Synthesis and Characterization of Controlled Nitric Oxide Release from S-Nitroso-N-Acetyl-D-Penicillamine Covalently Linked to Polyvinyl Chloride (SNAP-PVC)

bioengineering

Article Synthesis and Characterization of Controlled Nitric Oxide Release from S-Nitroso-N-Acetyl-D-Penicillamine Covalently Linked to Polyvinyl Chloride (SNAP-PVC)

Sean P. Hopkins and Megan C. Frost ,* Department of Biomedical Engineering, Michigan Technological University, Houghton, MI 49931, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-906-487-3350

Received: 10 August 2018; Accepted: 3 September 2018; Published: 5 September 2018

Abstract: Polyvinyl chloride (PVC) is one of the most widely used polymers in medicine but has very poor biocompatibility when in contact with tissue or blood. To increase biocompatibility, controlled release of nitric oxide (NO) can be utilized to mitigate and reduce the inflammatory response. A synthetic route is described where PVC is aminated to a specified degree and then further modified by covalently linking S-nitroso-N-acetyl-D-penicillamine (SNAP) groups to the free primary amine sites to create a nitric oxide releasing polymer (SNAP-PVC). Controllable release of NO from SNAP-PVC is described using photoinitiation from light emitting diodes (LEDs). Ion-mediated NO release is also demonstrated as another pathway to provide a passive mechanism for NO delivery. The large range of NO fluxes obtained from the SNAP-PVC films indicate many potential uses in mediating unwanted inflammatory response in blood- and tissue-contacting devices and as a tool for delivering precise amounts of NO in vitro.

Keywords: nitric oxide releasing polymer; controlled release; photoinitiated; PVC; biocompatibility

1. Introduction Nitric oxide (NO) is a free radical, bioregulatory molecule in the body with a variety of important functions such as controlling vasodilation of blood vessels, acting as a neurotransmitter, and other important cellular signaling pathways which are dependent on the NO flux being generated [1–3]. Within the vasculature, endogenous S-nitrosothiols (RSNOs) such as S-nitrosoglutathione also help regulate these signaling processes along with generating NO at various locations within the body [4]. The production of NO is accomplished through specific enzymatic pathways based on different isoforms of nitric oxide synthase (NOs), depending on the specific physiological location in the body. When NO is released into the bloodstream by endothelial cells, it interacts with platelets, contributing to the non-adhesive properties of vasculature by controlling intracellular calcium regulation [5,6]. If NO production is impaired in the endothelium, the risk for atherosclerosis and hypertension drastically increases. Macrophages are another cell type that utilize the benefits of NO by promoting cytotoxic activity specifically towards viruses, bacteria, protozoa, and tumor cells [7]. The popularity of NO has led to a wide variety of materials capable of mimicking these beneficial properties. Incorporation of a widely-used RSNO, S-nitroso-N-acetyl-D-penicillamine (SNAP), into different polymer matrices has been accomplished by either direct physical blending, impregnation through swelling in SNAP solutions, or covalent attachment to the polymer [8–10]. Each method has its own benefits and drawbacks. Blending NO donors within a polymer matrix can give you precise control over exactly how much NO is released from a specific material by varying your donor

Bioengineering 2018, 5, 72; doi:10.3390/bioengineering5030072 www.mdpi.com/journal/bioengineering Bioengineering 2018, 5, 72 2 of 11 concentration but has issues with potential leaching of the donor and by-products into the cellular environment which can be cytotoxic. Covalent attachment can prevent this issue but is more difficult to control in terms of total NO loading and release capacity. This has led to the development of a variety of polymers modified with SNAP to utilize the beneficial effects of NO. Polymers such as chitosan have been developed with covalently bound SNAP and macromolecules such as dendrimers have also been synthesized with the same strategy [11,12]. The hemocompatibility of several blended and swelled SNAP-based polymers have been tested in extracorporeal circuits (ECCs) to further demonstrate how NO-releasing materials can mimic healthy vasculature [8,13,14]. Simultaneously, these polymers have been demonstrated to be effective antimicrobial agents against a broad range of gram positive and negative bacteria, which is another popular use for NO [15–17]. As antibiotic resistant bacteria continue to be an issue within the medical field, the development of more novel NO-releasing polymers has become more prevalent as a possible solution. PVC is the most widely used polymeric material in medical applications including blood bags, heart lung bypass sets, and endotracheal tubes [18]. Around 25% of all biomedical devices contain some form of PVC due to its adjustable material properties and interactions with certain plasticizers and fillers [19]. PVC also has a very low cost to manufacture compared to other medical grade polymers and has a strong resistance to external chemical components. It is simple to chemically modify as the free chlorine reactive groups allow for the attachment of other chemical components to further enhance the surface chemistry of PVC. Previously, NO-releasing PVC was created by blending N-diazeniumdiolates within the polymer matrix and covalently binding it to a modified version of the polymer [20]. Herein, we describe the synthesis and characterization of a novel NO-releasing PVC based polymer (SNAP-PVC) and demonstrate its wide range of tunable NO-releasing properties which depend on the trigger mechanism. Primary amine groups are first attached to the PVC backbone, allowing SNAP groups to then modify the polymer to make it capable of NO release. Controlled release over a large range of NO fluxes was then demonstrated using light. The novel material described in this report imparts NO release from a covalently bound RSNO using a straightforward, cost-effective means to produce a robust, tunable NO releasing material. In contrast to N-diazeniumdiolates, NO release from RSNOs can be triggered through multiple different mechanisms, allowing SNAP-PVC to have a wide array of potential applications [21]. By using an RNSO functionalized polymer, SNAP-PVC is able to have an active tunable control of NO release for a variety of situations along with the possibility to create a passive release system.

