âThunderstruckâ: Plasma-Polymer-Coated Porous Silicon

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“Thunderstruck”: Plasma polymer coated porous silicon microparticles as a controlled drug delivery system Steven James Peter McInnes, Thomas D Michl, Bahman Delalat, Sameer A Al-Bataineh, Bryan Robert Coad, Krasimir Vasilev, Hans Joerg Griesser, and Nicolas H. Voelcker ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b12433 • Publication Date (Web): 02 Feb 2016 Downloaded from http://pubs.acs.org on February 10, 2016

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ACS Applied Materials & Interfaces

“Thunderstruck”: Plasma polymer coated porous silicon microparticles as a controlled drug delivery system

Steven J. P. McInnes,1,§ Thomas D. Michl,2,§ Bahman Delalat,1 Sameer A. AlBataineh2, Bryan R. Coad,2 Krasimir Vasilev,2 Hans J. Griesser2 and Nicolas H. Voelcker1*

1

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Future Industries Institute, University of South Australia, Adelaide, South Australia 5001, Australia.

2

Future Industries Institute, University of South Australia, Mawson Lakes, SA 5095, Australia.

§: These authors contributed equally

* Corresponding Author, Email: [email protected], Fax: +61 8 8302 5613, Tel: +61 8 8302 5508

ACS Paragon Plus Environment


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Abstract Controlling the release kinetics from a drug carrier is crucial to maintain a drug’s therapeutic window. We report the use of biodegradable porous silicon microparticles (pSi MPs) loaded with the anti-cancer drug camphothecin, followed by a plasma polymer over-coating using a loudspeaker plasma reactor. Homogenous “Teflon-like” coatings were achieved by tumbling the particles playing AC/DC’s song “Thunderstruck”. The over-coating resulted in a markedly slower release of the cytotoxic drug and this effect correlated positively with the plasma polymer coating times; ranging from 2-fold up to more than 100-fold. Ultimately, upon characterizing and verifying the pSi MPs’ production, loading and coating with analytical methods such as time-of-flight secondary ion mass spectrometry, scanning electron microscopy, thermal gravimetry, water contact angle measurements, fluorescence microscopy, human neuroblastoma cells were challenged with pSi MPs in an in vitro assay; revealing a significant time delay in cell death onset.

Keywords: plasma polymerization, fluorinated coating, particle coating, porous silicon, controlled drug release.


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Introduction “The dose makes the poison.”1 Hence attaining the “control” in controlled drug release is a current ongoing challenge since the alternatives are either diminished therapeutic effect or overdose.2 To avoid either, controlling the release kinetics is crucial to maintain the drug’s therapeutic window. Porous silicon (pSi) and microparticles formed from it (pSi MPs) are promising candidates for various biomedical applications ranging from biosensors, drug delivery to imaging, theranostic applications and even tissue engineering scaffolds.3-11 This wide scope of application is partly credited to the ease of fabrication of pSi and broad variety of pore structures possible, with the potential to be subsequently functionalized using a range of chemistries including oxidations6,

12

nitridization,13 silanization,11,

14

hydrosilylations,10,

15

carbonization reactions,16-17 hydrosilanization9 and surface-initiated/surfacegrafted polymerizations5 or combinations of thereof.3 This

combination of

chemical

and

physical

attributes

provides

the

prerequisites for pSi MPs to be used as the carrier matrix for controlled drug release as they, upon contact with aqueous solutions, degrade into non-toxic silicic acid and liberate the encapsulated drug. However, the issue has been to control the direct dissolution of drug loaded in the pores, which can lead to undesirably fast drug release. There are three general approaches to control the release of a payload from pSi based materials; covalent attachment, physical trapping and adsorption.18 Covalent attachment employs linkers such as alkenes19 or silanes20 to generate groups on the surface for further attachment of the drug payload, which is then released upon dissolution of the pSi. Physical trapping in turn makes use of the oxidation and subsequent pore shrinkage to entrap the drug,21 whilst adsorption-based approaches tune the chemistry of the pSi surface to best suit the properties of the drug and allow for maximal electrostatic adsortipon.6 An additional class of pSi-based materials are those that possess polymer layers to clog the pores and slow diffusion from the porous matrix underneath.22-24 Our hypothesis is that an over-coating with a thin, hydrophobic coating could delay direct drug dissolution and hence lead to a more sustained drug release. In addition, the over-coating does not diminish the pore volume available for drug loading.



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Plasma polymerization is an industrial grade process to deposit nanometer thin coatings and has many desirable properties such as substrate independence, speed, lack of solvents and scalability.25-27 To date, however, this technique has mainly been used for planar surfaces with little to no translation to 3-dimensional particles.28-30 Building upon this, we recently demonstrated the ability to coat polystyrene particles with a fluorinated plasma polymer.31 This hydrophobic overcoating led to an increase in the particle’s water contact angle. One implementation of this technique uses a loudspeaker, driven by acoustic waves, to agitate the particles in the plasma chamber to achieve a homogenous coating. In this work, we marry the two ideas of a hydrophilic porous silicon drug carrier and hydrophobic plasma over-coating to develop a novel class of drug release system. This drug release system combines the biocompatibility of pSi4, 7 with the tunability of release by varying the over-coating’s thickness. We have investigated the feasibility of this approach by loading the pSi MPs with the anti-cancer drug camptothecin (CPT), followed by plasma treatment with the fluorinated precursor perfluorooctane (PFO) while agitating the pSi MPs in the plasma by vibrational excitation from acoustic sound waves of hard rock music. While monotonic frequencies can be used to accomplish agitation, our experience with this technique has shown better agitation (and coatings) are obtained from chaotic motion when applied motion is produced non-monotonic frequencies. For this, music can be used. A song which produces chaotic motion and excellent “Teflon-like” overcoatings on the particles was “Thunderstruck” by AC/DC. The successful preparation, loading, coating and subsequent retarded release was verified by a suite of analytical methods, those being time-of-flight secondary ion mass spectrometry (ToFSIMS), scanning electron microscopy (SEM), thermal gravimetry (TGA), water contact angle (WCA) measurements, fluorimetry, fluorescence microscopy and colorimetric assays. Finally, we challenged human neuroblastoma cells (SH-SY5Y) in an in vitro assay with the coated/uncoated and loaded particles to investigate the timely onset of CPT’s cytotoxic properties.

