Nanoroughness, Surface Chemistry and Drug Delivery Control by Atmospheric Plasma Jet on Implantable Devices

Implantable devices need specific tailored surface morphologies and chemistries to interact with the living systems or to actively induce a biological response also by the release of drugs or proteins. These customized requirements foster technologies that can be implemented in additive manufacturing systems. Here, we present a novel approach based on spraying processes that allow to control separately topographic features in the submicron range (∼60 nm to 2 μm), ammine or carboxylic chemistry, and fluorophore release even on temperature-sensitive biodegradable polymers such as polycaprolactone (PCL). We developed a two-steps process with a first deposition of 220 nm silica and poly(lactic- co-glycolide) (PLGA) fluorescent nanoparticles by aerosol followed by the deposition of a fixing layer by an atmospheric pressure plasma jet (APPJ). The nanoparticles can be used to create the nanoroughness and to include active molecule release, while the capping layer ensures stability and the chemical functionalities. The process is enabled by a novel APPJ which allows deposition rates of 10-20 nm·s-1 at temperatures lower than 50 °C using argon as the process gas. This approach was assessed on titanium alloys for dental implants and on PCL films. The surfaces were characterized by Fourier transform infrared, atomic force microscopy, and scanning electron microscopy (SEM). Titanium alloys were tested with the preosteoblast murine cells line, while the PCL film was tested with fibroblasts. Cell behavior was evaluated by viability and adhesion assays, protein adsorption, cell proliferation, focal adhesion formation, and SEM. The release of a fluorophore molecule was assessed in the cell growing media, simulating a drug release. Osteoblast adhesion on the plasma-treated materials increased by 20% with respect to commercial titanium alloy implants. Fibroblast adhesion increased by a 100% compared to smooth PCL substrates. The release of the fluorophore by the dissolution of the PLGA nanoparticles was verified, and the integrity of the encapsulated drug model was confirmed.


S1 Samples preparation
Figure S3: a) Image obtained with a thermographic camera of the plasma device on a plastic substrate in stationary conditions (after 5 minutes) and 15W of RF power.The image clearly shows that plasma has no emissions in the infra-red spectral region since it is not thermal.
On the substrate the mirrored image of the jet can be observed.b) Electrical characterization on a substrate of the coupling of the HV and RF plasmas, obtained measuring the current on a copper substrate connected to ground with a resistance of 147 Ω.As can be seen by the electrical characterisation, the HV and RF coupling in fact is not just the overlapping of two different plasmas but they also interact with each other.The major effect is that the streamers created by the HV electrodes are blown up by the RF field that rapidly changes the polarity.As a consequence, if hundreds of mA streamers can be measured on a conductive substrate if HV is switched on when both power supplies are active, only a small current bumps of few tens of mA can be measured .In this way local heating is avoided and plasma treatments can be performed at room temperature.

S1.4 Plasma deposition process parameters
Table S1: Process parameters of the plasma jet for the layers deposition.The other parameters are kept constant for all processes: 17 kHz power supply at 8 W, the distance from the samples is 2 mm and the confinement N 2 atmosphere coming from the external duct at 10 slm.

S1.5 Thermal power load evaluation during APPJ deposition process
The thermal power load (P ) on the sample during the APPJ process was obtained by a calorimetry measurement 1,2 using Equation 1, A copper plate (10×10×2.5mm 3 , mass m = 2.39 mg, specific heat C = 385 mJ g −1 °C−1 ) was placed in front of the plasma jet in the same position of the samples treated but in static conditions on a thermally insulated sample-holder.All the parameters was set equal to the ones used in the deposition process.The jet 17 kHz power supply was set at 8 W, RF power supply at 14 W, the distance from the copper plate at 2 mm and the confinement N 2 atmosphere coming from the external duct at 10 slm.The temperature of the copper plate was recorded by a thermocouple put in contact with the surface just after switching off the plasma.The data was taken at different interval of exposure of the copper plate to the plasma.The variation of the temperature ∆T from the room temperature was plotted against the exposures intervals (fig.S7).The copper plate temperature rise up due to the plasma heating until it reaches an equilibrium with thermal losses.The data was fitted with an exponential decay curve and the derivative a time 0 d∆T /dt is considered for the calculation in 1.