2. Materials and Methods

2.1. Materials

Polyvinyl chloride (average Mw = 233,000, Mn = 99,000) (PVC), ethylenediamine, 5,5’-dithiobis (2-nitrobenzoic acid), triethylamine, 1,4,8,11-tetraazacyclotetradecane (cyclam), N-acetyl-D-penicillamine (Fluka), and concentrated hydrochloric acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). ATTO-TAGTM FQ Amine-Derivatization Kit was purchased from Invitrogen (Grand Island, NY, USA). Tert-butyl nitrite (90% technical grade, Acros Organics) was purchased from Fisher Scientiﬁc. Magnesium sulfate (MgSO4), copper (II) bromide (CuBr2), L-ascorbic acid sodium salt, and acetic anhydride (Alfa Aesar) were purchased from VWR (West Chester, PA, USA).

2.2. Synthesis of NAP-Thiolactone The procedure reported by Moynihan and Robert was followed to synthesize a self-protected N-acetyl-D-penicillamine (NAP) thiolactone [22]. Briefly, 5 g of N-acetyl-D-penicillamine was dissolved in 10 mL of pyridine while 10 mL of acetic anhydride and 10 mL of pyridine were mixed in a separate container. Then, both solutions were cooled in an ice bath for 1 h before being combined. The mixture Bioengineering 2018, 5, 72 3 of 11 was stirred at room temperature for approximately 24 h until the solution turned light red. The solution was rotary-evaporated at 45 ◦C to remove pyridine. The temperature was then increased to 60 ◦C until most of the remaining solvent was removed to obtain an orange viscous liquid. The resulting product was then dissolved in 20 mL of chloroform and washed and extracted three times with 20 mL of 1 M HCl. MgSO4 was added to the chloroform solution to remove any water and was then removed by Bioengineering 2018, 5, x FOR PEER REVIEW 3 of 11 vacuum filtration. The chloroform was removed by rotary evaporation at room temperature and the resultant crystalssolution was were rotary-evaporated collected and at 45 rinsed °C to remove with hexanes pyridine. andThe temperature filtered. Light was then yellow/white increased to colored crystals were60 °C recovered until most andof the vacuum remaining dried solvent overnight. was removed to obtain an orange viscous liquid. The resulting product was then dissolved in 20 mL of chloroform and washed and extracted three times 2.3. Synthesiswith of20 SNAP-PVC mL of 1 M HCl. MgSO4 was added to the chloroform solution to remove any water and was then removed by vacuum filtration. The chloroform was removed by rotary evaporation at room Figuretemperature1 shows theand overallthe resultant reaction crystals scheme were collected to synthesize and rinsed SNAP-PVC. with hexanes Aminatedand filtered. PVCLight was first synthesizedyellow/white using a modified colored crystals procedure were recovered from Tinkilic and vacuum et al. dried [23]. overnight. Briefly, 2.5 g of PVC was suspended in ◦ 50 mL of methanol2.3. Synthesis and of SNAP 11 mL‐PVC of triethylamine and heated to 60 C. 15.25 mL of ethylenediamine was then slowly addedFigure to1 shows the heated the overall solution. reaction The scheme reaction to synthesize mixture SNAP-PVC. was allowed Aminated to reflux PVC was for first a designated ◦ amount ofsynthesized time at 60 usingC. a Themodified resulting procedure light from yellow Tinkilic polymer et al. [23]. Briefly, powder 2.5 wasg of PVC then was filtered suspended and washed thoroughlyin with50 mL water, of methanol methanol, and 11 mL 1 M of HCl, triethylamine and water, and heated in that to order, 60 °C. 15.25 before mL being of ethylenediamine dried under vacuum. was then slowly added to the heated solution. The reaction mixture was allowed to reflux for a For NAP attachment, 200 mg of PVC-NH2 was first dissolved in 10 mL of anhydrous designated amount of time at 60 °C. The resulting light yellow polymer powder was then filtered and N,N-dimethylacetamidewashed thoroughly (DMAC). with water, 60 methanol, mg of 1 NAP-thiolactone M HCl, and water, in was that then order, added before being to the dried mixture and stirred overnight.under vacuum. 2 mL of the NAP-PVC solution was taken out and nitrosated using 0.5 mL of t-butyl nitrite.For The NAPt-butyl attachment, nitrite 200 was mg of first PVC-NH treated2 was to first chelate dissolved any in copper 10 mL of stabilizer anhydrous using N,N- a 30 mM aqueous cyclamdimethylacetamide solution (1,4,8,11-tetraazacyclotetradecane).(DMAC). 60 mg of NAP-thiolactone was then For added the to chelation the mixture step, and stirred approximately overnight. 2 mL of the NAP-PVC solution was taken out and nitrosated using 0.5 mL of t-butyl nitrite. 3 mL of t-butylThe t-butyl nitrite nitrite and was 5 first mL treated of 30 to mM chelate cyclam any copper were stabilizer mixed andusing stirreda 30 mM vigorously. aqueous cyclam The cyclam was thensolution removed (1,4,8,11-tetraazacyclotetradecane). and the t-butyl nitrite was For rewashed the chelation with step, moreapproximately cyclam 3 amL total of t-butyl of three times before thenitrite cleaned and 5t-butyl mL of 30 nitrite mM cyclam was were extracted mixed and stirred stored vigorously. at 2 ◦C. The The cyclam light was yellow/clear then removed solution of PVC-thiolactoneand the t turned-butyl nitrite a light wasgreen-yellow rewashed with more color cyclam after beinga total of allowed three times to reactbeforewith the cleanedt-butyl t- nitrite for butyl nitrite was extracted and stored at 2 °C. The light yellow/clear solution of PVC-thiolactone 30 min. Theturned SNAP-PVC a light green-yellow solution color was after then being cast allowed into polytetrafluoroethylene to react with t-butyl nitrite (PTFE)for 30 min. rings Theand left to dry underSNAP-PVC vacuum overnightsolution was tothen form cast ainto thin polytetrafluoroethylene polymer film. A plasticizer(PTFE) rings additiveand left to coulddry under be dissolved into the solutionvacuum beforeovernight casting to form the a thin film polymer to give film. more A plasticizer elastic properties additive could as purebe dissolved PVC caninto bethe somewhat brittle. Non-plasticizedsolution before casting PVC the was film used to give for more the elasti experimentsc properties being as pure described. PVC can be somewhat brittle. Non-plasticized PVC was used for the experiments being described.