Experimental Section


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Chemicals Hydrofluoric acid 48% (Merck), dichloromethane (CH2Cl2, Labserv, analytical grade, 99.5 %), Methanol (Merck, analytical grade, 99.5%), acetone (Ajax, analytical grade, 99.5%) and EtOH (Ajax, absolute, 100%) were used for etching and washing without further purification. Conductivity water 18.2 MΩ cm was obtained from an A10 MilliQ system (Merck Millipore). CPT (Sigma, 95%) was stored at 2 - 4

o

C and protected from light at all times.

Perfluoroctane (98%) was purchased from Sigma Aldrich and used as received. Phosphate buffered saline (PBS) was prepared from sodium chloride (NaCl, AR, Chemsupply 99.0%, (8 g/L)), potassium chloride (KCl, AR, Biolab

Scientific,

99.5%,

(0.2

g/L)),

disodium

phosphate

dihydrate

(Na2HPO4.2H2O, AR, Chemsupply, 99.0%, (1.12 g/L)) and potassium dihydrogen orthophosphate (KH2PO4, AR, Ajax, 99.0%, (0.24 g/L)). The pH was adjusted to 7.4 with 1 M solutions of NaOH (Ajax, analytical grade) or HCl (Aldrich, reagent grade) in conductivity water.

pSi MP preparation p++ Si wafers (boron doped, resistivity = 0.00055 - 0.001 Ωcm, ) purchased from Silicon Quest International were etched in 1:1 HF:EtOH on a MPSB150 wetbench, with cathodic cooling supplied by AMMT (Frankenthal, Germany). A total area of 132 cm2 was etched with a square waveform consisting of the following two steps: 1) an etch current density of 15 mAcm-2 for 28.25 min and 2) a perforation step of 227 mAcm-2 for 7.5 s. This square waveform etch was repeated 6 times, resulting in 114 µm of penetration into the approximately 500 µm Si wafer. The freshly etched pSi surface was washed with water and ethanol before allowing the surface to air dry. The resulting pSi film flaked off the Si substrate upon drying. Any pSi that did not flake off was pushed off with a flat metal blade. Once collected, the pSi flakes were sonicated for 2 h and sized via wet sieving (using ethanol) through a combination of 75 µm, 53 µm and 25 µm sieves. For this work, we only used the fraction of pSi MPs in the 25-53 µm size range.



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CPT loading of pSi pSi MPs were placed into an Eppendorf tube to which a known volume of (50200 µL) of the model drug (CPT) solution of approximately 5 mg/mL CPT in dry distilled DMF was added. This mixture was allowed to incubate for 2 h before the particles were dried under vacuum (10 mm Hg) in a desiccator. This procedure was performed before the deposition of the plasma polymer film. The total loadings were calculated based on the mass of pSi placed into the tube originally, for example a typical loading used 400 µL of 4.6 mg/mL CPT and 96.6 mg of pSi, resulting in a loading of 19 µg of CPT per mg of pSi. The average loading of the pSi MPs across all batches used was 20 ± 1 µg/mg of pSi. Each individual batch loading was used to convert the release amounts into percentages. The release amounts were calculated via the use of a CPT calibration curve.

Plasma polymerization The pSi MPs’ agitation was achieved by utilizing a commercially available loudspeaker (5 inch paper cone woofer CW2192, Jaycar) within the plasma chamber.31 Without interfering with the plasma, this method enabled full control over the particles’ agitation frequency and the amplitude during plasma exposure. The original plasma reactor design and its use to treat flat substrates have been reported previously.32-33 The tube shaped glass reactor contains two electrodes with the grounded electrode being circular and the powered upper electrode being U-shaped. The monomer vapors were introduced into the chamber via Swagelok fitted valves, connected to a vacuum inlet, which led to a Baratron (Model D27B01TCEC0B0, MKS instruments) to observe the pressure, and a cold trap.31 For a typical coating procedure, the pSi MPs were placed into an aluminum cupcake holder, which was stuck to the loudspeaker, followed by sealing the chamber. The cold trap was filled with liquid nitrogen and the system was evacuated to base pressure. Afterwards, PFO vapors were via an inlet line, controlled by a Swagelok valve. After adjusting the pressure 200 mTorr, the


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acoustic drive signal was turned on by playing AC/DC’s song “Thunderstruck” on a loop. The volume was slowly increased until the particles were visibly bouncing. To enable comparison, all samples were shaken at the same acoustic volume. Lastly, the plasma was ignited using a RF generator (RFX600, Advanced Energy) and matching network (ATX-600, Advanced Energy) at a power of 25 W and the particles were treated for the desired time while being constantly tumbled.

