- Email: [email protected]
Microneedle drug eluting balloon for enhanced drug delivery to vascular tissue
Journal Pre-proof Microneedle drug eluting balloon for enhanced drug delivery to vascular tissue
KangJu Lee, JiYong Lee, Seul Gee Lee, SeungHyun Park, Da Som Yang, Jung-Jae Lee, Ali Khademhosseini, Jung Sun Kim, WonHyoung Ryu PII:
S0168-3659(20)30089-4
DOI:
https://doi.org/10.1016/j.jconrel.2020.02.012
Reference:
COREL 10165
To appear in:
Journal of Controlled Release
Received date:
1 November 2019
Revised date:
3 February 2020
Accepted date:
5 February 2020
Please cite this article as: K. Lee, J. Lee, S.G. Lee, et al., Microneedle drug eluting balloon for enhanced drug delivery to vascular tissue, Journal of Controlled Release (2020), https://doi.org/10.1016/j.jconrel.2020.02.012
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier.
Journal Pre-proof
Microneedle Drug Eluting Balloon for Enhanced Drug Delivery to Vascular Tissue KangJu Lee1,2,3,4 , JiYong Lee1 , Seul Gee Lee5 , SeungHyun Park1 , Da Som Yang1 , Jung-Jae Lee6 , Ali Khademhosseini2,3,4,7,8,9,10 , Jung Sun Kim5 *, WonHyoung Ryu1 * 1
Department of Mechanical Engineering, Yonsei University, Seoul, 03722, Republic of Korea Department of Bioengineering, University of California-Los Angeles, Los Angeles, CA 90095, USA 3 Center for Minimally Invasive Therapeutics (C-MIT), University of California-Los Angeles, Los Angeles, CA 90095, USA 4 California NanoSystems Institute, University of California-Los Angeles, Los Angeles, CA 90095, USA 5 Division of Cardiology, College of Medicine, Yonsei University, Seoul, 03722, Republic of Korea 6 Graduate Program in Science for Aging, Yonsei University, Seoul, 03722, Republic of Korea 7 Jonsson Comprehensive Cancer Center, University of California-Los Angeles, Los Angeles, CA 90095, USA 8 Department of Radiology, University of California-Los Angeles, Los Angeles, CA 90095, USA 9 Department of Chemical Engineering, University of California-Los Angeles, Los Angeles, CA 90095, USA 10 Department of Biomolecular Engineering, University of California-Los Angeles, Los Angeles, CA 90095, USA
Corresponding authors: Jung Sun Kim ([email protected]), WonHyoung Ryu ([email protected])
e-
*
pr
oo
f
2
Abstract
Jo u
rn
al
Pr
High rates of restenosis and neointimal formation have driven increasing interest in the application of drug eluting balloons (DEB) as counteractive measures for intraluminal drug delivery. The use of DEBs eliminates the need for stents so that serious side effects including in-stent restenosis and stent thrombosis can be avoided and long-term medication of antiplatelet agent is not needed. Despite their benefits, DEBs have poor drug delivery efficiency due to short balloon inflation times (30~60 seconds) that limit the passive drug diffusion from the balloon surface to the luminal lesion. To increase drug delivery efficiency, a microneedle DEB (MNDEB) was developed by a conformal transfer molding process using a thin polydimethylsiloxane mold bearing a negative array of MNs of 200 μm in height. A MN array composed of UV curable resin was formed onto the surface of DEB, and drugs were coated onto the structure. The mechanical properties of the MN array were investigated and MN penetration into luminal vasculature was confirmed in vivo. An increase in drug delivery efficiency compared to a standard DEB was demonstrated in an in vivo test in a rabbit aorta. Finally, the superior therapeutic efficacy of MNDEBs was evaluated using an atherosclerosis rabbit model. Keywords: microneedle, drug eluting balloon, vascular disease, endovascular drug delivery, conformal transfer molding
1
Journal Pre-proof
pr
oo
f
1. Introduction Since the development of a balloon catheter for percutaneous trans- luminal angioplasty (PTCA) in 1978 [1], vascular intervention technology has continued to evolve from the bare metal stent (BMS) [2] to catheter-delivered drug eluting stents (DES) [3-5]. The original balloon for PTCA treatement was inflated at the blockage site to compress the plaque against the vessel wall. However, this therapy was only temporarily effective in many patients as over 30% suffered from restenosis, usually within 5 months of the original intervention [6]. In 5~10% of patients, elastic recoil occurred almost immediately (minutes-hours) after the procedure [7]. To address the problem of restenosis, the BMS was developed to permanently promote vascular integrity after implantation by supporting the vessel wall after deployment [2]. While recoil was no longer a concern, in-stent restenosis (ISR) was common as the stent was eliciting a foreign body reponse from the immune system [8]. Long term follow-up of BMS cases revealed a 20~30% incidence rate of ISR [9]. To address ISR, DESs emerged; these stents maintained the same basic structure of BMSs but were covered by a polymeric carrier loaded with a therapeutic agent. While DESs have decreased the rate of ISR signficantly through the local delivery of anti-proliferative drugs, metal stent-based intervention still suffers from other complications such as late onset thrombosis and hypersensitivity to the drug or polymer coating [10].
Jo u
rn
al
Pr
e-
The most certain way of preventing ISR is to avoid a foreign body response by not leaving any foreign material in the blood vessels. Drug-eluting balloons (DEB) are one solution that addresses the limitations presented by DESs. These devices work by inflating the balloon in the blood vessel so it is in contact with the lesion for 60 seconds. The drug coated on the surface of the DEB is transferred to the lesion and exerts its therapeutic effect [11]. Several trials have been conducted to treat both ISR [12-14] and de novo lesions in blood vessels using DEBs [15-17]. Most DEBs used today deliver paclitaxel or sirolimus [18] using different carriers and excipients. Beyond adhering the drug to the balloon, the drug carrier plays a central role in a drug transfer into the vessel wall. To improve drug delivery to the lumen, many researchers have developed various coating formulations; some of the leading commercial prodcuts include PACCOCATH®, based on iopromide [19]; DIOR®, based on microcrystals of paclitaxel [20]; IN.PACT™ (Medtronic, USA), containing a hydrophilic matrix and urea [21]; Lutonix® 035 (BARD, USA) consisting of either polysorbate or a sorbitol hydrophilic carrier [22]; and Pantera LUX® (BIOTRONIK AG, Switzerland), consisting of butyryl-trihexyl citrate loaded with paclitaxel [23]. Although a variety of products exist to optimize drug release, current DEBs still have limitations in terms of low drug delivery efficiency and limited sorts of drugs that cannot be solved by changing the drug carrier [24, 25]. The fundamental restriction imposed on the DEB is its passive mechanism of drug transfer from the DEB to the lumen. There have been a few trials that have tested various modifications to DEBs aimed at improving the efficiency of endovascular drug delivery. A porous balloon was designed to release the drug through the pores at the time of inflation [26]. Additionally, Lee et al. have developed linear-micropatterned drug eluting balloons (LMDEB) using customized balloon forming molds [27]. LMDEBs were fabricated to have 16 linear micropatterns (LMs) on the surface with a height of 130 μm. The goal of the LMs is to increase the contact force between the drug coated surface and lumen, which leads to the enhanced efficiency of endovascular drug delivery. Another solution developed by Mercator Medsystems, Inc. (Emeryville, CA) are the Bullfrog® and Cricket® Micro-infusion devices. These devices combine a 34G microscale needle with a balloon catheter. While the catheter is closed, the hollow needle is covered by the balloon so that it does not injure the vessel walls. When the catheter is opened, the needle extends out of the balloon and is used to inject drugs directly into the surrounding 2
Journal Pre-proof
e-
pr
oo
f
luminal tissue. Delivery with a needle penetrates the luminal layers of the vessel, facilitating better distribution through the tissue [28, 29]. The major drawback to these devices is the size of the 34G needles as they are too large for use in many blood vessels.
Pr
Figure 1. A graphical abstract of MNDEB in this study indicating that drug coated MNs onto balloon surface enhance the efficiency of endovascular drug delivery by MN insertion compared to conventional DEB.
Jo u
rn
al
Microneedles (MNs) may be a suitable solution for increasing endovascular drug delivery, just as it has been transdermally. In this study, we have fabricated a MN drug eluting balloon (MNDEB) by molding a MN array onto the surface of a DEB to improve drug delivery efficiency (Figure 1). The MN array was fabricated by wet etching and subsequent polydimethylsiloxane (PDMS) casting. We then devised a transfer molding process that fixed the MN structures to a conventional DEB by flexing the PDMS mold around the balloon and curing with UV light. This resulted in a strong bond between each MN and the surface of the balloon that minimized the risk of detachment. The MNDEB was proven to be suitable for intraluminal use as the MNs penetrated the tissue and reached the tunica media of the vascular tissue. Additionally, we adapted the conventional PACCOCATH technology to coat the drugs on the surface of both the MNDEB and DEB [27]. A unique mechanical property of the MNs was investigated using a deformation test and in vitro insertion test with vascular tissue phantom scaffolds. The enhanced drug delivery efficiency of the MNDEB was demonstrated using a model drug and fluorescent microscopy. The safety of the balloon was studied through histological analysis and scoring. In vivo therapeutic intervention was then conducted on an atherosclerotic rabbit model, and therapeutic efficacy was evaluated in a comparative study of the MNDEB and DEB.
2. Materials and Methods 2.1. MN array fabrication Male MNs (200 µm height) were fabricated by lithography, multi- layer deposition, and wet etching. The MN shape was achieved through manip ulating the size of the square micropattern on the photomask. First, an oxide layer was formed on a 4-inch wafer (in a furnace), followed by low-pressure chemical vapor deposition of nitride. The layer thicknesses were 300 Å and 1200 Å, respectively. Spin-coating was used to deposit a 100 µm3
Journal Pre-proof thick layer of SU-8 on the wafer. The material was crosslinked by irradiating the wafer with UV light passed through an aligned mask with the square pattern array (500×500 µm2 ). After development, the exposed Si3 N4 and SiO 2 layers were patterned by reactive ion etching. Then, the processed wafer was soaked in KOH solution (29 %, 79 °C) for 210 minutes and rinsed in a bubbling water for 30 minutes. The result of the fabrication process was an array of octagonal-based cones, 150 µm in length (Figure S1) with a 1.5 aspect ratio interval between MNs is 500 µm. Casting PDMS (Silicone Elastomer-184, Dow-Corning, USA) over the wafer formed a mold containing the negative MN pattern. The PDMS was cured at room temperature for 24 hours.
al
Pr
e-
pr
oo
f
2.2. MNDEB Fabrication We selected a commercially available, UV-curable adhesive (208-CTH-F, DYMAX, USA) as the material for the MNs. An MN array (200 µm- height and 800 µm- interval) was prepared and PDMS (Dow-Corning, Silicone Elastomer-184) MN cavities were molded subsequently with a uniform thickness of 1 mm. The curable resin was deposited on the flexible female mold and placed in a vacuum chamber for 2 minutes to eliminate trapped air from the MN cavities. Residual resin on the surface of the mold was removed by doctor blading (Figure 2). After removing the bubbles, we wrapped the mold around the surface of the inflated DEB. The entire apparatus was exposed to UV light for four minutes to fix the MN array onto the surface of the DEB. After removing the PDMS mold, the deflated MNDEBs were folded, and the drug was pipette-coated with a mixture of drug and shellac as matrix polymer instead of iopromide of the PACCOCATH® formulation. Rhodamine B (RB, Samchun Chemical, South Korea) and paclitaxel (PTX, Genoss, South Korea) were used as the model drugs in this study. 3 μg/mm2 of either RB or PTX was coated onto the surface of the MNDEB.
Jo u
rn
2.3. Mechanical characterization The mechanical behavior of MNs attached to a MNDEB was monitored using a stereomicroscope and a stainless-steel pillar, which was fixed to a customized z-axis auto-stage (STM-2-USB, Science Town Corp., South Korea) (Figure S2). For the testing, MNDEBs were cut longitudinally and attached to the wall of microstage so that the MNs were oriented horizontally. The MN and the steel pillar used for testing were aligned first in the horizontal plane. Once aligned, the pillar was lowered until it contacted the MN. The pillar deformed the MN as it was moved vertically at a speed of 100 µm/s. Once the MN had been deformed to 90 degrees, the pillar was retracted at a speed of 100 µm/s. The deformation and recovery of the MN was recorded by stereomicroscopy.