NH2

Cl Cl Cl Cl Cl H2N Cl Cl HN Cl Cl NH2

x Triethylamine x Methanol 60oC 1-4 hours HN N,N-Dimethylacetamide O O

O O

HN HN

O S O SH O NH N NH

t-butyl nitrite Cl Cl HN Cl Cl Cl Cl HN Cl Cl

x x

Figure 1. Synthesis route of the nitric oxide-releasing polymer (SNAP-PVC). Bioengineering 2018, 5, 72 4 of 11

2.4. Polymer Characterization The quantification of the degree of amination of the PVC was accomplished using the ATTO-TAG FQ test for primary amines. Fluorescence measurements were completed using a 96-well plate reader (BioTek Instruments, Winooski, VT, USA) and a glycine standard was used to form a calibration curve. The fluorescent tag was maximally excited at 450 nm with an emission maximum at 550 nm [24]. Once the aminated PVC reacted with NAP-thiolactone, the primary amine disappeared and a free thiol was exposed on the polymer backbone. Ellman’s test for thiols was done to quantify the amount of thiols grafted onto the polymer using 5,5’-dithiobis (2-nitrobenzoic acid) (DNTB), following a modified protocol developed by George Ellman [25]. Absorbance was then taken at 412 nm after the reaction with DTNB persisted for 1 h using a 96-well plate reader. FTIR was used to monitor the reaction progress by following the important polymer functional groups (Mattson Genesis II). CHN combustion analysis was performed on a Costech 4010 Elemental Combustion System to quantify the overall nitrogen content in the various aminated PVC iterations.

2.5. Nitric Oxide Release by Photoinitiation The light source used for photoinitiated NO release from SNAP-PVC ﬁlms was 460 nm blue VAOL-5GSBY4 LED obtained from Mouser Electronics Inc. (Mansﬁeld, TX, USA). A 130 Ω resistor was connected in series with the LED and a variable voltage source was applied to the system. The emission spectra and relative intensity of the LED with respect to drive current was shown to be linear over the currents being used to power the LED for the study and was characterized by Starrett et al. [24].

