and the absorbance was recorded at 570 nm for the evaluation of MRSA proliferation.
Statistical analysis All the experiments were performed in triplicate. Means and standard deviation were calculated and t-test was used for p value. Data were analyzed with EXCEL for Windows.
Cell-compatibility of antibiotic-loaded hydrogel
Results
Cell-compatibility was tested by the extraction medium method. Briefly, the extraction medium was prepared by incubating the antibiotic-loaded hydrogel with a standard culture medium for 72 h at 37 C. The extraction medium (200 mL) was transferred on a monolayer of cells in 96-well plates and cultured for 1 and 3 days at 37 C in the presence of 5% CO2 to evaluate the cell viability and cytotoxic effects on the L929, MC3T3 and mesenchymal stem cells. For cellcompatibility evaluation, water-soluble tetrazolium-8 (WST-8) assay was used to evaluate the cell viability. Briefly, 0.2 ml of water-soluble tetrazolium-8 (WST-8) working solution was transferred to each well. After 2 h incubation, the WST-8 working solution should show color change due to cleavage of the tetrazolium salt forming formazan by a cellular mitochondrial dehydrogenase. NP cell viability was quantitatively assessed by a spectrophotometer at 450 nm. The reference wavelength was 650 nm.
The release curve of vancomycin-containing HA hydrogels is shown in Fig. 1. As HA was gradually dissolved in the PBS environment, the release rate of vancomycin in the hydrogel was little faster than expected, nearly 4/5th of the antibiotic was released into the PBS on day 3. The average release percentage of vancomycin on day 3 was about 86%. The rate of HA gel degradation was about 20% on day 1, 30% on day 7, 40% on day 14, and complete degradation on day 21. For the biocompatibility assay, the cell viability was evaluated by WST-1 in the presence of the antibioticloaded hydrogel (Fig. 2AeC). HA gel hydrogel slightly affected mesenchymal stem cell (MSC) and MC3T3, but not L929. As for the effect of vancomycin, there was no significant difference between the HA hydrogel and HA hydrogel containing vancomycin groups. Even HA hydrogel containing 1% vancomycin carries little toxicity across three cell types. Most of the cells died in the negative control group. To test the antimicrobial activity of vancomycin-loaded hydrogel, we added 500X, 50X, and 10X minimum inhibitory concentration (MIC) of antibiotics inside, and called them HA hydrogel-Van1 (hydrogel with 500X MIC vancomycin, 625 mg/ml), HA hydrogel-Van2 (hydrogel with 50X MIC vancomycin, 62.5 mg/ml), HA hydrogel-Van3 (hydrogel with 10X MIC vancomycin, 12.5 mg/ml). Fig. 3 shows the results of the antimicrobial activity of vancomycin containing hydrogel. The average zone of inhibition of HA HydrogelVan1, -Van2, -Van3, and HA Hydrogel was about 21.4 0.7, 13.1 0.2, 9.4 0.7, and 5.7 0.6 mm after overnight culture, respectively. The zone diameter did not change on day 3 and day 7. In the MRSA contaminated Ti6Al4V implant model, HA hydrogel with different concentration of vancomycin (0.01e1%) showed excellent activity in reducing the number of MRSA cells as demonstrated by MTT assay (Fig. 4). The absorbance of all vancomycin containing hydrogel
Antimicrobial activity of vancomycin-loaded hydrogel The antimicrobial activity of the hydrogel was tested against methicillin-resistant S. aureus (MRSA), ATCC 33592 (vancomycin MIC, 0.5e2 mg/L). The bacterial suspension was cultured overnight in an adequate amount of broth to yield a slightly cloudy solution, and the turbidity was adjusted to McFarland 0.5e1.0 before use. Bacteria were inoculated on the dried surface of Mueller-Hinton agar plates by streaking with a swab, which allowed the bacterial suspension to be absorbed over the entire sterile agar surface. Five disks from each experimental group were gently placed on the aforementioned microbial plates. The plates were incubated at 37 C in a fully humidified atmosphere. After each 24-h period, the zone of inhibition (ZOI) was photographed and measured, and the disks were moved to fresh plates that were prepared in the same fashion. The plates were observed for 72 h. As the
Please cite this article as: Liao C-H et al., Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Biocompatibility, drug release, antimicrobial activity, and biofilm model, Journal of Microbiology, Immunology and Infection, https:// doi.org/10.1016/j.jmii.2019.08.008
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Figure 2. AeC. The cell viability evaluated by WST-1 in the presence of the different concentrations of vancomycin-loaded hydrogel. A: Mesenchymal stem cell (MSC), B: MC3T3, C: L929. HA Hydrogel 0% vancomycin, HA Hydrogel e Van1 0.01% vancomycin, HA Hydrogel e Van2 0.1% vancomycin, HA Hydrogel e Van3 0.5% vancomycin, HA Hydrogel e Van4 1% vancomycin. * indicate p < 0.05 compared with positive control group; # indicate p < 0.05 compared with HA hydrogel group.