X-ray photoelectron spectroscopy (XPS) XPS was performed using an AXIS Ultra DLD spectrometer (Kratos Analytical, UK) equipped with a monochromatic Al Kα radiation source (hν - 1486.6 eV) at a power of 225 W. The pass energy for survey spectra, recorded over the energy range 0 - 1000 eV, was 160 eV with 0.5 eV step size and the pass energy for high-resolution C 1s spectra was 20 eV with 0.1 eV step size. The analysis area was approximately 700 µm x 300 µm. The spectra were acquired at a take-off angle of 90°. Elements present on the surface were identified from survey spectra and quantified in atomic percentage (at.%) with CasaXPS Software (version 2.3.14, www.casaxps.com) using a Shirley-type background and applying the relative sensitivity factors supplied by the manufacturer of the instrument. In order to minimize X-ray-induced sample degradation, the exposure time was kept to the minimum required to obtain an adequate signal-to noise ratio. Charging effect of the samples during analysis was corrected using a reference value of 285.0 eV; the binding energy of the main C 1s component arising from neutral hydrocarbon (CHx).34 Two nonoverlapping areas of each surface were analyzed and the standard deviation (SD) calculated. The atomic percentages were rounded to one decimal after the comma.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) ToF-SIMS analysis was performed using a Physical Electronics Inc. PHI TRIFT V nano-ToF instrument equipped with a pulsed liquid metal

79

Au+

primary ion gun (LMIG), operated at 30 kV. The extractor current of the ion



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source was maintained at 3 µA. Surface analyses were performed using ‘bunched’ Au1 beam settings for surface imaging and spectroscopy. Positive ion images (500 x 500 µm2) were acquired for 5 min on the uncoated and PFO-coated µpSi particles. Positive ion mass spectra (50 x 50 µm2) were collected on the same surfaces with an acquisition time of 2 min each. Mass calibration of the spectra was done with CH3+, C2H5+, and C3H7+ ions. Experiments were performed under a vacuum of 7.5 x 10-9 Torr and in the static mode.

Scanning

electron

microscopy

(SEM)

and

energy-dispersive

X-ray

spectroscopy (EDX) FEI Quanta 450 FEG environmental scanning electron microscope equipped with an EDAX Apollo X energy dispersive X-ray (EDX) detector. Samples were analyzed uncoated to facilitate EDX analysis. EDX was performed under high vacuum, with the beam set at 5 kV, lifetime 100 s, takeoff angle ~35 ˚ and the sample tilt set at 0 ˚.

Fluorescence microscopy Fluorescence microscopy was performed on an Eclipse 50i microscope equipped with a D-FL universal epi-fluorescence attachment and a 100 W mercury lamp (Nikon Instruments, Japan). Fluorescence images were captured with a CCD camera (Nikon Instruments, Japan), using the following fluorescent filters. Blue channel (violet excitation, blue emission): excitation: 385 - 400 nm (bandpass, 393 CWL), dichromatic mirror: 435 - 470 nm (bandpass) and barrier filter wavelength: 450 - 465 nm (bandpass, 458 CWL). Green channel (blue excitation, green emission): excitation: 475 - 490 nm (bandpass, 483 CWL), dichromatic mirror: 500 - 540 nm (bandpass) and barrier filter wavelength: 505 - 535 nm (bandpass, 520 CWL). Red channel (green excitation, orange/red emission): excitation: 545 - 565 nm (bandpass, 555 CWL), dichromatic mirror: 570 - 645 nm (bandpass) and barrier filter wavelength: 580 - 620 nm (bandpass, 600 CWL). Images were analyzed using NIS-elements v3.07 software (Nikon Instruments, Japan).


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Thermogravimetric analysis (TGA) TGA experiments were conducted under N2 at a flow rate of 50 mL min-1 on a SDT 2960 (TA Instruments, USA). The temperature was ramped from room temperature to 500 °C at 10 °C/min. Each sample was accurately weighed by the TGA before measurements were performed and all samples weights introduced in the instrument ranged from 10 - 13 mg.

Water contact angle (WCA) measurements The WCA was measured by placing a 1 mL drop of water on the sample surface and capturing a digital image using a Panasonic Super Dynamic wvBP550 Closed Circuit TV camera. The contact angle measurements were analyzed by Scion Image for Windows Framegrabber software (Beta version 4.0.2).

Interferometric reflectance spectroscopy Interferometric reflectance spectroscopy was used to monitor the EOT of the pSi layer in time-lapse mode. The experiments were performed using a custom-built interferometer with an S2000 CCD detector (Ocean Optics, USA) as described in our earlier work.35 pSi substrates were placed inside a custom-built glass fluidic cell that allowed solutions to be flowed over the sample whilst monitoring the EOT in real time.