2.6. Nitric Oxide Measurements Nitric oxide release from the polymer was directly measured by the chemiluminescent reaction of NO with ozone using a Sievers 280i Nitric Oxide Analyzer (Zysense, LLC, Frederick, CO, USA). SNAP-PVC films with a diameter of 5.5 mm were placed inside an amber glass sample holder with an inlet nitrogen sweep gas flowing at 200 mL min−1. The LED was mounted in the top of the sample holder, 5 cm above the film to be tested. Varying voltage levels were used (0, 3, 4.5, 6, 7.5 volts) at 130 Ω to demonstrate the control of the NO flux from the polymer film. Total NO release of the polymer films was done by cleaving the sulfur-nitroso bond through copper-mediated decomposition following a procedure developed by Frost et al. [26]. A solution of 10 mM CuBr2 and a 100 mM solution of L-ascorbic acid sodium salt was used for the NO quantification. 2 mL of the CuBr2 solution were placed in a glass sample holder containing a weighed SNAP-PVC film. After 5 min, 500 µL of the 100 mM L-ascorbic acid solution was then added to reduce the Cu2+ ions to Cu+, enhancing the sulfur-nitroso bond cleaving. The test was continued until all of the NO reservoir was depleted.

3. Results

3.1. Synthesis of Covalent Bound SNAP to PVC Several strategies were implemented to optimize the amount of NO release from the SNAP-PVC materials. The overall goal was to covalently attach SNAP in the highest capacity, while still being able to maintain precise control of NO release via photoinitiation. The initial amination step with ethylenediamine was varied by reaction time for 1, 2, and 4 h. This was done to optimize the greatest amount of reactive primary amine groups on the PVC resin backbone. When reacted for too long, primary amine end groups react and crosslink with separate PVC polymer chains, eliminating the possibility for SNAP attachment and greatly reducing the solubility of the PVC. On the other hand, too short of a reaction time will limit the available primary amine sites, thereby reducing the overall amount of SNAP attached to the polymer backbone. Once completely synthesized, it was found that the 2 h ethylenediamine reaction time SNAP-PVC was optimal for maximal NO loading and was used for all full testing and characterization. Bioengineering 2018, 5, x FOR PEER REVIEW 5 of 11 the 2 h ethylenediamine reaction time SNAP-PVC was optimal for maximal NO loading and was usedBioengineering for all 2018full ,testing5, 72 and characterization. 5 of 11

3.2. FTIR Analysis 3.2. FTIR Analysis Each step of the reaction schematic shown in the Figure 1 for the SNAP-PVC synthetic route was verifiedEach using step FTIR of the (Figure reaction 2). After schematic attaching shown ethy inlenediamine the Figure 1to for the the PVC SNAP-PVC backbone, primary synthetic amines route andwas secondary veriﬁed using amines FTIR were (Figure present2). Afterin the attachingspectrum ethylenediaminewhich was seen in to the the N-H PVC bending backbone, at 1641 primary cm−1. Theamines attachment and secondary of NAP-thiolactone amines were to present the free in pr theimary spectrum amine which sites indicated was seen the in theformation N-H bending of NAP- at PVC1641 cmas −the1. Thepresence attachment of a carbon of NAP-thiolactoneyl peak is evident to the at free 1750 primary cm−1 along amine with sites a indicatedbroad, short the formationthiol S-H peakof NAP-PVC at around as 2550 the presencecm−1 and ofan aamide carbonyl N-H peak stretch is evident peak at at3270 1750 cm cm−1. −Additionally,1 along with the a broad, secondary short aminethiol S-H peaks peak also at increased around 2550 with cm NAP-PVC−1 and an because amide N-Hone primary stretch peakamine at was 3270 converted cm−1. Additionally, to a secondary the aminesecondary and amine there peaks was also an increasedadditional with secondary NAP-PVC amine because present one primary in the amine newly was convertedattached acetylpenicillamineto a secondary amine group. and there was an additional secondary amine present in the newly attached acetylpenicillamine group.

FigureFigure 2. FTIR of PolyvinylPolyvinyl chloride chloride (PVC) (PVC) (green), (green), PVC-NH PVC-NH2 (Blue),2 (Blue), and andN-acetyl- N-acetyl-D-penicillamine-PVCD-penicillamine- PVC(NAP-PVC) (NAP-PVC) (Red). (Red).

