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

Summary

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.

Abstract

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.

Introduction

Stimuli responsive hydrogels are of interest for their wide-ranging applications in enzyme / antibody immobilization, analytical techniques, nucleic acid and protein delivery, and cell encapsulation^{1,2,3,4,5,6,7}. 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 purposes^8,9,10. Previously, our group reported the synthesis of positively and negatively charged OPF hydrogels^11,12. 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 "red man syndrome". Additionally, serum concentrations of vancomycin correlate poorly with tissue exposure - and clinical outcome^13,14,15. 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 site^17,18. 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.

Protocol

1. OPF/SMA hydrogel synthesis

1. Prepare the OPF macromer beginning with 10,000 Da poly(ethylene glycol) according to methods described by Kinard, et al.²¹.
2. 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 °C), and gently stir to dissolve completely.
3. In a 50 mL conical tube, mix the desired amount of 10,000 Da molecular weight OPF macromer with 950 µL of ddH₂O and 750 µL of the photoinitiator solution. The proper amount of OPF depends on the desired ratio of OPF/SMA. For example, create a 10% SMA co-polymer by beginning with 900 mg of OPF, and adding 100 mg of SMA in step 1.4.
   1. For each desired OPF/SMA copolymer, prepare a separate 50 mL conical tube with the reaction mix. Stir the OPF, water, and photoinitiator solution in each conical tube vigorously with a spatula. Incubate at 37 °C for 10 min in a water bath.
4. Remove the conical tube containing the reaction mix from the water bath and add 300 µL of 1-vinyl-2-pyrrolidinone (NVP) by pipette, and add the desired amount of sodium methacrylate (SMA) powder. Mix with a clean metal spatula until the SMA is dissolved completely.
5. Centrifuge the 50 mL conical tube containing the reaction mix for 5 min at 3,000 x g to remove bubbles from the solution. The reaction mix is now ready to be molded and photo-crosslinked.
6. Pour the reaction mix into 1"x1" square, 1/32" thick PTFE molds covered by 1/8" thick soda lime window glass plates and seal with metal binder clips (two for each mold). Pour approximately 500 µL of reaction mix into each mold, or until the reaction mix has filled the boundaries of the mold. Typically, use one mold for each OPF/SMA copolymer film being produced.
7. Place the prepared molds into a room temperature UV oven for 30 min to crosslink the polymer hydrogel.
8. Once the crosslinking has finished, remove the samples from the oven, open the molds and cut the hydrogel film into 3 mm discs using a 3 mm diameter biopsy punch. The molds may be difficult to open as the hydrogel has crosslinked within it. A metal spatula may be used to help open the molds. More than thirty 3 mm discs may be cut from a single prepared hydrogel film.
9. Place the freshly cut hydrogel discs into 50 kDa exclusion standard dialysis tubing, separating each copolymer group into unique dialysis bags and labelling carefully. Allow the discs to dialyze in 1 L of ddH₂O for 72 h in order to remove any uncrosslinked, soluble material.
10. Using forceps, remove the dialyzed hydrogels from the tubing and allow to dry for 2 h at room temperature. Typically, place the hydrogels on 10 cm dishes covered with aluminum foil during the drying process. Place the hydrogels in a desiccating oven for 8 h (or overnight) in order to remove excess water from the samples. The clean hydrogels are now ready for use.

2. Hydrogel characterization

1. OPF/SMA hydrogel swelling ratio calculation
   1. Weigh the dried OPF/SMA hydrogels. It was typical to find that a dried 3 mm hydrogel disc weighed between 4.0 and 6.0 mg. Place each hydrogel to be used for swelling ratio calculations in a 50 mL conical tube containing a solution of ddH₂O, Dulbecco's phosphate buffered saline (DPBS), or 400 µg/mL vancomycin in ddH₂O.
   2. Allow hydrogels to incubate in the solution at room temperature for 24 h.
   3. Remove hydrogels from the solution with forceps and dry briefly to remove excess water. Measure the weight of the swollen hydrogels.
   4. Calculate the swelling ratio of the hydrogels by subtracting the dried mass (Md) from the swollen mass (Ms) and dividing by the dried mass, such that the swelling ratio = (Ms-Md) / Md.
2. Perform thermal gravimetric analysis of OPF/SMA hydrogels
   1. Load dialyzed and dried hydrogel discs onto the sample plate of a thermal gravimetric analyzer.
   2. Heat the samples at a ramp rate of 20 °C/min from 50 °C to 1000 °C under an inert atmosphere.
      NOTE: Increased degradation near 140 °C is observed in the samples containing higher mass percentage of SMA.