Silicic acid degradation Silicic acid produced during pSi degradation was determined by means of an ammonium molybdate colorimetric assay. Pre-weighed thermally-oxidized uncoated pSi MPs, PFOpp coated pSi MPs samples were placed into separate 50 mL solutions of 0.25 M Tris-HCl (pH 7.2). The pSi samples were agitated gently over five days. 40 µL of the silicic acid-containing solution was removed daily and acidified with 40 µL of 0.3 M HCl. 20 µL of a 42 mM



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ammonium molybdate solution was then added and the mixture was incubated for 10 min. 20 µL of 27 mM EDTA and 1.35 M sodium sulphite was subsequently added. After incubation at room temperature for 1 h, spectrophotometric analysis at 640 nm was performed on a microplate reader (VMax, Molecular Devices, USA). Silicic acid concentration standards were prepared with sodium metasilicate pentahydrate. The data were adjusted to show the % of pSi degradation.

Fluorescence-based drug release experiments CPT release was monitored via fluorimetry. Fluorimetry was performed on a Perkin Elmer Instruments LS55 luminescence spectrometer with an excitation wavelength of 340 nm and emission wavelength of 434 nm. The slit width was set to 3 nm and the photomultiplier voltage was set to 775 V. The cumulative release data of CPT into 3 mL of PBS was monitored over a 17-h period. Release rates were calculated from the slope of release curves obtained. The actual amount of CPT released was calculated with reference to a calibration curve and was normalized to the surface area of the sample to give the amount of CPT released per square centimeter. This allowed the CPT release data to be directly compared between each of the samples. A minimum of three release curves was averaged to produce the release curves. Release of CPT was performed in PBS at pH 7.4 and at pH 1.8. Despite the low solubility of CPT in aqueous solutions (14.2 ± 2.9 µM)36 sink conditions were maintained for all release experiments (maximum release concentrations were below 1.4 µM).

Cytotoxicity assay SH-SY5Y cells (human neuroblastoma cells), were cultured in DMEM supplemented with 10 % FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen) as previously described. SH-SY5Y cells were placed in wells of a 96-well plate at 15000 cells per well. After one day, the cultured cells were incubated with prepared CPT-loaded microparticles (the following set of samples was used: uncoated, coated for 15, 30 and 60 min with


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PFOpp). Microparticles at 100 µg/mL (100 µL/well) in DMEM (10 % FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin) were incubated for 24, 48 and 96 h at 37 °C and 5% CO2 prior to cell viability measurements. Controls were generated by incubating SH-SY5Y cells in DMEM without microparticles for an identical incubation period. To determine the effect of the microparticles treatment on cell viability, the percentage of live and dead cells, lactate dehydrogenase (LDH) released in culture supernatants was measured using an established assay (Abcam LDHCytotoxicity Assay Kit II) according to the manufacturer’s instructions. After 24, 48 and 96 h incubation with microparticles, 100 µL of the cell suspension was centrifuged at 600 x g for 10 min, and the supernatant was transferred into a 96-well plate. To each well, 100 µL LDH reaction mix (Abchem) was added. After 30 min of incubation at room temperature, the absorbance at 450 nm was measured. All experiments were repeated at least 3 times.



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3.

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Results and Discussion

pSi MPs have been previously fabricated from bulk silicon by electrochemical etching in ethanol and hydrofluoric acid (HF) mixtures, followed by the pSi film removal from the bulk silicon through electropolishing, achieved either at low HF

concentration

or

high

current

fragmentation, such as ball milling

37

density;

followed

by

mechanical

8

or sonication. In this work, we fabricated

pSi MPs using a multi-step square waveform etch for six cycles alternating between low and high current regimes where the high current regimes formed an electropolished region (sacrificial layer) in between the MP-forming layers. Finishing the etch with a high current (electropolishing) regime allowed easy harvesting of a multilayered pSi film in the form of flakes (Figure 1A). Those flakes were fractured into MPs by sonication. The resulting plate-shaped MPs were sized via the use of mechanical sieves resulting in a MP plate thickness and size of approximately 19 µm and 45 µm, respectively (Figure 1B-C). These particles are too large to be directly taken up by mammalian cells. However, they are not too large to deliver a payload to cells in their surrounding environment. Multilayers of particles, which had not separated along the sacrificial layer(s), were rare (Figure S1). The porosity of the pSi MPs was determined by gravimetric analysis as 84±2%. The pore size of the pSi MPs was 21±6 nm (Figure 2A). This procedure is similar to previous reports describing pSi NPs manufacture,38 but to the best of our knowledge has not previously been used for pSi MPs. Our fabrication method increases pSi MP production yield considerably since multiple MP layers can be etched repeatedly without the need to perform a separate electropolish and membrane harvesting after every etch. In this example, we etched into wafer to a depth of 114 µm. However, in principle it is possible to etch the entire 500 µm Si wafer.

Figure 1: A) Photograph of multilayered pSi film used for MP manufacture, B) SEM cross section of the multilayered pSi MP structure, the pSi layers (1 and 2) are clearly visible and are separated by the sacrificial layers (SL) and C) SEM of the sonicated and sieved pSi MPs.


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Whilst the etching current and time for the pSi MP in this manuscript were kept constant, these parameters can also be varied to generate different porous structures and pore sizes for the required application. It is important to note that pSi MPs were chosen over pSi NPs as pSi NPs tend to agglomerate upon drying which in turn prevents them from being coated individually in the plasma reactor due to it being a “dry” method. In addition, bouncing leads to loss of pSi NPs through the vacuum of the plasma system.

