Controlled Release on Cardiovascular Stents Using Plasma-Enhanced Adhesion of Biodegradable Nanoparticles

One of the most common solutions to treat ischemic heart disease nowadays is the implantation of drug eluting stents. Currently, new strategies are being developed to improve healing process, which includes pharmacological treatments or gene therapy. In this paper, we presented a proposal based on the use of poly(β-amino ester) (pBAE) nanoparticles. To enhance the release method, we used an approach based on attaching these nanoparticles on a polymeric coating. The process includes coating metallic cardiovascular stents with a thin and highly functionalized layer of pentafluorophenyl methacrylate (PFM) to which loaded nanoparticles are chemically bonded. Through this design, when a stent is expanded during its implantation, nanoparticles will stay attached to the polymer matrix. Nanocarriers will penetrate target adjacent cells, guaranteeing effective drug delivery. Results obtained show that this work opens a pathway for pharmacological and/or gene delivery systems based on the adhesion of pBAE nanoparticles prior to stent implantation.


INTRODUCTION
Coronary Artery Disease (CAD), also known as ischemic heart disease, is the most typical type of heart disease, killing more than 385,000 people annually according to Centres for Disease Control and Prevention (CDCP) [1]. It is a serious and common disease that can profoundly influence a patient's prognosis and quality of life due to the consequent restriction of blood supply (ischemia). The mechanism of coronary ischemia is described as the development of coronary atherosclerosis, while the atherosclerotic process is not well understood [2,3]. Atherosclerosis is associated with inflammatory processes in the endothelial cells of the vessel wall, and related with the deposition of fatty substances (cholesterol, fatty acids, etc.) in arteries, resulting in a narrowed blood vessel lumen (stenosis) and thus reduced myocardial oxygen supply [2,4].
To solve this issue, a common approach is to undergo a coronary stent implantation. This procedure is used to achieve a permanent dilation of a stenotic coronary artery in order to improve myocardial perfusion. During the process of reopening the artery lumen and restoring the exterior layer of the artery wall, there are various treatments used to ensure positive results. Although there are different approaches, these are far from being optimal. Besides the risk of subacute thrombosis, the main problem of stents is late intra-stent restenosis which can be caused by multiple factors. The stent acts as a platform for preventing acute vascular occlusion, removing the elastic recoil and reducing late remodelling.
To mitigate these effects, during a procedure of stent implantation, a simultaneous pharmacological treatment is often performed. In this case, there are different types of stents which can be used to treat the affected area [11,12].
The most common solutions, with overall good results, imply drug eluting stents (DES) [5][6][7][8][9][10]. The first DES developed were the Taxus and II and IV, SIRIUS, E-SIRIUS and C-SIRIUS studies) [11][12][13][14][15]. Drugs used in DES might include inhibitors of inflammation, platelet aggregation, cell migration and proliferation, or promoters of vascular healing and reendothelization. Although there are a variety of DES present in the market, none presents an optimal model which solves all the derived consequences of stent application.
Due to the lack of a unique solution, a variety of different methods based on drug delivery systems and gene therapy systems have been tested out. Results from these drug eluting stents have been obtained by elution analysis of pharmacological release [16][17][18]. A second approach, gene therapy, is a technique that uses nucleic acids to treat or prevent diseases by acting on the genetic roots of the illness [19,20]. The goal of gene therapy is to modify a gene, or genetic pathway, to prevent or treat a disease. Although gene therapy can be applied with DES treatment, it would render better results if eluted with a carrier that ensured a controlled delivery. There have been different techniques developed to treat inflammatory responses through gene modification, but these efforts have not been directed to achieve restenosis reduction through controlled release with DES. One of the most promising carriers used which enables safe and efficient delivery systems are nanoparticles [13,[21][22][23][24].
When applying nanoparticles locally, they can penetrate the vessel wall and form a depot which will allow a sustained release of drugs into the arterial wall. This method allows a focused treatment which targets affected cells [25]. Nanocarriers can be tailored to have specific sizes, shapes, densities or functionalities. Customization makes nanoparticles ideal as drug delivery vehicles when used to encapsulate desired drugs or gene vectors. It is of vital importance to ensure the non-toxicity of the nanoparticles used for drug delivery, as they are going to be used to penetrate the targeted cells [26]. As mentioned before, nanoparticles can be excellent carriers and can prove useful when developing a therapeutic strategy for DES. Achieving a controlled release of encapsulated contents is possible as long as adhesion of nanoparticles to a stent surface is guaranteed. A good range of biodegradable and biocompatible materials have been tried out to fulfil this task, showing there is no unique solution to this question. Up to date, DES have been made of different polymers, but some of the most used ones are poly(lactic acid) and poly(lactic-co-glycolic acid) [27][28][29][30][31][32].
In this study, a promising approach has been implemented to fulfil therapeutic delivery of DES through the adhesion of poly(β-amino ester) (pBAE) nanoparticles to the stent strut. If this system brings positive results, on one hand, it will be useful to deliver antiproliferative drugs. On the other hand, it will also be useful to ensure controlled and localized release of a large number of microRNA (miRNA). These endogenous, noncoding, single-stranded RNAs have been demonstrated to reduce proliferation of smooth muscle cells, which is a key factor for stent treatment. miRNAs are novel regulatory RNAs for neointimal lesion Med One. 2019;4:e190014. https://doi.org/10.20900/mo.20190014 formation, making them a useful tool for the treatment of proliferative vascular diseases such as atherosclerosis, postangioplasty restenosis, transplantation arteriopathy, and stroke [33]. Our approach can also enhance the therapeutic treatment, as it provides a regulated release of small interfering RNA (siRNA), which have an important role on regulating the inflammatory process [34]. This tailored release is able to span over an initial burst, producing a better controlled pharmacological liberation. pBAEs used in this study have previously shown high transfection efficiencies, biodegradability and biocompatibility. Furthermore, nanoparticles made from these polymers can be tailored in a cell-specific manner by changing their composition [35]. Recently, pBAEs have been used in different therapeutic applications and proven successful. Multiple applications have been described in stem cell modification or osteodifferentation, showing great promise to deliver interfering RNA (RNAi) in a safe way [36,37]. Since different backbone structures can be easily designed for pBAEs, there is a great variety of polymers to choose from [38,39]. In previous studies, results show that polymer structure has a direct influence on RNAi binding and delivery efficiency, concluding that the polymer structures C6 and C32 outperformed most of the rest [40].
These polymers show a high flexibility and can be easily tuned to fulfil specific purposes [37,[41][42][43]. Due to this flexibility, different strategies have been tried out in order to produce nanoparticles with pBAEs. Positive results have been achieved, demonstrating that they can act as functional nanocarriers with a low toxicity [44].
Research in this area has led to an approach which focuses on creating pBAE based nanoparticles for drug delivery/gene therapy systems designed to prevent in-stent restenosis. Controlled delivery mechanisms using pBAEs are achieved in this study through the chemical bonding of nanoparticles to an initial polymer layer adhered to the stent strut. This first layer is composed of a tailored pentafluorophenyl methacrylate (PFM) coating which enables the chemical adhesion of nanoparticles, avoiding the instantaneous total release of the carriers when in contact with the medium. Nanoparticles will bond to the PFM layer, therefore, it is of great importance to produce a coating which maintains the highest number of aromatic groups as possible to enhance adhesion. While polymerization is carried out on a stent strut, the luminal part remains protected to ensure the polymer will only be deposited on the abluminal side. Chemical adhesion of the nanoparticles and their usefulness in gene therapy are also tested. the esters of the PFM chains react with the amine group from the nanoparticles through a nucleophilic substitution reaction with pentafluorophenol acting as the leaving group [45]. This process is illustrated on Figure 1. We believe that through the bonding of carriers to a polymeric layer attached to a stent, a controlled and localized treatment can be achieved. This method will ensure the persistence of loaded nanoparticles on the stent, avoiding the carriers to be completely removed through the blood stream at first contact. When a stent is expanded at the affected area, nanoparticles will stay on the polymer matrix and mostly penetrate the target adjacent cells on to which they are pressed. This approach can render a higher nanocarrier efficiency due solely to the tailored release method. Oligopeptide-ended pBAE were synthetized in-house through a method previously described by our research group [35,46]. Briefly, poly(β-amino ester)s were synthesized following a two-step procedure. First, an acrylate-
As this first process is the one commonly used in our laboratory it was used as a reference to compare results. In this experiment tprocess refers to the time it takes to complete a polymerization, starting when plasma is generated inside the reactor and finishing when the reactor is turned off. As expected in plasma polymerization, = + , ton shows the time the plasma pulse is active and toff indicates the time the plasma pulse is shut down. Ppeak is the input power given to the system and Peq is the equivalent power applied taking in account the time that the plasma pulse is active. reduction of aromatic group signalling, causing the ratio (Carbonyl group/aromatic group) to increase its value. Figure 3 shows the results obtained after applying the procedure to glass and CrCo disks. On the two graphs, the same tendencies can be observed for both substrates. Plotted results were separated in three different groups labelled as A, B and C.

