High-surface-area porous coatings for implantable biomedical devices represent an intriguing solution to confer the device additional functionalities with respect to the uncoated implant [1
]. In this regard, drug-releasing devices featuring biocompatibility, biostability, antibacterial resistance properties, efficient drug loading and delivery should be pursued. For such purposes, the most commonly used materials are polymers [2
]; new generation carbon-based materials, such as graphene and graphene oxide [3
]; and other inorganic porous materials like anodized alumina [4
], titania nanotubes [5
], and porous silicon coatings [6
]. However, most of these are limited by their unpredictable rather than poor degradation behaviors, also resulting in toxic reaction products in some cases, thereby inducing undesirable inflammatory reactions in the body.
Nowadays, one of the main limitation affecting ureteral stents’ operation is the formation of progressive encrustations due to inorganic salt deposition and bacteria biofilm formation, resulting in the total occlusion of the stent lumen in the worst case, especially for prolonged indwelling times. Therefore, new practical solutions are strongly required to face the above-mentioned critical issues, that is, to fight the infections rising in patients because of bacteria biofilm formation and to guarantee a suitable urine drainage, finally avoiding the stent exchange at regular intervals. Within this scope, some solutions have been developed so far, and which have mostly involved the surface modification of the stent by the deposition of functional coatings such as antimicrobial silver [7
], hydrophilic coating with hydrogel (like Hydromer®
], heparin [9
], plasma-deposited diamond-like amorphous carbon coatings [10
], and many more. With these solutions, both the bacterial adhesion and the formation of inorganic encrustation have been limited to some extent. To further improve and intelligently render the ureteral stent performances, however, drug eluting capabilities [11
] and the complete biodegradation [14
] of the stent materials would be required. Presently, only some efforts in this direction are in place, with some satisfactory results in animal models using polylactic acid (PLA) polymers [15
]. However, the incomplete dissolution of the stent, leading to the permanence in the ureter to small polymer fragments [16
], causing an obstruction to the urine flow that is then impossible to be further removed without surgical intervention. Concerning drug eluting stents, some antibiotics, like triclosan, have not received approval from the Food and Drug Administration (FDA) because of their potential for developing antibiotic resistance [17
]. Thus, in this prospect, the use of intrinsically antimicrobial materials (i.e., nanoantibiotics) [18
] with a nanostructured surface able also to host therapeutic molecules will be of great interest.
Zinc oxide (ZnO) is attracting increasing interest for a wide plethora of application fields, including the biomedical one [20
], in particular as an antibacterial agent [22
]. ZnO is a wide band-gap metal oxide n-type semiconductor, showing a generous surface chemistry, interesting optical and piezoelectric properties, and promising photocatalytic activities at the same time. It has been successfully explored for new-generation energy harvesting systems [23
], sensors [24
], and photocatalytic systems [25
]. Moreover, ZnO in the bulk form has already been considered as safe and has been approved by the FDA [26
]. More interestingly, various ZnO morphologies and shapes showing high-surface areas and porous structures coupled with a generous surface chemistry may be easily prepared and functionalized [27
]. Some examples include porous thin films [28
], nanowires [29
], nanocrystals [30
], and flower-like structures [31
], which are easily obtainable by following numerous dry and wet synthetic approaches, like sol–gel strategies, hydrothermal routes, and vapor-phase deposition methods.
At first, the investigation of ZnO nanomaterials for biomedical applications was highly focused on their use in the fabrication of biosensing units [32
] able to detect various bio moieties, like proteins, glucose, and acid uric. More recently, ZnO nanomaterials have been successfully explored for tissue engineering [33
], drug-delivery [34
], and anticancer therapeutics [35
] as well. The active promotion of cell growth and proliferation, together with its proangiogenic, osteogenic, and antibacterial properties, have been reported [36
], and the efficacy of ZnO in promoting wound healing and the formation of new bone tissue has been successfully demonstrated. High-surface area ZnO morphologies have been investigated for drug delivery applications as well [34
]. For this purpose, the high sensitivity of ZnO against pH variations was successfully exploited and for different ZnO nanostructures, which exhibited pH-triggered release properties for various drug molecules. Moreover, ZnO can be successfully used in photodynamic therapy, thanks to its reactive oxygen species (ROS) generation properties under UV irradiation in aqueous media [35
]. Finally, the combination of a broad visible emission spectrum, strong luminescence, and the ability to generate ROS and work as a drug delivery system, also make ZnO nanostructures promising candidates for theranostic platforms with both imaging and therapeutically-active properties.
