A Multilayered Biomaterial for Vectorial Release of DHA-Derived RvD1 to Restrain Local Tissue Inflammatory Responses and Disorders

This article at a glance
  • A thin, pliable, biodegradable multi-layered material incorporating the DHA-derived lipid mediator resolvin D1 (RvD1) was developed.
  • The thin-film device allowed sustained diffusion of RvD1 from the surface with the lowest copolymer density, permitting one-sided release.
  • RvD1 could be released from the thin-film device into arterial tissue, and activated anti-inflammatory, anti-proliferative and anti-migratory activity in smooth muscle cells.
  • Thin-film devices eluting specific pro-resolving lipid mediators (SPMs), such as RvD1, may be further developed for applications in surgical and endovascular interventions to lower or resolve local inflammatory reactions and surgical complications.
A variety of implantable devices are currently being employed in interventional cardiology and other surgical fields, to repair and treat different health-threatening tissue lesions. For example, the placement of stents and bioresorbable scaffolds is a relatively common procedure in myocardial revascularization to restitute blood flow delivery to a poorly perfused part of the myocardium. Other relatively frequent revascularization interventions, such as in the lower extremities in patients with varying types and degrees of peripheral artery disease, employ different stents and prosthetic vascular grafts. Implants intended to have long residency times in the body, such as bone replacements, artificial mitral and tricuspid valves, and vascular stents, need to have a high degree of patency. They should adequately adhere to surrounding tissue, be compatible with the physical demands of the tissue in which they are implanted, and should not cause chemical and physical irritation that would trigger thrombosis (in intra-vascular implants) or inflammatory responses that would ultimately lead to failure of the device, or stimulate tissue remodeling. Restenosis (narrowing of the lumen of a blood vessel previously opened by some surgical intervention) and neo-atherosclerosis are frequently observed following the placement of an endovascular device or venous or prosthetic graft, and comprise a chronic inflammatory reaction with neo-intimal hyperplasia and fibrosis. In contrast to implants that should last, an implanted material may need to be fully resorbed, e.g. bioresorbable stents, sutures and some prosthetic materials. Additionally, the implant can be a drug-eluting device with the eluting drug contributing to device patency. For example, the implementation of vascular stents that release immunomodulating drugs has measurably lowered the incidence of restenosis. The sustained perivascular release of such drugs from an implanted biomaterial has also been shown to exert anti-restenotic activity in animal models. Given the variety of different tissues that are potentially subject to repair or treatment using biomaterials, and the distinct demands placed on device patency, interest and space for innovations abound for the delivery of substances that can improve the outcome of surgical interventions or as locally-delivered therapeutic agents. A particularly attractive prospect is learning how to harness the body’s own mechanisms to control local tissue inflammatory responses and stress reactions to achieve a high degree of device patency. In this regard, the omega-3 LCPUFA eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and DHA, generate a family of bioactive compounds known as the specialized pro-resolving mediators (SPMs), which have been shown to have important roles in modulating inflammatory responses. SPMs, such as DHA-derived D-series resolvins, potently activate the termination of inflammatory responses. Resolution of inflammation involves the modulation of a range of cellular activities of the innate immune system, such as neutrophil and macrophage migration, cytokine release, phagocytosis and apoptosis, as well as the activity of resident tissue cells (such as migration and proliferation). Given the involvement of SPMs in the self-limiting nature of inflammation, a growing body of research has addressed the ability of SPMs to modify the outcome of a range of acute and chronic inflammatory disorders. Employing SPMs in bioresorbable scaffolds may offer new prospects for device patency. One such SPM, resolvin D1 (RvD1; 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid), promotes tissue protection from