Scanning Electron Microscopy
Structural and morphological characterization of prepared TNT–Ti sample surfaces was performed using a field emission SEM (Philips XL 30). The samples were mounted on a holder with double-sided conductive tape and coated with a platinum layer 3-nm thick. SEM images from the top, bottom and cross-section were acquired.
Dynamic Light Scattering
The particle size distribution of AuNPs and TPGS micelles (before and after drug encapsulation) were evaluated by laser dynamic light scattering (DLS) using a Zetasizer Nano ZS, Malvern Instrument Ltd (Worcestershire, UK) from five repeated measurements.
The amount of loaded drug and drug carriers in TNT with and without AuNPs, was determined by thermogravimetric analysis (TGA; Hi-Res Modulated 2950, Q Series™ Thermal Analysis; TA Instruments, DE, USA), before and after the in vitro release studies. A TNT–Ti sample was mounted onto a platinum pan, placed inside a burning furnace and heated from 20 to 800°C at a scanning rate of 10°C/min under nitrogen gas flow of 50 ml/min. The obtained thermograms were analyzed using the software Universal Analysis 2000 (TA Instruments). TGA was performed after the release experiments to confirm the amount of released drug carriers and AuNPs from TNT–Ti. The amount of drug–micelle loading was obtained by measuring the mass of drug and micelles loaded inside the TNT from TGA graphs. The net amount of drug was quantified from the difference between empty TPGS micelles (no drug encapsulation) and indomethacin-loaded TPGS micelles, to obtain the weight loss difference, (i.e. drug mass). The loading capacity of drug and drug carriers was calculated from TGA results using the formula:
In Vitro Studies of Drug & Drug Carriers Released From TNT
Four TNT–Ti samples loaded with active agents including drugs with and without AuNPs, and drug–micelles with and without AuNPs were used for this study. An in vitro release experiment was performed by immersion of these samples in PBS (pH = 7.2) at room temperature in a 1.5-ml eppendorf tube with 1 ml of PBS. A 0.5-ml aliquot of eluted drug medium was removed for quantification and this volume was replaced with fresh buffer to prevent sink conditions. Drug release was quantified by measuring the absorbance of the release media diluted with fresh PBS (1:1) using an UV–visible (Vis) spectrophotometer. The release studies were carried out after drug loading, and the active agents were stored for 24 h in TNT in a vacuum desiccator, without any passive diffusion. The RF trigger was applied as soon as the samples were immersed in PBS in our first study to show the impact of RF and after 5 days of natural release in our second study, which demonstrated on-demand delivery at arbitrary time points. Drug release characteristics under the influence of a RF trigger for stimulated release and non-stimulated release based on free diffusion as a control were obtained. Another set of control experiments with only drug and drug carriers (without AuNPs) inside the TNTs triggered by RF were carried out independently. The trigger source was a custom-made RF system as shown in Figure 2. A variable frequency generator set to 1 GHz RF field was used (Wavetek 2500A Signal Generator 0.2 to 1100 MHz; CA, USA). The RF generator was connected to an amplifier, powered by an external source – the Lab Power Supply 0–30 V 2.5 Amp Digital Display (Dick Smith Australia Pty Ltd, Adelaide, Australia). A voltage of 25 V and a current of 2 Amps were found to be the optimal settings based on conducted preliminary experiments. The instrument generates a reciprocal power of 20 W at 1 GHz, measured by an in-line wattmeter that reads forward and reverse power (Bird Electronic Model 43 RF Wattmeter, accuracy ±5% full scale; average mode; Bird Technologies, OH, USA). From these two measurements, it is possible to calculate the standing wave ratio (SWR) from the RF load (amplifier), which is also the industry standard and benchmark for performance and reliability in routine RF power measurements. The SWR is the ratio of the maximum amplitude of the standing wave to the minimum amplitude (i.e., the ratio of antinode voltage:node voltage of the feed line standing wave). It is calculated using the following equation:
Radiofrequency generator set-up. The radiofrequency set-up for in vitro drug and drug carrier release, consisting of (from left to right): a signal generator to control radiofrequency (power supply); connected to an amplifier; followed by a wattmeter; and a solenoid copper coil for holding the titania nanotube–titanium sample loaded with drug or polymer micelles mixed with gold nanoparticles and the entire drug-eluting implant immersed in the buffer solution at pH 7.2.
in which Γ is the reflection coefficient and E the amplitude. The generated RF signals from the copper coil surrounding the tube carrying the TNT sample was used as an energy source to induce the AuNPs loaded at the bottom-most corner of the nanotubes for release of the drug or drug carriers. The release is based on heating AuNPs inside TNT via RF, which provides the excitation energy to stimulate drug or drug carrier release. External nitrogen gas cooling was used to control the temperature of the coil.
The RF triggering was performed during two stages of drug release: the initial (burst) release and after 5 days of release. Three different RF exposure times (2, 5 and 10 min) were investigated in order to explore the influence of these durations and the impact of the RF trigger on the release characteristics of the drug and drug carrier with respect to time. The concentration of released drug or drug carriers from TNT was determined by UV–Vis spectroscopic analysis using a Cary 60 (Varian Ltd, Crawley, UK) instrument. The UV detection wavelength was set at λ = 250 nm (drug–micelles from TNT) and 320 nm (pure drug from TNT). The samples were collected consecutively and periodically every 5–10 min for up to 2.5 h (initial trigger), or 5–7 days in the case of control samples (normal/nontriggered, RF trigger on day 5 and with RF trigger but no AuNPs), when complete release was reached. After each release experiment, TNT–Ti samples were characterized by TGA to confirm that no remaining drug, drug carriers or AuNPs were left at the end of the experiment. The concentration of AuNPs was not specifically monitored during drug release. The DLS characterization of released drug carriers was also conducted to probe the integrity of polymer micelles after release and to compare the results with samples before their release.
Nanomedicine. 2014;9(8):1263-1275. © 2014 Future Medicine Ltd.