Plasma deposited nano-capsules containing coatings for drug delivery

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Applications of Polymer, Composite, and Coating Materials

Plasma deposited nano-capsules containing coatings for drug delivery applications Fabio Palumbo, Annalisa Treglia, Chiara Lo Porto, Francesco Fracassi, Federico Baruzzi, Gilles Frache, Dana El Assad, Bianca Rita Pistillo, and Pietro Favia ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11504 • Publication Date (Web): 19 Sep 2018 Downloaded from http://pubs.acs.org on September 20, 2018

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ACS Applied Materials & Interfaces

Plasma deposited nano-capsules containing coatings for drug delivery applications Fabio Palumboa*, Annalisa Tregliab, Chiara Lo Portob, Francesco Fracassia,b, Federico Baruzzic, Gilles Frached, Dana El Assadd, Bianca Rita Pistillod, Pietro Faviaa,e*

a. Institute of Nanotechnology, National Research Council of Italy, c/o Department of Chemistry, University of Bari “Aldo Moro”, Via Orabona 4, 70126 Bari, Italy b. Department of Chemistry, University of Bari “Aldo Moro”, Via Orabona 4, 70126 Bari, Italy, c. Institute of Sciences of Food Production, National Research Council of Italy, Via Amendola, 122/O, 70126 Bari, Italy d. Luxembourg Institute of Science and Technology, Material Research & Technology Department, 41, rue du Brill, L-4422 Belvaux, Luxembourg e. Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari “Aldo Moro”, Via Orabona 4, 70126 Bari, Italy

Author Contributions Fabio Palumbo and Annalisa Treglia equally contributed in designing the research work, planning the activity and elaborating the data The other authors all contributed to the research activity necessary to elaborate this manuscript. In particular: -Federico Baruzzi coordinated the drug delivery and antibacterial assessment. -LIST people mostly collaborated in the characterization of the material. -Researchers at the University of Bari mostly contributed to the development of the material.

Contact Authors: Pietro Favia, Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari “Aldo Moro”, Via Orabona 4, 70126 Bari, Italy, Email: [email protected] Fabio Palumbo, Institute of Nanotechnology, National Research Council of Italy, c/o Department of Chemistry, University of Bari “Aldo Moro,” Via Orabona 4, Bari 70126, Italy. Email: [email protected]

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ABSTRACT Coatings consisting in gentamicin-containing nano-capsules have been synthetized by means of an aerosol-assisted atmospheric pressure plasma deposition process. The influence of different parameters affecting the process has been extensively investigated by means of a morphological and chemical characterization of the coatings. Scanning electron microscopy highlighted the presence of nano-capsules whose size and abundance depend on power input and deposition time. A detailed analysis carried out with Matrix Assisted Laser Desorption Ionization coupled to High Resolution Mass Spectrometry allowed to detect and identify the presence of gentamicin embedded in the coatings and its rearrangement, as a result of the interaction with the plasma. The release of gentamicin in water has been monitored by means of UV-vis Fluorescence Spectroscopy, and its biological activity has been evaluated as well by the disk diffusion assay against Staphylococcus aureus and Pseudomonas aeruginosa. It is confirmed that the antibacterial activity of gentamicin is preserved in the plasma deposited coatings. Preliminary cytocompatibility investigations indicated that eukaryotic cells well tolerate the release of gentamicin from the coatings.

KEYWORDS: Aerosol-Assisted atmospheric pressure Plasma, Gentamicin, Drug Release, Nanocapsules, plasma deposition, composite coatings