inflammatory injury in various settings of inflammation, including vascular injury, ischemia-reperfusion injury, acute kidney injury and inflammatory bowel disease. Studies addressing the in vivo activity of SPMs in animal models generally involve the administration of the test substance directly into the circulation, by oral administration (through the drinking water, or by gavage), or topically (onto the skin surface). SPMs are considered to generally function as autacoids, i.e. they exert local hormone-like activity on neighboring cells to activate a range of inflammation-resolving activities. Specific enzymes rapidly degrade SPMs into biologically inactive metabolites that presumably preclude spill-over beyond the cells and tissues where these mediators are formed. Although SPMs are capable of exerting systemic effects upon oral and systemic administration, a way to locally deliver SPMs specifically to tissues where resolution activation is desired in a controlled fashion has not been addressed. Previous reports have shown that local activity of SPMs in the context of biomaterials may be useful. For example, in experiments with a porous chitosan-based acetylated biomaterial in a murine inflammation model, RvD1 was able to modulate the functional phenotype of macrophages towards one involved in inflammation resolution. Nanoparticles prepared from human neutrophil microparticles enriched with SPMs have also been shown to have anti-inflammatory and proresolving properties. However, a well-defined biomaterial that allows the local release of SPMs in a sustained manner while maintaining biological activity over a long time remains to be achieved. A recent study has addressed this possibility. The development of such a new biomaterial was carried out by Lance and colleagues from the UCSF Graduate Group in Bioengineering at University California Berkeley, San Francisco, CA, in collaboration with colleagues at the Department of Bioengineering and Therapeutic Sciences, and the Cardiovascular Research Institute and Department of Surgery, at the University of California, San Francisco, and the Department of Anesthesiology, Perioperative, and Pain Medicine at Brigham and Women’s Hospital at Harvard Institutes of Medicine, Boston, MA, USA. The study reports on the development of a thin-film device composed of two or three layers of poly(lactic-co-glycolic acid) (PLGA) each with a different density. Bilayered and trilayered composite films were created by sequential layering of thin films of co-polymer solution using the spin coating technique. Film layers with distinct densities were achieved by using solutions of PLGA in which the ratio of lactic acid to glycolic acid used for the formation of the co-polymer was different. PLGA layers with ratios of lactic to glycolic acid co-polymers of 50:50, 72:25 and 85:15, had average layer thicknesses of 12, 9.9 and 26 µm, respectively. After assembly into a trilayer, an average film thickness of 50.9 µm was obtained. Such devices were transparent and pliant. RvD1 was placed in between layers and patches of multilayered materials were obtained that would theoretically allow vectorial diffusion of the lipid mediator (100 ng and 200 ng for bilayered and trilayered devices, respectively) towards and out of the side of lowest density. PLGA is biodegradable since it slowly hydrolyzes in the body, and is biocompatible as its aqueous hydrolysis products lactic acid and glycolic acid are endogenous metabolites. The expected mass transfer behavior was evaluated in practice by several approaches. First, it was confirmed that RvD1 could diffuse from the composite materials, by measuring release over time into buffer or serum-free medium when placed in test tubes at 37oC. Maximum recovery of entrapped RvD1 was determined by homogenization in ethanol. The directional elution of RvD1 was studied by placement of the device in a two-sided diffusion chamber that allowed sampling of medium in contact with either side of the trilayered film. Release of biologically-active RvD1 from the thin-film device was determined in various ways. The effect of RvD1 on primary human vascular smooth muscle cells isolated from saphenous vein explants was determined by placement of the thin-film device in a permeable transwell insert placed over the cells grown in culture. Modulation of NF-kB activity (as a readout of inflammatory activity) following the addition of tumor necrosis factor- α (TNF-α), smooth muscle cell proliferation, cell viability, and cell