2.4. Polyvinyl alcohol (PVA) phantom vascular tissue PVA phantom was fabricated according to reported research [30, 31]. In brief, PVA, M w 146,000-186,000 (Sigma Aldrich), was dissolved in DI water to make a 10% (w/w) solution. The solution was heated to 120 ◦ C on a hotplate and was stirred for 2 hours until the PVA powder was fully dissolved. Homogeneous PVA solution was poured into the petri dish and solidified at room temperature. The solidified PVA samples were polymerized by freeze-thaw cycles to adjust the mechanical properties. The freeze-thaw cycle consisted of two steps: a freezing step for 8 hours at -20 ◦ C, and a thawing step for 16 hours at room temperature (approximately 20 ◦ C). During freeze-thaw cycles, hydrogen bonds are formed between the PVA chains. As more hydrogen bonds are formed, the elastic modulus of the material increases; material stiffness increases proportionally with the number of freeze-thaw cycles. The number of cycles was selected to yield material properties mimicking those of the normal 4
Journal Pre-proof vessel (1 cycle) and atherosclerotic vessel (8 cycles). After the last cycle, the samples were tested using a universal tensile machine (UTM, OTT-001, Oriental TM, Republic of Korea) to validate the elastic moduli of 52 kPa and 183 kPa, respectively.
rn
al
Pr
e-
pr
oo
f
2.5. In vivo animal study In this study, 12 male New Zealand White rabbits (3.0-3.5 kg weight) were used. 3 rabbits were used for each of the following experiments: 1) drug distribution and MN insertion test, 2) atherosclerosis modeling, 3) therapeutic efficacy test, and 4) safety test. All rabbits were acclimatized for 1 week before in vivo test. The study protocol was approved by the local Institutional Animal Care and Use Committee of Yonsei University Health System (IACUC: 2017-0269). All animals received human care in compliance with the Animal Welfare Act and the "Principles of Laboratory Animal Care" formulated by the Institute of Laboratory Animal Resources (National Research Council, NIH Publication No. 85-23, revised 1996). Animals were anesthetized by intramuscular injection with Zoletil (10mg/kg, Virvac, USA) and Rompun (5mg/kg, Bayer, Germany), followed by 2% of isoflurane (Forane ®, JW Pharm, South Korea) and oxygen. The iliac artery was accessed via the carotid artery. Heparin (150 units/kg) was injected to maintain an activated clotting time within 250 seconds before catheterization. For the drug distribution and MN insertion tests, both conventional DEBs and MNDEBs were inflated in the iliac artery for 60 seconds to 1.2 times the size of the artery. To develop the atherosclerotic model, each rabbit injured its iliac artery by two times of balloon over- inflation for 30 seconds was fed a high cholesterol diet (1% cholesterol, DooYeol Biotech, South Korea) for one week. The surgical intervention included the inflation of both MNDEBs and DEBs (Genoss Co., Ltd., South Korea) to 1.2 times the size of the artery (based on quantitative angiography) for 60 seconds. Following the operation, the rabbits were maintained on a high cholesterol diet for 4 weeks; optical coherence tomography (OCT) and quantitative coronary angiography (QCA) were taken prior to the euthanization of the animal. There was no animal death during the entire in vivo study.
Jo u
2.6. Fluorescent microscopy and histological analysis Vessels treated by RB-coated MNDEB and DEB were cryosectioned and imaged with fluorescent microscopy to visualize drug distribution. The cultured tissue was embedded in optimum cutting temperature compound (Sakuta Finetek USA Inc., USA) and frozen. The frozen samples were cut by a microtome with a thickness of 10 µm and analyzed by a fluorescent microscope (BX53, Olympus, Japan) under identical parameters (light sensitivity=ISO 100, light exposure time=1 second and light intensity=6). Afterwards, the fluorescent intensity of each samples was quantified by measuring integrated density (ID) as described [32]. Since the sectional area of vessels is different to each other, the meas ured fluorescent value was normalized to the area of the blood vessel to secure normalized integrated density (NID). To analyze the efficacy of the intervention, the target vessel was fixed by continuous perfusion of normal buffered formalin (10%) for one week. A 5 mm- long vascular sample was placed into a cassette and treated with alcohols and xylenes. After that, the sample was embedded in a paraffin block. The paraffinized samples were sectioned to a thickness of 4 µm and stained with hematoxylin and eosin (H&E, Merck, Germany) and Masson’s trichrome (BBC Biochemical, USA). The sectioned area was measured by an optical microscope (SCN400, Leica, Germany) and then histomorphometry was conducted using LAS 4.2 software (Leica, Germany). Additional sections were labeled with a mouse monoclonal anti-rabbit macrophage antibody (RAM11, Dako, USA), Interleukin (IL)-1 (Abbkine, USA), IL-6 (Santa Cruz Biotechnology, USA), tumor necrosis factor (TNF)- (Abbkine, USA) to evaluate the degree of inflammation and with vascular endothelial growth 5
Journal Pre-proof factor (VEGF, Abcam, United Kingdom), intercellular adhesion molecule 1 (ICAM1, Abcam, United Kingdom), vascular cell adhesion molecule 1 (VCAM1, Santa Cruz Biotechnology, USA) to test the expression of pro- inflammatory atherosclerosis factors. Immunostaining was performed and quantified by calculating the percentage of labeled area with respect to total tissue area. The color detection threshold was chosen for the DAB chromogen (brown staining) in tissue sections. Measurements were performed using QWin Image Analysis software (Leica, Germany).
e-
pr
oo
f
2.7. Vascular stenosis evaluation In addition to histological analysis, the degree of stenosis was evaluated by scanning the 10 mm- long vessel following intervention with the DEB and MNDEB. QCA and OCT were conducted after delivering drugs and after 4 weeks to monitor the progress of intimal proliferation in terms of diameter stenosis (DS) and area stenosis (AS). OCT was conducted using the C7-XR imaging system (Abbott Vascular, CA, USA). First, 16 ml of contrast media was infused through a guiding catheter for 4 seconds. Meanwhile, the OCT catheter was pulled back (20 mm/s), and OCT images were acquired (100 frames/s). All OCT images were analyzed at Cardiovascular Research Center (Republic of Korea) by blinded analysts. All histological images were provided by the histological laboratory in Yonsei University Hospitial and compared to proximal side branches from the edge of the injury.
Jo u
rn
al
Pr
3. Results and Discussion 3.1. Conformal transfer molding to transfer the MN array onto the DEB surface We first fabricated an array of MNs with an aspect ratio of 1.5, 200 µm height, and 800 µm spacing interval as shown Figure S1. A thin nickel oxide layer was deposited on a silicon wafer by low pressure chemical vapor deposition and etched by photolithography into a square shape. This patterned wafer was dipped into KOH solution and then processed with undercut etching which removed material below nickel oxide mask patterns. The wet etching continued until the nickel oxide mask was detached as a result of the undercut etching. MNs of various height were formed by varying the size of the square mask. The square nitride masks were patterned at 250, 500, 750, and 1000 µm; this resulted in MN with a height of 100, 200, 300, 400 µm, respectively with an average etch rate of 0.96 ± 0.02 µm/sec. The relationship between the height (h) of the MN and width (l) of square mask was calculated to be approximately l:h = 2.5:1. To be used clinically, the MN array should be firmly fixed to the surface of the DEB. We have developed a novel micromolding process, called conformal transfer molding, to transfer the MN array to the curved surface of a conventional DEB. The concept and process of conformal transfer molding is shown in Figure 2. We first fabricated a uniform, 1 mmthick PDMS mold using the previously fabricated silicon MNs; a thin and flexible mold is required for this process in order to make conformal contact with the curved surface. Then, UV curable adhesive was spread onto the PDMS mold and trapped air in the MN cavities was eliminated in a vacuum chamber for 2 minutes. Residual UV curable resin on the surface of the mold was removed by doctor blading. Next, the mold filled with resin was rolled around an inflated DEB (2.75 mm diameter, 10 mm length). To transfer and solidify the MN array on the surface of DEB, the device was exposed to UV light for 4 minutes and the flexible mold was detached. The UV curable resin we used was developed and commericalized as adhesive to bond PET-based polymer in implantable medical devices. We hypothesized that UV curing during conformal transfer molding would enable the MNs to solidify and attach to the DEB surface consising of a PET-based polymer. Through this process, more than 95% of the MNs 6
Journal Pre-proof transferred onto the surface of the DEB (as confirmed by optical microscopic analysis from Figure 2G, data not shown). In addition, seamless boundary at the inteface between MN and the balloon surface indicated the firm integration of the MN.
Jo u
rn
al
Pr
e-
pr
oo
f
To our knowledge, this is the first use of MNs for endovascular drug delivery. Thus far, MNs have remained in the form of patches due to the manufacturing limitations of conventional molding techniques such as centrifugation [33] or vacuum suction [34]. Recently, several groups have developed transfer molding [35, 36], pressure assisted molding [37], and thermal welding [38] processes to integrate MNs to macroscale applicators. Fixing these needles to easy-to-handle materials, such as a flexible mesh and an injection-aid pen, has facilitated the application of MNs to the peri- vascular [38] and ocular drug delivery [3537]. Additionally, our work is novel as MN arrays have not yet been tethered to curved threedimensional surfaces. In this study, we created a MNDEB through conformal transfer molding. This process is not restricted by the height, spacing, or shape of the MNs as long as PDMS molds can be fabricated thin enough to be conformally attached over the curvature of a three-dimensional surface. Just as MN patches have revolutionized transdermal drug delivery, we believe it is possible to do the same for endovascular drug delivery through the creation of the MNDEB.
Figure 2. Schematic images of conformal transfer molding process consisting of (A-C) premolding with UV curable adhesive, (D) alignment of flexible planar PDMS mold, (E) curing process and (F) completed MNDEB. (G) Stereo- and optical microscopic images of MNDEB surface. An inset is the balloon surface before conformal transfer molding. (scale bar = 300 µm) (H) A scanning electron microscopic image of a MN onto MNDEB surface. (scale bar = 100 µm)
3.2. Mechanical behavior of conformally molded MN array and vascular tissue insertion In order for the MNs tethered to the DEB to reach the lesion through the blood vessels, the array must be firmly attached. Additionally, if the MN is too brittle, contact with the luminal 7
Journal Pre-proof
Jo u
rn
al
Pr
e-
pr
oo
f
surface of the vessel may cause fragmentation of MN tips and leave debris in the lumen. For these reasons, the MNs must be flexible enough to sustain lateral MN bending during the delivery process. While a certain degree of flexibility is required, the MNs must also be longitudinally rigid enough to penetrate the vascular tissue upon inflation of the DEB. To ensure that our design met these criteria, we investigated the mechanical behavior of the MN array. To test the bending properties, we aligned the MNDEB vertically. A stainless-steel pillar (100 µm diameter) was attached to micro z-axis stage and aligned to the MN (Figure 3A). The metal pillar was lowered down to the MN at a speed of 100 µm/s and subsequently retracted with an identical speed once the needle bent 90°. As shown in Figure 3A, the tip of the needle deformed 100 µm vertically and recovered more than 50% within 3 seconds. The MN recovered more than 75% after 1 minute, and nearly 100% after 4 minutes. The video of this experiment is available as Supplementary Movie 1. This test also confirmed that the MN would not detach from the balloon surface or deform as confirmed by stereomicroscopic analysis.
Figure 3. (A) Mechanical deformation of the MN attached to the MNDEB surface. After deformation, the deformed MN was 50% recovered in 3 seconds and 100 % recovered over 4 minutes. (scale bar = 100 µm) (B) Representative fluorescent confocal microscopic images of MN insertion marks within 50 and 200 kPa PVA tissue phantom scaffolds. (scale bar = 100 µm) (C) Plotted MN incisions in 50 and 200 kPa PVA scaffolds from the confocal microscopic images. (D) Insertion marks (red arrow) of MNDEB into lumen through in vivo 8
Journal Pre-proof application. Blue dotted lines indicate the endothelium and asterisks (*) indicate the lumen side. Right images are sectional view of DEB treated vessels with no insertion mark. (scale bar = 200 µm (big), 20 µm (small)) (E) Images of MN tip of MNDEB before and after in vivo test. (scale bar = 100 µm)
Pr
e-
pr
oo
f
To test the ability of the device to insert MNs into the tissue, we performed an insertion test with vascular tissue phantom. We prepared PVA scaffolds with elastic moduli of 52 and 183 kPa, analogs of normal or calcified and narrowed vessels in hypersensitive patients respectively [39]. MN patches were fabricated using the same resin and molds as those created for the MNDEB. The PVA scaffold was wetted with 0.1% (w/w, in DI water) RB solution for 1 minute before the MNs were inserted for fluorescent staining. Subsequently, we manually applied the MN patch to the PVA scaffolds and analyzed the insertion marks using a confocal microscope. We measured MN incision width at the midpoint of the MN penetration depth and averaged the measured widths in both the x- and y-axes. As shown in Figure 3B&C, the MN patch penetrated both PVA scaffolds at a depth of approximately 75~100 µm. Puncturing the 183 kPa scaffold resulted in greater MN deformation compared to the 52 kPa material due its higher elastic modulus. Since the 52kPa scaffold was soft, it was slightly incised after being stretched. However, the 183 kPa scaffold yielded a different result as the MNs easily penetrated the surface. Because of the varied substrate properties, the incision depths varied; in the 183 kPa scaffold the MN penetrated to a depth of 102 ± 11 µm, while the MNs reached a depth of 72 ± 6 µm in the 52 kPa scaffold. Despite of the variation of MN incision depths, both MNs could penetrate both vascular tissue phantom to a depth sufficient to overcome the endothelium (< 10 µm) without compromising the media layer (< 500 µm).
Jo u
rn
al
Finally, we conducted an in vivo insertion test with healthy rabbit tissue. The MNDEB was prepared and deployed in the illiac arteries, a commonly damaged region in rabbits with atherosclerosis. The MNDEB was inflated and maintained at a diameter of the iliac artery for 60 seconds. After retraction and removal, the rabbit was sacrificed and the illiac artery was harvested. The harvested vascular tissue was then sectioned and stained to observe the MN insertion marks. As shown in Figure 3D, MN marks were found in the lumen of the treated vessels after H&E staining. Image analysis displayed an insertion depth of approximately 100 µm maintaining the endothelium (blue dotted line, Figure 3D). Secondary analysis of the MNDEB structure after the procedure was a lso investigated and no structural difference in the MN shape or size was observed (Figure 3E).
