We thank the Mayo Clinic Pharmacology Shared Resource for supplying critical reagents, HPLC equipment and analytical expertise.

1. OPF/SMA hydrogel synthesis
<ol>
	<li>Prepare the OPF macromer beginning with 10,000 Da poly(ethylene glycol) according to methods described by Kinard, et al.21.</li>
	<li>Create a 10% w/v solution of photoinitiator by mixing with double-distilled water (ddH2O). For example, add 100 mg of photoinitiator to 10 mL of ddH2O. Add low heat (55 &#176;C), and gently stir to dissolve completely.</li>
	<li>In a 50 mL conical tube, mix the desired amount of 10,000...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positively+Charged+Oligo[Poly(Ethylene+Glycol)+Fumarate]+Scaffold+Implantation+Results+in+a+Permissive+Lesion+Environment+after+Spinal+Cord+Injury+in+Rat.">Positively Charged Oligo[Poly(Ethylene Glycol) Fumarate] Scaffold Implantation Results in a Permissive Lesion Environment after Spinal Cord Injury in Rat.</a> Tissue Eng. Part A. 21 (13), (2015).</li><li>Maran, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel-PLGA+delivery+system+prolongs+2-methoxyestradiol-mediated+anti-tumor+effects+in+osteosarcoma+cells.">Hydrogel-PLGA delivery system prolongs 2-methoxyestradiol-mediated anti-tumor effects in osteosarcoma cells.</a> J Biomed Mater Res - Part A. 101 (9), 2491-2499 (2013).</li><li>Dadsetan, M., Knight, A. 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L., Nicolau, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vancomycin+serum+concentrations+do+not+adequately+predict+tissue+exposure+in+diabetic+patients+with+mild+to+moderate+limb+infections.">Vancomycin serum concentrations do not adequately predict tissue exposure in diabetic patients with mild to moderate limb infections.</a> J Antimicrob Chemother. 70 (7), 2064-2067 (2014).</li><li>Fish, R., Nipah, R., Jones, C., Finney, H., Fan, S. L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+vancomycin+concentrations+during+peritoneal+dialysis-associated+peritonitis:+Correlation+with+serum+levels.">Intraperitoneal vancomycin concentrations during peritoneal dialysis-associated peritonitis: Correlation with serum levels.</a> Perit Dial Int. 32 (3), 332-338 (2012).</li><li>Rubinstein, E., Keynan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vancomycin+revisited+-+60+years+later.">Vancomycin revisited - 60 years later.</a> Front PPublic Health. 2 (October), 217 (2014).</li><li>Strom, R. G., Pacione, D., Kalhorn, S. P., Frempong-Boadu, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumbar+laminectomy+and+fusion+with+routine+local+application+of+vancomycin+powder:+Decreased+infection+rate+in+instrumented+and+non-instrumented+cases.">Lumbar laminectomy and fusion with routine local application of vancomycin powder: Decreased infection rate in instrumented and non-instrumented cases.</a> Clin Neurol Neurosurg. 115 (9), 1766-1769 (2013).</li><li>Hill, B. W., Emohare, O., Song, B., Davis, R., Kang, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+vancomycin+powder+reduces+surgical+reoperation+in+posterior+instrumented+and+noninstrumented+spinal+surgery.">The use of vancomycin powder reduces surgical reoperation in posterior instrumented and noninstrumented spinal surgery.</a> Acta Neurochirurgica. 156 (4), 749-754 (2014).</li><li>Bakhsheshian, J., Dahdaleh, N. S., Lam, S. K., Savage, J. W., Za Smith, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+vancomycin+powder+in+modern+spine+surgery:+systematic+review+and+meta-analysis+of+the+clinical+evidence.">The use of vancomycin powder in modern spine surgery: systematic review and meta-analysis of the clinical evidence.</a> World Neurosurg. , 1-8 (2014).</li><li>Gustafson, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+delivery+of+vancomycin+via+charged+hydrogels.">Controlled delivery of vancomycin via charged hydrogels.</a> PLoS ONE. 11 (1), (2016).</li><li>Kinard, L. a., Kasper, F. K., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+oligo(poly(ethylene+glycol)+fumarate.">Synthesis of oligo(poly(ethylene glycol) fumarate.</a> Nature Protocols. 7 (6), 1219-1227 (2012).</li><li>Aminabhavi, T. M., Deshmukh, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Polymeric Hydrogels as Smart Biomaterials. , (2015).</li><li>Choi, J. S., Yoo, H. 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The methods described here outline a facile method to synthesize OPF/SMA charged hydrogels, and to quantify the elution of a drug (vancomycin) from the hydrogel matrix. While the protocol is relatively straightforward, it may be useful to note several pitfalls which should be avoided during the synthesis, loading, and release in order to achieve more accurate results. During the hydrogel synthesis (see step 1.6), it is important to avoid the incorporation of air pockets in the co-polymer while pouring into a mold. This c...