migration into an experimental injury site (modelled by “scratching” a thick line of cells growing on the culture dish), were measured to assess the release of biologically active RvD1. An ex vivo flow chamber was designed to test RvD1 release from the lower-density side facing an inner piece of rabbit aorta, which was in turn enclosed by a larger diameter section of aorta. The inner vessel was then perfused in a closed circuit with pulsed flow of serum-free media, in which the accumulation of RvD1 could be measured. Finally, in vivo device functionality was determined in a rat model in which the trilayered material was wrapped around the left carotid artery with the lowest density side facing the artery. After one hour, RvD1 levels were measured by enzyme-linked immunoassay in arterial tissue of the left and right carotid artery. Release of RvD1 that had been enclosed between the layers reached a maximum rate after three weeks when tested in vitro, with rates staying constant up to seven weeks. Release rates were approximately twice as high into a serum-free medium than in a phosphate-buffered salt solution (PBS), indicating that the environment has a marked effect on RvD1 diffusion. The physical disintegration of the thin-film device observed at 10 weeks was visibly more marked in serum-free medium than in PBS, and may also contribute to the higher RvD1 release. The amount of RvD1 that could maximally be released was determined after homogenization of the device into ethanol and constituted 84% of the total dose loaded within the film structure. In non-homogenized devices, release into ethanol did not surpass 35% of total dose, suggesting that total surface area plays an important role in releasing the bulk of the incorporated RvD1. In PBS, the amount eluted did not surpass 14% of total RvD1 load. Employing the two-sided diffusion chamber, directional RvD1 release occurred after one day, with significantly higher release of RvD1 from the low-density side. After two weeks, 97.9% of the total released RvD1 had been released on the low-density side, demonstrating that the high-density PLGA layer effectively blocked diffusion. RvD1 that diffused from the device was shown to be chemically stable when PBS was used, whereas in serum-free medium at least two other structurally-related substances of unreported identify were observed in addition to RvD1 itself. Assays of vascular smooth muscle cell responses to trilayered thin-film devices showed that RvD1-loaded devices markedly inhibited a cellular inflammatory response (determined after 20 hours) initiated by TNF-α exposure, nearly abolished serum-induced cell proliferation (determined after 10 days), and significantly reduced cell migration (determined after 24 hours). These effects were observed at RvD1 concentrations of 5-50 nanomolar in the medium. No effect was observed on endothelial cell migration in a similar scratch injury model. In the ex vivo aorta perfusion model, RvD1 release from the low-density side towards the inner vessel was 0.41 picogram per mg artery in 24 hours. In contrast, release towards the outer vessel was 4.4-fold lower (0.094 picogram per mg tissue), showing that vectorial release could be achieved within a biological tissue. Continuous removal of RvD1 from the inner aortal tissue into the perfusion medium may likely have occurred, and the absolute dose of RvD1 released from the low-density side is likely higher than calculated from tissue content alone. Finally, employing the thin-film device as a perivascular wrap showed that release of RvD1 into the left carotid arterial tissue (0.63 picogram per mg tissue) was significantly higher than the levels measured in the right carotid artery that was used as control tissue (with basal tissue levels of 0.12 picogram RvD1 per mg tissue). This study has reported on the development of a new multi-layered thin-film drug delivery device made from bioresorbable material that allows vectorial diffusion of an omega-3 LCPUFA-derived lipid mediator with anti-restenotic and inflammation-resolving activity. Release from the thin-film biodegradable device was sustained over several weeks and could be directed towards one side of the device by using graded layer densities. Biological activity was confirmed and occurred at medium concentrations in the range where RvD1 is expected to act as an agonist (high picomolar to low nanomolar). Restenosis and thrombosis constitute significant sources of risk to endovascular interventions. Continued efforts are being