9
oo
f
Journal Pre-proof
e-
pr
Figure 4. (A) Fluorescent microscopic images of rhodamine B (model drug) distribution in vessels treated with the MNDEB and the DEB (scale bar = 500 µm). (B) Normalized integrated density (NID) of DEB vs. MNDEB using model drug RB (n=3). *p < 0.05 on twotailed T-test. All data were presented as the mean ± SEM.
Jo u
rn
al
Pr
3.3. In vivo drug distribution in vascular tissue We next investigated if the MNDEB was able to increase the drug delivery efficiency compared to the standard DEB. Fluorescent model drugs were used which facilitated facile study of drug distribution through fluorescent microscopy imaging. Both the MNDEB and DEB were coated with RB and were used to deliver the model drug to the iliac artery. In vivo tests were performed using the same procedure as the in vivo insertion tests; analysis was performed used fluorescent microscopy and tissue sectioning. Model drug was delivered by both the MNDEB and DEB and the treated area was cultured for 2 days. The tissue was cryosectioned (instead of paraffinization) to avoid quenching the fluorescent signal [27]. The intensity of the fluorescent signal was quantified from still images by measuring ID using ImageJ. The measured reading was normalized to the area of vascular tissue to secure the NID. The results of this experiment demonstrated that the MNDEB enhanced drug delivery efficiency compared to the conventional DEB (Figure 4). A higher fluorescent signal was observed in MNDEB treated samples compared to the conventional DEB treated samples (Figure 4A). The mean NID value in the MNDEB treated tissue (NID=2.7) was 2.4 times higher than that of the DEB treated tissue (NID=1.17).
3.4. Safety of MNDEB Even though the MNDEB achieved superior drug delivery efficiency compared to the conventional DEB, the integrity of the vascular tissue should not be impaired by MN insertion. To check this safety issue, we applied both drug- free conventional balloons and drug- free MN balloons to the normal vessel in vivo. The degree of inflammation and vessel injury was measured 4 weeks after the procedure. The degree of inflammation was investigated to check for possible damage to the vessel by staining the tissue sections with RAM11. Inflammation was scored according to Kornowski [40], allocating values of 0 (none), 1 (mild with minimal infiltrated inflammatory cells), 2 (moderate), or 3 (severe, with large clusters of inflammatory cells with granulomatousmorphology). Injury scoring was performed based on H&E and 10
Journal Pre-proof trichrome stained slides according to Schwartz [41]. The criteria for injury scoring is as follows: 0, no injury; 1, internal elastic lamia (IEL) lacerated; 2, media injury; or 3, laceration of external elastic lamia (EEL). IEL and EEL areas were assessed with ImageJ. Additionally, morphological assessment of endothelialization was described as percentage endothelial coverage of the arterial circumference: 0 < 25%, 1 = 25~75%, 2 = 76~99%, 3 = 100% (complete) [42].
oo
f
As shown in Figure 5A&B, RAM11 staining and inflammation scoring revealed no statistically significant difference in tissues treated with either the drug- free MN balloon and drug- free conventional balloon. This indicates that MN insertion did not elicit a larger immune response in the blood vessel compared to the balloon with usual surface. As for the injury scores, 6 slide sections of each group (drug-free MN balloon or conventional balloon) showed no statistical significance (Figure 5C), which indicates that MN insertion does not affect the physical integrity of the blood vessel. In addition, there was no statistical significance in endothelization score between drug- free MN balloon or conventional balloon. Therefore, the insertion of MN to endovascular tissue may be considered as safe as conventional DEB intervention. Details of the values are provided in Figure S3A.
Jo u
rn
al
Pr
e-
pr
The primary benefit of MNs in the context of transdermal drug delivery is that they are minimally invasive. This property is advantageous as the small puncture size minimizes pain during application [43] in addition to reducing damage to the tissue [44]. While the application of MNs to skin has been thoroughly studied, the effects of insertion into other tissues are not well characterized. In the case of vascular endothelium, maintaining the integrity of the blood vessel is a major concern. For perivascular application of MNs (first penetrating the adventitia then the media layers [32]), the tunica media may be ruptured by MN insertion; we investigated endovascular MN insertion to ensure that MNs on the DEB would not damage the overall vessel structure. Both normal and diseased vessels showed no histopathologic differences when the balloon was inflated with or without the MNs [32, 45]. The height of the perivascular MNs was 650 µm, long enough to penetrate the adventitia, but short enough not to reach the endothelium and puncture through the vessel wall. In this study, we designed the height of the MNs based on this criterion and after histopathological analysis, did not find any evidence of vascular damage due to MN insertion into the vascular endothelium. Additionally, in vivo testing did not show any adverse effects or animal death as a result of MN insertion. The results of these two studies (perivascular and endovascular MN drug delivery) demonstrate the safety of vascular micro-incision by MN insertion.
11
al
Pr
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
Figure 5. (A) Representative RAM11 stained histopathological images of normal vessels treated non-drug coated MNDEB and DEB. (Scale bars = 500 μm) (B) Quantitative data of RAM11 area in the normal vessels treated non-drug coated MNDEB and DEB. (C) Scoring data of inflammation, vessel injury and endothelization in the normal vessels treated non-drug coated MNDEB and DEB. All data were presented as the mean ± SEM. p < 0.05 on twotailed T-test.
3.5. Therapeutic efficacy of MNDEB compared to DEB Therapeutic efficacy of the MNDEB was demonstrated by an in vivo test. Atherosclerosis was induced in the iliac arteries of a rabbit by balloon injury and consumption of a high- fat diet for 4 weeks. The diseased rabbits were operated with both a MNDEB and a standard DEB coated with 3 µg/mm2 of PTX; the balloons were inflated for 60-seconds to apply the drug to the lumen of the vessel. The four experimental groups used in this comparative study were defined as follows: normal rabbit (n=3, negative control); atherosclerosis model (n=3, positive control); MNDEB treatment (n=3); DEB treatment (n=3). Subsequently, we monitored the progress of patency by measuring AS and DS using QCA and OCT. In addition, all vascular tissues were cultured and stained with H&E and trichrome for analysis of vascular integrity and intima. We evaluated the neointimal growth after each intervention. Significant proliferation was shown in the positive control group, which indicates the disease model (atherosclerosis) exhibited the disease phenotype (Figure 6A, positive). The values of AS and DS were estimated from OCT and QCA analysis by averaging the cross-sectional area along 12
Journal Pre-proof
e-
pr
oo
f
the whole vessel where PTX was delivered. The cross-sectional area of the vessel, plaque and lumen were calculated with OCT to estimate the AS for each group (Figure S3B). As shown in Figure 6B, the AS of the samples treated by MNDEB was smaller than both the DEB and control (positive) groups. While the difference between the MNDEB groups and positive control group was statistically significant, the difference between the MNDEB and DEB groups was not. Minimal lumen diameter (MLD), mean radial diameter (RD) and DS were measured from QCA data (Figure S3B). In Figure 6C, the MNDEB-treatment yielded a statistically significant decrease in DS compared to both the positive control and DEB-treated group. The DEB treatment did not produce a statistically significant difference in DS compared to the positive control. Stenosis in each group was quantified by measuring the area of the media, intima, and plaque. The intima-plaque ratio (I/P) was calculated as a metric of disease progression (Figure S3B); this ratio was used to normalize intimal formation with respect to the media layer. As indicated in Figure 6A&D, the thickness and area of intimal layer decreased with statistical significance as a result of treatment with both the DEB and MNDEB. The MNDEB-treated tissues also demonstrated a statistically significant higher patency than their DEB-treated counterparts (Figure 6D). Expressions of pro-inflammatory atherosclerosis factors including ICAM1, VCAM1 and VEGF were investigated as well (Figure S4A). Even though there is no statistical significance in the difference between MNDEB and DEB groups in the VCAM1 expression, the average VCAM1 area of MNDEB was lower than that of DEB. In addition, MNDEB samples showed statistically significant decrease of ICAM1 and VEGF expressions compared to DEB samples.
Jo u
rn
al
Pr
Inflammation were scored after the in vivo test for therapeutic efficacy by RAM11 staining as well (Figure 7A). As shown in Figure 7A&B, there was no statistical significance for safety profiles between the DEB and MNDEB in histologic images of atherosclerotic rabbit models. All scoring data including inflammation, injury and endothelization revealed no statistically significant difference in tissues treated with either MNDEB and DEB after treatment, while both groups treated by MNDEB and DEB have statistical significance compared to positive control group (diseased models) (Figure 7C and S3A). We further tested inflammatory markers (IL-1, IL-6, and TNF-) (Figure S4B). Briefly, the results showed the reduction of all the markers in case of MNDEBs, which is consistent with the findings from RAM 11 tests. The MNDEB treated samples showed statistically significant decrease of IL-1 and IL-6 expressions compared to the DEB treated ones. Moreover, while the DEB group has no statistical significance with the positive group, the MNDEB group has the statistically significant decrease of TNF- expression compare to the positive group. This indicates that MN insertion did not elicit a larger immune response compared to the balloon with usual surface especially in the diseased blood vessel as well. In addition, this result showed MN insertion does not affect the physical integrity of the diseased blood vessel and does not limit the endothelization.
13
Jo u
rn
al
Pr
e-
pr
oo
f
Journal Pre-proof
Figure 6. (A) Optical coherence tomography (OCT), quantitative coronary angiograp hy (QCA) and trichrome stained histopathological images of vessels treated with positive, negative, DEB and MNDEB. (EEL: external elastic layer, IEL: internal elastic layer, Scale bars = 500 μm) (B) Percentage of area stenosis (AS) of positive group (n = 3): 52.80 ± 4.93%, negative group (n=3): 9.38 ± 0.37%, DEB group (n=3): 44.03 ± 1.60%, and MNDEB group (n=3): 31.26 ± 0.82% (p = 0.001 by ANOVA). (C) Percentage of diameter stenosis (DS) of positive group: 41.40 ± 2.58%, negative group: 4.28 ± 1.16%, DEB group: 35.49 ± 0.65% and MNDEB group: 19.63 ± 6.77% (p = 0.001 by ANOVA). (D) Intima to plaque ratios of positive group: 62.26 ± 2.44%, negative group: 3.25 ± 1.27%, DEB group: 46.34 ± 8.42% and MNDEB group: 25.57 ± 3.18%. (p = 0.009 by ANOVA). All data were presented as the mean ± SEM. *p < 0.001, compared with positive group. **p < 0.05, compared with DEB group.
14
al
Pr
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
Figure 7. (A) Representative RAM11 stained histopathological images and (B) quantitative data of RAM11 area of in vivo result. (Scale bars = 500 μm) (C) Scoring data of inflammation, vessel injury and endothelization of in vivo result. All data were presented as the mean ± SEM. *p < 0.05, compared with positive group.
4. Conclusions In this study, we have developed the novel concept of MNDEB. It is the first device to use a MN array to perform endovascular drug delivery. The MN array was transferred and fixed to the inflated surface of a PTCA balloon through our process termed conformal transfer molding. Analysis of the mechanical behavior of the MNs demonstrated sufficient flexibility for each needle to yield to the external stimuli that may be encountered upon catheter insertion into the lesion area. In addition to flexibility, the needles had sufficient mechanical strength to penetrate the inner layer of the vessel. By doing so, the MNs on the MNDEB surface were able to make micro- incisions which facilitate improved drug delivery to the stromal area of the blood vessel. We demonstrate the superior drug delivery efficiency of the MNDEB through a comparative study with conventional DEB. The safety of endovascular MN insertion was confirmed in both normal and diseased vessels. Finally, we developed an atherosclerotic rabbit model to test the in vivo efficacy of the device. Animals treated with the MNDEB showed statistically significant reduction in atherosclerosis compared to both the untreated and conventional DEB-treated groups. The MNDEB device is an innovative drug delivery technology that shows great promise for improved treatment of vascular diseases by endovascular delivery of therapeutics. 15
Journal Pre-proof
Jo u
rn
al
Pr
e-
pr
oo
f
Acknowledgements This work was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry for Health & Welfares, Republic of Korea (HI08C2149), by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIT) (2015R1A5A1037668 and 2017R1A2B2003191), and by Mid-career Researcher Program through NRF grant funded by the MEST (2016R1A2B4010487). We specially thank to Genoss Co. Ltd for their generous donation of drug eluting balloons. We would also like to thank Peyton Tebon for his assistance in the revision of this article.