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INTRODUCTION In recent years, aerosol assisted Atmospheric Pressure Plasma (APP) deposition processes have emerged as a novel approach to the plasma deposition of nano/bio-composite coatings1,2, leading to lower fabrication costs and easier procedures with respect to low-pressure plasma deposition processes, and higher versatility toward biomedical applications3,4. The synthesis of bio-composite coatings represents, indeed, an important technological issue: undoubtedly, these coatings bring benefit to the substrate they are deposited on, without affecting its bulk characteristics5. Biocomposite coatings usually consist of two fundamental components: a bioactive molecule and a matrix the molecule is embedded in. The nature of the molecule and of the matrix can be properly selected in order to obtain different coatings for a very wide range of applications, including food packaging6,7, biosensors8,9, and bioreactors10,11. In the field of biomaterials, particularly for regenerative medicine and tissue engineering applications, bio-composite coatings are being tested as drug delivery systems, with the aim of providing anti-bacterial12, or enhanced cell-growth properties either on 3D scaffolds13 or on flat substrates14-15. In this particular plasma deposition process, a bioactive molecule dispersed in water (or other suitable solvent/buffer solution) is injected in form of an aerosol into a Dielectric Barrier Discharge (DBD) through an atomizer, and an additional gas/vapor can be added as a precursor of the matrix. When this complex feed passes through the discharge, the precursor is fragmented and plasmapolymerized, forming the matrix. Simultaneously, the biomolecule is incorporated in the plasma polymer during its growth16,17. Damages to the biomolecule are limited thanks to mild fragmentation conditions typical of atmospheric pressure discharges, and by a thin protective solvent shell around the biomolecule, which can be effectively included in the coating preserving its structure and functionalities18. Interestingly, this one-step deposition process can lead to the formation of a coating composed of nanometric capsules19-21 containing the active species dissolved in the aerosol, as it has been shown in the case of vancomycin22. This finding represents an absolute novelty since, for the first time22, a one-step, easy-to-handle, with low environmental impact, plasma process is presented for the synthesis of nano-capsules embedding a bioactive molecule. In this work, the aerosol assisted APP process has been optimized in order to produce gentamicincontaining coatings. Gentamicin is a broad-spectrum aminoglycoside antibiotic produced by Micromonospora purpurea, active against a wide range of gram-positive and gram-negative bacteria23. It is a mixture of four major molecules, defined as congeners and designated as C1, C2, C1a and C2a (Fig. S1, in Supporting Information file, SI), differing in their degree of methylation on the purpurosamine ring24. Gentamicin binds irreversibly to the bacterial 30S ribosomal subunit, 4 ACS Paragon Plus Environment

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causing the misreading of t-RNA, and leaving the bacterium unable to synthesize its own proteins necessary to its growth25. The use of this molecule in drug delivery systems is very common in literature26-28. The aerosol assisted plasma deposition of gentamicin containing coatings has been optimized, and their feasibility for drug releasing purposes has been evaluated. Ethylene was chosen as the plasma polymerization precursor, since the film deposition of this monomer is well known in plasma processing and it is easy to handle. A multi-diagnostic approach has been carried out for the chemical and morphological characterization of the coatings. In particular, an accurate identification of the drug and the characterization of eventual modification of the structure induced by the plasma has been performed with an Atmospheric Pressure- Matrix Assisted Laser Desorption Ionization coupled with an Orbitrap High Resolution Mass Spectrometer (AP-MALDI HRMS). Finally, both the antibacterial activity and the non-lethal influence on eukaryotic cells of the gentamicin containing coating have been assessed.

MATERIALS AND METHODS Materials Helium 99.999% and ethylene 99.95% (Air Liquide) were used as feed gas in the APP deposition process. 10 mg/mL (corresponding to a concentration of 6.7 mM) solution of gentamicin Sulfate (powder, Apollo Scientific), in water of Milli-Q quality (Millipore, Bedford, MA) was used to generate the aerosol. Shards (1x1 cm2) of 710 µm thick double face polished crystalline silicon (100) wafers (MicroChemicals GmbH) were used as substrates for the deposition experiments and MALDI-HRMS analyses. Thermo Scientific™ Oxoid™ Blank Antimicrobial Susceptibility Disks were used as substrate for antimicrobial tests. Polycarbonate films (1.5x3 cm2, 0.25 mm thickness, GoodFellow) were chosen as substrate, upon washing in isopropyl alcohol, for in vitro drug release tests and cytocompatibility assays. Water Milli-Q quality (Millipore, Bedford, MA), analytical grade methanol (MeOH), o-phthaldialdehyde (OPA), 2-mercaptoethanol, sodium tetraborate (Na2B4O7*10 H2O, 0.1 mol/L) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used for in vitro drug release tests.