Stimuli responsive hydrogels are of interest for their wide-ranging applications in enzyme / antibody immobilization, analytical techniques, nucleic acid and protein delivery, and cell encapsulation1,2,3,4,5,6,7. Many such materials are polymer networks which contain charged pendant chains - thus electric field, temperature, pH, and ionic strength modulate the physical characteristics of the hydrogel. Thus, response to changes in physical stimuli can be tailored to suit desired applications for hydrogels. Oligo(poly(ethylene glycol)fumarate) (OPF) polymers and co-polymer films or particulate formulations have been used for a number of chemical and biological applications. Their ease of use makes them attractive candidate polymers for crosslinking with other synthetic or naturally found polymer chains, or functionalization with pendant groups. OPF polymers have been shown to be useful in regenerative applications, as well as for drug delivery purposes8,9,10. Previously, our group reported the synthesis of positively and negatively charged OPF hydrogels11,12. This led us to consider the possibility of exploiting an affinity based interaction between the polymer and a charged drug for an easily produced, bioresorbable drug delivery device.
Vancomycin, is a broadly active, positively charged, glycopeptide antimicrobial drug which was first isolated from Amycolatopsis orientalis. Vancomycin efficacy is limited by a number of factors: systemic delivery often results in an immunologic adverse event termed &#34;red man syndrome&#34;. Additionally, serum concentrations of vancomycin correlate poorly with tissue exposure - and clinical outcome13,14,15. This unpredictability has steered some clinicians away from vancomycin use when other antibiotics may be chosen. Despite such troubles, vancomycin use has gradually increased recently16. In an attempt to avoid dose-limiting systemic side effects, a number of surgical teams have reported the use of powdered vancomycin hydrochloride as an intra-operative wound dressing for prevention of bacterial colonization of the wound site17,18. This means of delivering concentrated doses of vancomycin to the wound site has been demonstrated to be especially effective in spine surgery19. Unfortunately, the high aqueous solubility, poor absorption, and quick elimination of vancomycin reduces its ability to distribute throughout local tissue, and limits its efficacy when administered topically. Thus, local delivery via a bioresorbable, implantable material would improve local tissue exposure to vancomycin. Environmentally responsive biopolymers such as OPF would provide an ideal solution to control dosing of antibiotics for such applications. This study focuses on the use of OPF due to its previously characterized biocompatibility in cellular and animal models, and facile synthesis and customization.
The wide-spectrum of applications for OPF, and derived co-polymers, highlight the utility of efficient methods for polymer and hydrogel synthesis and purification. Vancomycin, as well as other charged molecules, may be loaded onto hydrogel matrices containing pendant charges through affinity based interactions. Additional functionalization of such hydrogels is possible through incorporation of micro- or nanospheres loaded with bioactive molecules, or by producing a porous hydrogel matrix through various means, such as salt leaching. Thus, fully customizable hydrogel matrices for a large spectrum of applications are achievable from the same starting hydrogel material, the synthesis of which we describe here, and have utilized previously20. After drug loading, the OPF/SMA co-polymer depends on an ion-exchange mechanism in the presence of physiologic salt concentrations to release the drug. In this study, we demonstrate an efficient method to synthesize an array of OPF/SMA charged co-polymers for use as implantable, drug eluting devices. Additionally, we demonstrate a simple, reverse-phase high performance liquid chromatography (HPLC)-based method for detection and quantification of vancomycin.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sodium methacrylate</td>
			<td>Sigma</td>
			<td>408212</td>
		</tr>
		<tr>
			<td>Polyethylene glycol 10,000MW</td>
			<td>Sigma</td>
			<td>81280</td>
		</tr>
		<tr>
			<td>50kDa exclusion dialysis tubing</td>
			<td>Spectrum Labs</td>
			<td>132129</td>
		</tr>
		<tr>
			<td>1-Vinyl-2-pyrrolidinone</td>
			<td>Sigma</td>
			<td>95060</td>
		</tr>
		<tr>
			<td>Irgacure 2959</td>
			<td>BASF</td>
			<td>106797-53-9</td>
		</tr>
		<tr>
			<td>50mL Conical tubes</td>
			<td>Corning</td>
			<td>352098</td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher Scientific</td>
			<td>01-213-104</td>
		</tr>
		<tr>
			<td>10cm dishes</td>
			<td>Corning</td>
			<td>353003</td>
		</tr>
		<tr>
			<td>2mL low affinity microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>02-681-321</td>
		</tr>
		<tr>
			<td>Vancomycin hydrochloride</td>
			<td>Sigma</td>
			<td>V1130</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Corning</td>
			<td>21031</td>
		</tr>
		<tr>
			<td>Thermogravimetric Analyzer</td>
			<td>TA Instruments</td>
			<td>TGA5500</td>
		</tr>
		<tr>
			<td>1/8&#34; thickness soda lime glass plate (1&#34;x1&#34;)</td>
			<td>Ace Hardware</td>
			<td>11529</td>
		</tr>
		<tr>
			<td>1/32&#34; thickness white PTFE sheet&#160;</td>
			<td>McMaster-Carr</td>
			<td>8545K11</td>
		</tr>
	</tbody>
</table>

