Design and testing of hydrophobic nano/micro particles for drug-eluting stent coating

In this study, we designed a novel drug-eluting coating for vascular implants consisting of a core coating of the anti-proliferative drug docetaxel (DTX) and a shell coating of the platelet glycoprotein IIb/IIIa receptor monoclonal antibody SZ-21. The core/shell structure was sprayed onto the surface of 316L stainless steel stents using a coaxial electrospray process with the aim of creating a coating that exhibited a differential release of the two drugs. The prepared stents displayed a uniform coating consisting of nano/micro particles. In vitro drug release experiments were performed, and we demonstrated that a biphasic mathematical model was capable of capturing the data, indicating that the release of the two drugs conformed to a diffusion-controlled release system. We demonstrated that our coating was capable of inhibiting the adhesion and activation of platelets, as well as the proliferation and migration of smooth muscle cells (SMCs), indicating its good biocompatibility and anti-proliferation qualities. In an in vivo porcine coronary artery model, the SZ-21/DTX drug-loaded hydrophobic core/hydrophilic shell particle coating stents were observed to promote re-endothelialization and inhibit neointimal hyperplasia. This core/shell particle-coated stent may serve as part of a new strategy for the differential release of different functional drugs to sequentially target thrombosis and in-stent restenosis during the vascular repair process and ensure rapid re-endothelialization in the field of cardiovascular disease.

Introduction

Cardiovascular diseases are among the most common diseases that give rise to disability and mortality. Currently, the use of coronary stents is commonplace in the treatment of such diseases¹. Extensive data from experimental and clinical studies confirm that although drug-eluting stents (DESs) reduce the rate of restenosis compared with bare-metal stents (BMSs), there remains an increased risk of late stent thrombosis with DESs^2,3,4,5,6. Percutaneous coronary intervention (PCI), especially involving stents, usually results in injury to the endothelium, consequently inducing the activation and gathering of blood platelets to form thrombosis, and the stent may become a site for the adhesion of platelets before they are completely covered by neointima^4,7. Other long-term clinical studies have reported that DESs loaded with anti-proliferative agents suppressed the growth of neointima, delayed the re-endothelialization of stents, lengthened the time for activation of platelets, and increased the risk of late thrombosis⁸. Hence, the design of DESs in the future must consider these critical issues.

The anti-proliferative drugs rapamycin (also known as sirolimus) and paclitaxel were the first-generation drugs coated with DES approved by the US FDA. Subsequently, as newer-generation stents have emerged, sirolimus analogues such as zotarolimus and everolimus have been utilized. Despite these advancements, today’s DES have not completely eradicated restenosis and still lead to a higher incidence of late stent thrombosis. Drug docetaxel (DTX) is a paclitaxel analogue with enhanced anti-proliferative ability and water solubility and has been shown to be a good candidate drug for the prevention of restenosis^9,10. Thrombosis remains a concern after stent implantation, whether at the early or very late stage. The exposure of damaged endothelial cells to the blood activates platelet adhesion and aggregation. The binding of the monoclonal antibody (mAb) to the platelet GP IIb/IIIa receptor¹¹ has potent anti-platelet and anti-thrombotic characteristics that have been shown to reduce thrombosis-related major complications after coronary angioplasty^12,13,14.