S1. 1 Figure S1 :
Figure S1: Surface topography of the PCL moulded film (left) and of a T1 substrate (right) measured by AFM in non-contact mode.

Figure S2 :
Figure S2: (a) Scheme of the novel atmospheric plasma jet (Nadir Stylus Noble).In violet is highlighted the plasma area in the principal alumina tube where Ar process gas is supplied.Externally to this alumina tube are visible the electrodes rings of the HV power supply (∼17kHz) and the RF electrodes (27.12 MHz).Two other ducts are present: an inner capillary for precursors inlet in the vapour or aerosol phase and an outer duct for atmosphere control at the exit of the torch, where air or nitrogen are generally used.The jet works with Ar to avoid energy thermalisation by molecular roto-vibrational motion and by dissociation/recombination reactions, allowing to keep the temperatures of plasma as low as possible.(b) A photograph of the device.
Figure S4: (a) SEM image of the SiO 2 nanoparticles produced using the Stöber process deposited on a silicon wafer.(b) Size distribution of the particles derived from the ImageJ (https://imagej.net/Welcome)analysis of the SEM image.

Figure S5 :
Figure S5: SEM cross section on a nanostructured coating deposited on silicon, where can be highlighted the fixing of the particles to the substrate by the plasma polymer.

Figure S6 :
Figure S6: SEM cross section on a nanostructured coating deposited on silicon, where can be highlighted the fixing of the particles among each other by the top plasma polymer.

Figure S7 :
Figure S7: The thermal power load on the sample during the APPJ process was obtained calorimetrically.

Figure S8 :
Figure S8: AFM measured topographies on 20×20 µm and 40×40 µm areas for PCL substrate (a,b); APTES plasma polymer coating fixing silica nanoparticles on Si substrate (c,d); MMA plasma polymer coating fixing silica nanoparticles on Si substrate (e,f).Dome size is the combination of the silica particle of ∅ 220 nm and the 100-150 nm coating on top, leading to a size close to 500 nm.
Figure S9: SEM images highlighting the focal adhesion points of the osteoblasts filipodia on the surfaces (a) on T1 -surface as worked, the filipodia is laying on the surface ; (b) on T2 -SBAE surface, the filipodia is anchoring to microasperities; (c) on T2 with nanoparticles and a fixing plasma polymer starting from APTES precursor, the filipodia are so adherent to the substrate that are merely recognised; (d) T2 with nanoparticles and plasma polymer starting from MMA precursor, as in (c) the filipodia are difficult to detect and focal adhesion points have the same size of the nanoparticles.

Figure S10 :
Figure S10: SEM images of cells morphology with the Backscattering detector on T1 -as worked substrate: (a,b) T1 as is; (c,d) T1 with silica nanoparticles and fixing plasma polymer starting from APTES precursor; (e,f) T1 with silica nanoparticles and fixing plasma polymer starting from MMA precursor.

Figure S11 :
Figure S11: Cell proliferation assays -confocal microscopy: cells were labelled with CFSE cell tracer (Carboxyfluorescein succinimidyl ester; ThermoFisher), a green fluorescent dye retained within cells for long periods.Once incorporated within cells the dye is not transferred to adjacent cells but it halves within daughter cells thus following each cell division up to 7-8 divisions.Cells were fixed and CFSE-related green fluorescent signal was observed at confocal microscopy).

Figure S12 :
Figure S12: Cell proliferation assays -flow cytometry: cells were labelled with CFSE cell tracer (Carboxyfluorescein succinimidyl ester; ThermoFisher),a green fluorescent dye retained within cells for long periods.Once incorporated within cells the dye is not transferred to adjacent cells but it halves within daughter cells thus following each cell division up to 7-8 divisions.Percentage of positive cells were quantified by FACS analysis.