The pSi MPs were introduced in a plasma reactor on a loudspeaker and after applying vacuum and acoustic drive signal was turned on by playing AC/DC’s song “Thunderstruck” on a loop. The volume was slowly increased until the particles were visibly bouncing. The particles were then treated in a PFO plasma for 4 to 60 min to generate a “Teflon”-like coating while being constantly bounced to generate PFO plasma polymer (PFOpp) coated pSiMPs. “Teflon”-like coatings are often used in medical applications where an

inert

material

is

required,

such

as

stents39

and

catheters.40

Perfluorocarbons can be used as non-toxic fluids for liquid ventilation.41-42 SEM images show that a polymer film formed and pore clogging increased with increasing plasma treatment (Figure 2A-D).

Figure 2: SEM micrographs showing the as etched pSi MPs generated from multilayered pSi film (A) and pSi MPs coated with PFOpp for 15 (B), 30 (C) and 60 (D) min.

The bouncing of the pSi MPs during plasma polymerization ensured that all surfaces of the pSi MPs are coated with polymer. To highlight how important the use of the loud-speaker is to obtain a homogeneous coating an untumbled control was also run for 60 min and investigated by SEM (Figure S2). Pores on the under-side of the non-bounced PFOpp coated pSi MPs remained open whist those on the top are substantially clogged with PFOpp.



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TGA analysis of the pSi MPs coated with PFOpp showed 3 broad decomposition peaks at 255 oC, 373 oC and 459 oC (Figure 3). These peaks are likely due to the decomposition of various length PFOpp polymer chains generated by the plasma polymer deposition process. Due to the random nature of this process we would expect a distribution of polymer chain lengths, leading to multiple broad decomposition peaks.

Figure 3: Comparison of TGA of A) blank pSi MPs and B) PFOpp-coated pSi MPs.

XPS analysis (Table 1) provided further evidence for a polymer coating. The blank pSi MPs showed some small carbon (2.5 at.%) and fluorine (5.6 at.%) contamination from the etching process. The 15 min coating contained substantially more carbon (12.2 at.%) and fluorine (19.3 at.%) than the uncoated pSi MPs, indicating a successful coating with PFOpp. As the coating time progressed, more silicon and oxygen from the underlying pSi was eclipsed and more fluorine was observed as evidenced by the F/Si ratio increasing for each plasma polymerization time. Although the 60 min coated sample has a lower C content, the difference is a mere 1.3 at.% and may easily be the result of adventitious carbon deposition on the high-surface energy porous silicon. Importantly, the 60 min sample does have the highest F signal and the lowest Si and O signals. EDX was also used to support the XPS analysis and is supplied in the Supporting Information, Table S1 and Figure S3.

Table 1: Summary of atomic percentages and F/Si ratios by XPS for blank and PFOpp coated pSi MPs (n = 3). Sample Blank pSi MPs

Si (at.%)

O (at.%)

C (at.%)

F (at.%)

F/Si

28.8

63.2

2.5

5.6

0.19


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PFOpp (15 min)

22.4

46.2

12.2

19.3

0.86

PFOpp (30 min)

20.2

40.3

15.9

23.5

1.16

PFOpp (60 min)

19.3

37.7

13.6

29.4

1.52

ToF-SIMS imaging was used to corroborate the TGA and XPS data. Figure 4 presents positive ion ToF-SIMS images of the uncoated pSi MPs and the 4 and 60 min PFOpp-coated pSi MPs. The total intensity of the selected positive ion fluorocarbon fragments, CF2+, CF3+, C2F5+, and C3F7+ (denoted CxFy) appear at the noise level on the uncoated particles, whereas the total intensity of these fragments on the plasma polymer coated particles was high. This suggests that after plasma treatment the particles were coated with a thin layer of fluorocarbon coating. Furthermore, as can be seen from the image, the fluorine signal traces well the particles’ contours, a strong indicator for a homogenous coverage. Positive mass spectra over the mass range 0-200 m/z of the uncoated and coated particles are given in Figure S4. Selected positive ion fluorocarbon fragments were assigned to their peaks to highlight the differences between the two mass spectra.

Figure 4. ToF-SIMS images (500 x 500 µm2) acquired on the uncoated pSi MPs, the 4 min PFOpp-coated pSi MPs and the 60 min PFOpp-coated pSi MPs. Scale bar is 100 µm. CxFy represents CF2+, CF3+, C2F5+, and C3F7+ fragments.

Figure S4 shows a comparison between pSi MPs that were static during the PFOpp coating (60 min) with those that were bounced by playing “Thunderstruck”. The presence of regions that lacked CxFy signals in the static pSi MPs again indicated that particle bouncing was essential for homogenous coating.

Surface wettability and degradation rate are important parameters for drug



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Page 16 of 33

release from composite materials. We determined the sessile drop WCAs for equivalent flat silicon and pSi films (MP etching conditions, without the electropolishing step) coated with a PFOpp coating (Table 2).

Table 2: WCAs of uncoated and 4 min PFOpp-coated flat silicon and pSi (n = 6). WCA (o ± SD)

Material Flat silicon

62 ± 6

Flat silicon with PFOpp

101 ± 2

pSi-Ox

6±1

pSi-Ox with PFOpp

103 ± 4

We observed that the WCA increased significantly (to approximately 100o) after the PFOpp coating on both flat and porous surfaces; confirming the hydrophobic nature of the plasma polymer coating. The samples in Table 2 were prepared on un-tumbled planar pSi films. This was necessary to facilitate the analysis via WCA, which is not possible on pSi MPs as their roughness negates the ability to obtain precise WCA measurement. In the case of the pSi-Ox, the WCA increased by over 90o from 6o to 103o. These contact angles compare well with other literature values for thermally oxidized pSi (approx. 10 o)22, 43 and hydrophobically coated pSi (> 112 o).44 Whilst quite hydrophobic, the coated pSi MPs easily dispersed into cell culture media without any shaking and only required some gentle shaking to overcome the surface tension and get them wetted in PBS solutions.