DBD Reactor The homemade DBD reactor used in this research has been described in detail elsewhere, in ref 22,29. As shown in the scheme reported in Figure S2 (in SI file), it consists of two parallel plates silver electrodes, 5x8 cm2 wide, 3 mm apart, both covered with 0.63 mm thick alumina sheets. The power generator is connected to the upper electrode, while the lower electrode is ground. The electrode set up is placed in a sealed Plexiglas chamber, as wide as the electrode assembly, in a way that its walls 5 ACS Paragon Plus Environment


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can confine the gas flow in the gap. The aerosol of gentamicin aqueous solution was generated with a pneumatic atomizer (mod. 3076, TSI) working with He at a flow rate of 5 slm. As stated by different authors30,31 the diameter range of the droplets produced in similar conditions by the atomizer used in the present work is typically sub-micrometric. Ethylene was also fed in the chamber at a flow rate range of 5-200 sccm (between 0.25 and 4% of buffer He gas); when not specified in the text, ethylene flow rate was 20 sccm. The gas flow rate was controlled by means of electronic mass flow controllers (MSK instruments). The gas/aerosol feed was let from the shorter side of the reactor, through the plasma zone between the electrodes, and was pumped out by an aspirator located on the opposite side. Discharges were ignited in Continuous Wave (CM) or Pulsed Mode (PM) using a corona power supply (PVM500, Information Unlimited). The electrical properties of the plasma were investigated, measuring the voltage and the current delivered to the system with a high-voltage (P6015A, Tektronix) probe and a resistance type current probe, both connected to an oscilloscope (TDS 2014C, Tektronix). Experiments were carried out at two different input power level: namely High (HP) and Low Power (LP), corresponding to 0.8 Wcm-2 and 0.3 Wcm-2, respectively. The applied peak-to peak voltage was kept at 4 kV at a frequency of 24 kHz in the experimental conditions used in this work. Before each deposition experiment, a cleaning run of the electrodes was performed by igniting for 5 min a discharge fed with a water aerosol carried by He at a flow rate of 5 slm. Substrates were positioned on the bottom electrode, always within 3 cm close to the inlet of the feed, and the chamber was purged with 5 slm He stream for 5 min. In order to better optimize the deposition process, the discharge duration has been tuned in the range 5-30 min. Discharges ignited in PM were also investigated; in this case a pulse generator controlled the power supply with an ON time of 60 ms and an OFF time of 60 ms, for a Duty Cycle (DC) of 50%.

Chemical and morphological characterization of the samples Fourier Transform Infrared Spectroscopy (FT-IR) was carried out to characterize the bulk of the coatings. FT-IR spectra (32 scans per analysis, 4 cm-1 resolution) were obtained in transmission mode with a Vertex 70V Bruker spectrometer. The spectrometer was evacuated to less than 150 Pa for 10 min before each acquisition. Spectra were normalized to the maximum intensity of the CHx stretching band at 2935 cm-1. Scanning Electron Microscopy (SEM) analysis were carried out to investigate the morphology of the samples with a Zeiss Supra 40 SEM instrument equipped with a Gemini field-effect emission gun. Analysis were carried out at an extraction voltage of 3 kV, onto samples sputter coated with a 20 nm thick Cr layer. 6 ACS Paragon Plus Environment

