Drug Release and Biocompatibility of a Paclitaxel-Coated Balloon Prepared Using the Electrostatic Spray Method

Abstract

Paclitaxel-coated balloons (PCBs) have become effective treatment options for vascular disease, but long-term drug release and biocompatibility are influenced by the drug patterns. In this work, paclitaxel coatings were prepared via electrostatic spraying, and the effect of D-tartaric acid additives was investigated. Microstructures and surface morphology were studied using Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM), respectively. Drug release was measured in vitro, and biocompatibility was evaluated using the haemolysis rate, platelet adhesion and activation, protein adsorption, cell adhesion, and cell proliferation. Our results showed that a uniform crystalline paclitaxel drug coating was obtained, and that the pattern and release of paclitaxel was influenced by the content of D-tartrate. The contact angle of all coatings was less than that of nylon 12. The drug coatings prepared at a mass ratio of paclitaxel to D-tartaric acid of 2:1 had the highest drug release in a brief period of time. The haemolysis rate of the drug coating was less than 5%. Compared with the control samples, platelet adhesion and activation were significantly reduced, albumin adsorption was increased, and the adsorption of fibrinogen was reduced on the surface of the drug coating. Endothelial cells demonstrated good proliferation after three days of cell culture. Therefore, PCBs with specific patterns have good biocompatibility and drug release, with potential clinical applications in vascular disease.

1. Introduction

Cardiovascular disease (CVD) is the leading cause of death worldwide and has a high incidence. According to the World Health Organization, the number of people who will die from CVD will reach 22 million worldwide in 2030 [1]. Drug-eluting stents (DESs) are currently the most widely used drug-delivery strategies globally [2,3,4]. However, their use is often associated with problems, including thrombosis or in-stent restenosis. This is most likely due to the associated release of foreign objects which remain in the blood vessels for extended periods, often requiring the long-term use of anticoagulants. Furthermore, the biocompatibility of the material used affects the long-term treatment efficacy, and stent restenosis has become the main complication of stent implantation [5,6,7]. A restenosis rate of 5%–15% has been reported [8], highlighting that the long-term efficacy after stent interventional therapy requires further investigation [9,10]. Drug-coated balloons (DCBs) have emerged as a new means of treating in-stent restenosis and small vessel lesions. The surface of the balloon interacts with the vascular wall after the balloon is implanted and expanded in vivo. The drug is then released from the surface of the balloon, entering the vascular wall to treat stenotic lesions. Hence, it is important that DCBs release the drugs instantaneously upon inflation. Compared to vascular stents, DCBs do not release foreign objects into the blood vessels, thereby reducing the occurrence of complications [11]. Different methods and drug formulations can be used to prepare drug coatings to improve the controllability of drug release, such as those that are tightly bound to the surface by chemical bonds, enabling continuous drug release [12]. However, chemically bonding the drug molecules on the balloon’s surface usually requires a unique and complex method [13]. Some coating methods such as dip coating, electro-treatment coating [14,15], plasma treatment coating [16], and spray coating [17] are commonly used to prepare DCBs, but these cannot regulate drug release, and their biosafety and biocompatibility limit clinical applications.

PA12 is a commonly used balloon material owing to its good thermoplasticity, dimensional and chemical stability, and easy processing, among other characteristics [18]. Paclitaxel (PTX) is a complex secondary metabolite of the redbud genus [19] which can be employed in DCBs due to its high efficiency, durability, and restorative effects. Its molecular structure is depicted in Figure 1, and comprises a benzene ring, an amino functional group (–NH), and multiple hydroxyl functional groups (–OH) [20]. PTX inhibits the occurrence of mitosis by binding to free tubulin proteins, inducing the assembly of stable microtubule structures, and simultaneously preventing microtubule depolymerization. This mechanism disrupts the connection between centrosomes and microtubule organizing centres within the cell, effectively suppressing the proliferation of vascular endothelial and smooth muscle cells [21]. Notably, the use of PTX on the surface of cardiovascular materials mainly inhibits the excessive proliferation or migration of smooth muscle cells, thereby preventing the occurrence of vascular restenosis. The instantaneous release of PTX is closely related to its crystalline state or microscopic morphology. To ensure the effectiveness and transfer rate of drug release from DCBs, the drug coating on the surface of the balloon is crucial.