3.3.3.3. Quantification Quantification of of Functional Functional Groups Groups QuantificationQuantification of of the the amination amination of of PVC PVC was was verified verified through through doing doing the theATTO-TAG ATTO-TAG FQ FQtest testfor primaryfor primary amines. amines. Once Oncethe NAP-thiolactone the NAP-thiolactone ring opened ring opened and reacted and reacted with the with primary the primary amine sites amine on thesites polymer on the polymerbackbone, backbone, free thiol free groups thiol were groups exposed. were exposed.To identify To the identify extent the to extentwhich tothis which reaction this wasreaction occurring, was occurring, Ellman’s Ellman’stest for thiols test for was thiols done. was The done. quantification The quantification of primary of primary amine amineand thiol and functionalthiol functional groups groups were normalized were normalized per milligram per milligram of polymer. of polymer. Primary Primary amine quantification amine quantification showed 0.201showed ± 0.0130.201 µmol/mg± 0.013 ofµmol/mg PVC-NH of2. Thiol PVC-NH quantification2. Thiol quantification showed 0.153 showed ± 0.009 0.153µmol/mg± 0.009 of NAP-PVC.µmol/mg Thisof NAP-PVC. was a 76.1% This conversion was a 76.1% rate conversion of primary rate amine of primaryto react aminewith the to reactthiolactone. with the The thiolactone. resultant exposedThe resultant free thiol exposed groups free where thiol groups then nitrosated where then to nitrosated form the toSNAP form group. the SNAP To verify group. the To verifytotal NO the loadingtotal NO capacity, loading copper capacity, mediated copper mediatedNO release NO wa releases performed was performed on the nitrosated on the nitrosated polymer polymer(SNAP- PVC)(SNAP-PVC) and showed and showed the NO the released NO released to be 0.0392 to be 0.0392 ± 0.0039± 0.0039µmol µpermol milligram per milligram of polymer, of polymer, which which is a totalis a total of 25.6% of 25.6% NO NOloaded loaded for the for potential the potential thiol thiol groups groups present. present.

3.4.3.4. NO NO Release Release from from SNAP SNAP-PVC‐PVC The physiological NO flux for healthy vasculature is estimated to be in the range of 0.5–4 × 10−10 mol·cm−2·min−1, therefore this is the target surface flux goal from NO-releasing Bioengineering 2018, 5, x FOR PEER REVIEW 6 of 11 Bioengineering 2018, 5, 72 6 of 11 The physiological NO flux for healthy vasculature is estimated to be in the range of 0.5–4 × 10−10 mol·cm−2·min−1, therefore this is the target surface flux goal from NO-releasing materials. The specific materials.NO flux generated The specific varies NO depending flux generated on the varies physiologi dependingcal status on the of physiologicalthe patient and status the location of the patient in the andbody. the Locations location in the body.vasculature Locations with in higher the vasculature sheer stress with will higher promote sheer higher stress local will NO promote release higher from localvascular NO endothelial release from cells vascular [27]. After endothelial damaging cells the [27 vascular]. After walls damaging from thean implanted vascular walls device, from more an implantedNO release device, may be more initially NO required release mayto prevent be initially unwanted required thrombosis to prevent and unwanted inflammation, thrombosis potentially and inflammation,leading to and neointimal potentially hyperplasia leading to and in the neointimal affected area hyperplasia [28]. Having in the a material affected that area is [28 able]. Having to adjust a materialthe NO flux that isincreases able toadjust the number the NO of flux applications increases the a hydrophobic number of applications polymer coating a hydrophobic could have polymer in a coatingmedical could setting. have in a medical setting. FigureFigure3 3shows shows the the NO NO release release profile profile from from SNAP-PVC SNAP-PVC films films (diameter (diameter 5.5 mm, 5.5 mm, thickness thickness 0.1 mm) 0.1 atmm) varying at varying applied applied voltage voltage levels usinglevels ausing 460 nm a 460 wavelength nm wavelength LED with LED no with bathing no bathing solution solution present (i.e.,present dry films(i.e., exposeddry films to exposed light). Nitrogen to light). sweep Nitrogen gas carried sweep the gas NO carried released the by photoinitiationNO released by to thephotoinitiation chemiluminescence to the chemiluminescence detector to result detector in a stair-step to result profile in a stair-step based on profile the voltage based supplied on the voltage to the LED.supplied The to profile the LED. showed The profile the wide showed range the of control wide rang in NOe of fluxcontrol that in the NO polymer flux that was the able polymer to obtain was −10 −2 −1 (~0–12able to ×obtain10 (~0–12mol·cm × 10·−min10 mol·cm). As−2·min the light−1). As emitted the light from emitted the LED from was the adjusted LED was (by adjusted varying the(by rangevarying of appliedthe range potential of applied between potential 0–7.5 between V), there 0– was7.5 littleV), there decay was in the little NO decay being in released the NO at being each level,released showing at each the level, stability showing of the the NO stability reservoir of the within NO reservoir the polymer. within Being the ablepolymer. to control Being the able NO to fluxcontrol of athe material NO flux can of bea material a useful can tool be for a inuseful vitro toolexperiments. for in vitro Since experiments. cells in theSince body cells interact in the body with NOinteract at specific with NO flux at rangesspecificof flux periods ranges of of time, period directlys of time, adding directly a soluble adding NO a soluble donor NO such donor as SNAP such wouldas SNAP not would accurately not accurately mimic what mimic is seen whatin is vivo seen. Thesein vivo donors. These continually donors continually decompose decompose over time over in solution,time in solution, resulting resulting in unpredictable in unpredictable burst releases burst releases of NO that of NO are that difficult are difficult to predict to [predict29]. Cell [29]. culture Cell experimentsculture experiments would benefitwould frombenefit a materialfrom a material that can that deliver can precisedeliver fluxesprecise of fluxes NO over of NO time over as time certain as therapeuticcertain therapeutic administrations administrations of NO may of NO be may necessary be necessary to achieve to achieve the desired the desired end result. end result.