Hydrophilic 2-hydroxyethyl methacrylate polymers (pHEMA) are widely used in biomedical applications because of their biodegradability and biocompatibility [37
]. Moreover, tunable swelling behaviors can be easily obtained with the addition of specific monomers like acrylic acid [38
]. Several works demonstrated the successful use of pHEMA as drug delivery systems [40
] in ophthalmology [41
] and in plastic surgery.
In this work, the combination of the above-mentioned properties of porous ZnO, that is, its drug delivery, biodegradation, and prospective anti-microbial effects, were combined with a soft biodegradable polymeric matrix intended as a smart coating for ureteral stents. In particular, mesoporous ZnO coatings were obtained by a facile two-step synthetic approach, combining the sputtering technique and thermal oxidation. The corresponding molecule loading and release properties were evaluated by considering calcein (i.e., a fluorescent dye) as a model drug molecule. The as-prepared ZnO films exhibited interesting molecule loading capacities because of their high surface area and generous surface chemistry, but they also displayed fast and uncontrolled release kinetics. To overcome this limitation, biocompatible 2-hydroxyethyl methacrylate (pHEMA) and p(HEMA-co-acrylic acid (AA)) polymer coatings were deposited by vacuum infiltration and drop-casting techniques, atop of the calcein-loaded mesoporous ZnO matrix. The release behavior of the resulting ZnO/pHEMA and ZnO/p(HEMA-co-AA) bilayer structures was evaluated in vitro, and the effect of switching the pH conditions from neutral to slightly acidic on the release properties was evaluated.
2. Materials and Methods
2.1. Preparation of Porous ZnO Thin Films
Porous ZnO thin films were prepared by following a two-step synthetic approach [34
], involving the deposition of metallic Zn films by sputtering, and a thermal oxidation process. In the first step, porous Zn layers were deposited at room temperature on silicon (Si; 100-oriented; p-type) substrates (~1 cm2
area) by radio-frequency (RF, 13.56 MHz) magnetron sputtering (from Elettrorava, Venaria, Italy; see Figure S1
of Supporting Information (SI)
for further details on sputtering machine geometry). Si was selected as a reference substrate material because of its smooth and flat surface, and the absence of any porosity, thus avoiding any artifact during the loading and release experiments. Moreover, it facilitates the preparation of samples for cross-section imaging, because it can be easily cut along a specific crystallographic direction. Zn depositions were carried out starting from a four-inch diameter metallic Zn target, in a pure Ar atmosphere, with a fixed deposition pressure of 5 × 10−3
Torr, and a RF power density of 0.66 W·cm−2
. The overall deposition time was set to 4 h and the final average thickness of the Zn layer was 10 μm. After the deposition, the Zn/Si samples were thermally oxidized in a muffle furnace (L-401K2RN from Nabertherm™, Lilienthal, Germany) at 380 °C (ramp rate 150 °C/h), in air for 2 h. Before starting the Zn deposition, the Si substrates were properly cleaned in an ultrasonic bath of acetone and ethanol (10 min for each washing cycle), and were dried under a nitrogen flow (99.999% purity).
2.2. Preparation of PolyHEMA (pHEMA) and Poly(HEMA-co-AA)
2-hydroxyethyl methacrylate (HEMA, 99%), acrylic acid (AA), and 2,2′-azobis(2-methylpropionitrile) (AIBN, 98%) were obtained from Sigma-Aldrich (Darmstadt, Germany). Ethanol and methanol were from POCh (Gliwice, Poland). The monomers (HEMA and AA) were purified from inhibitors using vacuum distillation, and were stored in a refrigerator until use. The other reagents were used as received, without further purification. The synthesis of the homopolymer (pHEMA) and copolymer p(HEMA-co-AA) was performed with the use of radical polymerization in the toluene solution. The appropriate amounts of monomer HEMA or mixture of HEMA and AA (2.75:1 molar mixture) were mixed with the initiator, AIBN (2 wt %). The polymerization reaction was carried out under a nitrogen atmosphere at 70 °C for 8 h. After the completion of the polymerization, the obtained polymers were filtered and dried under a vacuum. The weight-average molar mass (Mw) and molar-mass dispersity (PDI), determined by the gel permeation chromatography (GPC) method were as follows: 12,500 Da and 1.41 for pHEMA, respectively, and 8500 Da and 1.35 for p(HEMA-co-AA) respectively.