synthesis and characterization of charged hydrogels for the extended delivery of vancomycin

Drug eluting polymers are of great interest due to their utility in cancer treatment, tissue regeneration, and infection prevention. It is desirable for these materials to offer tunable structural characteristics and molecular affinities, as well as excellent biocompatibility. In the context of surgery, infection of the wound site remains a significant risk for any procedure. It is standard practice to deliver systemic antimicrobial agents to combat infection of the surgical wound. To complement systemic therapy, an implantable, local delivery vehicle would be ideal. Our group investigated the utility of oligo(poly(ethylene glycol)fumarate) / sodium methacrylate (OPF/SMA) charged copolymers as a local delivery vehicle for the antimicrobial drug vancomycin. Here, we describe the methods of OPF synthesis, and OPF/SMA hydrogel production. Additionally, we demonstrate a facile chromatographic method for quantification of vancomycin loading and release from the OPF/SMA hydrogels. OPF-based biopolymers have proven to be biocompatible in vivo and have wide-ranging applications due to their ability to be crosslinked chemically or via ultra-violet irradiation. OPF is readily functionalized with charged groups, or mixed with other polymers to create co-polymers with optimal structural properties or unique responses to environmental stimuli. This study utilizes an array of charged OPF/SMA co-polymers to develop a controlled release vehicle for the broad spectrum antibiotic vancomycin based upon charge-charge interactions. Hydrogel loading of vancomycin can be quantified with a simple high performance liquid chromatography method. Vancomycin release from the hydrogels is initiated in the presence of free ions in solution, and release is buffered by free charges on the pendant chains within the crosslinked hydrogel. In sum, we report an efficient method for synthesis and characterization of the OPF/SMA hydrogels, and quantification of their ability to load and release vancomycin.

This method describes the synthesis and characterization of OPF/SMA hydrogels, and the quantification of their vancomycin loading and release properties. In Figure 1, the general structure of the hydrogels is depicted with their idealized loading of vancomycin. OPF macromer chains are crosslinked with SMA, resulting in free negative charges within the hydrogel co-polymer network. Figure 2 establishes the change in hydrogel physical properties due to charg...

Here we describe the synthesis and use of oligo(poly(ethylene glycol)fumarate) / sodium methacrylate (OPF/SMA) charged copolymers as an affinity based delivery system for vancomycin.

Synthesis and Characterization of Charged Hydrogels for the Extended Delivery of Vancomycin

Drug eluting polymers are of great interest due to their utility in cancer treatment, tissue regeneration, and infection prevention. It is desirable for ...

Synthesis and Characterization of Charged Hydrogels for the Extended Delivery of Vancomycin

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