A molybdic acid assay was used to determine the silicon degradation rate of coated and uncoated pSi MP. A comparison between the degradation rate over 12 days between the coated and uncoated pSi MPs can be seen in Figure 5. The comparison confirms that the PFOpp-coated particles markedly decreased degradation rate after PFOpp coating at just 0.48 %/day versus


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1.85 %/day for the uncoated blank pSi. This result also confirms that the coated pSi MPs remain biodegradable.

Figure 5: Molybdic acid assay measurements for blank pSi MPs and pSi MPs coated with PFOpp for 4 min incubated in PBS buffer at 37 oC and pH 7.4 (n = 3).

CPT loading was performed by vacuum drying CPT from a DMF solution into a dispersed pSi MPs. Estimated loading values were calculated to be 20 ± 1 µg CPT / mg pSi MPs. The 2 wt% loadings achieved here in this manuscript, compares with previous work with pSi MP loaded PLLA monoliths where we have successfully loaded at 1 mg / 50 mg of pSi (or 2 wt%).45 The average in vitro IC50 value (across 32 different cell lines) is 15.3 ng/mL.46-47 Assuming that the release experiments in PBS give a reliable estimate for the release curve in cell culture media then the maximum release values (after 3 days) obtained during the in vitro release experiments were approximately, 2 µg/mL, 2 µg/mL, 1.47 µg/mL, 1.21 µg/mL and 0.29 µg/mL for the uncoated, 4 min, 15 min, 30 min and 60 min PFOpp pSi MP samples, respectively, well above the IC50 of CPT. The CPT retention after plasma polymer coating was confirmed in a qualitative manner by means of fluorescence microscopy. Figure 6 shows a comparison between blank pSi MPs, CPT loaded pSi MPs, CPT loaded + coated pSi MPs and loaded + coated pSi MPs after 3 days of release in PBS. It is evident that the blank pSi MPs and those that have been in PBS for 3 days (Figure 6 A+D) lacked blue fluorescence from CPT while loaded and loaded + coated (Figure 6 B+C) exhibited strong fluorescence signals.

Figure 6: Fluorescence microscopy images of A) blank pSi MPs, B) CPT loaded pSi MPs, C) CPT-loaded pSi MPs coated with PFOpp for 4 min and D) CPT loaded pSi MPs coated with PFOpp for 4 min after 3 days of release in PBS at pH 7.4 and 37 oC. Scale bar is 200 µm.



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Figure 7 depicts CPT release kinetics from coated and uncoated pSi MPs. The reference uncoated particles displayed a rapid release rate of 25.36 %/h; releasing over 97.5% of the loaded CPT within half a day and giving complete release in less than 24 h. This release rate is attributed to the CPT being a hydrophobic water insoluble drug that diffuses slowly from the porous silicon network, and is similar to previous work with CPT loaded pSi films (74% in 16 h23 and 100% in 18 h22).

By comparison, the pSi MPs that were coated for a short 4 min with PFOpp exhibited an initial delay in release and slower release rate of 10.97 %/h; yet complete release was still observed in less than a day. Longer coating times (15, 30 and 60 min) resulted in progressively slower release rates (1.02 %/h, 0.84 %/h and 0.18 %/h). Particles coated for 15 min released 50% of CPT in approximately 1 day, whereas 30 min coated samples took 1.5 days for the same amount to be released. As for the 60 min coated particles, no more than 10% of CPT was released even after 3 days. Note that after the 3 day incubation at 37 oC in PBS there are still large regions of the PFOpp on the coated pSi MPs (Figure S6).

These release kinetics demonstrate that the benefits of these coated samples are twofold regarding CPT release retardation. Firstly, the release rates fully correlate inversely with overcoating times and the 5 examples show how the release rate can be adjusted by prolonged coating times. Second, longer coating times lead to a pronounced delay in release. For example, whereas the release was only mildly slowed for 4 min coated pSi MPs-PFOpp, it was strongly supressed in case of 60 min pSi MPs-PFOpp. However, this may not be a disadvantage as markedly different release rates are often desirable for different drug types; for example a faster but sustained one for pain medication but a slow and steady one for sub-dermal hormonal contraception.

Modeling of the release data was performed and the results are presented in


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Table 3 below. The uncoated pSi MPs released according to a first order kinetics whilst the release from coated pSi MPs fitted best to the Higuchi model. The good fit to classical Higuchi equation demonstrates that the release is diffusion controlled. Table 3: Comparison of R2 values for CPT release for different PFOpp coating times of pSi MPs. Sample

Zero-Order

First-Order

Higuchi

Hixon-Crowell

Uncoated

0.52746

0.93357

0.77109

0.2857

4 min 15 min

0.79085 0.87715

0.9433 0.97009

0.95147 0.98546

0.45317 0.5056

30 min 60 min

0.85087 0.81385

0.93491 0.8305

0.9854 0.97497

0.49726 0.46366

Figure 7: Comparison of CPT release for pSi-Ox and pSi-PFOpp MPs (n ≥ 2).

