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Characterizing Drug Release from Nonfouling Polyampholyte Hydrogels Marcos Barcellona, Nicholas Johnson, and Matthew T. Bernards Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b03597 • Publication Date (Web): 25 Nov 2015 Downloaded from http://pubs.acs.org on December 2, 2015
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Submitted to Langmuir, 2015 Characterizing Drug Release from Nonfouling Polyampholyte Hydrogels Marcos N. Barcellona1, Nicholas Johnson2, Matthew T. Bernards1,2,*
1
Department of Bioengineering and 2Department of Chemical Engineering University of Missouri, Columbia, MO, 65211
*Corresponding Author: Matthew Bernards: Email:
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Submitted to Langmuir, 2015 Abstract Controlled delivery of bioactive signaling molecules and drugs is essential for the development of the next generation of tissue regeneration scaffolds. However, these molecules must be delivered from a nonfouling platform, so that the therapeutic role is not masked by the naturally occurring foreign body response. Therefore, the purpose of this study is to characterize the release profiles of three pseudo-drug molecules from a nonfouling polyampholyte hydrogel to gain insight into the potential for this platform to serve as a tissue regeneration scaffold. Hydrogels composed of equimolar concentrations of [2-(acryloyloxy)ethyl] trimethylammonium chloride (TMA) and 2-carboxyethyl acrylate (CAA) monomers were synthesized in the presence of caffeine, methylene blue, or metanil yellow. Then the release of these three molecules was tracked as a function of the hydrogel cross-linker density, the solution pH, and the solution ionic strength. The results suggest that the release of the neutral caffeine molecule is dictated by diffusion alone, while the release of the two charged pseudo-drug molecules are controlled by their interactions with the charged regions of the TMA and CAA monomer subunits. These interactions are clearly impacted by solution pH and ionic strength leading to clear changes in the rate of release and extent of release for metanil yellow and methylene blue. Additionally, an enzyme-linked immunosorbent assay was used to confirm that the TMA:CAA hydrogels retain their nonfouling characteristics following the release of the pseudo-drug molecules. When these results are combined with the literature related to TMA:CAA hydrogels it is concluded that this system represents a promising multi-functional platform for both short-term and long-term delivery of bioactive molecules for tissue regeneration.
Keywords: Polyampholytes, Hydrogels, Multi-functional, Drug Delivery
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Submitted to Langmuir, 2015 1. Introduction. Biocompatible drug delivery and biomolecule delivery systems have many applications in biomedical fields including targeted drug delivery vehicles, controlled release platforms, and tissue regeneration scaffolds.1 Given this diversity of applications, there is interest in developing multiple delivery systems with controllable release profiles. In tissue regeneration applications, this delivery may include both a short term release to modulate the immune response as well as a long term release to influence the native tissue integration. However, in this type of application, protein fouling plays a major role because nonspecific protein adsorption can trigger immune system responses which lead to encapsulation of the device and ultimately failure to integrate with the native tissue.2,3 Consequently, nonfouling materials, or materials that are resistant to nonspecific protein adsorption, are crucial for implantable devices as they have been shown to significantly reduce the immune system response.4,5 Polyethylene glycol (PEG) hydrogels are the most widely investigated nonfouling platforms for use as a tissue engineering and biomolecule delivery scaffold.6,7 However, PEG has a few drawbacks that have led to an increase in investigators searching for alternative nonfouling hydrogel platforms. The first drawback is the fact that additional chemistry must be used to incorporate bioactive functional groups into a PEG hydrogel.8-10 However, this modification to the PEG backbone reduces its nonfouling nature. The second drawback relates to the vulnerability of PEG to oxidation, which hinders its long-term use in slow healing tissue engineering applications.8,11,12 Finally, there is increasing evidence that some humans have begun producing antibodies to PEG, which suggests that it cannot serve as a universal tissue engineering platform.13,14
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Submitted to Langmuir, 2015 Zwitterionic functional groups have come to the forefront as the leading nonfouling alternative to PEG.15-17 For example phosphorylcholine (PC) based polymers have risen from the seminal paper by Ishihara15 to be utilized in a wide range of applications where their excellent nonfouling properties are beneficial.4,18,19 Other zwitterionic functional groups include sulfobetaine (SB) and carboxybetaine (CB), which have been widely studied by Jiang and colleagues.16,20 A related, but less well known subgroup of the zwitterionic family of materials is polyampholyte polymers, which are composed of mixtures of positively and negatively charged monomer subunits.17,21 Polyampholyte polymers hold a unique niche for tissue engineering applications because of two characteristics. First, polyampholyte polymers have been demonstrated to be capable of covalently attaching bioactive molecules without