16
Journal Pre-proof
Jo u
rn
al
Pr
e-
pr
oo
f
References [1] A.R. Gruntzig, A. Senning, W.E. Siegenthaler, Nonoperative Dilatation of CoronaryArtery Stenosis - Percutaneous Transluminal Coronary Angioplasty, New England Journal of Medicine, 301 (1979) 61-68. [2] U. Sigwart, J. Puel, V. Mirkovitch, F. Joffre, L. Kappenberger, Intravascular Stents to Prevent Occlusion and Re-Stenosis after Transluminal Angioplasty, New England Journal of Medicine, 316 (1987) 701-706. [3] P.W. Serruys, M.J.B. Kutryk, A.T.L. Ong, Drug therapy - Coronary-artery stents, New England Journal of Medicine, 354 (2006) 483-495. [4] O.C. Marroquin, F. Selzer, S.R. Mulukutla, D.O. Williams, H.A. Vlachos, R.L. Wilensky, J.-F. Tanguay, E.M. Holper, J.D. Abbott, J.S. Lee, C. Smith, W.D. Anderson, S.F. Kelsey, K.E. Kip, A comparison of bare-metal and drug-eluting stents for off-label indications, New England Journal of Medicine, 358 (2008) 342-352. [5] M. Morice, P.W. Serruys, J.E. Sousa, J. Fajadet, E.B. Hayashi, M. Perin, A. Colombo, G. Schuler, P. Barragan, G. Guagliumi, F. Molnar, R. Falotico, R.S. Grp, A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization, New England Journal of Medicine, 346 (2002) 1773-1780. [6] D.R. Holmes Jr, R.E. Vlietstra, H.C. Smith, G.W. Vetrovec, K.M. Kent, M.J. Cowley, D.P. Faxon, A.R. Gruentzig, S.F. Kelsey, K.M. Detre, Restenosis after percutaneous transluminal coronary angioplasty (PTCA): a report from the PTCA Registry of the National Heart, Lung, and Blood Institute, The American journal of cardiology, 53 (1984) C77-C81. [7] C. Bauters, T. Meurice, M. Hamon, E. McFadden, J.-M. Lablanche, M.E. Bertrand, Mechanisms and prevention of restenosis: from experimental models to clinical practice, Cardiovascular Research, 31 (1996) 835-846. [8] P.W. Serruys, B.H. Strauss, K.J. Beatt, M.E. Bertrand, J. Puel, A.F. Rickards, B. Meier, J.J. Goy, P. Vogt, L. Kappenberger, Angiographic follow-up after placement of a selfexpanding coronary-artery stent, New England Journal of Medicine, 324 (1991) 13-17. [9] R. Hoffmann, G.S. Mintz, G.R. Dussaillant, J.J. Popma, A.D. Pichard, L.F. Satler, K.M. Kent, J. Griffin, M.B. Leon, Patterns and mechanisms of in-stent restenosis: a serial intravascular ultrasound study, Circulation, 94 (1996) 1247-1254. [10] M. Joner, A.V. Finn, A. Farb, E.K. Mont, F.D. Kolodgie, E. Ladich, R. Kutys, K. Skorija, H.K. Gold, R. Virmani, Pathology of drug-eluting stents in humans - Delayed healing and late thrombotic risk, Journal of the American College of Cardiology, 48 (2006) 193-202. [11] R. Waksman, R. Pakala, Drug-eluting balloon: the comeback kid?, Circulation: Cardiovascular Interventions, 2 (2009) 352-358. [12] B. Scheller, C. Hehrlein, W. Bocksch, W. Rutsch, D. Haghi, U. Dietz, M. Boehm, U. Speck, Treatment of coronary in-stent restenosis with a paclitaxel-coated balloon catheter, New England Journal of Medicine, 355 (2006) 2113-2124. [13] B. Scheller, C. Hehrlein, W. Bocksch, W. Rutsch, D. Haghi, U. Dietz, M. Boehm, U. Speck, Two year follow-up after treatment of coronary in-stent restenosis with a paclitaxelcoated balloon catheter, Clinical Research in Cardiology, 97 (2008) 773-781. [14] M. Unverdorben, C. Vallbracht, B. Cremers, H. Heuer, C. Hengstenberg, C. Maikowski, G.S. Werner, D. Antoni, F.X. Kleber, W. Bocksch, M. Leschke, H. Ackermann, M. Boxberger, U. Speck, R. Degenhardt, B. Scheller, Paclitaxel-Coated Balloon Catheter Versus Paclitaxel-Coated Stent for the Treatment of Coronary In-Stent Restenosis, Circulation, 119 (2009) 2986-2994. [15] G. Tepe, T. Zeller, T. Albrecht, S. Heller, U. Schwarzwaelder, J.-P. Beregi, C.D. Claussen, A. Oldenburg, B. Scheller, U. Speck, Local delivery of paclitaxel to inhibit restenosis during angioplasty of the leg, New England Journal of Medicine, 358 (2008) 689699. 17
Journal Pre-proof
Jo u
rn
al
Pr
e-
pr
oo
f
[16] M. Werk, S. Langner, B. Reinkensmeier, H.-F. Boettcher, G. Tepe, U. Dietz, N. Hosten, B. Hamm, U. Speck, J. Ricke, Inhibition of restenosis in femoropopliteal arteries - Paclitaxelcoated versus uncoated balloon: Femoral paclitaxel randomized pilot trial, Circulation, 118 (2008) 1358-1365. [17] A. Micari, A. Cioppa, G. Vadala, E. Stabile, F. Castriota, P. Pantaleo, C. Grattoni, A. Cremonesi, A. Marchese, P. Rubino, G. Biamino, A new paclitaxel-eluting balloon for angioplasty of femoropopliteal obstructions: acute and midterm results, Eurointervention, 7 (2011) K77-K82. [18] Y.P. Clever, D. Peters, J. Calisse, S. Bettink, M.-C. Berg, C. Sperling, M. Stoever, B. Cremers, B. Kelsch, M. Böhm, Novel Sirolimus–Coated Balloon Catheter: In Vivo Evaluation in a Porcine Coronary Model, Circulation: Cardiovascular Interventions, 9 (2016) e003543. [19] S. Habara, K. Mitsudo, K. Kadota, T. Goto, S. Fujii, H. Yamamoto, H. Katoh, N. Oka, Y. Fuku, S. Hosogi, A. Hirono, T. Maruo, H. Tanaka, Y. Shigemoto, D. Hasegawa, H. Tasaka, M. Kusunose, S. Otsuru, Y. Okamoto, N. Saito, Y. Tsujimoto, H. Eguchi, K. Miyake, M. Yoshino, Effectiveness of Paclitaxel-Eluting Balloon Catheter in Patients With SirolimusEluting Stent Restenosis, Jacc-Cardiovascular Interventions, 4 (2011) 149-154. [20] J.C. Fanggiday, P.R. Stella, S.H. Guyomi, P.A. Doevendans, Safety and efficacy of drugeluting balloons in percutaneous treatment of bifurcation lesions: The DEBIUT (Drug-Eluting Balloon in BIfurcaton UTrecht) registry, Catheterization and Cardiovascular Interventions, 71 (2008) 629-635. [21] F. Nijhoff, P.R. Stella, M.S. Troost, A. Belkacemi, H.M. Nathoe, M. Voskuil, M. Samim, P.A. Doevendans, P. Agostoni, Comparative assessment of the antirestenotic efficacy of two paclitaxel drug-eluting balloons with different coatings in the treatment of in-stent restenosis, Clinical Research in Cardiology, 105 (2016) 401-411. [22] K. Rosenfield, M.R. Jaff, C.J. White, K. Rocha-Singh, C. Mena-Hurtado, D.C. Metzger, M. Brodmann, E. Pilger, T. Zeller, P. Krishnan, R. Gammon, S. Mueller-Huelsbeck, M.R. Nehler, J.F. Benenati, D. Scheinert, I. Levant, Trial of a Paclitaxel-Coated Balloon for Femoropopliteal Artery Disease, New England Journal of Medicine, 373 (2015) 145-153. [23] P. Bondesson, B. Lagerqvist, S.K. James, G.K. Olivecrona, D. Venetsanos, J. Harnek, Comparison of two drug-eluting balloons: a report from the SCAAR registry, Eurointervention, 8 (2012) 444-449. [24] S. Sharma, N. Kukreja, C. Christopoulos, D.A. Gorog, Drug-eluting balloon: new tool in the box (vol 7, pg 381, 2010), Expert Review of Medical Devices, 7 (2010) 576-576. [25] B. Cortese, A. Bertoletti, Paclitaxel coated balloons for coronary artery interventions: A comprehensive review of preclinical and clinical data, International Journal of Cardiology, 161 (2012) 4-12. [26] S.M. Seedial, S. Ghosh, R.S. Saunders, P.A. Suwanabol, X. Shi, B. Liu, K.C. Kent, Local drug delivery to prevent restenosis, Journal of Vascular Surgery, 57 (2013) 1403-1414. [27] K. Lee, S.G. Lee, I. Jang, S.H. Park, D. Yang, I.H. Seo, S.-K. Bong, D.H. An, M.K. Lee, I.K. Jung, Y.H. Jang, J.S. Kim, W. Ryu, Linear Micro-patterned Drug Eluting Balloon (LMDEB) for Enhanced Endovascular Drug Delivery, Scientific Reports, 8 (2018). [28] C.D. Owens, W.J. Gasper, J.P. Walker, H.F. Alley, M.S. Conte, S.M. Grenon, Safety and feasibility of adjunctive dexamethasone infusion into the adventitia of the femoropopliteal artery following endovascular revascularization, Journal of Vascular Surgery, 59 (2014) 1016-1023. [29] A. Peppas, K. Seward, K. Van Wygerden, G. Conditt, A. Tellez, S. Rousselle, G. Kaluza, J. Granada, Prevention of Restenosis with Adventitial Delivery of Torisel via Mercator MedSystems Bullfrog Catheter in Femoral Arteries of Swine, Journal of the American College of Cardiology, 70 (2017) B323-B323. 18
Journal Pre-proof
Jo u
rn
al
Pr
e-
pr
oo
f
[30] J. Fromageau, J.-L. Gennisson, C. Schmitt, R.L. Maurice, R. Mongrain, G. Cloutier, Estimation of polyvinyl alcohol cryogel mechanical properties with four ultrasound elastography methods and comparison with gold standard testings, IEEE transactions on ultrasonics, ferroelectrics, and frequency control, 54 (2007) 498-509. [31] T.H. Kim, D.B. An, S.H. Oh, M.K. Kang, H.H. Song, J.H. Lee, Creating stiffness gradient polyvinyl alcohol hydrogel using a simple gradual freezing–thawing method to investigate stem cell differentiation behaviors, Biomaterials, 40 (2015) 51-60. [32] K.J. Lee, S.H. Park, J.Y. Lee, H.C. Joo, E.H. Jang, Y.-N. Youn, W. Ryu, Perivascular biodegradable microneedle cuff for reduction of neointima formation after vascular injury, Journal of controlled release, 192 (2014) 174-181. [33] J.W. Lee, J.-H. Park, M.R. Prausnitz, Dissolving microneedles for transdermal drug delivery, Biomaterials, 29 (2008) 2113-2124. [34] J. Lee, S.H. Park, I.H. Seo, K.J. Lee, W. Ryu, Rapid and repeatable fabrication of high A/R silk fibroin microneedles using thermally-drawn micromolds, European Journal of Pharmaceutics and Biopharmaceutics, 94 (2015) 11-19. [35] H.B. Song, K.J. Lee, I.H. Seo, J.Y. Lee, S.-M. Lee, J.H. Kim, J.H. Kim, W. Ryu, Impact insertion of transfer-molded microneedle for localized and minimally invasive ocular drug delivery, Journal of Controlled Release, 209 (2015) 272-279. [36] S.H. Park, D.H. Jo, C.S. Cho, K. Lee, J.H. Kim, S. Ryu, C. Joo, J.H. Kim, W. Ryu, Depthwise-controlled scleral insertion of microneedles for drug delivery to the back of the eye, European Journal of Pharmaceutics and Biopharmaceutics, 133 (2018) 31-41. [37] K. Lee, H.B. Song, W. Cho, J.H. Kim, J.H. Kim, W. Ryu, Intracorneal injection of a detachable hybrid microneedle for sustained drug delivery, Acta biomaterialia, 80 (2018) 4857. [38] J. Lee, D.-H. Kim, K.J. Lee, I.H. Seo, S.H. Park, E.H. Jang, Y. Park, Y.-N. Youn, W. Ryu, Transfer-molded wrappable microneedle meshes for perivascular drug delivery, Journal of Controlled Release, 268 (2017) 237-246. [39] R.N. Isnard, B.M. Pannier, S. Laurent, G.M. London, B. Diebold, M.E. Safar, Pulsatile diameter and elastic modulus of the aortic arch in essential hypertension: a noninvasive study, Journal of the American College of Cardiology, 13 (1989) 399-405. [40] R. Kornowski, M.K. Hong, F.O. Tio, O. Bramwell, H. Wu, M.B. Leon, In-stent restenosis: contributions of inflammatory responses and arterial injury to neointimal hyperplasia, Journal of the American College of Cardiology, 31 (1998) 224-230. [41] P.R. Stella, A. Belkacemi, R. Waksman, S. Stahnke, R. Torguson, R.P. von Strandmann, P. Agostoni, G. Sangiorgi, S. Silber, I. Valentine, The Valentines Trial: results of the first one week worldwide multicentre enrolment trial, evaluating the real world usage of the second generation DIOR paclitaxel drug-eluting balloon for in-stent restenosis treatment, Eurointervention, 7 (2011) 705-710. [42] P.P. Buszman, K. Milewski, A. Żurakowski, J. Pajak, M. Jelonek, P. Gasior, A. Peppas, A. Tellez, J.F. Granada, P.E. Buszman, Experimental evaluation of pharmacokinetic profile and biological effect of a novel paclitaxel microcrystalline balloon coating in the iliofemoral territory of swine, Catheterization and Cardiovascular Interventions, 83 (2014) 325-333. [43] S. Kaushik, A.H. Hord, D.D. Denson, D.V. McAllister, S. Smitra, M.G. Allen, M.R. Prausnitz, Lack of pain associated with microfabricated microneedles, Anesthesia & Analgesia, 92 (2001) 502-504. [44] J. Gupta, H.S. Gill, S.N. Andrews, M.R. Prausnitz, Kinetics of skin resealing after insertion of microneedles in human subjects, Journal of controlled release, 154 (2011) 148155. [45] D.-H. Kim, E. Jang, K. Lee, J. Lee, S. Park, I. Seo, S. Lee, W. Ryu, Y.-N. Youn, A biodegradable microneedle cuff for comparison of drug effects through perivascular delivery to balloon-injured arteries, Polymers, 9 (2017) 56. 19
Jo u
rn
al
Pr
e-
pr
oo
f
Journal Pre-proof
20
KangJu Lee, JiYong Lee, Seul Gee Lee, SeungHyun Park, Da Som Yang, Jung-Jae Lee, Ali Khademhosseini, Jung Sun Kim, WonHyoung Ryu PII:
S0168-3659(20)30089-4
DOI:
https://doi.org/10.1016/j.jconrel.2020.02.012
Reference:
COREL 10165
To appear in:
Journal of Controlled Release
Received date:
1 November 2019
Revised date:
3 February 2020
Accepted date:
5 February 2020
Please cite this article as: K. Lee, J. Lee, S.G. Lee, et al., Microneedle drug eluting balloon for enhanced drug delivery to vascular tissue, Journal of Controlled Release (2020), https://doi.org/10.1016/j.jconrel.2020.02.012
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier.