In this work, PTX-coated balloons (PCBs) with a needle-like pattern of PTX were prepared using the electrostatic spraying method, and the microscopic morphology of the PTX coatings was adjusted by varying the content of the additives. Further research was conducted on the drug release of PTX drug coatings with different amounts of D-tartaric acid additives. In addition, blood compatibility and biocompatibility of PCBs were investigated in vitro in order to establish the foundation for future in vivo research and clinical applications.

2. Materials and Methods

2.1. Preparation of PCBs

PCBs were prepared on a nylon 12 surface (PA12, Yuxin Plastic Materials Co., Ltd, Yuxin, China.) using the electrostatic spraying method. The material used was a thin 10 × 10 × 1 mm sheet. Several PCBs were designed using different contents of D-tartaric acid. In order to enhance the adhesion of the coating to the material’s surface, the PA12 surface was hydrophilically pretreated. The PA12 was ultrasonically cleaned in acetone and anhydrous ethanol for 10 minutes and immersed in a solution of PVP K30 (Chengdu Cologne Chemical Co., Ltd, Chengdu, China) at a mass concentration of 2% for 10 additional minutes. The PA12 was then removed and dried vertically in an oven. This process was repeated one additional time. The PA12 was subsequently placed in the receiver and connected to the negative ground of the high-voltage power supply (DW-P503-1ACDF, Tianjin Dongwen High Voltage Power Supply Co., Ltd, Tianjin, China), and the nozzle was connected to the positive terminal. The distance between the PA12 and the nozzle was 80 mm, and the spraying voltage was 10 kV. The PTX mixture was poured into a syringe and added to the nozzle at a uniform rate. After this process, all samples were dried in an oven at 45 °C. The parameters used and data are shown in

Table 1. Preparation parameters used to manufacture PCBs.

Sample No.	PTX Concentration (w/v)	PTX: D-Tartrate (w/w)	Solvents
a	4%	1:1	Acetonitrile(v): Deionized Water(v) = 10:1
b	4%	2:1	Acetonitrile(v): Deionized Water(v) = 10:1
c	4%	3:1	Acetonitrile(v): Deionized Water(v) = 10:1
d	4%	4:1	Acetonitrile(v): Deionized Water(v) = 10:1

. The preparation process is illustrated in Figure 2.

2.2. Coating Characterisation

The surface morphology and microstructure of the coatings were characterized using field emission scanning electron microscopy (SEM, JSM-6360, Electronics Co., Ltd, Shimadzu, Japan) and Fourier transform infrared spectroscopy (FT-IR, BRUKER V70, Bruker Co., Ltd, Bruker, Germany). To study the wettability of the coating’s surface, a liquid–solid wettability angle analyser (SDC-200, SINDIN, Dongguan, China) was used to test the hydrophobicity of the coating.

2.3. Drug Release In Vitro

The peaks of different concentrations of PTX standard solutions were tested using high-performance liquid chromatography (HPLC, Agilent 1260, Palo Alto, USA) at 227 nm, and the fitted curves were obtained. The PCBs were immersed in 5 mL of a mixture at a constant temperature (volume ratio of phosphate buffer solution (PBS) to methanol of 9:1) buffer at 37 °C. The leach liquor of various PCBs was collected after 2 h, 12 h, 1 d, 3 d, and 7 d. Drug release was analysed via the fitted curve.

2.4. Biocompatibility

2.4.1. Haemolysis Rate

A haemolysis assay was performed according to standard [22]. Blood was diluted by adding 16 mL of anticoagulant to 20 mL of 0.9% sodium chloride injection solution and mixing with gentle shaking. Absorbance was measured at 545 nm using a UV spectrophotometer. The haemolysis rate was calculated using the formula:

HR (%) = (A − A₁)/(A₂ − A₁) × 100%

(1)

where HR is the haemolysis rate (%), A is the absorbance of the sample, A₁ is the absorbance of the negative control, and A₂ is the absorbance of the positive control. Triplicate samples were tested for each group.

2.4.2. Platelet Attachment and Activation

One set of experimental blood samples was sterilized with UV light and centrifuged, and supernatants were collected. The samples were then incubated in platelet-rich plasma (PRP) in a constant temperature water bath at 37 °C for 2.5 h. The samples were removed, rinsed with PBS, fixed with 2% glutaraldehyde for 4 h, and then sequentially dehydrated with 50%, 70%, 90%, and 100% ethanol concentrations for 15 min each time. The number and morphology of platelet adhesions were observed using SEM.