FigureFigure 3.3. RepresentativeRepresentative nitric oxide (NO) release profileprofile of a SNAP-PVCSNAP-PVC filmfilm (5.5(5.5 mmmm dia.,dia., 0.10.1 mmmm thickthick disc)disc) fromfrom photoinitiatedphotoinitiated NONO releaserelease usingusing aa 460 nm LED with varied applied voltages, resulting inin variedvaried levelslevels ofof lightlight energyenergy impingingimpinging uponupon thethe filmfilm whilewhile thethe resistanceresistance waswas heldheld atat 130 130Ω Ω..

NO release of SNAP-PVC in phosphate buffered saline (PBS) at 37 °C was also tested to show NO release of SNAP-PVC in phosphate buffered saline (PBS) at 37 ◦C was also tested to show the release at physiological conditions. Trace transition metal ions are able to penetrate the polymer the release at physiological conditions. Trace transition metal ions are able to penetrate the polymer film and interact with the reactive SNAP groups (PVC is a popular polymer used in ion selective ﬁlm and interact with the reactive SNAP groups (PVC is a popular polymer used in ion selective electrodes) [30]. Most hydrophobic polymers tend to resist ion diffusion, which is seen with medical electrodes) [30]. Most hydrophobic polymers tend to resist ion diffusion, which is seen with medical grade silicone rubbers that are popular for in vivo applications [31]. The ability of transition metal grade silicone rubbers that are popular for in vivo applications [31]. The ability of transition metal ions ions and ascorbate to diffuse into PVC provides an additional pathway to stimulate the NO response and ascorbate to diffuse into PVC provides an additional pathway to stimulate the NO response for for SNAP-PVC, as light mediated NO release will not always be possible. In some scenarios, having SNAP-PVC, as light mediated NO release will not always be possible. In some scenarios, having a a level of passive release over time may be the optimal route for NO generation. This follows a similar level of passive release over time may be the optimal route for NO generation. This follows a similar release profile as seen with N-diazeniumdiolates NO donors, but is able to release lower amounts release proﬁle as seen with N-diazeniumdiolates NO donors, but is able to release lower amounts over a longer period of time. The passive release of SNAP-PVC in PBS at 37 °C is shown in Figure 4. over a longer period of time. The passive release of SNAP-PVC in PBS at 37 ◦C is shown in Figure4. The amount of NO generated in this manner could be controlled by adjusting the diffusion of ions Bioengineering 2018, 5, 72 7 of 11

Bioengineering 2018, 5, x FOR PEER REVIEW 7 of 11 The amount of NO generated in this manner could be controlled by adjusting the diffusion of ions into theinto PVC the by PVC applying by applying certain certain polymer polymer topcoats topcoats and/or and/or varying varying the amount the amount of SNAP-PVC of SNAP-PVC present present by controllingby controlling the thickness the thickness of the of SNAP-PVC the SNAP-PVC ﬁlms films or blending or blending SNAP-PVC SNAP-PVC with with standard standard PVC. PVC.

FigureFigure 4. 4.Passive Passive NO NO release release profile profile of of a a representative representative SNAP-PVC SNAP-PVC film film (5.5 (5.5 mm mm dia., dia., 0.1 0.1 mm mm thick thick ◦ disc)disc) placed placed in in PBS PBS at at 37 37C °C for for 24 24 h h (inset (inset shows shows the the cumulative cumulative NO NO release release from from the the film). film). The The NO NO releaserelease was was initiated initiated by by trace trace levels levels of of transition transition metal metal ions ions present present in in PBS. PBS.

TheThe NO NO capacity capacity and and release release demonstrated demonstrated by by SNAP-PVC SNAP-PVC could could potentially potentially be be much much larger larger as as therethere were were many many unreacted unreacted chlorine chlorine sites sites on theon backbonethe backbone of the of polymer. the polymer. A longer A longer reaction reaction time with time ethylenediaminewith ethylenediamine could be could implemented be implemented to achieve to a higherachieve degree a higher of amination degree butof complicationsamination but withcomplications crosslinking with and crosslinking solubility occur and solubility as PVC becomes occur as more PVC dechlorinated becomes more [32 dechlorinated]. This limits [32]. the NO This storagelimits the capacity NO storage of SNAP-PVC capacity of through SNAP-PVC using thro a componentugh using a like component ethylenediamine like ethylenediamine as an aminating as an agent.aminating PVC alsoagent. becomes PVC also darker becomes in color darker the morein colo dechlorinatedr the more dechlorinated it becomes, whichit becomes, would which hinder would the NOhinder release the byNO photoinitiation release by photoinitiation as light penetration as light throughpenetration the filmthrough would the be film limited. would As be mentioned limited. As previously,mentioned NO previously, release from NO films release reacting from withfilms ethylenediamine reacting with ethylenediamine for 1, 2, and 4 h for were 1, tested,2, and and4 h were 2 h wastested, found and to 2 have h was the found highest to have capacity the highest of NO capacity while still of allowing NO while photolytic still allowi cleavageng photolytic of the cleavage RSNO functionalof the RSNO group. functional A separate group. LED studyA separate varying LED the st leveludy ofvarying light impinging the level onof thelight polymer impinging films on was the performedpolymer films on all was three performed types of SNAP-PVCon all three filmstypes while of SNAP-PVC at 37 ◦C and films is shownwhile at in 37 Figure °C and5. is shown in Figure 5. Bioengineering 2018, 5, 72 8 of 11 Bioengineering 2018, 5, x FOR PEER REVIEW 8 of 11