Please cite this article as: Liao C-H et al., Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Biocompatibility, drug release, antimicrobial activity, and biofilm model, Journal of Microbiology, Immunology and Infection, https:// doi.org/10.1016/j.jmii.2019.08.008
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Figure 3. Antimicrobial activity of vancomycin containing hydrogel evaluated by diffusion disc method. HA hydrogel-Van1 (hydrogel with 500X MIC vancomycin, 625 mg/ml), HA hydrogel-Van2 (hydrogel with 50X MIC vancomycin, 62.5 mg/ml), HA hydrogel-Van3 (hydrogel with 10X MIC vancomycin, 12.5 mg/ml). 2.5
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Figure 4. MRSA contaminated Ti6Al4V implant model, HA hydrogel with different concentration of vancomycin (0.01e1%), evaluated by MTT assay. * indicate p < 0.05 compared with Hydrogel group; # indicate p < 0.05 compared with No hydrogel group.
groups at 570 nm was lower than that of 0% vancomycin hydrogel group (p < 0.05). The McFarland value of 0% vancomycin hydrogel was similar to that of the group without containing hydrogel.
Discussion In this study, we demonstrated that vancomycin could be loaded into oxidized HA and adipic acid dihydrazide. The vancomycin-containing HA hydrogels exhibited excellent drug release and in vitro antimicrobial activity with minimal cell toxicity. Hydrogel is a novel material that can be mixed with antibiotics and applied easily to either wound or
prosthesis surface.13 The compositions of different types of hydrogel vary and each component has its own characteristic feature. For example, dextran-PEG hydrogel had been mixed with polymyxin B and vancomycin designed for wound dressing, PEG-based monomer coupling PEG with vancomycin, and chitosan/glycerophosphate thermosensitive hydrogel with microparticles containing vancomycin for local antibiotics delivery use.20e22 The price of HA is higher than that of dextran, PEG, and chitosan, but HA is a natural biomolecule, which is distributed widely in the connective, epithelial, and neural tissues. It can be degraded by hyaluronidases, and hence, it is biocompatible and biodegradable and more suitable material for antibiotic hydrogel preparation.