Figure 8 shows the cell viability in a LDH assay for all pSi-MP samples over a 4 day time course. The control (untreated) nuroblastoma cells and the cells treated with unloaded and uncoated pSi MPs, as well as unloaded and PFOpp coated (60 min) showed virtually no decline in viability over the 4 day time period. This confirms that neither the pSi nor the PFOpp coating by themselves exhibit a cytotoxic effect (at least for the LDH assay). In stark contrast, the uncoated pSi MPs loaded with CPT showed a rapid onset of decreased viability within the first day (35% viability). Viability was reduced to 5% after 2 days and to 0% after 4 days. For the coated pSi MPs, the delayed CPT release from the 15, 30 and 60 min coated particles translated into reduced and later onset cytotoxicity. The 15 and 30 min samples caused a comparable decrease in viability (to around 70% after 1 day and around 10% after 2 days). In comparison, the 60 min coated particles showed the slowest decrease in viability, which as not significant after 1 day, and the highest viability of all loaded samples after 2 days (25%). After 4 days all loaded samples resulted in complete cell death. These biological results coincide with the release kinetics discussed in the previous section (Figure 8) where longer coating times slowed down the drug release.



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Figure 8: Viability of SH-SY5Y human neuroblastoma cells upon incubation with uncoated pSi MPs with or without CPT loading, pSi MPs PFOpp-coated for 60 min without CPT loading and CPT loaded pSi MPs PFOpp coated for 15, 30 and 60 min (n ≥ 3).

Conclusions In conclusion, we have used biodegradable pSi MPs, which degrade upon contact with aqueous solutions, loaded with a drug of choice (for demonstration purposes in this study the cytotoxic CPT) and then plasma polymer coated them with a hydrophobic, “Teflon-like” over layer. This over layer was formed in a novel plasma reactor utilizing a loudspeaker to tumble the particles, thereby achieving a homogenous coating by playing AC/DC’s song “Thunderstruck”. The thickness of this hydrophobic coating could be controlled by the plasma polymerization time leading to the possibility to adjust the drug release kinetics from the pSi MPs. The pSi MPs preparation, loading, coating and subsequent drug release was verified using SEM, TGA, ToF-SIMS, water contact angle measurements, colorometric assay and fluorescence microscopy. All methods congruently confirm the successful CPT loading into the particles and subsequently the homogenous coating with a fluorinated plasma polymer. This over coating manifests itself in a markedly slowed drug release, which can be further adjusted by altering the plasma deposition time. A short plasma coating led to a 2-fold slower CPT release (10.97 %/hour vs 25.36 %/hour for the uncoated pSi MPs), whereas stepwise longer polymerization times led up to more than a 100-fold slower release (0.18 %/hour vs 25.36 %/hour). This slowed release was then shown in vitro by the delayed cell death onset in human neuroblastoma for all coated particles. This work has shown the feasibility of plasma-coated pSi MPs as a drug delivery system with adjustable release kinetics. This was demonstrated by the reduced release rate of CPT and resulted in a delayed cell death onset of human neuroblastoma in vitro.


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Hence, this novel controlled drug release type could span a wide range of applications; due to the adjustable release kinetics these may potentially range from short term applications such as sedation and pain relief all the way to longer term applications such as sub-dermal insulin or hormone delivery. The release rate’s fine adjustment was not this study’s aim but a mere proof of principle. However, a finer adjustment could be envisioned by using a more hydrophilic plasma polymer or a mixture between of hydrophobic and hydrophilic plasma polymers. This, among the study of loading and releasing other drug types as well as in vivo testing are aims for future work.

Acknowledgements The authors would like to thank Dr. John Denman for assistance with acquiring ToF-SIMS spectra and the authors acknowledge the facilities, and scientific

and technical

assistance of

the Australian

Microscopy &

Microanalysis Research Facility. The authors would like to acknowledge the use of the song “Thunderstruck” written and performed by AC/DC: Brian Johnson, Angus Young, Malcolm Young, Cliff Williams and Chris Slade. Published by Albert Productions. © 1990.

References 1. 2. 3. 4.