Figure 5. SNAP-PVC photoinitiated NO release at different synthetic reaction times with Figure 5. SNAP-PVC photoinitiated NO release at different synthetic reaction times with ethylenediamine at 37 ◦C in nitrogen where 2 h (green) demonstrated a higher NO release potential ethylenediamine at 37 °C in nitrogen where 2 h (green) demonstrated a higher NO release potential than 1 h (red) and 4 h (blue). NO release from a SNAP-PVC ﬁlm (5.5 mm dia., 0.1 mm thick) was than 1 h (red) and 4 h (blue). NO release from a SNAP-PVC film (5.5 mm dia., 0.1 mm thick) was initiated using a 460 nm LED with a 130 Ω resistor and varying applied potentials as noted on the face initiated using a 460 nm LED with a 130 Ω resistor and varying applied potentials as noted on the face of the graph. of the graph.

InIn order order to to optimize optimize the the amination amination reaction reaction time, time, PVC PVC polymers polymers were were reacted reacted for 1, for 2 and1, 2 4and h and 4 h thenand convertedthen converted to SNAP-PVVC to SNAP-PVVC by reaction by reaction with the with thiolactone, the thiolactone, followed byfollowed nitrosation. by nitrosation. The polymers The werepolymers cast intowere films cast into and films irradiated and irradiated with high with intensity high intensity UV light UV to light force to complete force complete release release of NO. of TheNO. total The NO total release NO fromrelease the from resultant the resultant polymers polymers was recorded was withrecorded chemiluminescence with chemiluminescence detection. Figuredetection.6 shows Figure that 6 shows the 1 h that reaction the 1 timeh reaction SNAP-PVC time SNAP-PVC (blue trace) (blue released trace) thereleased lowest the levels lowest of levels NO whileof NO the while 2 h reactionthe 2 h reaction time SNAP-PVC time SNAP-PVC (green trace)(green released trace) released the highest the highest level of level NO forof NO the for same the illumination.same illumination. The 4 hThe amination 4 h amination time (red time trace) (red released trace) released a greater a greater total load total of load NO thanof NO the than 1 h, the but 1 wash, but significantly was significantly less total less NO total than NO the than 2 h the animation 2 h animation time. Antime. explanation An explanation for this for could this could be that be thethat amination the amination crosslinking crosslinking at 4 h at reaction 4 h reaction time time excessively excessively interferes interferes with with the ultimatethe ultimate formation formation of theof the SNAP SNAP groups groups on theon the PVC PVC backbone. backbone. This This was was further further confirmed confirmed by by CNH CNH analysis analysis that that showed showed thethe increase increase inin ethylenediamineethylenediamine reaction time time corresp correspondsonds to to an an increase increase in inthe the nitrogen nitrogen content content but buta decrease a decrease in primary in primary amines amines at the at the4 h 4reaction h reaction time time (see (see Table Table 1),1 where), where the the 4 h 4 reaction h reaction time time has has1/10 1/10 the primary the primary amines amines present present (0.018 (0.018 µmol/mgµmol/mg of polymer of polymer compared compared to 0.201 to µmol/mg 0.201 µmol/mg of polymer of polymerfor the 2 for h reaction the 2 h reactiontime) even time) though even it though has a high it has total a high nitrogen total nitrogencontent by content mass by(0.20% mass N (0.20% for 4 h Nreaction for 4 h time reaction compared time compared to 0.17% N to for 0.17% 2 h reaction N for 2 time). h reaction A higher time). level A higherof amination level of with amination primary withamines primary could amines possibly could be possiblyachieved, be thereby achieved, increasing thereby increasingthe capacity the to capacity form SNAP to form by SNAP using by a usingprotected a protected aminating aminating agent agentinstead instead of an of anaccess accessibleible difunctional difunctional aminating aminating agent agent suchsuch asas ethylenediamine.ethylenediamine. ThisThis wouldwould allowallow thethe attachmentattachment ofofthe the reactive reactive molecule molecule to to the the backbone backbone of of the the PVCPVC chains chains with with no no crosslinking. crosslinking. The The polymer polymer could could then then be be deprotected deprotected to to expose expose the the reactive reactive group group thatthat could could then then be be modified modified with with SNAP. SNAP. Bioengineering 2018, 5, 72 9 of 11 Bioengineering 2018, 5, x FOR PEER REVIEW 9 of 11