requiring a modification of their base chemistry and without reducing their underlying nonfouling properties away from the attached molecule.22,23 This is a property that is shared only with CB based systems and it provides an easy approach for the long-term delivery of bioactive molecules from a tissue engineering platform. Second, the physical properties of polyampholyte hydrogels strongly depend on their monomer subunits.22,24 This provides a toolbox for controlling the properties of a tissue engineering platform through monomer selection that is not available with the single monomer zwitterionic systems. The foci of this investigation are to 1) characterize the release of non-bound pseudo-drug molecules from a polyampholyte hydrogel platform to gain fundamental insight into the roles that electrostatic interactions have on the short-term release of bioactive molecules from this mixed-charge platform and 2) demonstrate that the nonfouling properties of the polyampholyte chemistry are not impacted by the inclusion of biomolecules during the hydrogel synthesis process. This subject has seen little work to date,25,26 and it will allow for a better understanding
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Submitted to Langmuir, 2015 of the potential for polyampholyte polymers to serve as a tissue regeneration platform. Polyampholyte hydrogels were formed from equimolar quantities of positively charged [2(acryloyloxy)ethyl] trimethylammonium chloride (TMA) and negatively charged 2-carboxyethyl acrylate (CAA), with varying concentrations of triethylene glycol dimethacrylate (TEGDMA) as the cross-linker. The structure of these monomers can be seen in Figure 1a. TMA:CAA hydrogels were preloaded with one of three different model drugs including positively charged methylene blue, negatively charged metanil yellow, and neutral caffeine, and the release was tracked as a function of cross-linker density, buffer pH, and buffer salt concentration. The structures of these molecules can be seen in Figure 1b. The release profiles were analyzed relative to the swelling of the hydrogels under the different conditions. Finally, the nonfouling properties of the TMA:CAA hydrogels following a majority of the molecule release were characterized using enzyme-linked immunosorbent assays (ELISAs). The results demonstrate that the underlying hydrogel pore structure can be adjusted via chemistry in order to control the rate of release of different molecules, and that external stimuli can either enhance or hinder the release of bioactive molecules based on the influence of the stimuli on the swelling and charge state of the polyampholyte hydrogel. When these results are combined with other known features of polyampholyte polymers, such as their multi-functional properties, it can be seen that polyampholyte hydrogels show great potential for use as a tissue regeneration platform.
2. Materials and Methods. 2.1 Materials. Ethylene glycol, caffeine (lab grade, MW = 194.2), metanil yellow (78.6% dye, MW = 375.38), methylene blue (95% pure, MW = 373.89), phosphate buffered saline (PBS, 150 mM,
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Submitted to Langmuir, 2015 pH 7.4), and sodium metabisulfate (SMS) were purchased from Fisher Scientific (Pittsburgh, PA). TMA, CAA, TEGDMA, lysozyme (LYZ) from chicken egg white (≥90%), Ophenylenediamine (OPD), sodium phosphate dibasic, potassium dihydrogen phosphate, phosphate-citrate buffer tablets, urea, and hydrogen peroxide were purchased from Sigma Aldrich (St. Louis, MO). Solid sodium hydroxide and ammonium persulfate (APS) were purchased from Acros Organics (Pittsburgh, PA). Human plasma fibrinogen (FBG) was purchased from CalBiochem (San Diego, CA). Horse radish peroxidase (HRP) conjugated antifibrinogen (HRP-anti-FBG) and HRP-conjugated anti-lysozyme (HRP-anti-LYZ) were purchased from United States Biological (Swampscott, MA). Ethanol was purchased from Decon Laboratories, Inc. (King of Prussia, PA). Ultrapure water (18.2 MΩ * cm) was taken from a Millipore Synergy UV water purification system (Billerca, MA) and it was used for all experiments.
2.2 Hydrogel Synthesis. The procedures for the TMA:CAA hydrogel preparation were adapted from previous work with slight modification.22,24,27 Briefly, 500 µL of solvent containing a 1.5:1:1.5 volume ratio of ethylene glycol:ethanol:3 M NaOH was mixed with 1 mmol of TMA, 1 mmol of CAA, and one of the three powdered pseudo-drug molecules. Caffeine, metanil yellow, and methylene blue were added at concentrations of 102.99 µM, 19.98 µM, and 10.16 µM, respectively, based on a series of trial and error investigations to determine the maximum amount of pseudo-drug that could be added before the hydrogel polymerization was noticeably affected. Next, the TEGDMA cross-linker was added to the solution and finally the hydrogel polymerization was initiated through the addition of 11.75 µL of 40 wt% ammonium persulfate in water. The
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Submitted to Langmuir, 2015 solution was well mixed and placed in a hydrogel mold consisting of two glass microscope slides separated by a 0.79 mm thick polytetrafluoroethylene spacer. The reaction proceeded for 1 hour at 60 ˚C, at which point the gels were allowed to cool to room temperature for 1 hour before being used for testing. The TEGDMA cross-linker was added at either 0.076 mmol (1×), 0.152 mmol (2×), or 0.304 mmol (4×), depending on the desired cross-linker density. Additionally, control hydrogels were formed with identical procedures, with the exception that no pseudo-drug molecules were included.