Please cite this article as: Liao C-H et al., Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Biocompatibility, drug release, antimicrobial activity, and biofilm model, Journal of Microbiology, Immunology and Infection, https:// doi.org/10.1016/j.jmii.2019.08.008
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6 The advantage of oxi-HA/ADH gel is that it could be gelled within a few minutes at room temperature, and forms a thin layer on the surface of the implant. In this study, the release of vancomycin from the gel was rapid and the gel degraded completely in 3 weeks under in vitro condition, and this would be much faster in the human body. The target rate of drug release depends on the purpose of the gel. If prevention of immediate infection is the purpose, early and rapid release might be the best design. Furthermore, osteointegration into prosthesis will not be affected since its degradation happens within weeks. On the contrary, for treatment of established infection, such as osteomyelitis, a prolonged release might be more appropriate. Currently, the release rate of the drug from the different gels is manageable by adding microparticles,22 charged copolymers,14 or adjusting of pH of the gel.23 Moreover, Lakes et al. showed that vancomycin release and material degradation were tunable via hydrophobic/hydrophilic content of the hydrogel matrix.21 There is no standard test to assess the efficacy of antibiotic-loaded gels. In this study, we used two models to examine the in vitro efficacy of the gel. Due to the plasticity of the gel, we first apply traditional disc diffusion method by molding vancomycin-loaded oxi-HA/ADH gel as the size of the standard disc and test antimicrobial activity of the disc.24 After overnight culture, the zone of inhibition of the hydrogel was easily observable. There was a dosedependent effect of vancomycin load gel as 500X MIC vancomycin hydrogel had an inhibition zone of 21 mm. Even a blank gel had an inhibition zone of 5 mm. HA is reported to have some anti-bacterial and fungal activities.25 The standard vancomycin paper disc (30 mg) used in the clinical microbiology lab could have an inhibition zone over 15 mm against MRSA, while our hydrogel with a 50X MIC of vancomycin (62.5 mg/ml) had an inhibition zone of 13 mm, which is quite comparable. This methodology could be easily applied to other kinds of hydrogels or drugs adding into hydrogels. The second in vitro test was a biofilm model modified from the study of Drago et al.18 In our MRSA-contaminated Ti6Al4V implant model, HA hydrogel with different concentration of vancomycin (0.01e1%) showed excellent activity in reducing the number of MRSA cells as measured by MTT assay. The results suggest that rapid releasing vancomycin-loaded oxi-HA/ADH gel has good effects on the surface-contaminated biofilm model even with a low concentration of vancomycin. The real challenge lies with deep and complicated infection, such as nails used in open contaminated wound. In an animal study, Giavaresi et al. showed that vancomycin-loaded hydrogel coating can reduce bacterial load from 72% to 99% on the prosthesis.26 Adding antibiotic into cement is a common practice nowadays to reduce subsequent prosthesis infection.24 Drug release from cement is generally slower than from hydrogel. A study with cement and calcium polyphosphate hydrogel combination showed that adding 10% CPP gel to cement led to a much lower burst release of vancomycin and extended duration of drug release.27 However, the degradation nature of HA gel makes it an unsuitable candidate for this type of design. On the other hand, some surgeons use antimicrobial powder directly while performing orthopedic surgery. It is not feasible to evaluate the
C.-H. Liao et al. pharmacodynamics and toxicity of drugs by this kind of approach. To sum up, oxi-HA/ADH hydrogel-loaded with vancomycin has an excellent bio-compatibility, rapid and complete drug release, and measurable in vitro antimicrobial activity and is quite effective in a biofilm model to prevent immediate bacterial colonization. With its bio-compatibility, 1% vancomycin-loaded gel can be used since high vancomycin concentration carries higher inhibition in the in vitro model.
Declaration of Competing Interest None.
Acknowledgements We would like to thank the Ministry of Science and Technology, Taiwan (MOST-105-2221-E-418-001, MOST-106-2221E-418-001), Far Eastern Memorial Hospital, Taiwan (FEMH2016-C-005, 105FN14) and AOTRAUMA (AOTAP16-08) for financial support. The Far Eastern Memorial Hospital Core Laboratories I & II for providing facilities and instruments.
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Please cite this article as: Liao C-H et al., Vancomycin-loaded oxidized hyaluronic acid and adipic acid dihydrazide hydrogel: Biocompatibility, drug release, antimicrobial activity, and biofilm model, Journal of Microbiology, Immunology and Infection, https:// doi.org/10.1016/j.jmii.2019.08.008