FigureFigure 6. Total 6. Total nitric nitric oxide oxide release release from from SNAP-PVC SNAP-PVC polymer polymer synthesized synthesized with with different different reaction reaction times times for initialfor initial amination. amination. Chemiluminescence Chemiluminescence detection detection was used was toused record to record total NO total release NO triggeredrelease triggered with UVwith light. UV The light. SNAP-PVC The SNAP-PVC were reacted were with reacted ethylenediamine with ethylenediamine for 1 h (blue), for 1 2h h(blue), (green), 2 h and(green), 4 h (red). and 4 h The(red). measurements The measurements were made were with made cast ﬁlms with ofcast SNAP-PVC films of SNAP-PVC that were allowed that were to cureallowed under to ambientcure under conditionsambient protected conditions from protected light. from light.

TableTable 1. Primary 1. Primary amine amine content content using using the the ATTO-TAG ATTO-TAG FQ FQ Amine-Derivitization Amine-Derivitization test test and and nitrogen nitrogen contentcontent via CHNvia CHN analysis analysis of PVC of PVC aminated aminated for 1,for 2 1, and 2 and 4 h 4 to h observe to observe the the primary primary amine amine and and nitrogen nitrogen contentcontent as reaction as reaction time time with wi ethylenediamineth ethylenediamine increases. increases. Sample Primary Amine Content (µ mol/mg) % N (by Mass) µ PVCSample 1 h reaction time Primary Amine 0.036 Content ± 0.016 ( mol/mg) % N (by 0.15 Mass) PVCPVC 1 h reaction 2 h reaction time time 0.036 0.20± 0.016 ± 0.013 0.15 0.167 PVC 2 h reaction time 0.20 ± 0.013 0.167 PVC 4 h reaction time 0.019 ± 0.0023 0.20 PVC 4 h reaction time 0.019 ± 0.0023 0.20

4. Conclusions 4. Conclusions The simple, robust synthesis of NO-releasing SNAP-PVC that contained covalently bound The simple, robust synthesis of NO-releasing SNAP-PVC that contained covalently bound SNAP SNAP functional groups was fully characterized. Optimization was completed to identify what functional groups was fully characterized. Optimization was completed to identify what degree of degree of amination to the chlorine backbone of PVC promotes the most SNAP attachment. Using a amination to the chlorine backbone of PVC promotes the most SNAP attachment. Using a 460 nm LED, 460 nm LED, controlled photoinitiated NO release was achieved. The material demonstrated its controlled photoinitiated NO release was achieved. The material demonstrated its ability to cover a ability to cover a wide range of NO fluxes (0–35 × 10−10 mol·cm−2· min−1) that could be useful for wide range of NO ﬂuxes (0–35 × 10−10 mol·cm−2· min−1) that could be useful for potential in vitro potential in vitro studies that require c = varied levels of controlled NO delivery. This could greatly studies that require c = varied levels of controlled NO delivery. This could greatly improve the potential improve the potential applications for blood- and tissue-contacting PVC-based materials that are applications for blood- and tissue-contacting PVC-based materials that are already used in medical already used in medical settings. The material could also be used as implantable device coatings to settings. The material could also be used as implantable device coatings to improve biocompatibility improve biocompatibility or to prevent neointimal hyperplasia of vessels from its passive NO release or to prevent neointimal hyperplasia of vessels from its passive NO release capabilities. Future studies capabilities. Future studies are being done to extend the life of NO-releasing polymers. are being done to extend the life of NO-releasing polymers. Author Contributions: Conceptualization, M.C.F.; Data curation, S.P.H.; Formal analysis, S.P.H. and M.C.F.; Author Contributions: Conceptualization, M.C.F.; Data curation, S.P.H.; Formal analysis, S.P.H. and M.C.F.; FundingFunding acquisition, acquisition, M.C.F.; M.C.F.; Investigation, Investigation, S.P.H. S.P.H. and and M.C.F.; M.C.F.; Methodology, Methodology, S.P.H. S.P.H. and and M.C.F.; M.C.F.; Project Project administration,administration, M.C.F.; M.C.F.; Supervision, Supervision, M.C.F.; M.C.F.; Validation, Validation, S.P.H.; S.P.H.; Writing—original Writing—orig draft,inal draft, S.P.H.; S.P.H.; Writing—review Writing—review & editing,& editing, S.P.H. S.P.H. and M.C.F. and M.C.F.

ConﬂictsConflicts of Interest: of Interest:The The authors authors declare declare noconﬂict no conflict of interest. of interest.

Bioengineering 2018, 5, 72 10 of 11

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