Journal Pre-proof
Microneedle Drug Eluting Balloon for Enhanced Drug Delivery to Vascular Tissue KangJu Lee1,2,3,4 , JiYong Lee1 , Seul Gee Lee5 , SeungHyun Park1 , Da Som Yang1 , Jung-Jae Lee6 , Ali Khademhosseini2,3,4,7,8,9,10 , Jung Sun Kim5 *, WonHyoung Ryu1 * 1
Department of Mechanical Engineering, Yonsei University, Seoul, 03722, Republic of Korea Department of Bioengineering, University of California-Los Angeles, Los Angeles, CA 90095, USA 3 Center for Minimally Invasive Therapeutics (C-MIT), University of California-Los Angeles, Los Angeles, CA 90095, USA 4 California NanoSystems Institute, University of California-Los Angeles, Los Angeles, CA 90095, USA 5 Division of Cardiology, College of Medicine, Yonsei University, Seoul, 03722, Republic of Korea 6 Graduate Program in Science for Aging, Yonsei University, Seoul, 03722, Republic of Korea 7 Jonsson Comprehensive Cancer Center, University of California-Los Angeles, Los Angeles, CA 90095, USA 8 Department of Radiology, University of California-Los Angeles, Los Angeles, CA 90095, USA 9 Department of Chemical Engineering, University of California-Los Angeles, Los Angeles, CA 90095, USA 10 Department of Biomolecular Engineering, University of California-Los Angeles, Los Angeles, CA 90095, USA
Corresponding authors: Jung Sun Kim ([email protected]), WonHyoung Ryu ([email protected])
e-
*
pr
oo
f
2
Abstract
Jo u
rn
al
Pr
High rates of restenosis and neointimal formation have driven increasing interest in the application of drug eluting balloons (DEB) as counteractive measures for intraluminal drug delivery. The use of DEBs eliminates the need for stents so that serious side effects including in-stent restenosis and stent thrombosis can be avoided and long-term medication of antiplatelet agent is not needed. Despite their benefits, DEBs have poor drug delivery efficiency due to short balloon inflation times (30~60 seconds) that limit the passive drug diffusion from the balloon surface to the luminal lesion. To increase drug delivery efficiency, a microneedle DEB (MNDEB) was developed by a conformal transfer molding process using a thin polydimethylsiloxane mold bearing a negative array of MNs of 200 μm in height. A MN array composed of UV curable resin was formed onto the surface of DEB, and drugs were coated onto the structure. The mechanical properties of the MN array were investigated and MN penetration into luminal vasculature was confirmed in vivo. An increase in drug delivery efficiency compared to a standard DEB was demonstrated in an in vivo test in a rabbit aorta. Finally, the superior therapeutic efficacy of MNDEBs was evaluated using an atherosclerosis rabbit model. Keywords: microneedle, drug eluting balloon, vascular disease, endovascular drug delivery, conformal transfer molding
1
Journal Pre-proof
pr
oo
f
1. Introduction Since the development of a balloon catheter for percutaneous trans- luminal angioplasty (PTCA) in 1978 [1], vascular intervention technology has continued to evolve from the bare metal stent (BMS) [2] to catheter-delivered drug eluting stents (DES) [3-5]. The original balloon for PTCA treatement was inflated at the blockage site to compress the plaque against the vessel wall. However, this therapy was only temporarily effective in many patients as over 30% suffered from restenosis, usually within 5 months of the original intervention [6]. In 5~10% of patients, elastic recoil occurred almost immediately (minutes-hours) after the procedure [7]. To address the problem of restenosis, the BMS was developed to permanently promote vascular integrity after implantation by supporting the vessel wall after deployment [2]. While recoil was no longer a concern, in-stent restenosis (ISR) was common as the stent was eliciting a foreign body reponse from the immune system [8]. Long term follow-up of BMS cases revealed a 20~30% incidence rate of ISR [9]. To address ISR, DESs emerged; these stents maintained the same basic structure of BMSs but were covered by a polymeric carrier loaded with a therapeutic agent. While DESs have decreased the rate of ISR signficantly through the local delivery of anti-proliferative drugs, metal stent-based intervention still suffers from other complications such as late onset thrombosis and hypersensitivity to the drug or polymer coating [10].
Jo u
rn
al
Pr
e-
The most certain way of preventing ISR is to avoid a foreign body response by not leaving any foreign material in the blood vessels. Drug-eluting balloons (DEB) are one solution that addresses the limitations presented by DESs. These devices work by inflating the balloon in the blood vessel so it is in contact with the lesion for 60 seconds. The drug coated on the surface of the DEB is transferred to the lesion and exerts its therapeutic effect [11]. Several trials have been conducted to treat both ISR [12-14] and de novo lesions in blood vessels using DEBs [15-17]. Most DEBs used today deliver paclitaxel or sirolimus [18] using different carriers and excipients. Beyond adhering the drug to the balloon, the drug carrier plays a central role in a drug transfer into the vessel wall. To improve drug delivery to the lumen, many researchers have developed various coating formulations; some of the leading commercial prodcuts include PACCOCATH®, based on iopromide [19]; DIOR®, based on microcrystals of paclitaxel [20]; IN.PACT™ (Medtronic, USA), containing a hydrophilic matrix and urea [21]; Lutonix® 035 (BARD, USA) consisting of either polysorbate or a sorbitol hydrophilic carrier [22]; and Pantera LUX® (BIOTRONIK AG, Switzerland), consisting of butyryl-trihexyl citrate loaded with paclitaxel [23]. Although a variety of products exist to optimize drug release, current DEBs still have limitations in terms of low drug delivery efficiency and limited sorts of drugs that cannot be solved by changing the drug carrier [24, 25]. The fundamental restriction imposed on the DEB is its passive mechanism of drug transfer from the DEB to the lumen. There have been a few trials that have tested various modifications to DEBs aimed at improving the efficiency of endovascular drug delivery. A porous balloon was designed to release the drug through the pores at the time of inflation [26]. Additionally, Lee et al. have developed linear-micropatterned drug eluting balloons (LMDEB) using customized balloon forming molds [27]. LMDEBs were fabricated to have 16 linear micropatterns (LMs) on the surface with a height of 130 μm. The goal of the LMs is to increase the contact force between the drug coated surface and lumen, which leads to the enhanced efficiency of endovascular drug delivery. Another solution developed by Mercator Medsystems, Inc. (Emeryville, CA) are the Bullfrog® and Cricket® Micro-infusion devices. These devices combine a 34G microscale needle with a balloon catheter. While the catheter is closed, the hollow needle is covered by the balloon so that it does not injure the vessel walls. When the catheter is opened, the needle extends out of the balloon and is used to inject drugs directly into the surrounding 2
Journal Pre-proof
e-
pr
oo
f
luminal tissue. Delivery with a needle penetrates the luminal layers of the vessel, facilitating better distribution through the tissue [28, 29]. The major drawback to these devices is the size of the 34G needles as they are too large for use in many blood vessels.
Pr
Figure 1. A graphical abstract of MNDEB in this study indicating that drug coated MNs onto balloon surface enhance the efficiency of endovascular drug delivery by MN insertion compared to conventional DEB.
Jo u
rn
al
Microneedles (MNs) may be a suitable solution for increasing endovascular drug delivery, just as it has been transdermally. In this study, we have fabricated a MN drug eluting balloon (MNDEB) by molding a MN array onto the surface of a DEB to improve drug delivery efficiency (Figure 1). The MN array was fabricated by wet etching and subsequent polydimethylsiloxane (PDMS) casting. We then devised a transfer molding process that fixed the MN structures to a conventional DEB by flexing the PDMS mold around the balloon and curing with UV light. This resulted in a strong bond between each MN and the surface of the balloon that minimized the risk of detachment. The MNDEB was proven to be suitable for intraluminal use as the MNs penetrated the tissue and reached the tunica media of the vascular tissue. Additionally, we adapted the conventional PACCOCATH technology to coat the drugs on the surface of both the MNDEB and DEB [27]. A unique mechanical property of the MNs was investigated using a deformation test and in vitro insertion test with vascular tissue phantom scaffolds. The enhanced drug delivery efficiency of the MNDEB was demonstrated using a model drug and fluorescent microscopy. The safety of the balloon was studied through histological analysis and scoring. In vivo therapeutic intervention was then conducted on an atherosclerotic rabbit model, and therapeutic efficacy was evaluated in a comparative study of the MNDEB and DEB.
2. Materials and Methods 2.1. MN array fabrication Male MNs (200 µm height) were fabricated by lithography, multi- layer deposition, and wet etching. The MN shape was achieved through manip ulating the size of the square micropattern on the photomask. First, an oxide layer was formed on a 4-inch wafer (in a furnace), followed by low-pressure chemical vapor deposition of nitride. The layer thicknesses were 300 Å and 1200 Å, respectively. Spin-coating was used to deposit a 100 µm3
Journal Pre-proof thick layer of SU-8 on the wafer. The material was crosslinked by irradiating the wafer with UV light passed through an aligned mask with the square pattern array (500×500 µm2 ). After development, the exposed Si3 N4 and SiO 2 layers were patterned by reactive ion etching. Then, the processed wafer was soaked in KOH solution (29 %, 79 °C) for 210 minutes and rinsed in a bubbling water for 30 minutes. The result of the fabrication process was an array of octagonal-based cones, 150 µm in length (Figure S1) with a 1.5 aspect ratio interval between MNs is 500 µm. Casting PDMS (Silicone Elastomer-184, Dow-Corning, USA) over the wafer formed a mold containing the negative MN pattern. The PDMS was cured at room temperature for 24 hours.
al
Pr
e-
pr
oo
f
2.2. MNDEB Fabrication We selected a commercially available, UV-curable adhesive (208-CTH-F, DYMAX, USA) as the material for the MNs. An MN array (200 µm- height and 800 µm- interval) was prepared and PDMS (Dow-Corning, Silicone Elastomer-184) MN cavities were molded subsequently with a uniform thickness of 1 mm. The curable resin was deposited on the flexible female mold and placed in a vacuum chamber for 2 minutes to eliminate trapped air from the MN cavities. Residual resin on the surface of the mold was removed by doctor blading (Figure 2). After removing the bubbles, we wrapped the mold around the surface of the inflated DEB. The entire apparatus was exposed to UV light for four minutes to fix the MN array onto the surface of the DEB. After removing the PDMS mold, the deflated MNDEBs were folded, and the drug was pipette-coated with a mixture of drug and shellac as matrix polymer instead of iopromide of the PACCOCATH® formulation. Rhodamine B (RB, Samchun Chemical, South Korea) and paclitaxel (PTX, Genoss, South Korea) were used as the model drugs in this study. 3 μg/mm2 of either RB or PTX was coated onto the surface of the MNDEB.
Jo u
rn
2.3. Mechanical characterization The mechanical behavior of MNs attached to a MNDEB was monitored using a stereomicroscope and a stainless-steel pillar, which was fixed to a customized z-axis auto-stage (STM-2-USB, Science Town Corp., South Korea) (Figure S2). For the testing, MNDEBs were cut longitudinally and attached to the wall of microstage so that the MNs were oriented horizontally. The MN and the steel pillar used for testing were aligned first in the horizontal plane. Once aligned, the pillar was lowered until it contacted the MN. The pillar deformed the MN as it was moved vertically at a speed of 100 µm/s. Once the MN had been deformed to 90 degrees, the pillar was retracted at a speed of 100 µm/s. The deformation and recovery of the MN was recorded by stereomicroscopy.