Polymer particles can be employed to deliver medication in a rate-controlled and sometimes targeted manner by drug leaching from the polymer or by degradation of the polymer matrix¹⁵. The advancement and convergence of particle drug delivery systems have recently been explored to optimize stent coatings. For example, drugs exhibiting different therapeutic effects were encapsulated in bi-layered PLGA particles containing vascular endothelial growth factor (VEGF) plasmid and paclitaxel; stents with this particle coating promoted early endothelium healing and inhibited smooth muscle cell (SMC) proliferation^16,17,18. Local particle-based drug delivery was hypothesized to facilitate high regional concentrations of therapeutic agents with prolonged retention at low doses, leading to reduced systemic toxicity^19,20. For drug loading and release of particles, there already exist several approaches²¹. Traditional particle preparation methods cannot adequately control the burst release of drugs²². Electrospraying, on the other hand, provides a superior alternative to producing stent coatings with different nano/micro particle sizes and assembly methods. Thian et al.²³ used electrospraying deposition to produce a uniform coating consisting of hydroxyapatite nanocrystals on a titanium substrate, which triggered an early apatite precipitation process in cellular-simulated body fluid. Almería et al.²⁴ demonstrated a multiplexed electrospraying process for the single-step synthesis of stabilized polymer particles for drug delivery, but the release rate of the drugs was found to be rapid, reaching approximately 70% within the first 4 h. To the best of our knowledge, only a limited number of studies have reported the application of electrospraying for the fabrication of vascular DES coatings. In particular, Dong et al. fabricated and controlled the release of the electrosprayed ReoPro-loaded metal vascular stent. Hydrophilic ReoPro release was controlled in the hydrophobic PLGA-based release system and was effective in suppressing platelet adhesion and SMC overgrowth for vascular stents²⁵. Zamani’s research showed that multi-drug coatings could easily be fabricated using electrospraying. The electrosprayed Montelukast/poly(lactic-co-glycolic acid) particle-based coating is a potential new therapeutic approach toward the prevention of in-stent restenosis²⁶.

Ideal vascular implant coatings should exhibit a number of properties. These properties include biocompatibility, anti-thrombogenicity, the ability to interface with anti-proliferative drugs to reduce platelet activation level and time, restenosis prevention and a favorable surface topography for re-endothelialization^27,28. To achieve dual-functionalized vascular stents with dual drugs, researchers have chosen to combine various coating methods and multiple-step methods for coating preparation^29,30. To the best of our knowledge, there is no published work that considers multi-hydrophilic/hydrophobic drug coating preparation for coronary stents with a single step. In our previous research, we reported the detection of sequential drug release of polymer-controlled core/shell nanoparticles prepared by coaxial electrospraying³¹. Here, we designed a novel DES coating consisting of particles with a core of the anti-proliferative drug DTX and a shell of SZ-21, which was the platelet glycoprotein IIb/IIIa receptor monoclonal antibody^32,33. The particles were produced and deposited on the stent by coaxial electrospraying (Fig. 1).

Fig. 1: Schematic diagrams of material design and function hypothesis.

The PLGA solution with DTX and chitosan solution with SZ-21 were affused in two syringes separately and placedon the coaxial electrospraying device. Injecting the solutions at a constant rate, the coaxial nozzle was given high voltage, and the dissolvents of the two solutions were volatilized to form dual core/shell drug particles. The dual drug particles were deposited on the surface of the stent to form the DPS. The differential release of SZ-21/DTX was assumed, and the mechanism of their function was shown. The differential releasing pattern allows rapid re-endothelialization in the early days and the later inhibition of smooth muscle cell proliferation

Materials and methods

Materials

Three hundred sixteen liters (316L) stainless steel (SS) stents (3.0 × 17 mm) and 316 L SS sheets (Ø 10 × 1.5 mm) were purchased from Beijing AmsinoMed Medical Device Co., Ltd., China. SZ-21 was supplied by Prof. Changgeng Ruan in the Jiangsu Institute of Hematology (First Affiliated Hospital of Suzhou University, Suzhou, China). Human umbilical vein endothelial cells (HUVECs) and Human umbilical arterial smooth muscle cells (HUASMCs) were a generous gift from Dr. Lushan Liu (Nan Hua University, China). DTX (Xieli Pharmaceutical Co., Ltd, Sichuan, China), chitosan (CS) M_w = 600,000 (Boxin Biotech Co., Ltd, Tianjin, China) and poly [(50% lactic acid) (50% glycolic acid)] (PLGA) M_w = 20,000 (Sigma Inc., St. Louis, USA) were also used in these studies.

