Dennis.Murray

Nitric Oxide-Releasing Biomaterial Development and Characterization
=Abstract=

Overcoming biocompatibility is an issue that is under on-going investigation for implantable biomedical devices. One strategy is to implore biomaterials in these devices that release Nitric Oxide (NO) in a controlled fashion to reduce thrombus formation and foreign body response of the immune system. Donor molecules can be covalently attached to biomaterials to generate this property of quantifiable, controlled NO release. Research has been conducted on many of these materials including but not limited to: polyesters, polyurethanes, polysaccharides, silica nanoparticles, Tygon© and xerogel films. 2 donor molecules were used in these studies: S-nitrosothiols and N-diazeniumdiolates. Using chemiluminescense nitric oxide assays, the flow of NO out of the modified materials can be quantified

=Introduction=


 * Nitric Oxide (NO) Therapy**

Nitric oxide (NO) is a diatomic free radical signaling molecule that exhibits many potentially therapeutic effects and has thus become a topic for biomedical applications. It is involved in a wide array of physiological processes including wound healing, immune response, and cancer pathology. NO was discovered to be the endothelium-derived relaxation factor (EDRF). It has also been shown that NO in a natural anti-thrombotic agent as it a regulator of platelet adhesion [1]. In the human body, NO is produced from L-arginine in the presence of nitric oxide synthase (NOS.) [2] When produced in the vascular endothelium, NO can go on to effect smooth muscle cells, platelets and immune cells. After NO diffuses into smooth muscle cells, it activates guanylate cyclase which results in the production of cyclic guanosine monophosphate (cGMP.)[1-3] This results in activation of cGMP-regulated kinases and phosphodiesterases, which play a role in neurotransmission, vasodilation, inhibition of platelet aggregation, and relaxation of smooth muscle [4,5]. cGMP production then results in vasodilation of these cells promoting healing in injured areas. NO deficiencies can occur in a number of cardiovascular diseases including: atherosclerosis, heart failure, hypertension and coronary heart disease. [6-8] Introduction of NO exogenously has become an interesting topic for therapy for NO deficient conditions. However, large concentration of NO can result in many ill-effects including cytotoxicity so regulation of NO release is of utmost importance when considering therapeutic application [9]. Tumor cell proliferation can also be mediated by NO therapy. This type of therapy appears to be concentration-dependent however, as higher concentrations result in tumor cell apoptosis while low concentrations result in increased survival. [10] NO is a signaling molecule that can create a pro or anti-apoptotic response.

Hemoglobin readily uptakes NO in the blood resulting in a short lifetime of about 3 seconds [6]. This short half-life can also be attributed to the gaseous nature of the molecule at physiological pH. Because of this, developing a technique for delivering NO consistently to an effected area over time has become topic of interest among researchers. Advances in material science and engineering have created mechanisms for addressing the issue of drug delivery. Creating matrices that contain NO-donors can allow for controlled release of the drug. Scaffolds such as hydrogels, polymers and nanoparticles all provide a means for delivering NO in a tunable manner. The end result is to increase plasma retention time of NO local to affected areas [8]. Biocompatibility is an important concept when developing any implantable medical device. In order for the device to be effective, the body must not reject the foreign material. This type of rejection is known as foreign body response and can be minimized by introducing NO-donors in biomaterials used in implantable devices.


 * NO-Releasing Moities**

S-nitrosothiols These compounds release NO in the presence of heat, light and some metals. They are generated by reacting a parent thiol with a nitrous acid. They are known to release NO under copper-ion mediated decomposition, reaction with ascorbate and homeolytic cleavage when exposed to light. Two common S-nitrosothiols used are S-nitrosoglutathione (GSNO) (1) and S-nitroso-N-acetylpenicillamine (SNAP) (2) because they are easily synthesized and relatively stable. These compounds were effectively as vehicles for NO delivery



NO can be released spontaneously or assisted by lymphocyte cleavage. S-Nitrosocysteine (3), and S-nitrosoglutathione (1) have been found in humans and are suspected to serve as NO-reservoirs for the body [1,2]

N-diazeniumdiolates N-diazniumdiolates (NONOates) (4) are formed by the reaction of NO with primary or secondary amines under basic conditions at low temperature. They are unique in that they allow for controlled NO delivery regulated by pH. These groups undergo proton-mediated decomposition which leads to the release of 2 equivalents NO per N-diazniumdiolate (4).