2.4. Polyvinyl alcohol (PVA) phantom vascular tissue PVA phantom was fabricated according to reported research [30, 31]. In brief, PVA, M w 146,000-186,000 (Sigma Aldrich), was dissolved in DI water to make a 10% (w/w) solution. The solution was heated to 120 ◦ C on a hotplate and was stirred for 2 hours until the PVA powder was fully dissolved. Homogeneous PVA solution was poured into the petri dish and solidified at room temperature. The solidified PVA samples were polymerized by freeze-thaw cycles to adjust the mechanical properties. The freeze-thaw cycle consisted of two steps: a freezing step for 8 hours at -20 ◦ C, and a thawing step for 16 hours at room temperature (approximately 20 ◦ C). During freeze-thaw cycles, hydrogen bonds are formed between the PVA chains. As more hydrogen bonds are formed, the elastic modulus of the material increases; material stiffness increases proportionally with the number of freeze-thaw cycles. The number of cycles was selected to yield material properties mimicking those of the normal 4
Journal Pre-proof vessel (1 cycle) and atherosclerotic vessel (8 cycles). After the last cycle, the samples were tested using a universal tensile machine (UTM, OTT-001, Oriental TM, Republic of Korea) to validate the elastic moduli of 52 kPa and 183 kPa, respectively.
rn
al
Pr
e-
pr
oo
f
2.5. In vivo animal study In this study, 12 male New Zealand White rabbits (3.0-3.5 kg weight) were used. 3 rabbits were used for each of the following experiments: 1) drug distribution and MN insertion test, 2) atherosclerosis modeling, 3) therapeutic efficacy test, and 4) safety test. All rabbits were acclimatized for 1 week before in vivo test. The study protocol was approved by the local Institutional Animal Care and Use Committee of Yonsei University Health System (IACUC: 2017-0269). All animals received human care in compliance with the Animal Welfare Act and the "Principles of Laboratory Animal Care" formulated by the Institute of Laboratory Animal Resources (National Research Council, NIH Publication No. 85-23, revised 1996). Animals were anesthetized by intramuscular injection with Zoletil (10mg/kg, Virvac, USA) and Rompun (5mg/kg, Bayer, Germany), followed by 2% of isoflurane (Forane ®, JW Pharm, South Korea) and oxygen. The iliac artery was accessed via the carotid artery. Heparin (150 units/kg) was injected to maintain an activated clotting time within 250 seconds before catheterization. For the drug distribution and MN insertion tests, both conventional DEBs and MNDEBs were inflated in the iliac artery for 60 seconds to 1.2 times the size of the artery. To develop the atherosclerotic model, each rabbit injured its iliac artery by two times of balloon over- inflation for 30 seconds was fed a high cholesterol diet (1% cholesterol, DooYeol Biotech, South Korea) for one week. The surgical intervention included the inflation of both MNDEBs and DEBs (Genoss Co., Ltd., South Korea) to 1.2 times the size of the artery (based on quantitative angiography) for 60 seconds. Following the operation, the rabbits were maintained on a high cholesterol diet for 4 weeks; optical coherence tomography (OCT) and quantitative coronary angiography (QCA) were taken prior to the euthanization of the animal. There was no animal death during the entire in vivo study.
Jo u
2.6. Fluorescent microscopy and histological analysis Vessels treated by RB-coated MNDEB and DEB were cryosectioned and imaged with fluorescent microscopy to visualize drug distribution. The cultured tissue was embedded in optimum cutting temperature compound (Sakuta Finetek USA Inc., USA) and frozen. The frozen samples were cut by a microtome with a thickness of 10 µm and analyzed by a fluorescent microscope (BX53, Olympus, Japan) under identical parameters (light sensitivity=ISO 100, light exposure time=1 second and light intensity=6). Afterwards, the fluorescent intensity of each samples was quantified by measuring integrated density (ID) as described [32]. Since the sectional area of vessels is different to each other, the meas ured fluorescent value was normalized to the area of the blood vessel to secure normalized integrated density (NID). To analyze the efficacy of the intervention, the target vessel was fixed by continuous perfusion of normal buffered formalin (10%) for one week. A 5 mm- long vascular sample was placed into a cassette and treated with alcohols and xylenes. After that, the sample was embedded in a paraffin block. The paraffinized samples were sectioned to a thickness of 4 µm and stained with hematoxylin and eosin (H&E, Merck, Germany) and Masson’s trichrome (BBC Biochemical, USA). The sectioned area was measured by an optical microscope (SCN400, Leica, Germany) and then histomorphometry was conducted using LAS 4.2 software (Leica, Germany). Additional sections were labeled with a mouse monoclonal anti-rabbit macrophage antibody (RAM11, Dako, USA), Interleukin (IL)-1 (Abbkine, USA), IL-6 (Santa Cruz Biotechnology, USA), tumor necrosis factor (TNF)- (Abbkine, USA) to evaluate the degree of inflammation and with vascular endothelial growth 5
Journal Pre-proof factor (VEGF, Abcam, United Kingdom), intercellular adhesion molecule 1 (ICAM1, Abcam, United Kingdom), vascular cell adhesion molecule 1 (VCAM1, Santa Cruz Biotechnology, USA) to test the expression of pro- inflammatory atherosclerosis factors. Immunostaining was performed and quantified by calculating the percentage of labeled area with respect to total tissue area. The color detection threshold was chosen for the DAB chromogen (brown staining) in tissue sections. Measurements were performed using QWin Image Analysis software (Leica, Germany).
e-
pr
oo
f
2.7. Vascular stenosis evaluation In addition to histological analysis, the degree of stenosis was evaluated by scanning the 10 mm- long vessel following intervention with the DEB and MNDEB. QCA and OCT were conducted after delivering drugs and after 4 weeks to monitor the progress of intimal proliferation in terms of diameter stenosis (DS) and area stenosis (AS). OCT was conducted using the C7-XR imaging system (Abbott Vascular, CA, USA). First, 16 ml of contrast media was infused through a guiding catheter for 4 seconds. Meanwhile, the OCT catheter was pulled back (20 mm/s), and OCT images were acquired (100 frames/s). All OCT images were analyzed at Cardiovascular Research Center (Republic of Korea) by blinded analysts. All histological images were provided by the histological laboratory in Yonsei University Hospitial and compared to proximal side branches from the edge of the injury.
Jo u
rn
al
Pr
3. Results and Discussion 3.1. Conformal transfer molding to transfer the MN array onto the DEB surface We first fabricated an array of MNs with an aspect ratio of 1.5, 200 µm height, and 800 µm spacing interval as shown Figure S1. A thin nickel oxide layer was deposited on a silicon wafer by low pressure chemical vapor deposition and etched by photolithography into a square shape. This patterned wafer was dipped into KOH solution and then processed with undercut etching which removed material below nickel oxide mask patterns. The wet etching continued until the nickel oxide mask was detached as a result of the undercut etching. MNs of various height were formed by varying the size of the square mask. The square nitride masks were patterned at 250, 500, 750, and 1000 µm; this resulted in MN with a height of 100, 200, 300, 400 µm, respectively with an average etch rate of 0.96 ± 0.02 µm/sec. The relationship between the height (h) of the MN and width (l) of square mask was calculated to be approximately l:h = 2.5:1. To be used clinically, the MN array should be firmly fixed to the surface of the DEB. We have developed a novel micromolding process, called conformal transfer molding, to transfer the MN array to the curved surface of a conventional DEB. The concept and process of conformal transfer molding is shown in Figure 2. We first fabricated a uniform, 1 mmthick PDMS mold using the previously fabricated silicon MNs; a thin and flexible mold is required for this process in order to make conformal contact with the curved surface. Then, UV curable adhesive was spread onto the PDMS mold and trapped air in the MN cavities was eliminated in a vacuum chamber for 2 minutes. Residual UV curable resin on the surface of the mold was removed by doctor blading. Next, the mold filled with resin was rolled around an inflated DEB (2.75 mm diameter, 10 mm length). To transfer and solidify the MN array on the surface of DEB, the device was exposed to UV light for 4 minutes and the flexible mold was detached. The UV curable resin we used was developed and commericalized as adhesive to bond PET-based polymer in implantable medical devices. We hypothesized that UV curing during conformal transfer molding would enable the MNs to solidify and attach to the DEB surface consising of a PET-based polymer. Through this process, more than 95% of the MNs 6
Journal Pre-proof transferred onto the surface of the DEB (as confirmed by optical microscopic analysis from Figure 2G, data not shown). In addition, seamless boundary at the inteface between MN and the balloon surface indicated the firm integration of the MN.
Jo u
rn
al
Pr
e-
pr
oo
f
To our knowledge, this is the first use of MNs for endovascular drug delivery. Thus far, MNs have remained in the form of patches due to the manufacturing limitations of conventional molding techniques such as centrifugation [33] or vacuum suction [34]. Recently, several groups have developed transfer molding [35, 36], pressure assisted molding [37], and thermal welding [38] processes to integrate MNs to macroscale applicators. Fixing these needles to easy-to-handle materials, such as a flexible mesh and an injection-aid pen, has facilitated the application of MNs to the peri- vascular [38] and ocular drug delivery [3537]. Additionally, our work is novel as MN arrays have not yet been tethered to curved threedimensional surfaces. In this study, we created a MNDEB through conformal transfer molding. This process is not restricted by the height, spacing, or shape of the MNs as long as PDMS molds can be fabricated thin enough to be conformally attached over the curvature of a three-dimensional surface. Just as MN patches have revolutionized transdermal drug delivery, we believe it is possible to do the same for endovascular drug delivery through the creation of the MNDEB.
Figure 2. Schematic images of conformal transfer molding process consisting of (A-C) premolding with UV curable adhesive, (D) alignment of flexible planar PDMS mold, (E) curing process and (F) completed MNDEB. (G) Stereo- and optical microscopic images of MNDEB surface. An inset is the balloon surface before conformal transfer molding. (scale bar = 300 µm) (H) A scanning electron microscopic image of a MN onto MNDEB surface. (scale bar = 100 µm)
3.2. Mechanical behavior of conformally molded MN array and vascular tissue insertion In order for the MNs tethered to the DEB to reach the lesion through the blood vessels, the array must be firmly attached. Additionally, if the MN is too brittle, contact with the luminal 7
Journal Pre-proof
Jo u
rn
al
Pr
e-
pr
oo
f
surface of the vessel may cause fragmentation of MN tips and leave debris in the lumen. For these reasons, the MNs must be flexible enough to sustain lateral MN bending during the delivery process. While a certain degree of flexibility is required, the MNs must also be longitudinally rigid enough to penetrate the vascular tissue upon inflation of the DEB. To ensure that our design met these criteria, we investigated the mechanical behavior of the MN array. To test the bending properties, we aligned the MNDEB vertically. A stainless-steel pillar (100 µm diameter) was attached to micro z-axis stage and aligned to the MN (Figure 3A). The metal pillar was lowered down to the MN at a speed of 100 µm/s and subsequently retracted with an identical speed once the needle bent 90°. As shown in Figure 3A, the tip of the needle deformed 100 µm vertically and recovered more than 50% within 3 seconds. The MN recovered more than 75% after 1 minute, and nearly 100% after 4 minutes. The video of this experiment is available as Supplementary Movie 1. This test also confirmed that the MN would not detach from the balloon surface or deform as confirmed by stereomicroscopic analysis.
Figure 3. (A) Mechanical deformation of the MN attached to the MNDEB surface. After deformation, the deformed MN was 50% recovered in 3 seconds and 100 % recovered over 4 minutes. (scale bar = 100 µm) (B) Representative fluorescent confocal microscopic images of MN insertion marks within 50 and 200 kPa PVA tissue phantom scaffolds. (scale bar = 100 µm) (C) Plotted MN incisions in 50 and 200 kPa PVA scaffolds from the confocal microscopic images. (D) Insertion marks (red arrow) of MNDEB into lumen through in vivo 8
Journal Pre-proof application. Blue dotted lines indicate the endothelium and asterisks (*) indicate the lumen side. Right images are sectional view of DEB treated vessels with no insertion mark. (scale bar = 200 µm (big), 20 µm (small)) (E) Images of MN tip of MNDEB before and after in vivo test. (scale bar = 100 µm)
Pr
e-
pr
oo
f
To test the ability of the device to insert MNs into the tissue, we performed an insertion test with vascular tissue phantom. We prepared PVA scaffolds with elastic moduli of 52 and 183 kPa, analogs of normal or calcified and narrowed vessels in hypersensitive patients respectively [39]. MN patches were fabricated using the same resin and molds as those created for the MNDEB. The PVA scaffold was wetted with 0.1% (w/w, in DI water) RB solution for 1 minute before the MNs were inserted for fluorescent staining. Subsequently, we manually applied the MN patch to the PVA scaffolds and analyzed the insertion marks using a confocal microscope. We measured MN incision width at the midpoint of the MN penetration depth and averaged the measured widths in both the x- and y-axes. As shown in Figure 3B&C, the MN patch penetrated both PVA scaffolds at a depth of approximately 75~100 µm. Puncturing the 183 kPa scaffold resulted in greater MN deformation compared to the 52 kPa material due its higher elastic modulus. Since the 52kPa scaffold was soft, it was slightly incised after being stretched. However, the 183 kPa scaffold yielded a different result as the MNs easily penetrated the surface. Because of the varied substrate properties, the incision depths varied; in the 183 kPa scaffold the MN penetrated to a depth of 102 ± 11 µm, while the MNs reached a depth of 72 ± 6 µm in the 52 kPa scaffold. Despite of the variation of MN incision depths, both MNs could penetrate both vascular tissue phantom to a depth sufficient to overcome the endothelium (< 10 µm) without compromising the media layer (< 500 µm).
Jo u
rn
al
Finally, we conducted an in vivo insertion test with healthy rabbit tissue. The MNDEB was prepared and deployed in the illiac arteries, a commonly damaged region in rabbits with atherosclerosis. The MNDEB was inflated and maintained at a diameter of the iliac artery for 60 seconds. After retraction and removal, the rabbit was sacrificed and the illiac artery was harvested. The harvested vascular tissue was then sectioned and stained to observe the MN insertion marks. As shown in Figure 3D, MN marks were found in the lumen of the treated vessels after H&E staining. Image analysis displayed an insertion depth of approximately 100 µm maintaining the endothelium (blue dotted line, Figure 3D). Secondary analysis of the MNDEB structure after the procedure was a lso investigated and no structural difference in the MN shape or size was observed (Figure 3E).
9
oo
f
Journal Pre-proof
e-
pr
Figure 4. (A) Fluorescent microscopic images of rhodamine B (model drug) distribution in vessels treated with the MNDEB and the DEB (scale bar = 500 µm). (B) Normalized integrated density (NID) of DEB vs. MNDEB using model drug RB (n=3). *p < 0.05 on twotailed T-test. All data were presented as the mean ± SEM.