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Multilayer coating with hydrophobic core/hydrophilic shell nano/micro particles by electrospraying

Coating experiments were carried out on 316 L SS stents (3.0 × 17 mm) and 316 L SS sheets (Ø 10 × 1.5 mm) utilizing coaxial electrospraying. The design and experimental setup of the coating procedure are shown in Fig. 1.
Dichloromethane and ethylic acid solution were used as the solvents for PLGA and CS, respectively. In the electrospray coating process, 10 mL syringes containing DTX-PLGA solution (DTX concentration at 1 mg/mL) or SZ-21-CS solution (SZ-21 concentration at 2 mg/mL) were connected with a coaxial nozzle. The reception distance between the tip of the needle to the coating surface was maintained at 5–15 cm; the voltage and inner/outer flow rate were changed to obtain a uniform coating (Table S1). The SZ-21/DTX drug-loaded hydrophobic core/hydrophilic shell particle coating (DPC) and no drug core/shell particle coating (BPC) were prepared according to the following selected parameters: 0.2 mL/h (inner) and 0.6 mL/h (outer), a PLGA concentration of 10 mg/mL, a CS concentration of 2.5 mg/mL, a reception distance of 5 cm, and a voltage of 14 kV. The morphology, core/shell structure, and particle size distribution were detected through a fluorescence microscope (IX81, Olympus, Japan), scanning electron microscope (SEM; Vega-LMH, Tescan, Czech), confocal microscope (SP8, Leica, German) and transmission electron microscope (TEM, JEM-2100F, JEOL, Japan).

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Morphometric analysis of the SZ-21/DTX drug-loaded hydrophobic core/hydrophilic shell particle coating stent (DPS)

Fluorescence characterization of DPS

DPS was prepared by coaxial electrospraying. To identify the hydrophobic core/hydrophilic shell particle nature of the coating, 1 mg/mL fluorescein isothiocyanate (FITC) was added to the shell solution and 1 mg/mL rhodamine B was added to the core solution, allowing for observation by fluorescence microscope (IX81, Olympus, Japan).

SEM and EDS test of DPS

After coating, DPS was expanded three times for 30 s each time by balloon (2.75 mm × 18 mm MAVERJCK2, Boston scientific) at 10 atm in vitro; the changes in morphology and element contents of BMS, DPS and after expansion were all determined using a scanning electron microscope (SEM; Vega-LMH, Tescan, Czech) and energy dispersive spectrometer (EDS; INCA x-sight 7557, Oxford Instruments, UK), respectively.

Atomic force microscopy and water contact angle analysis of DPS

The morphology changes of stents with different coatings were characterized using an atomic force microscope (AFM; SPI3800N, SEIKO, Japan). The hydrophilicity alteration of different coating surfaces, as determined by the distilled water contact angle, was measured with a contact angle tester (DSA30, Krüss, Germany) at room temperature. Three different sites were selected randomly for each group.

Fourier transform infrared spectroscopy (FTIR) detection of drugs and polymers for DPS

FTIR measurement was performed to analyze the chemical structure of the coaxial sprayed coatings before and after coaxial electrospraying. Using the squash method, KBr (100–200 mg) and solid samples (1–2 mg) were ground into micron powder and pressed into sheets (Ø 13 × 1.0 mm) for analysis.

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Drug release analysis in vitro

Detection of drug release in vitro

For drug release analysis, DPSs were transferred in 1 mL phosphate-buffered saline (PBS) in a constant temperature shaking incubator at 60 rpm/min and 37 °C. The concentrations of released drugs were monitored at pre-determined time points, and the fresh PBS was replaced each time. Moreover, the stent morphology after 0, 7, and 28 days was characterized by SEM. The SZ-21 release rate in vitro was tested using mice immunoglobulin IgG Elisa kits. DTX was assayed by HPLC (Agilent 1100, USA) with a C18 250 mm × 4.6 mm column. The mobile phase was acetonitrile: water (65:35). The sample was eluted at 1 mL/min at room temperature, and the absorbance was measured at 230 nm.