made in material development to reduce and eliminate these risks. The use of RvD1 in a bioresorbable device that permits sustained localized release may have specific advantages in vascular lesions involving neointimal hyperplasia, such as in restenosis after venous grafting and in-stent restenosis. RvD1 triggers the active down-regulation of vascular smooth muscle migration and proliferation, the activation of local tissue inflammatory response to injury or cytokine stimulation, as well as oxidative stress. The advantage of this new thin-film device, in particular its ability of directional SPM release, for specific applications remains to be demonstrated. The authors indicate that many applications that are surgically-adaptable may be evaluated, for example in bypass-grafting, and reducing neointimal hyperplasia of carotid angioplasty. The observation that the type of matrix around the thin-film device affected the rates of release, the stability of the device itself, and the formation of structurally-related analogues of RvD1, indicate that the precise kinetics of the release of the enclosed RvD1, or other SPMs, will be challenging to predict in different in vivo applications. Depending on the purpose, sustained release over several weeks may be sufficient to address local therapeutic delivery of an SPM, after which the device is gradually degraded and absorbed. The vectorial diffusion that initially directs RvD1 release towards the intima and vascular lumen when placed around a vessel, may be lost over time as the device progressively disintegrates. How resolvins affect the resorption of the otherwise biodegradable PLGA thin-film device will need to be tested. Combinations of this material to improve the grafting of stable implants may also be envisaged. The capacity of SPMs to promote microbial clearance, and improve device patency by lowering risk of infections associated with biomaterial implantation, may also constitute an additional advantage. The road of medical device development is very long, but it will be very interesting to watch how this new functional biomaterial may find its way into clinical practice.   Lance KD, Chatterjee A, Wu B, Mottola G, Nuhn H, Lee PP, Sansbury BE, Spite M, Desai TA, Conte MS. Unidirectional and sustained delivery of the proresolving lipid mediator resolvin D1 from a biodegradable thin film device. J. Biomed. Mater. Res. A 2017;105(1):31-41. [PubMed]   Worth Noting Buccheri D, Piraino D, Andolina G, Cortese B. Understanding and managing in-stent restenosis: a review of clinical data, from pathogenesis to treatment. J. Thorac. Dis. 2016;8(10):E1150-E1162. [PubMed] Chiang N, Fredman G, Bäckhed F, Oh SF, Vickery T, Schmidt BA, Serhan CN. Infection regulates pro-resolving mediators that lower antibiotic requirements. Nature 2012;484(7395):524-528. [PubMed] Farb A, Weber DK, Kolodgie FD, Burke AP, Virmani R. Morphological predictors of restenosis after coronary stenting in humans. Circulation 2002;105(25):2974-2980. [PubMed] Miyahara T, Runge S, Chatterjee A, Chen M, Mottola G, Fitzgerald JM, Serhan CN, Conte MS. D-series resolvin attenuates vascular smooth muscle cell activation and neointimal hyperplasia following vascular injury. FASEB J. 2013;27(6):2220-2232. [PubMed] Norling LV, Spite M, Yang R, Flower RJ, Perretti M, Serhan CN. Humanized nano-proresolving medicines mimic inflammation-resolution and enhance wound healing. J. Immunol. 2011;186(10):5543-5547. [PubMed] Pires NM, van der Hoeven BL, de Vries MR, Havekes LM, van Vlijmen BJ, Hennink WE, Quax PH, Jukema JW. Local perivascular delivery of anti-restenotic agents from a drug-eluting poly(epsilon-caprolactone) stent cuff. Biomaterials 2005;26(26):5386-5394. [PubMed] Steg PG, Serruys PW, Abdelghani M, Wijns W. The year in cardiology 2015: coronary intervention. Eur. Heart J. 2016;37(4):335-343. [PubMed] Thin film spin coating: http://louisville.edu/micronano/files/documents/standard-operating-procedures/SpinCoatingInfo.pdf Vartanian SM, Conte MS. Surgical intervention for peripheral arterial disease. Circ. Res. 2015;116(9):1614-1628. [PubMed] Vasconcelos DP, Costa M, Amaral IF, Barbosa MA, Aguas AP, Barbosa JN. Development of an immunomodulatory biomaterial: using resolvin D1 to modulate inflammation. Biomaterials 2015;53:566-573. [PubMed] Wu B, Mottola G, Chatterjee A, Lance KD, Chen M, Siguenza IO, Desai TA, Conte MS. Perivascular delivery of resolvin D1 inhibits neointimal hyperplasia in a rat model of arterial injury. J. Vasc. Surg. 2017;65(1):207-217. [PubMed]