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Morphology was also assessed by AFM analysis, onto samples deposited for 20 min in CM at HP and LP and pulsed mode, by means of a XE-70 microscope (Park Systems) in non-contact mode using PPP-NCHR probes from Nanosensors with typical resonance frequency around 320 kHz. Root mean square (RRMS) roughness was determined by sampling three different spots for each film. Static Water Contact Angle (WCA) measurements were performed with a Rame´-Hart Inc, 100 goniometer (5µl drops, double distilled water). X-ray Photoelectron Spectroscopy (XPS) analysis was carried out with a PHI-5600 VersaProbe spectrometer, using a monochromatic Al X-ray source operating at 150 W. Wide scan and highresolution C1s, O1s, N1s, S2p spectra were acquired at 115 eV and 23.5 eV pass energy, respectively. Flood gun (18 mA emission current; 40 eV electron energy) was used for charge compensation of the samples. The hydrocarbon component of C1s spectra was set at 284.8 ± 0.2 eV as reference of the Binding Energy scale. Spectral data were processed with Multipack software. The thickness of the coatings has been evaluated by means of a alpha-step profilometer D120 (Tencor-Instruments), after deposition onto polished crystalline silicon and scratching part of the coating with a scalpel.

AP-MALDI-HRMS analyses AP-MALDI-HMRS analyses were carried out to detect and identify the presence and the degradation products of gentamicin on 1 µm thick coatings deposited in CM and PM HP conditions. Two kinds of approaches have been considered: a) analysis of extracts obtained after immersion of the samples in water, and b) direct analysis of the coatings. For the former, 1x1 cm2 samples were dipped into 1 ml distilled water in 6-well polystyrene plates, for 24 h under gentle stirring at 250 rpm at 25°C. The extracted solutions were then analysed. A matrix solution consisting of 1:1 (v/v) mixture of ethanol/water of α-cyano-4-hydroxycinnamic acid (CHCA), was prepared at a concentration of 10 mg/mL. Equal volumes of extracts and of CHCA solution were mixed and stirred. A 2 µL drop of this mixture was dropped off and dried on a goldcoated MALDI plate. In the second approach, the direct sample analysis, a drop of 2 µL CHCA matrix solution was directly dropped off on the coatings and dried. Mass spectrometry experiments were performed with an Orbitrap Elite™ Hybrid Ion Trap-Orbitrap Mass Spectrometer from Thermo Scientific (San Jose, CA) coupled with an AP-MALDI PDF+ source (MassTech In, Columbia, MA) with a 355 nm Nd: YAG laser. Each sample was desorbed by the laser for 40 seconds with the following MALDI source parameters: Ion Source Voltage 2000 V, 7 ACS Paragon Plus Environment


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Capillary Temperature 200 °C, 60 % Laser energy for the extracted samples and 65 % for the direct film analysis.

Gentamicin release tests Plasma processed samples, 1.5x3 cm2 wide and 1 µm thick, were placed into 6-well Iwaki polystyrene plates (Sterilin Limited, Newport, UK), covered with 2 mL of water, and incubated at room temperature. The amount of gentamicin released was monitored collecting 750 µL of supernatant, at different soaking time, up to 24h. Every withdrawn aliquot of liquid was refurbished with fresh distilled water, to keep the dipping volume constant. Release solutions were derivatized with OPA. 40 mg OPA were dissolved in 1 mL methanol and mixed with 5 mL of sodium tetraborate solution (0.1 M). Then, 50 µL of 2-mercaptoethanol were added and mixed for 1 minute. 250 µL of such OPA solution were vigorously mixed with the release aliquot for 1 minute and transferred in a cuvette. The analysis was carried out with a Fluorescence Spectrophotometer (Varian Cary Eclipse; excitation 335 nm, emission 440 nm). Calibration was carried out with gentamicin standard solutions in the range 1 - 50 µg/mL.

Antibacterial activity of the coatings The antimicrobial activity of gentamicin containing coatings was evaluated against Pseudomonas aeruginosa DSM939 and Staphylococcus aureus DSM799 (Leibniz Institute - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). Two kind of coatings, both 1µm thick, were considered, deposited in CM and PM modes at HP. Antimicrobial disk susceptibility assays were carried out as defined by CLSI32 with slight modification. About 105 of fresh microbial cells of target strains, incubated and standardized as previously reported33,34 were plated onto Plate Count Agar (Biolife Italiana srl, Milan, Italy) before disk diffusion assays. Positive control filter paper disks loaded with 10 µg gentamicin (6 mm, KAIROSafe srl, TS, Italy), defined as the breakpoint amount able to distinguish sensitive or resistant P. aeruginosa and S. aureus strains35 as well as sterile filter paper blank disks were used. In order to define the antimicrobial activity of the samples, an antibiotic regression curve was drawn. Briefly, each disk was soaked with 20 µl of a gentamicin solution in order to load disks with 0.6 to 20 µg active gentamicin/disk. Within 10 min after the soaking, disks were placed over the inoculated agar surface; after 2 h at 4°C, plates were incubated at 37 °C for 24 h. Three replicates per disk were positioned onto three different agar plates. The antimicrobial activity was evaluated by measuring the area of inhibition of bacterial growth rather than their diameter. In order to 8 ACS Paragon Plus Environment