Mathematical modeling of drug release in vitro

The large number of core/shell particles on the coating made it impractical to mathematically model each individual particle. Instead, we adopted a continuum approach and approximated the core/shell coating layer as a biphasic material, providing two independent routes of transport for each drug. These two routes could result from surface preparation methods and may correspond to the drug phases being either fully embedded within the coating layer (slow release) or connected to the surface by pores or other defects (fast release)³⁴.
Each route of transport was associated with a different effective transport speed characterized by a rate constant k_i, i = 1,2. Assuming that the drug was initially uniformly distributed within each respective route, the strut was impermeable to drug transport, the release medium acted as an infinite sink and the drugs were dilute, and then considering drug transport within each route as a diffusion-dominated process, we derived an expression for the mass of each drug within the coating as a function of time, M(t):

M(t)=∑n=1∞A(n){re−K1(n)t+(1−r)e−K2(n)t}

(1)

where A(n)=8M0π2(2n−1)2,K1(n)=(2n−1)2π2k14,K2(n)=(2n−1)2π2k24
, and r is the initial fraction of the total drug loading (M₀) within each route.
Using the knowledge of the total drug loading for each drug and Eq. (1), we calculated the cumulative fraction of each drug that had been released as a function of time.

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Blood compatibility evaluation

Blood was obtained from healthy volunteers in accordance with the protocol approved by the Blood Donation Law of the People’s Republic of China. To isolate fresh human platelet-rich plasma (PRP), the blood was centrifuged at 1200 rpm for 10 min, and the supernatant was collected. All sheets were incubated in 0.5 mL of PRP for 30 min at 37 °C, rinsed with PBS, fixed with 2.5% glutaraldehyde for 12 h at 4 °C and dehydrated for SEM³⁵. The expression of platelet granule membrane glycoprotein CD62P was detected to evaluate the anti-platelet activation of the coating. The deformability of adherent platelets (type I-V) on coatings was analyzed, and the morphological indices were calculated via the following formula:³⁶

morphologicalindex=(No.typeI×1+No.typeII×2+No.typeIII×3+No.typeIV×4+No.typeV×5)totalnumberofadherentplatelets

(2)

The hemolysis ratio of  DPC was tested following the Chinese national standard (Biological Evaluation of Medical Devices—Part 4: Selection of Tests for Interactions with Blood, GB/T 16886.4-2003). The activated partial thrombin time (APTT) and prothrombin time (PT) were applied to evaluate the anti-thrombogenicity of stents in vitro³⁷.

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The proliferation and migration of HUVEC and HUASMC on coatings

Cell culture

HUVECs and HUASMCs were cultured in RPMI 1640 and DMEM/F12 media (HyClone, USA), respectively, supplemented with 10% fetal bovine serum (FBS; Cambrex Corp, USA) and 1% P/S (Gibco Industries Inc, USA) at 37 °C under 5% CO₂.

HUVECs seeding and cell viability assay

A rotation culture device after autoclave sterilization was used to seed cells on the stent surface. BMS, BPS, and DPS were transferred to the rotation culture tubes after undergoing ultraviolet irradiation sterilization. Then, HUVECs were seeded in the tubes at 5 × 10⁴ cells/tube density with 10 rpm/min for 12 h at 37 °C. Finally, the stents were gently taken out in an aseptic condition and cultured in 12-well plates at 37 °C under 5% CO₂³⁸.
After a 5-day culture, the stents were immobilized with 2.5% glutaraldehyde and dehydrated for morphological observation by SEM. The proliferation rates of cells on the stent surfaces were investigated with Cell Titer 96^® Aqueous One Solution Cell Proliferation Assay after 1, 3, and 5 days. The relative growth of rate (RGR) was calculated by this formula:

RGR=(ODtODnc)×100%

(3)

(where OD_t represents the experimental group, and OD_nc represents the negative control group).
After co-culturing for 1, 3, and 5 days, the culture supernatant was collected by centrifuging at 3000 rpm for 10 min. The nitric oxide (NO) release levels and total antioxidant capacity (T-AOC) of HUVECs were tested.

The migration of HUVECs and HUASMCs on coatings

HUVECs and HUASMCs were cultured separately. Both HUVEC and HUASMC migration assays were carried out with the classical scarification method. Different coatings were sprayed on 316 L SS sheets by coaxial electrospraying. Then, the sheets of three groups (316 L SS, BPC, DPC) were placed in 24-well plates after undergoing ultraviolet irradiation sterilization for 12 h. One milliliter of cell suspension (1 × 10⁵ cells/ml) was seeded and grown on the coating surfaces. After being starved in media with 1% serum for 4 h, confluent monolayers of HUVECs were wounded by scraping a pipet tip (200 μL) across the monolayer to produce initial wounds with a constant diameter; they were then washed three times with PBS. The migration distance of the scratch was measured at 0, 6, 12, and 24 h using ImageJ (NIH, US). The migration lengths were counted following the methods in our previous research³⁸.