2.3. Preparation of ZnO/Polymer Bilayer Coatings
The pHEMA and p(HEMA-co-AA) polymer solutions (10 wt %) were prepared by mixing each polymer (396 mg) in methanol (5 mL), under magnetic stirring at room temperature. The pHEMA and p(HEMA-co-AA) films were obtained starting from the as-prepared solutions, by following two separate deposition approaches: (i) vacuum infiltration and (ii) drop-casting technique. In the first case, the ZnO/Si samples were placed at the bottom of a glass round-bottom flask connected to a rotary pump for creating low vacuum conditions. Then, 100 μL of the polymer solution was injected inside the reactor using a syringe through a rubber septum, and was left in static vacuum pumping conditions for 30 min. Then, the vacuum was set for an additional 30 min to promote solvent evaporation and the stabilization of the polymer film. A schematic representation of the apparatus used for vacuum infiltration is shown in Figure S2
of SI. In the second approach, the polymer solution (100 μL) was drop-casted directly atop the porous ZnO surface. Then, the solvent evaporation was obtained by drying the samples overnight at room temperature.
2.4. Calcein Loading and Release Experiments
The loading experiments were performed in simulated body fluid (SBF; pH 7.4), prepared according to Kokubo’s protocol [42
]. Then, 9.34 mg of fluorescent calcein dye (Carl Roth, 622.55 molar mass) was dissolved in 15 mL SBF, at room temperature, under continuous stirring (200 rpm) for 30 min. The calcein was loaded by soaking the porous ZnO/Si samples for 2 h in 2 mL of the loading solution (1 × 10−3
M), in orbital shaking conditions (200 rpm) at room temperature. After the calcein loading, all of the samples were stored in the dark and were air-dried overnight. Then, each sample was diced in two equal parts (each one with dimension of ~0.5 cm2
). The release experiments were carried out in duplicate, by soaking the samples in 5 mL of the release solution, that is, SBF or 5.8-buffered solution, in orbital conditions (200 rpm) at 37 °C. Then, 100 μL of aliquot was collected from each release solution at specific points of time (5 min, 15 min, 30 min, 60 min, 120 min, 24 h, 48 h, 72 h, and 7 days), and analyzed using UV-Vis spectroscopy. The molecule release profile was then constructed by considering the characteristic calcein UV absorbance peak at 473 nm. This was compared with a calibration curve obtained by evaluating the UV absorbance value at 473 nm for a series of calcein dilutions (from 1 × 10−6
M to 1 × 10−3
M), prepared both in the SBF and pH 5.8 buffered solutions. The amount of calcein at each release time and the corresponding cumulative release profiles were obtained according to the literature [34
]. All of the loading and release experiments were performed in dark conditions so as to prevent any degradation of the fluorescent dye.
2.5. Characterization Setup
The attenuated total reflectance (ATR)-FTIR spectra of the polymers were recorded using the Tensor 27 spectrometer (Bruker, Bremen, Germany), equipped with a diamond crystal. The spectrum was made in the spectral range of 4000–600 cm−1, with a resolution of 4 cm−1, and with 16 scans per spectrum. The weight-average molar mass (Mw) and the molar-mass dispersity (PDI) of the polymer and copolymer samples were determined using the gel permeation chromatography (GPC) method, with the use of Shimadzu LC-20AD liquid chromatograph equipped with an ELSD (Evaporative Light Scattering Detector) detector (Shimadzu Kyoto Corporation, Kyoto, Japan), using two Phenogel columns (pore sizes of 100 Å and 50 Å in series) and THF (tetrahydrofuran) as eluent with a flow rate of 1 mL min−1 at 35 °C. Monodistributed poly(methyl methacrylate) was used as the calibration standards. Before the analyses, the studied polymers were dissolved in methanol/acetonitrile (50/50 v/v). The 1H NMR spectra were obtained using a Bruker Avance 300 MSL spectrometer (Bruker, Bremen, Germany) at the field strength of 300 MHz. The polymer solutions were prepared in deuterated methanol. The morphology of the samples was evaluated by means of a field-emission scanning electron microscope (FESEM, Merlin Carl Zeiss AG, Oberkochen, Germany). The ATR-FTIR spectra of the ZnO/polymer samples were acquired with a 4 cm−1 resolution and 16 scans per spectrum were accumulated, using a Nicolet 5700 FTIR Spectrometer (ThermoFisher, Waltham, MA, USA), and were background subtracted. The UV-VIS absorbance spectra were collected in the range of 200–800 nm, by means of a microplate reader (Multiskan™ FC Microplate Photometer, from ThermoFisher Scientific, Waltham, MA, USA). All of the UV spectra were background subtracted.