Scheme 1: Proton mediated N-diazeniumdiolate (4) decomposition [] Therefore if the degree of functionalization can be quantified when designing a material, then a precise measure of the amount of NO to be released can be determined. Also, the rate of NO release can be determined as a function of the rate constant for the decomposition of the diazinumdiolate (4). Protecting groups can be added that can be cleaved to release NO in a more controlled fashion. One example of this uses β-galactosidase to cleave NONOate (4) protected with galactose. Therefore NO can be released as a function of β-galactosidase concentration leading to a higher degree of regulation [1,2].

=NO-Releasing Biomaterials= It has been shown that flux of NO can be tailored using functionalized, cross-linked polyesters. In addition, these polymers can be designed to be degradable and resorbable in a nontoxic manner. This kind of approach is very appealing because of the extensive applications that degradable materials can have. By degrading into nontoxic metabolites, the result is reduced healing time for patients undergoing surgical implantation [11]. Also, foreign body response is minimized as the device naturally degrades to be removed by the patient’s own body. This also reduces morbidity and the cost associated with follow-up surgeries to remove the implant.
 * Polyesters**

Such polyesters have been generated that exemplify these desired characteristics. In one study, N-diaziumdiolate- and S-nitrosothiol- functionalized polyesters were developed that were shown to do a few important things. Firstly, they achieved a maximum release of 0.81 μmol NO cm-2 for up to 6 days. These materials showed an % 80 reduction of P. aeruginosa, a common bacteria associated with implant infection [11, 12]. Also, these materials could be fully degraded in vitro in a minimum of nine weeks. An important feature of these materials however is their ability to be modified for desire results.



Figure 1. Degradation profiles of NO-releasing polyesters PE1-PE6 [11]

Figure 1 shows that over the course of 10 weeks the six polyesters synthesized resulted in a large range of degradation rates. All of the polyesters can degrade into nontoxic metabolites and contain NO-releasing functional groups. The degradation rates were tailored by the prepolymer mixtures used. Here, PE4 was synthesized using adipic acid and pentaerythritol (almost no degradation) and glutaric acid and glycol (greatest degradation rate.) In general, glutaric acid-based polyesters exhibited greater degradation rates than their adipic acid-based counterparts. Another beneficial characteristic of these materials is that all have glass transition temperatures well below physiological temperature indicated that they are flexible at 37⁰C. This property can be very important for devices that could utilize this technology as a coating increases the amount of potential applications.


 * Polyurethanes**

NO-releasing polyurethanes are another potential mode of controlled NO delivery. In general, increased hydrophobicity of a material leads to better blood biocompatibility. This is because as the hydrophobicity increases, plasma protein absorption of the material decreases. Polyurethanes possess both hydrophilic “soft” segments and hydrophobic “hard” segments but they undergo surface restructuring in the presence of water that align all hard segments on the outside resulting in a material that is hydrophobic on the outside when exposed to blood [12]. This makes polyurethanes an intriguing class of materials when designing biomedical devices. The problem is that protein and platelet adhesion remains an issue in polyurethane-based devices. In essence, it seems then that if a polyurethane could exploit the anti-protein and platelet adhesion properties of NO by releasing it in a controlled manner, this issue of adhesion could be addressed. Additional research must be conducted to determine how such a material could be used clinically but these polyurethanes have been synthesized and NO release has been characterized [12].