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Stent implantation in vivo

Two groups of drug-coated stents were prepared using coaxial electrospraying: DPSs containing 50 ± 2.36 μg DTX (DPS-L) and DPSs containing 100 ± 3.88 μg DTX (DPS-H). The DTX content on the two stent groups was detected in the drug release after complete elution. Three control groups, BMS, BPS, and SES (DES with Sirolimus), were also prepared.
Twenty male Bama minipigs (30–40 kg) fed a normal diet were used in this study in accordance with the guidelines of the Chinese Animal Care and Use Committee standards. Three days before surgery, 150 mg aspirin and 300 mg clopidogrel were administered orally with feed. Before surgery, an intramuscular injection of xylazine hydrochloride (0.15 mL/kg, 30 mg/1.5 mL) was given to each minipig for muscle relaxation. The venous channel was set up at the edge of the ear vein, and blood was taken simultaneously for a routine blood examination and serum biochemical examination, followed by ear intravenous administration of propofol at 8 ml/h for anesthesia. Minipigs were fixed to the operating table in a lateral position, and a 6F introducer sheath was positioned in the right femoral artery under surgical exposure. Heparin sodium (200 IU/kg) was injected via the sheath. All catheters were subsequently introduced through the sheath and advanced to the left anterior descending (LAD)/left circumflex (LCX)/right coronary artery (RCA) through a guide wire. Baseline angiograms of each artery were obtained for each pig through quantitative coronary analysis (QCA). Prepared stents were deployed in the suitable vessel region during angiographic guidance. It was ensured that no branches were present in the stented region. Stents were dilated at 10 atm. At the level of the balloon dilatation, the diameter of the aorta was approximately 2.5 mm and the expansion ratio of the aorta was 1:1.1. Two different stents were deployed per pig, and pre-deployment and post-deployment digital subtraction angiography was recorded. Gentamicin sulfate (5 mg/kg) was given as intramuscular injection in the next three days, and 150 mg aspirin and 300 mg clopidogrel were administered orally with feed every day. Animals were fed a normal diet after the intervention.

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Acquisition and observation of stent implantation samples

Pigs were euthanized at 1, 3, and 6 months (n = 3 at each time point for each stent group). At each time point, aortic specimens were excised transversely into two parts. One part was fixed in 4% paraformaldehyde and embedded in light-cured resin with hematoxylin and eosin stain (HE) for pathology organization analysis. Another part was fixed in 0.25% glutaraldehyde for 48 h, and the morphology of the intima surface was observed by SEM. The proximal part and distal part of each stented vessel, myocardium, liver, spleen, lung, and kidney of pigs were fixed and sliced to check the safety of stents. Histomorphometric analysis was conducted on each section, including the vessel area (mm²), internal elastic lamina (IEL) area (mm²), lumen area (mm²), neointima area (mm²), and the neointima thickness (mm) by Image Tool. The percentage of restenosis was calculated using the following equation:

Restenosisrate(%)=1−lumenareainternalelasticlaminaarea×100%

(4)

Venous blood of pigs was collected for routine blood examination and serum biochemical examination.

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Statistical analysis

At least three independent experiments were performed for the tests described above. Statistical analyses were conducted with GraphPad Prism 6. The differences between experimental groups were considered statistically significant when p < 0.05. Unless indicated, the values are expressed as the mean ± SD.

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Authors:

• Ruolin Du,
• Yazhou Wang,
• Yuhua Huang,
• Yinping Zhao,
• Dechuan Zhang,
• Dingyuan Du,
• Yuan Zhang,
• Zhenggong Li,
• Sean McGinty,
• Giuseppe Pontrelli,
• Tieying Yin &
• Guixue Wang

Electronic supplementary material

1. Design and Testing of Hydrophobic core/ hydrophilic shell nano/micro particles for drug-eluting stent coating

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