PRP was obtained via centrifugation of fresh whole blood containing sodium citrate. The PRP was separated into three parallel sample surfaces, incubated at 37 °C for 2 h, and washed three times with a bovine serum albumin solution. Subsequently, mouse anti-human CD62P antibody (primary antibody) was added and incubated for 1 h at 37 °C, followed by horseradish peroxidase-labelled goat anti-mouse polyclonal antibody for 1 h at 37 °C. Developing reagents were added for 5–10 min, and then the colour development solution was transferred to a microtitration plate. Colour development was stopped by the addition of a sulfuric acid solution and placed in an enzyme standardizer. The concentration of platelet granule membrane protein 140 (GMP-140) was calculated using the optical density (OD) value.

2.4.3. Protein Adsorption

A protein quantification kit (BCA Assay) was used to measure the adsorption of two major proteins (albumin and fibrinogen) in plasma on to the surface of the material. A 1 mg/mL protein solution was first prepared in PBS solution, and then 3 mL was added to the surface of the experimental samples at 37 °C for 2 h. The coating was immersed in 2% sodium dodecyl sulphate (SDS) solution and stirred at 37 °C for 2 h to fully dissolve the adsorbed proteins on the surface into the SDS solution. The absorbance of the adsorbed proteins was tested using the corresponding reaction and colour development kits, and the protein concentrations were calculated using the standard curves. Triplicate samples were tested for each group.

2.4.4. Cell Adhesion and Proliferation

All samples were sterilized with UV light and placed in 24-well cell culture plates after the treatment was completed. Subsequently, 2 mL of endothelial cell suspensions at a concentration of 5 × 10⁴ cells/mL were inoculated onto the substrate and incubated at 37 °C for 1 d and 3 d, in a humid environment containing 5% CO₂. After a predetermined warming time, the samples were washed 3 times with PBS. Cells were stained sequentially with rhodamine and 4,6-diamidino-2-phenylindole (DAPI). Next, 200 μL of rhodamine (1:1000 dilution in PBS) was added to the surface of each sample, incubated for 30 min, and then rinsed with PBS 3 times. Finally, 200 μL DAPI (1:400 in PBS solution) was added to the sample surface for 4 min, and then rinsed with PBS 3 times. The stained samples were viewed using fluorescence microscopy.

Endothelial cells were cultured as described above, and after incubation with the samples for 1 d and 3 d respectively, 0.5 mL of CCK-8 solution (CCK-8: cell culture medium = 9:1) was added to each sample and incubated at 37 °C for 3.5 h. The absorbance of the medium was measured at 450 nm using an enzyme marker. Triplicate samples were measured, and the mean value of each sample was calculated. Triplicate samples were tested for each group.

3. Results and Discussion

3.1. Microstructure and Surface Morphology

The FT-IR spectra of different PCBs are shown in Figure 3. It can be observed that the IR peak at 3440 cm⁻¹ was caused by the –OH stretching vibration of PTX in the drug coating, and the peak at 2945 cm⁻¹ was caused by the –OH vibration in the –COOH. The peaks of the four PCBs were weakened when compared to PTX due to the addition of D-tartaric acid. The absorption peak near 1717 cm⁻¹ was caused by the C=O of PTX, the change at 1260 cm⁻¹ caused by the C–C vibration, and the vibrational peaks at 682 cm⁻¹ and 548 cm⁻¹ caused by the deformation vibration of the C–H of the ring structure. The drug coating showed almost no difference in its IR spectrum, indicating that the addition of D-tartaric acid did not affect the structure of PTX. Compared to PTX, the positions of the peaks were comparable, indicating that this method did not change the structure or properties of PTX.