Figure 2. nitrosated polyurethane NO-release profiles [12]

Here, a series of nitrosated polyurethanes were synthesized that exhibited a maximum NO-release profile of 0.20μmol NO cm-2 which is comparable to the 0.18 μmol NO cm-2 seen with polyesters. Also, antithrombotic flux (0.4 pmol cm-2 s-1) was achieved for nitrosated polyurethanes NTPU1 and NPU-POMT for greater than 30 hours.



Figure 3. NO flux v. Time of NTPU1 compared with antithrombotic threshold

It has been shown that nitrosated polyurethanes can release NO in similar quantities to those in polyesters. Further development can be made to design potential NO-releasing biomaterials based on this polyurethane structure.

One group from the University of North Carolina is investigating the use of such a material to coat implantable biosensors [13]. Namely, they investigated using electrospun polyurethane fibers doped with NO-releasing dentrimers. These fibers encapsulated a needle-type implantable glucose sensor that could be used by diabetics. This membrane coating could help mitigate the issue of foreign body resistance in such a device.

Dextran is a naturally occurring polysaccharide that is used to reduce cardiopulmonary inflammation, blood plasma volume expansion and in blood isotonic electrolyte solutions. This, in conjunction with NO-releasing moieties could provide interesting clinical applications and biomedical device development. One study investigated this material and showed that S-nitrosated dextran thiomers could be synthesized that achieved appreciable NO flux [14,15]. In addition, since it is well documented that dextran is enzymatically degraded by dextranase at the 1,6-α-glycosidic linkage, it was shown that this NO-releasing material could be degraded by the relatively simple means of introducing an enzyme [10]. This study investigated just two of these potential biomaterials and characterized their ability to load NO and enzymatically degrade.
 * Polysaccharides**

Nanoparticles are of particular interest when discussing NO-releasing biomaterials. If NO donor molecules can be incorporated into nanoparticles, these particles can be introduced into a scaffold for NO delivery. Silica nanoparticles have been shown be potential candidates for this type of design. These particles show promise because of their ability to be functionalized in addition to the great surface area generated by nanoparticles [16]. In addition, they are inherently biocompatible and can be made more biocompatible by further functionalization. For instance, it has been shown that coating silica nanoparticles with polyethylene glycol (PEG), hydrophobicity is increased which results in decreased immune response and increased blood circulation ratio [17-19]. Size of particles and functionalization can be controlled by the solution chemistry and emulsion parameters to create specific particles for specific needs. Therefore, these particles can be designed to “fit” inside a given matrix and release NO at a specified rate through functionalization.
 * Silica Nanoparticles**

Tygon© is a proprietary plasticized poly(vinyl chloride) material used in many extracorporeal circuits (ECC). Along with many other polymeric materials used for implantable devices, blotting clotting and infection are seen often as complications to surgical procedures. Platelet accumulation starts immediately after this material is exposed to blood. Typically, a systemic anticoagulant drug such as heparin is administered in conjunction with this type of implant. In order to address this issue, one group examined doping Tygon© with S-nitrosoglutothione (GSNO) [20]. This dopant was chosen because GSNO and the parent thiol, glutathione occur naturally in the human body so any consequent leaching would not produce harmful byproducts. The GSNO-Tygon© material was generated and exihibited the following NO-release profile
 * GSNO-Tygon©**



Figure 4. NO-Release Profile for GSNO-Tygon© [20]

What they found was that at 20 w/w% GSNO, the modified Tygon© released 0.64 x 10-10mol NO cm-2min-1. This group was therefore able to tune this material to release NO in a fashion similar to natural endothelial release. This was done on a polymer that is already used in biomedical devices so the results are intriguing. They found this flux was relatively steady for a period of 5 hours making it a potential candidate for ECC device development.