Jo u
rn
al
Pr
3.3. In vivo drug distribution in vascular tissue We next investigated if the MNDEB was able to increase the drug delivery efficiency compared to the standard DEB. Fluorescent model drugs were used which facilitated facile study of drug distribution through fluorescent microscopy imaging. Both the MNDEB and DEB were coated with RB and were used to deliver the model drug to the iliac artery. In vivo tests were performed using the same procedure as the in vivo insertion tests; analysis was performed used fluorescent microscopy and tissue sectioning. Model drug was delivered by both the MNDEB and DEB and the treated area was cultured for 2 days. The tissue was cryosectioned (instead of paraffinization) to avoid quenching the fluorescent signal [27]. The intensity of the fluorescent signal was quantified from still images by measuring ID using ImageJ. The measured reading was normalized to the area of vascular tissue to secure the NID. The results of this experiment demonstrated that the MNDEB enhanced drug delivery efficiency compared to the conventional DEB (Figure 4). A higher fluorescent signal was observed in MNDEB treated samples compared to the conventional DEB treated samples (Figure 4A). The mean NID value in the MNDEB treated tissue (NID=2.7) was 2.4 times higher than that of the DEB treated tissue (NID=1.17).
3.4. Safety of MNDEB Even though the MNDEB achieved superior drug delivery efficiency compared to the conventional DEB, the integrity of the vascular tissue should not be impaired by MN insertion. To check this safety issue, we applied both drug- free conventional balloons and drug- free MN balloons to the normal vessel in vivo. The degree of inflammation and vessel injury was measured 4 weeks after the procedure. The degree of inflammation was investigated to check for possible damage to the vessel by staining the tissue sections with RAM11. Inflammation was scored according to Kornowski [40], allocating values of 0 (none), 1 (mild with minimal infiltrated inflammatory cells), 2 (moderate), or 3 (severe, with large clusters of inflammatory cells with granulomatousmorphology). Injury scoring was performed based on H&E and 10
Journal Pre-proof trichrome stained slides according to Schwartz [41]. The criteria for injury scoring is as follows: 0, no injury; 1, internal elastic lamia (IEL) lacerated; 2, media injury; or 3, laceration of external elastic lamia (EEL). IEL and EEL areas were assessed with ImageJ. Additionally, morphological assessment of endothelialization was described as percentage endothelial coverage of the arterial circumference: 0 < 25%, 1 = 25~75%, 2 = 76~99%, 3 = 100% (complete) [42].
oo
f
As shown in Figure 5A&B, RAM11 staining and inflammation scoring revealed no statistically significant difference in tissues treated with either the drug- free MN balloon and drug- free conventional balloon. This indicates that MN insertion did not elicit a larger immune response in the blood vessel compared to the balloon with usual surface. As for the injury scores, 6 slide sections of each group (drug-free MN balloon or conventional balloon) showed no statistical significance (Figure 5C), which indicates that MN insertion does not affect the physical integrity of the blood vessel. In addition, there was no statistical significance in endothelization score between drug- free MN balloon or conventional balloon. Therefore, the insertion of MN to endovascular tissue may be considered as safe as conventional DEB intervention. Details of the values are provided in Figure S3A.
Jo u
rn
al
Pr
e-
pr
The primary benefit of MNs in the context of transdermal drug delivery is that they are minimally invasive. This property is advantageous as the small puncture size minimizes pain during application [43] in addition to reducing damage to the tissue [44]. While the application of MNs to skin has been thoroughly studied, the effects of insertion into other tissues are not well characterized. In the case of vascular endothelium, maintaining the integrity of the blood vessel is a major concern. For perivascular application of MNs (first penetrating the adventitia then the media layers [32]), the tunica media may be ruptured by MN insertion; we investigated endovascular MN insertion to ensure that MNs on the DEB would not damage the overall vessel structure. Both normal and diseased vessels showed no histopathologic differences when the balloon was inflated with or without the MNs [32, 45]. The height of the perivascular MNs was 650 µm, long enough to penetrate the adventitia, but short enough not to reach the endothelium and puncture through the vessel wall. In this study, we designed the height of the MNs based on this criterion and after histopathological analysis, did not find any evidence of vascular damage due to MN insertion into the vascular endothelium. Additionally, in vivo testing did not show any adverse effects or animal death as a result of MN insertion. The results of these two studies (perivascular and endovascular MN drug delivery) demonstrate the safety of vascular micro-incision by MN insertion.
11
al
Pr
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
Figure 5. (A) Representative RAM11 stained histopathological images of normal vessels treated non-drug coated MNDEB and DEB. (Scale bars = 500 μm) (B) Quantitative data of RAM11 area in the normal vessels treated non-drug coated MNDEB and DEB. (C) Scoring data of inflammation, vessel injury and endothelization in the normal vessels treated non-drug coated MNDEB and DEB. All data were presented as the mean ± SEM. p < 0.05 on twotailed T-test.
3.5. Therapeutic efficacy of MNDEB compared to DEB Therapeutic efficacy of the MNDEB was demonstrated by an in vivo test. Atherosclerosis was induced in the iliac arteries of a rabbit by balloon injury and consumption of a high- fat diet for 4 weeks. The diseased rabbits were operated with both a MNDEB and a standard DEB coated with 3 µg/mm2 of PTX; the balloons were inflated for 60-seconds to apply the drug to the lumen of the vessel. The four experimental groups used in this comparative study were defined as follows: normal rabbit (n=3, negative control); atherosclerosis model (n=3, positive control); MNDEB treatment (n=3); DEB treatment (n=3). Subsequently, we monitored the progress of patency by measuring AS and DS using QCA and OCT. In addition, all vascular tissues were cultured and stained with H&E and trichrome for analysis of vascular integrity and intima. We evaluated the neointimal growth after each intervention. Significant proliferation was shown in the positive control group, which indicates the disease model (atherosclerosis) exhibited the disease phenotype (Figure 6A, positive). The values of AS and DS were estimated from OCT and QCA analysis by averaging the cross-sectional area along 12
Journal Pre-proof
e-
pr
oo
f
the whole vessel where PTX was delivered. The cross-sectional area of the vessel, plaque and lumen were calculated with OCT to estimate the AS for each group (Figure S3B). As shown in Figure 6B, the AS of the samples treated by MNDEB was smaller than both the DEB and control (positive) groups. While the difference between the MNDEB groups and positive control group was statistically significant, the difference between the MNDEB and DEB groups was not. Minimal lumen diameter (MLD), mean radial diameter (RD) and DS were measured from QCA data (Figure S3B). In Figure 6C, the MNDEB-treatment yielded a statistically significant decrease in DS compared to both the positive control and DEB-treated group. The DEB treatment did not produce a statistically significant difference in DS compared to the positive control. Stenosis in each group was quantified by measuring the area of the media, intima, and plaque. The intima-plaque ratio (I/P) was calculated as a metric of disease progression (Figure S3B); this ratio was used to normalize intimal formation with respect to the media layer. As indicated in Figure 6A&D, the thickness and area of intimal layer decreased with statistical significance as a result of treatment with both the DEB and MNDEB. The MNDEB-treated tissues also demonstrated a statistically significant higher patency than their DEB-treated counterparts (Figure 6D). Expressions of pro-inflammatory atherosclerosis factors including ICAM1, VCAM1 and VEGF were investigated as well (Figure S4A). Even though there is no statistical significance in the difference between MNDEB and DEB groups in the VCAM1 expression, the average VCAM1 area of MNDEB was lower than that of DEB. In addition, MNDEB samples showed statistically significant decrease of ICAM1 and VEGF expressions compared to DEB samples.
Jo u
rn
al
Pr
Inflammation were scored after the in vivo test for therapeutic efficacy by RAM11 staining as well (Figure 7A). As shown in Figure 7A&B, there was no statistical significance for safety profiles between the DEB and MNDEB in histologic images of atherosclerotic rabbit models. All scoring data including inflammation, injury and endothelization revealed no statistically significant difference in tissues treated with either MNDEB and DEB after treatment, while both groups treated by MNDEB and DEB have statistical significance compared to positive control group (diseased models) (Figure 7C and S3A). We further tested inflammatory markers (IL-1, IL-6, and TNF-) (Figure S4B). Briefly, the results showed the reduction of all the markers in case of MNDEBs, which is consistent with the findings from RAM 11 tests. The MNDEB treated samples showed statistically significant decrease of IL-1 and IL-6 expressions compared to the DEB treated ones. Moreover, while the DEB group has no statistical significance with the positive group, the MNDEB group has the statistically significant decrease of TNF- expression compare to the positive group. This indicates that MN insertion did not elicit a larger immune response compared to the balloon with usual surface especially in the diseased blood vessel as well. In addition, this result showed MN insertion does not affect the physical integrity of the diseased blood vessel and does not limit the endothelization.
13
Jo u
rn
al
Pr
e-
pr
oo
f
Journal Pre-proof
Figure 6. (A) Optical coherence tomography (OCT), quantitative coronary angiograp hy (QCA) and trichrome stained histopathological images of vessels treated with positive, negative, DEB and MNDEB. (EEL: external elastic layer, IEL: internal elastic layer, Scale bars = 500 μm) (B) Percentage of area stenosis (AS) of positive group (n = 3): 52.80 ± 4.93%, negative group (n=3): 9.38 ± 0.37%, DEB group (n=3): 44.03 ± 1.60%, and MNDEB group (n=3): 31.26 ± 0.82% (p = 0.001 by ANOVA). (C) Percentage of diameter stenosis (DS) of positive group: 41.40 ± 2.58%, negative group: 4.28 ± 1.16%, DEB group: 35.49 ± 0.65% and MNDEB group: 19.63 ± 6.77% (p = 0.001 by ANOVA). (D) Intima to plaque ratios of positive group: 62.26 ± 2.44%, negative group: 3.25 ± 1.27%, DEB group: 46.34 ± 8.42% and MNDEB group: 25.57 ± 3.18%. (p = 0.009 by ANOVA). All data were presented as the mean ± SEM. *p < 0.001, compared with positive group. **p < 0.05, compared with DEB group.
14
al
Pr
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
Figure 7. (A) Representative RAM11 stained histopathological images and (B) quantitative data of RAM11 area of in vivo result. (Scale bars = 500 μm) (C) Scoring data of inflammation, vessel injury and endothelization of in vivo result. All data were presented as the mean ± SEM. *p < 0.05, compared with positive group.
4. Conclusions In this study, we have developed the novel concept of MNDEB. It is the first device to use a MN array to perform endovascular drug delivery. The MN array was transferred and fixed to the inflated surface of a PTCA balloon through our process termed conformal transfer molding. Analysis of the mechanical behavior of the MNs demonstrated sufficient flexibility for each needle to yield to the external stimuli that may be encountered upon catheter insertion into the lesion area. In addition to flexibility, the needles had sufficient mechanical strength to penetrate the inner layer of the vessel. By doing so, the MNs on the MNDEB surface were able to make micro- incisions which facilitate improved drug delivery to the stromal area of the blood vessel. We demonstrate the superior drug delivery efficiency of the MNDEB through a comparative study with conventional DEB. The safety of endovascular MN insertion was confirmed in both normal and diseased vessels. Finally, we developed an atherosclerotic rabbit model to test the in vivo efficacy of the device. Animals treated with the MNDEB showed statistically significant reduction in atherosclerosis compared to both the untreated and conventional DEB-treated groups. The MNDEB device is an innovative drug delivery technology that shows great promise for improved treatment of vascular diseases by endovascular delivery of therapeutics. 15
Journal Pre-proof
Jo u
rn
al
Pr
e-
pr
oo
f
Acknowledgements This work was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry for Health & Welfares, Republic of Korea (HI08C2149), by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIT) (2015R1A5A1037668 and 2017R1A2B2003191), and by Mid-career Researcher Program through NRF grant funded by the MEST (2016R1A2B4010487). We specially thank to Genoss Co. Ltd for their generous donation of drug eluting balloons. We would also like to thank Peyton Tebon for his assistance in the revision of this article.