The SEM images of various PCBs are shown in Figure 4. It can be clearly observed that the pattern of PTX is a needle-like crystal, which can be adjusted via the ratio of PTX and D-tartaric acid. The drug crystallization was not uniform when the ratio of drug to additive mass was 1:1 (Figure 4a), with evidence of adhesion and agglomeration of PTX observed, indicating that an excessive amount of D-tartaric acid may affect the quality of the coating. Notably, uniform crystals and good surfaces were obtained when the ratio of drug to additive mass was 2:1 (Figure 4b). However, as the ratio increased, the PTX crystals of and distribution uniformity were poor. This was most likely because there is only one amide group in the PTX molecule, which can serve as a protonation site, signifying that each molecule can only accept one proton. D-tartaric acid has two acidic protonation sites with different pKa values (approximately 3.00 and 4.35), indicating that its two successive dissociations were equivalent but within a pKa range of around 4.0. From the perspective of the acid–base equilibrium, each mole of D-tartaric acid should be capable of protonating 2 moles of PTX to maximize the utilization of its two protonation sites, thereby achieving optimal reaction efficiency.

3.2. Surface Performance

Cell adhesion, proliferation, and migration on the PCB’s surface are affected by the wettability of the coating. The hydrophilicity of medical materials can be characterized through contact angles, which are the basis for evaluating biocompatibility. The water contact angles of PA12 and the different coatings are shown in Figure 5. The highest water contact angle (98°) was found for PA12, while the drug coatings were considerably lower. Increasing the proportion of D-tartaric acid will reduce the water contact angle of the PCBs and enhance their hydrophilicity. D-tartaric acid is easily soluble in water, and its structure contains hydrophilic groups; hence, increasing the content of D-tartaric acid should enhance the hydrophilicity of drug coatings and increase the crystalline particle size and surface area.

3.3. Drug Release In Vitro

The drug release measures of the different PCBs are shown in Figure 6. The drug release of the four coatings was different, with the drug coating of a 2:1 mass ratio of PTX to D-tartaric acid having the largest cumulative release at 1st day. The drug coating with the least amount of D-tartaric acid had the greatest drug release at 3rd day. At 7th day, the cumulative drug release decreased as the D-tartaric acid content increased. This is most likely because the relative concentration of PTX is high at a low D-tartaric acid content, and therefore the drug release was greater. According to Figure 6B, an initial increase and subsequent decrease in drug release was observed with the increase in D-tartaric acid content in a brief period of time. In particular, the drug release rate was greatest at a 2:1 mass ratio of PTX to D-tartaric acid at 1st day. It is likely that the addition of D-tartaric acid increased the solubility of PTX, which resulted in a higher drug release rate in a short period of time. The crystallinity of sample b was the best, which affected the release of PTX. Therefore, the short- and long-term release properties of PTX can be controlled by adjusting the additive’s content. Electrospray technology not only enhances the uniformity of drug distribution, but also exhibits strong controllability. Additionally, it facilitates the production of minute drug particles or droplets, resulting in micrometre-sized drugs. This contributes to improved drug adsorption and permeability, thereby enhancing the drug’s efficacy in the target area. Finally, the use of this technique enables the application of trace amounts of drugs.

3.4. Haemolysis Rate

The haemolysis rate of PA12 and various PCBs are shown in Figure 7. The haemolysis rate of PA12 was 1.53%. The haemolysis rate of PA12 after preparation of the drug coating increased, as PTX is a diterpenoid alkaloid which affects red blood cells. The haemolysis rate also increased with the increase in the D-tartaric acid content. Nonetheless, the haemolysis rate on all PCB surfaces was less than 5%, which meets the requirements of biomedical materials according to standard [22].

3.5. Platelet Adhesion and Activation

The microscopic morphology of platelet adhesion on PA12 and different PCB coating surfaces is shown in Figure 8. The platelets on the PA12 surface showed more dendritic pseudopods and extensions, a small amount of spreading, and a slight agglomeration, indicating that the platelets were activated on this surface. The number of spreading platelets was reduced on the surface of PCBs. When the ratio of drug to D-tartaric acid was 1:1 and 2:1, there were a small number of discoid or round platelets and a small number of dendritic pseudopod platelets on the PCB surface, indicating that fewer platelets were activated. With the decrease in D-tartaric acid content, the shape of the platelets changed to dendritic pseudopod platelets when the ratio of PTX to D-tartaric acid was 3:1 or above, particularly for sample d, where platelets were more dendritic pseudopod-like and extended. No samples showed evidence of pseudopodia or platelet aggregation, indicating a low level of blood activation and anticoagulation.