Duty Cycles (DC) tprocess/min
Group A shows the highest ratio and therefore, the poorest quality PFM   As shown in Figure 4, surfaces obtained show reduced values for average roughness. These results are desirable when producing a stent surface in order to avoid inflammatory response [48][49][50]. AFM characterization of the PFM layers showed roughness results much lower than 1 µm (Ra = 0.550 nm, Rq = 0.644 nm and Rz = 2.023 nm), producing optimum quality and smooth coatings. Once the surfaces obtained had been analysed, the optimized process was ready to be used throughout the experimentation.
As a next step, a method to attach nanoparticles on to the plasma polymerized surfaces was necessary. Initial experiments were carried out on glass discs to ensure cells could grow easily on a well-known substrate.
Glass waffles were coated with PFM, later taken out of the reactor, and placed in a 24 well plate. All samples were prepared as described previously in the "In Vitro Transfection Assay" section. Nanoparticles were deposited over the PFM layers and incubated for 24 and 48 h, aiming to check for cell morphology and transfection. In a previous article from our group, PFM has proven to become cytotoxic six hours after its polymerization due to the elution of pentafluoro phenol [45]. Its release must be avoided, as this is a serious concern for an implantable device. For that reason, images shown in the third row of Figure 5 aim to solve this issue. This last row shows samples which have been treated to ensure there is no liberation of pentafluoro phenol from the PFM layer. Absence of pentafluoro phenol elution was achieved through the treatment of PFM layers with BSA for one hour, followed by a gentle rinse. Once this process was completed, the PFM surface was prepared to be exposed to the cells. Normal growth rates and typical morphologies for this cell line are observed after 24 and 48 h.
Results obtained conclude that this treatment allows the elimination of pentafluoro phenyl groups, found in excess on the PFM surface, avoiding cytotoxicity. This procedure renders a suitable coating for cells to grow on.  CrCo stents were treated with PFM and then incubated with C32 nanoparticles and BSA (as described above). Part of these stents were analysed by SEM. PFM treated stents which did not undergo an immersion process with nanoparticles were used as controls. SEM pictures of both, CrCo discs and stents, are presented in Figure 8.
When CrCo disks samples were prepared, their surface was partially masked with a silicon wafer. As shown in Figure 8,   AP, VR and Sb wrote and corrected the paper.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.