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compare the halos produced over different agar plates, jpg format pictures were imported into the Adobe Photoshop CS2 image analysis software (Adobe Systems Incorporated, San Jose, CAL, USA). The number of pixel of each halo (excluding the pixels of the disk) was converted into square millimeters after normalization with the average pixel number of all disks (about 30 mm2).

Cytocompatibility assay with eukaryotic cells Coatings deposited in CM HP mode on polycarbonate substrates, with or without aerosol feed, were tested for their cytocompatibility in terms of eukaryotic cell adhesion and viability. The SAOS-2 line (ATCC, HTB-85), derived from primary osteosarcoma, has been used for this study. Besides their worldwide availability, their well-documented characterization data set is among the advantages of using the Saos-2 cell line in this kind of tests. SAOS-2 cells were routinely grown in McCoy’s 5A Medium (ATCC) supplemented with 10% FBS, glutamine and penicillin-streptomicin solution (Complete McCoy’s 5A). SAOS-2 cells were cultured at 37 °C in saturated humid 5% CO2/air atmosphere in cell culture flasks (Barloworld Scientific, UK). Cells were detached from flasks with a trypsin/EDTA solution (Sigma–Aldrich) and collected in centrifuge tubes (Falcon). Cell pellets were suspended in the medium, counted and seeded onto native and plasma-coated PC substrates (3 × 104 cells in 0.8 mL medium) in 48-multiwell plates. Cells were incubated for 48 h at 37°C, in 5% CO2 air. The cytocompatibility of the plasma-deposited coatings was were tested through the analysis of cell morphology and cell viability. Morphology was tested by visualization of the actin cell cytoskeleton with AlexaFluor488 conjugated phalloidin (Molecular Probes). After 48 h of incubation, cells were fixed in a 4% formaldehyde/PBS solution at room temperature (RT) for 20 min. Later, they were permeabilized with PBS containing 0.1% Triton X-100, and incubated with Alexa Fluor488 conjugated phalloidin (RT, 30–40 min). Counterstaining with DAPI (Sigma–Aldrich) allowed to visualize cells nuclei. Fluorescence images were acquired with an Axiomat Zeiss AM1 microscope. The viability of Saos-2 cells was determined with the MTT colorimetric assay. The cell growth was stopped at 48 h. The complete medium was aspirated and 1 ml of fresh complete medium with 100 µl of 5 mg/ml MTT solution was added. The solution was prepared by dissolving 100 mg of MTT powder (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, M5655-100 mg, Sigma Aldrich) in 20 ml of DMEM without serum and phenol red. The resulting medium was incubated (37 °C, 2 h) to allow MTT to penetrate the cells, react with the mitochondrial succinate dehydrogenase, and form the dark blue formazan product. Then, 1 ml of the MTT solubilization solution (M8919-125 ml, Sigma Aldrich) was added to dissolve formazan and the optical density of the resulting solution was read with a spectrophotometer (Agilent Cary 60 UV-Vis) at 570 nm after 9 ACS Paragon Plus Environment


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subtracting the background absorbance at 690 nm. Since reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells. Statistical analysis was performed using one-way ANOVA followed by Tukey's posthoc test by means of the Graph Pad Prism Software (GraphPadSoftware,Inc.). Differences were considered statistically significant with p

Plasma deposited nano-capsules containing coatings for drug delivery

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