3. Hydrogel loading with vancomycin

1. Prepare a fresh, stock solution of concentrated vancomycin in water (e.g. 10 mg/mL) by weighing powdered vancomycin into a low affinity microcentrifuge tube and adding an appropriate volume of ddH₂O. Use this solution to create the loading solutions by standard dilution. Be sure to mix well by vortexing before use.
2. Add 1 mL of working concentration (e.g. 400 µg/mL) of vancomycin solution to a 2 mL, low-affinity microcentrifuge tube. Keep a stock of this solution for further use as a quantification standard. Prepare enough vancomycin solution, hydrogels, and tubes for all groups and replicates.
3. Weigh clean, dried OPF/SMA hydrogels, and place individual hydrogels into the microcentrifuge tubes containing the working concentration vancomycin solution. Mix well and allow to incubate up to 72 h to allow vancomycin to load onto the hydrogel scaffold. The hydrogels will also swell during this period.
4. After loading, remove the hydrogels from the microcentrifuge tubes with a forceps and allow to dry at room temperature for 2 h by placing on 10 cm dishes covered with aluminum foil. Retain the loading solutions for determination of vancomycin concentration in solution, and calculation of loading efficiency.
5. Place the drug loaded hydrogels in a desiccating oven for 8 h in order to completely remove excess liquid from the hydrogels.
6. Measure initial and final concentrations of loading solutions (see step 5 for quantification method) to determine the mass of vancomycin loaded per scaffold. Calculate loading efficiency by determining the unit mass of vancomycin achieved per unit mass of hydrogel (e.g. µg VCM/mg hydrogel).

4. Vancomycin release from OPF/SMA hydrogels

1. Place the loaded and dried OPF/SMA hydrogels into fresh 2 mL, low-affinity microcentrifuge tubes containing 1 mL of DPBS. At this time, Step 5.1 may be begun as well. Keep the vancomycin solutions to be used as standards at 37 °C for the entire release period in order to control for degradation of the drug over time.
2. Incubate the hydrogels in DPBS at 37 °C for the desired release period.
3. Following drug elution, remove samples from the tubes, retaining the elution solution. Keep the solution containing eluted drug for analysis via HPLC.

5. High performance liquid chromatography quantification of solution vancomycin

1. Prepare a standard curve of vancomycin from stock solution used to prepare the vancomycin loading solution (as in Step 3.1, except diluted with DPBS). A recommended standard curve may include 8 standard concentrations ranging from 1 µg/mL to 1000 µg/mL.
2. Equilibrate a reverse-phase C₁₈ HPLC column (150 x 4.6 mm i.d., 2.1 µm particle size) with pH 5.0 dibasic potassium phosphate buffer (5 mM, pH = 2.8) and acetonitrile at a ratio of 80/20, respectively. Equilibrate the column for at least 10 min prior to any samples being run.
3. Set the mobile phase flow rate to 0.5 mL/min, and prepare ultra-violet detection of eluents at 282 nm.
4. Load the standard samples of vancomycin in water onto the HPLC (for drug release solutions, prepare standards in DPBS). Under the described conditions, vancomycin elution time is approximately 4.9 min.
5. Run all samples through the HPLC, ordering the samples such that solutions expected to contain less vancomycin are run first. This prevents contamination from higher concentration samples in the event that all vancomycin does not elute or rinse properly. Calculate signal peak area at 282 nm wavelength detection. Allow for 10 min per sample in order to remove any excess drug or polymer degradation materials, and to re-equilibrate the column prior to the next sample.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Sodium methacrylate	Sigma	408212
Polyethylene glycol 10,000MW	Sigma	81280
50kDa exclusion dialysis tubing	Spectrum Labs	132129
1-Vinyl-2-pyrrolidinone	Sigma	95060
Irgacure 2959	BASF	106797-53-9
50mL Conical tubes	Corning	352098
Aluminum foil	Fisher Scientific	01-213-104
10cm dishes	Corning	353003
2mL low affinity microcentrifuge tubes	Fisher Scientific	02-681-321
Vancomycin hydrochloride	Sigma	V1130
Dulbecco's Phosphate Buffered Saline	Corning	21031
Thermogravimetric Analyzer	TA Instruments	TGA5500
1/8" thickness soda lime glass plate (1"x1")	Ace Hardware	11529
1/32" thickness white PTFE sheet	McMaster-Carr	8545K11