GMP-140 is expressed on platelets and can promote adhesion and coagulation during blood vessel injuries, which in turn leads to thrombosis at the vessel site. Hence, GMP-140 can be used to assess the activation and coagulation of platelets in blood vessels.

The concentration of GMP-140 across the different samples is shown in Figure 9. It was observed that the GMP-140 content on PCBs was lower than that on the PA12 surface. This indicated that the activation of platelets on the PCB surfaces was lower than that on the PA12, and that the drug-coated surface had an inhibitory effect on the activation of platelets. This result is consistent with the SEM findings in Figure 8. However, GMP-140 decreased following the increase in D-tartaric acid, although this effect was not significant when the mass ratio of PTX to D-tartaric acid exceeded 2:1.

3.6. Protein Adsorption

Protein adsorption was also investigated using a protein quantification kit (BCA Assay). The adsorption of albumin decreases platelet adhesion and activation, thus inhibiting the coagulation of platelets on the surface of materials. The albumin adsorption on the surface of PA12 was 0.69 mg/mL (Figure 10A), which increased on the different PCB surfaces. The concentration of albumin in sample a was 1.40 mg/mL, which was double that of PA12. In addition, the concentration of albumin tended to decrease as the content of D-tartaric acid decreased. Albumin was more easily adsorbed on the surface of the hydrophilic material, and the hydrophilicity of PCBs was greater than that of the raw material surface. This result is consistent with the SEM and GMP-140 results of platelet activation, indicating that the coating had good blood compatibility.

The adsorption of fibrinogen (FIB) decreases the hemocompatibility of materials [22,23]. The adsorption concentration of FIB on the surface of PA12 was 1.10 mg/mL (Figure 10B), which decreased on the surface of different PCBs, but increased as the content of D-tartaric acid increased. The decrease in FIB adsorption was accompanied by an increase in the hydrophilicity of the PCBs, which was further influenced by the D-tartaric acid content [24]. The lowest concentration of FIB on the different PCB surfaces was at the ratio of 2:1 of drug to additive mass, which indicated that sample b was the most biocompatible sample under these preparation conditions.

3.7. Endothelial Cell Adhesion and Proliferation

Endothelial cell adhesion and proliferation can reflect effects between materials and cells of organisms, assessing biocompatibility. The fluorescent staining of endothelial cells adhered to PA12 and different PCB surfaces is shown in Figure 11. The number of adherent endothelial cells was low and the distribution was not uniform at 1 d due to the drug properties of PTX. Cell proliferation was observed at 3 d, most likely due to the decreased concentration of PTX on the surface of PCBs.

The results of cell activity at 1 d and 3 d of culturing endothelial cells on the surface of various samples are shown in Figure 12. The results showed that the OD value of CCK-8 was lower at 1 d, indicating that few cells adhered to the sample surface. The OD values of the different PCB surfaces did not differ from PA12. These values were significantly higher at 3 d, indicating that the endothelial cells on all surface proliferated well. In addition, the OD values were low when the content of D-tartaric acid addition was low, and the OD value increased and then decreased as the D-tartaric acid content increased. This indicated that excessive D-tartaric acid may have an inhibitory effect on the proliferation of endothelial cells. The OD value of CCK-8 was highest when the mass ratio of PTX and D-tartaric acid was 3:1, which indicated that the cytocompatibility of the PCBs under this condition was optimal.

4. Conclusions

PCBs were successfully prepared using the electrostatic spraying method, and their surface morphology, wettability, drug release, and biocompatibility were influenced by the content of D-tartaric acid. The pattern and crystallinity of PTX was mainly determined by the addition of D-tartaric acid, and the uniformity and denseness of the coating were regulated via the content of D-tartaric acid. An increase in the content of D-tartaric lead to an increase in the hydrophilicity of all PCB surfaces. The cumulative drug release increased with the increase in D-tartaric acid content. The maximum drug release in a brief period of time was at the ratio of 2:1 of PTX to tartaric acid, and the cumulative drug release increased with the increase in D-tartaric acid content. Platelet adhesion and activation were significantly reduced on the surface of PCBs, which also showed increased adsorption of albumin and decreased adsorption of FIB. Good proliferation of endothelial cells was detected at 3 d. These results show that D-tartaric acid can regulate PTX release, which has potential applications in the field of CVD.