16
Journal Pre-proof
Jo u
rn
al
Pr
e-
pr
oo
f
References [1] A.R. Gruntzig, A. Senning, W.E. Siegenthaler, Nonoperative Dilatation of CoronaryArtery Stenosis - Percutaneous Transluminal Coronary Angioplasty, New England Journal of Medicine, 301 (1979) 61-68. [2] U. Sigwart, J. Puel, V. Mirkovitch, F. Joffre, L. Kappenberger, Intravascular Stents to Prevent Occlusion and Re-Stenosis after Transluminal Angioplasty, New England Journal of Medicine, 316 (1987) 701-706. [3] P.W. Serruys, M.J.B. Kutryk, A.T.L. Ong, Drug therapy - Coronary-artery stents, New England Journal of Medicine, 354 (2006) 483-495. [4] O.C. Marroquin, F. Selzer, S.R. Mulukutla, D.O. Williams, H.A. Vlachos, R.L. Wilensky, J.-F. Tanguay, E.M. Holper, J.D. Abbott, J.S. Lee, C. Smith, W.D. Anderson, S.F. Kelsey, K.E. Kip, A comparison of bare-metal and drug-eluting stents for off-label indications, New England Journal of Medicine, 358 (2008) 342-352. [5] M. Morice, P.W. Serruys, J.E. Sousa, J. Fajadet, E.B. Hayashi, M. Perin, A. Colombo, G. Schuler, P. Barragan, G. Guagliumi, F. Molnar, R. Falotico, R.S. Grp, A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization, New England Journal of Medicine, 346 (2002) 1773-1780. [6] D.R. Holmes Jr, R.E. Vlietstra, H.C. Smith, G.W. Vetrovec, K.M. Kent, M.J. Cowley, D.P. Faxon, A.R. Gruentzig, S.F. Kelsey, K.M. Detre, Restenosis after percutaneous transluminal coronary angioplasty (PTCA): a report from the PTCA Registry of the National Heart, Lung, and Blood Institute, The American journal of cardiology, 53 (1984) C77-C81. [7] C. Bauters, T. Meurice, M. Hamon, E. McFadden, J.-M. Lablanche, M.E. Bertrand, Mechanisms and prevention of restenosis: from experimental models to clinical practice, Cardiovascular Research, 31 (1996) 835-846. [8] P.W. Serruys, B.H. Strauss, K.J. Beatt, M.E. Bertrand, J. Puel, A.F. Rickards, B. Meier, J.J. Goy, P. Vogt, L. Kappenberger, Angiographic follow-up after placement of a selfexpanding coronary-artery stent, New England Journal of Medicine, 324 (1991) 13-17. [9] R. Hoffmann, G.S. Mintz, G.R. Dussaillant, J.J. Popma, A.D. Pichard, L.F. Satler, K.M. Kent, J. Griffin, M.B. Leon, Patterns and mechanisms of in-stent restenosis: a serial intravascular ultrasound study, Circulation, 94 (1996) 1247-1254. [10] M. Joner, A.V. Finn, A. Farb, E.K. Mont, F.D. Kolodgie, E. Ladich, R. Kutys, K. Skorija, H.K. Gold, R. Virmani, Pathology of drug-eluting stents in humans - Delayed healing and late thrombotic risk, Journal of the American College of Cardiology, 48 (2006) 193-202. [11] R. Waksman, R. Pakala, Drug-eluting balloon: the comeback kid?, Circulation: Cardiovascular Interventions, 2 (2009) 352-358. [12] B. Scheller, C. Hehrlein, W. Bocksch, W. Rutsch, D. Haghi, U. Dietz, M. Boehm, U. Speck, Treatment of coronary in-stent restenosis with a paclitaxel-coated balloon catheter, New England Journal of Medicine, 355 (2006) 2113-2124. [13] B. Scheller, C. Hehrlein, W. Bocksch, W. Rutsch, D. Haghi, U. Dietz, M. Boehm, U. Speck, Two year follow-up after treatment of coronary in-stent restenosis with a paclitaxelcoated balloon catheter, Clinical Research in Cardiology, 97 (2008) 773-781. [14] M. Unverdorben, C. Vallbracht, B. Cremers, H. Heuer, C. Hengstenberg, C. Maikowski, G.S. Werner, D. Antoni, F.X. Kleber, W. Bocksch, M. Leschke, H. Ackermann, M. Boxberger, U. Speck, R. Degenhardt, B. Scheller, Paclitaxel-Coated Balloon Catheter Versus Paclitaxel-Coated Stent for the Treatment of Coronary In-Stent Restenosis, Circulation, 119 (2009) 2986-2994. [15] G. Tepe, T. Zeller, T. Albrecht, S. Heller, U. Schwarzwaelder, J.-P. Beregi, C.D. Claussen, A. Oldenburg, B. Scheller, U. Speck, Local delivery of paclitaxel to inhibit restenosis during angioplasty of the leg, New England Journal of Medicine, 358 (2008) 689699. 17
Journal Pre-proof
Jo u
rn
al
Pr
e-
pr
oo
f
[16] M. Werk, S. Langner, B. Reinkensmeier, H.-F. Boettcher, G. Tepe, U. Dietz, N. Hosten, B. Hamm, U. Speck, J. Ricke, Inhibition of restenosis in femoropopliteal arteries - Paclitaxelcoated versus uncoated balloon: Femoral paclitaxel randomized pilot trial, Circulation, 118 (2008) 1358-1365. [17] A. Micari, A. Cioppa, G. Vadala, E. Stabile, F. Castriota, P. Pantaleo, C. Grattoni, A. Cremonesi, A. Marchese, P. Rubino, G. Biamino, A new paclitaxel-eluting balloon for angioplasty of femoropopliteal obstructions: acute and midterm results, Eurointervention, 7 (2011) K77-K82. [18] Y.P. Clever, D. Peters, J. Calisse, S. Bettink, M.-C. Berg, C. Sperling, M. Stoever, B. Cremers, B. Kelsch, M. Böhm, Novel Sirolimus–Coated Balloon Catheter: In Vivo Evaluation in a Porcine Coronary Model, Circulation: Cardiovascular Interventions, 9 (2016) e003543. [19] S. Habara, K. Mitsudo, K. Kadota, T. Goto, S. Fujii, H. Yamamoto, H. Katoh, N. Oka, Y. Fuku, S. Hosogi, A. Hirono, T. Maruo, H. Tanaka, Y. Shigemoto, D. Hasegawa, H. Tasaka, M. Kusunose, S. Otsuru, Y. Okamoto, N. Saito, Y. Tsujimoto, H. Eguchi, K. Miyake, M. Yoshino, Effectiveness of Paclitaxel-Eluting Balloon Catheter in Patients With SirolimusEluting Stent Restenosis, Jacc-Cardiovascular Interventions, 4 (2011) 149-154. [20] J.C. Fanggiday, P.R. Stella, S.H. Guyomi, P.A. Doevendans, Safety and efficacy of drugeluting balloons in percutaneous treatment of bifurcation lesions: The DEBIUT (Drug-Eluting Balloon in BIfurcaton UTrecht) registry, Catheterization and Cardiovascular Interventions, 71 (2008) 629-635. [21] F. Nijhoff, P.R. Stella, M.S. Troost, A. Belkacemi, H.M. Nathoe, M. Voskuil, M. Samim, P.A. Doevendans, P. Agostoni, Comparative assessment of the antirestenotic efficacy of two paclitaxel drug-eluting balloons with different coatings in the treatment of in-stent restenosis, Clinical Research in Cardiology, 105 (2016) 401-411. [22] K. Rosenfield, M.R. Jaff, C.J. White, K. Rocha-Singh, C. Mena-Hurtado, D.C. Metzger, M. Brodmann, E. Pilger, T. Zeller, P. Krishnan, R. Gammon, S. Mueller-Huelsbeck, M.R. Nehler, J.F. Benenati, D. Scheinert, I. Levant, Trial of a Paclitaxel-Coated Balloon for Femoropopliteal Artery Disease, New England Journal of Medicine, 373 (2015) 145-153. [23] P. Bondesson, B. Lagerqvist, S.K. James, G.K. Olivecrona, D. Venetsanos, J. Harnek, Comparison of two drug-eluting balloons: a report from the SCAAR registry, Eurointervention, 8 (2012) 444-449. [24] S. Sharma, N. Kukreja, C. Christopoulos, D.A. Gorog, Drug-eluting balloon: new tool in the box (vol 7, pg 381, 2010), Expert Review of Medical Devices, 7 (2010) 576-576. [25] B. Cortese, A. Bertoletti, Paclitaxel coated balloons for coronary artery interventions: A comprehensive review of preclinical and clinical data, International Journal of Cardiology, 161 (2012) 4-12. [26] S.M. Seedial, S. Ghosh, R.S. Saunders, P.A. Suwanabol, X. Shi, B. Liu, K.C. Kent, Local drug delivery to prevent restenosis, Journal of Vascular Surgery, 57 (2013) 1403-1414. [27] K. Lee, S.G. Lee, I. Jang, S.H. Park, D. Yang, I.H. Seo, S.-K. Bong, D.H. An, M.K. Lee, I.K. Jung, Y.H. Jang, J.S. Kim, W. Ryu, Linear Micro-patterned Drug Eluting Balloon (LMDEB) for Enhanced Endovascular Drug Delivery, Scientific Reports, 8 (2018). [28] C.D. Owens, W.J. Gasper, J.P. Walker, H.F. Alley, M.S. Conte, S.M. Grenon, Safety and feasibility of adjunctive dexamethasone infusion into the adventitia of the femoropopliteal artery following endovascular revascularization, Journal of Vascular Surgery, 59 (2014) 1016-1023. [29] A. Peppas, K. Seward, K. Van Wygerden, G. Conditt, A. Tellez, S. Rousselle, G. Kaluza, J. Granada, Prevention of Restenosis with Adventitial Delivery of Torisel via Mercator MedSystems Bullfrog Catheter in Femoral Arteries of Swine, Journal of the American College of Cardiology, 70 (2017) B323-B323. 18
Journal Pre-proof
Jo u
rn
al
Pr
e-
pr
oo
f
[30] J. Fromageau, J.-L. Gennisson, C. Schmitt, R.L. Maurice, R. Mongrain, G. Cloutier, Estimation of polyvinyl alcohol cryogel mechanical properties with four ultrasound elastography methods and comparison with gold standard testings, IEEE transactions on ultrasonics, ferroelectrics, and frequency control, 54 (2007) 498-509. [31] T.H. Kim, D.B. An, S.H. Oh, M.K. Kang, H.H. Song, J.H. Lee, Creating stiffness gradient polyvinyl alcohol hydrogel using a simple gradual freezing–thawing method to investigate stem cell differentiation behaviors, Biomaterials, 40 (2015) 51-60. [32] K.J. Lee, S.H. Park, J.Y. Lee, H.C. Joo, E.H. Jang, Y.-N. Youn, W. Ryu, Perivascular biodegradable microneedle cuff for reduction of neointima formation after vascular injury, Journal of controlled release, 192 (2014) 174-181. [33] J.W. Lee, J.-H. Park, M.R. Prausnitz, Dissolving microneedles for transdermal drug delivery, Biomaterials, 29 (2008) 2113-2124. [34] J. Lee, S.H. Park, I.H. Seo, K.J. Lee, W. Ryu, Rapid and repeatable fabrication of high A/R silk fibroin microneedles using thermally-drawn micromolds, European Journal of Pharmaceutics and Biopharmaceutics, 94 (2015) 11-19. [35] H.B. Song, K.J. Lee, I.H. Seo, J.Y. Lee, S.-M. Lee, J.H. Kim, J.H. Kim, W. Ryu, Impact insertion of transfer-molded microneedle for localized and minimally invasive ocular drug delivery, Journal of Controlled Release, 209 (2015) 272-279. [36] S.H. Park, D.H. Jo, C.S. Cho, K. Lee, J.H. Kim, S. Ryu, C. Joo, J.H. Kim, W. Ryu, Depthwise-controlled scleral insertion of microneedles for drug delivery to the back of the eye, European Journal of Pharmaceutics and Biopharmaceutics, 133 (2018) 31-41. [37] K. Lee, H.B. Song, W. Cho, J.H. Kim, J.H. Kim, W. Ryu, Intracorneal injection of a detachable hybrid microneedle for sustained drug delivery, Acta biomaterialia, 80 (2018) 4857. [38] J. Lee, D.-H. Kim, K.J. Lee, I.H. Seo, S.H. Park, E.H. Jang, Y. Park, Y.-N. Youn, W. Ryu, Transfer-molded wrappable microneedle meshes for perivascular drug delivery, Journal of Controlled Release, 268 (2017) 237-246. [39] R.N. Isnard, B.M. Pannier, S. Laurent, G.M. London, B. Diebold, M.E. Safar, Pulsatile diameter and elastic modulus of the aortic arch in essential hypertension: a noninvasive study, Journal of the American College of Cardiology, 13 (1989) 399-405. [40] R. Kornowski, M.K. Hong, F.O. Tio, O. Bramwell, H. Wu, M.B. Leon, In-stent restenosis: contributions of inflammatory responses and arterial injury to neointimal hyperplasia, Journal of the American College of Cardiology, 31 (1998) 224-230. [41] P.R. Stella, A. Belkacemi, R. Waksman, S. Stahnke, R. Torguson, R.P. von Strandmann, P. Agostoni, G. Sangiorgi, S. Silber, I. Valentine, The Valentines Trial: results of the first one week worldwide multicentre enrolment trial, evaluating the real world usage of the second generation DIOR paclitaxel drug-eluting balloon for in-stent restenosis treatment, Eurointervention, 7 (2011) 705-710. [42] P.P. Buszman, K. Milewski, A. Żurakowski, J. Pajak, M. Jelonek, P. Gasior, A. Peppas, A. Tellez, J.F. Granada, P.E. Buszman, Experimental evaluation of pharmacokinetic profile and biological effect of a novel paclitaxel microcrystalline balloon coating in the iliofemoral territory of swine, Catheterization and Cardiovascular Interventions, 83 (2014) 325-333. [43] S. Kaushik, A.H. Hord, D.D. Denson, D.V. McAllister, S. Smitra, M.G. Allen, M.R. Prausnitz, Lack of pain associated with microfabricated microneedles, Anesthesia & Analgesia, 92 (2001) 502-504. [44] J. Gupta, H.S. Gill, S.N. Andrews, M.R. Prausnitz, Kinetics of skin resealing after insertion of microneedles in human subjects, Journal of controlled release, 154 (2011) 148155. [45] D.-H. Kim, E. Jang, K. Lee, J. Lee, S. Park, I. Seo, S. Lee, W. Ryu, Y.-N. Youn, A biodegradable microneedle cuff for comparison of drug effects through perivascular delivery to balloon-injured arteries, Polymers, 9 (2017) 56. 19
Jo u
rn
al
Pr
e-
pr
oo
f
Journal Pre-proof
20