2.3 Swelling Characterization. The swelling behaviors of the TMA:CAA hydrogels were measured after preparing hydrogels with the procedures outlined above. The hydrogels were placed in an oven with desiccant at 60°C and left for 48 hours to fully dehydrate. The hydrogels were then removed from the oven, cut into disks with a 5 mm biopsy punch, and placed into the appropriate buffer. The samples were allowed to rehydrate over a 48 hour period, at which point the dimensions of the hydrogels were measured using a digital caliper with an accuracy of ± 0.03 mm. The percent volume increase was determined from the ratio of the final volume to the initial volume. Swelling studies were conducted under all of the release conditions described below. The swelling results are presented as the mean ± standard deviation for all of the conditions investigated.
2.4 Absorbance Measurements. Following synthesis, TMA:CAA hydrogels with one of the three pseudo-drug molecules were placed into Sørensen’s buffer of varying pH and ionic strength. The release of the
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Submitted to Langmuir, 2015 methylene blue, metanil yellow, and caffeine from the preloaded hydrogels was directly quantified by measuring the intensity of color released into the buffer solutions.25 Aliquots of buffer solution were taken at predetermined time intervals, pipetted into a 96 well plate and placed in a BioTek PowerWave XS2 spectrophotometer (BioTek, Winooski, VT), where the color intensity in the solutions was measured at 663 nm for methylene blue, 430 nm for metanil yellow, and 290 nm for caffeine using Gen5 1.07.5 software (BioTek). The entire volume of buffer was replaced at each measurement time point. The amount of pseudo-drugs released at each time point was determined by subtracting the results for each sample from the results for an unloaded control, followed by conversion to concentration based on calibration curves that were obtained using predetermined concentrations of each molecule in the base case buffer. The base case buffer was 1/15 M Sørensen’s Buffer with a pH of 7.41. Additional buffer conditions that were tested include pH 4.51 and pH 9.51 buffers with a salt concentration of 1/15 M and pH 7.41 buffer with salt concentrations of 2/15 M and 3/15 M. The buffer conditions were prepared using various ratios of monopotassium phosphate and disodium phosphate. The release measurements are presented as the mean ± standard error of the mean for all of the conditions investigated.
2.5 Enzyme-Linked Immunosorbent Assay. ELISA experiments were conducted to determine the degree of fouling resistance by adapting procedures from our previous work.22,24 Briefly, the hydrogels were preloaded with the pseudo drug and placed in buffer, which was regularly replaced over 360 hours. After the 360 hour release period, the hydrogels were cut into disks using a 5 mm biopsy punch and placed in 1 mg/mL of either FBG or LYZ in PBS for 1.5 hours. Simultaneously, protein solution was added
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Submitted to Langmuir, 2015 to separate wells of a 24-well tissue culture polystyrene (TCPS) well plate and also allowed to incubate for 1.5 hours. TCPS is included as a fouling control surface and it is assumed to provide a monolayer of adsorbed protein. After the protein adsorption step, the disks and TCPS control wells were extensively rinsed with PBS to wash away any non-bound protein. The hydrogel disks and TCPS control wells were then incubated in the corresponding antibody solution (either 1.25 µg/mL HRP-anti-FBG in PBS or 10 µg/mL HRP-anti-LYZ in PBS) for 1.5 hours. After 1.5 hours, the samples were extensively washed and then the disks were transferred to clean wells of a 24 well plate and 800 µL of substrate solution containing OPD was added to the wells. Absorbance was monitored at 492 nm over 30 minutes and then a final measurement was taken. A set of control hydrogels without pseudo-drug molecules were also evaluated using identical procedures as a nonfouling control. All of the results are normalized to the fouling TCPS control, which represents a monolayer of adsorbed protein. The results are presented as the normalized mean ± propagated error for all of the conditions investigated.
2.6 Statistical Analysis. A minimum of three independent samples were conducted for each experiment, and each data point represents the mean of all of the data collected for each experiment. Statistical analysis was conducted using one-way analysis of variance (ANOVA) and results were considered to be statistically significant at 95% confidence interval (p