Creating a NO-releasing film has many potential uses. Any implantable that could be coated with a film that releases NO in a controlled manner could substantially increase the biocompatibility of that device. In a study by Storm et al, aminosilane monomers modified with N-diazeniumdiolate were synthesized. These monomers were then polymerized to create a xerogel film that was capable of releasing NO 0.39-3.13 μmol cm-2 over a period of 11.1-90.8 hours. The three aminosilane precursors used were N-2-(aminoethyl)-aminopropyltrimethoxysilane (AEAP) (5), N-ethylaminoisobutyltrimethoxysilane (EAiB) and N-methylaminopropyltrimethoxysilane (MAP). NONOate modification followed for the three aminosilanes followed scheme 2:
 * Xerogel Film**



Scheme 2: NONOate modification of AEAP (5) [21]

The three N-diazeniumdiolate-modified silanes were taken on for characterization:



Xerogels synthesized from 5, 10 and 15 mol% of each silane were generated that resulted In the NO release profiles seen in Figure 5. A Chemiluminesence Sievers 280 Nitric Oxide Analyzer was used to test the films.



Figure 5: NO release in N-diazeniumdiolate-modified Xerogels [21]

Total NO release ranged from 0.39-3.13 μmol cm-2. Simply by changing the composition of the silanes prior to xerogel formation, this group showed effectively that NO release over time could be controlled. In all models, a 5 mol% increase resulted in an increase of total NO released and duration of release. In the same study, the 15 mol% AEAP xerogel films were used to coat the sensing layer of insulated platinum disc macroelectrodes in a glucose sensor. They found that the coated senor remained sensitive enough for clinical applications. When submersed for 7 days in PBS, the sensors exhibited a sensitivity and dynamic range of 3.5nA mM-1 and 1-30mM, respectively



Figure 6: 15mol% AEAP xerogel film coated glucose sensor

When compared to the noncoated sensor, they found 3.6% decrease glucose sensitivity. There was also an increase in dynamic range and response time but were deemed acceptable when compared to other methods and materials. Other studies from Gifford et al. suggest that a glucose sensors remain functionally reliable with sensitivity of 4.88-6.77nAmM-1 and response time is adequate for up to ~10min.[21]

=Conclusion= A wide array of NO-releasing biomaterials provide significant promise in the development of biomedical devices. NO is a naturally occurring signaling molecule that has important roles in cell survival and proliferation. This molecule has been shown to reduce platelet aggregation locally as well as kill bacteria and prevent foreign body response. Developments must be made to characterize biomaterials that contain NO-donor moieties so that specific needs can be tailored. Another challenge that researchers may face is that the assays used for measuring NO release are under investigation. One study suggests that the commonly used methods of Greiss assay and chemiluminesence assays may not provide accurate results. These were the assays used in the studies outlined in this review. Perhaps further studies should incorporate another method of detection when characterizing these materials. One such assay is an amperometric NO sensor that utilizes and electrochemical sensor with much lower detection limits than traditional NO assays [22]. In the end, in vitro analysis provides only prediction for how the materials will perform in the human body. Many NO-releasing biomaterials have already been investigated including polyesters, polyurethanes, polysaccharides, silica nanoparticles, Tygon© and xerogel film that seem to exploit the desired characteristics of such a material. Many medical devices already utilize such materials and could greatly benefit from antithrombotic and antimicrobial properties. Ultimately, the goal is to create a material that can be tailored to load a desired amount of NO and release it at a specified rate. In this way, an implantable device could remain in the body without being attacked by the immune system. Then for many potential applications it would be desirable for the material to degrade and be resorbed into the body over time. This allows for an implantable device that does not need to be removed surgically that also has improved biocompatibility. Also, by reducing the risk of thrombosis, patients may not need to take anticoagulants which would decrease the risk of hypertension and internal bleeding associated with these medications. Further studies must be done to get this type of technology to the public, however. Any implantable medical device must go through the rigors of FDA regulation. For the safety of the user, materials like these would need to be extensively characterized to determine exact synthesis parameters. From there, the materials would require years of testing in animal models and eventually clinical testing on humans. If any counter-indications developed suggested side effects or mechanical failure of the materials, development could be halted. In these early stages of research, however, the materials surveyed in this review seem promising for application in biomedical devices. No matter how the development process continues from here, these studies seem to prove the concept that NO-donor molecules, covalently attached to biomaterials may provide a means of making better, more biocompatible medical devices.

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