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22. McInnes, S. J. P.; Szili, E. J.; Al-Bataineh, S. A.; Vasani, R. B.; Xu, J.; Alf, M. E.; Gleason, K. K.; Short, R. D.; Voelcker, N. H., Fabrication and Characterization of a Porous Silicon Drug Delivery System with an Initiated Chemical Vapor Deposition Temperature-Responsive Coating. Langmuir 2016, 32, 301-308. 23. McInnes, S. J. P.; Szili, E. J.; Al-Bataineh, S. A.; Xu, J.; Alf, M. E.; Gleason, K. K.; Short, R. D.; Voelcker, N. H., Combination of iCVD and Porous Silicon for the Development of a Controlled Drug Delivery System. ACS Appl. Mater. Interfaces 2012, 4, 3566-3574. 24. Wu, J.; Sailor, M. J., Chitosan Hydrogel-Capped Porous SiO2 as a pH Responsive Nano-Valve for Triggered Release of Insulin. Adv. Funct. Mater. 2009, 19, 733-741. 25. Desmet, T.; Morent, R.; Geyter, N. D.; Leys, C.; Schacht, E.; Dubruel, P., Nonthermal Plasma Technology as a Versatile Strategy for Polymeric Biomaterials Surface Modification: A Review. Biomacromolecules 2009, 10 (9), 2351-2378. 26. Chu, P. K.; Chen, J. Y.; Wang, L. P.; Huang, N., Plasma-Surface Modification of Biomaterials. Mater. Sci. Eng., R 2002, 36 (5-6), 143-206. 27. Shi, F. F., Recent Advances in Polymer Thin Films Prepared by Plasma Polymerization Synthesis, Structural Characterization, Properties and Applications. Surf. Coat. Technol. 1996, 82 (1-2), 1-15. 28. Akhavan, B.; Jarvis, K.; Majewski, P., Plasma Polymer-Functionalized Silica Particles for Heavy Metals Removal. ACS App. Mater. Interfaces 2015, 7 (7), 4265-4274. 29. Akhavan, B.; Jarvis, K.; Majewski, P., Plasma Polymerization of SulfurRich and Water-Stable Coatings on Silica Particles. Surf. Coat. Technol. 2015, 264, 72-79. 30. Akhavan, B.; Jarvis, K.; Majewski, P., Development of Negatively Charged Particulate Surfaces through a Dry Plasma-Assisted Approach. RSC Adv. 2015, 5 (17), 12910-12921. 31. Michl, T. D.; Coad, B. R.; Hüsler, A.; Vasilev, K.; Griesser, H. J., Laboratory Scale Systems for the Plasma Treatment and Coating of Particles. Plasma Processes Polym. 2014, 12 (4), 305-313. 32. Griesser, H. J., Small Scale Reactor for Plasma Processing of Moving Substrate Web. Vacuum 1989, 39 (5), 485-488. 33. Coad, B. R.; Lu, Y.; Meagher, L., A Substrate-Independent Method for Surface Grafting Polymer Layers by Atom Transfer Radical Polymerization: Reduction of Protein Adsorption. Acta Biomater. 2012, 8 (2), 608-618. 34. Beamson, G.; Briggs, D., High Resolution XPS of Organic Polymers. John Wiley and Sons: Chichester, UK, 1992. 35. Szili, E. J.; Jane, A.; Low, S. P.; Sweetman, M.; Macardle, P.; Kumar, S.; Smart, R. S. C.; Voelcker, N. H., Interferometric Porous Silicon Transducers Using an Enzymatically Amplified Optical Signal. Sens. Actuators, B 2011, 160 (1), 341-348. 36. Sætern, A. M.; Nguyen, N. B.; Bauer-Brandl, A.; Brandl, M., Effect of Hydroxypropyl-Cyclodextrin-Complexation and pH on Solubility of Camptothecin. Int. J. Pharm. 2004, 284, 61-68. 37. Kafshgari, M. H.; Cavallaro, A.; Delalat, B.; Harding, F. J.; McInnes, S. J. P.; Mäkilä, E.; Salonen, J.; Vasilev, K.; Voelcker, N. H., Nitric Oxide-



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Supporting Information: SEM images of incorrectly formed pSi MPs. SEM images of static PFOpp coatings on pSi MPs. EDX analysis of PFOpp coated pSi MPs. ToF-SIMS spectra of uncoated and coated pSi MPs. ToF-SIMS image overlays of static and bounced PFOpp coated pSi MPs. SEM of pSi MPs and PFO coated pSi MPs after incubation in PBS for 3 days at 37 oC.


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TOC Image



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Figure 1: A) Photograph of multilayered pSi film used for MP manufacture, B) SEM cross section of the multilayered pSi MP structure, the pSi layers (1 and 2) are clearly visible and are separated by the sacrificial layers (SL) and C) SEM of the sonicated and sieved pSi MPs. 420x239mm (300 x 300 DPI)


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Figure 2: SEM micrographs showing the as etched pSi MPs generated from multilayered pSi film (A) and pSi MPs coated with PFOpp for 15 (B), 30 (C) and 60 (D) min. 435x308mm (300 x 300 DPI)



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Figure 3: Comparison of TGA of A) blank pSi MPs and B) PFOpp-coated pSi MPs. 187x257mm (300 x 300 DPI)


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Figure 4. ToF-SIMS images (500 x 500 µm2) acquired on the uncoated pSi MPs, the 4 min PFOpp-coated pSi MPs and the 60 min PFOpp-coated pSi MPs. Scale bar is 100 µm. CxFy represents CF2+, CF3+, C2F5+, and C3F7+ fragments. 495x382mm (300 x 300 DPI)



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Figure 5: Molybdic acid assay measurements for blank pSi MPs and pSi MPs coated with PFOpp for 4 min incubated in PBS buffer at 37 oC and pH 7.4 (n = 3). 287x201mm (150 x 150 DPI)


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Figure 6: Fluorescence microscopy images of A) blank pSi MPs, B) CPT loaded pSi MPs, C) CPT-loaded pSi MPs coated with PFOpp for 4 min and D) CPT loaded pSi MPs coated with PFOpp for 4 min after 3 days of release in PBS at pH 7.4 and 37 oC. Scale bar is 200 µm. 190x143mm (300 x 300 DPI)



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Figure 7: Comparison of CPT release for pSi-Ox and pSi-PFOpp MPs (n ≥ 2). 287x201mm (150 x 150 DPI)


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Figure 8: Viability of SH-SY5Y human neuroblastoma cells upon incubation with uncoated pSi MPs with or without CPT loading, pSi MPs PFOpp-coated for 60 min without CPT loading and CPT loaded pSi MPs PFOpp coated for 15, 30 and 60 min (n ≥ 3). 287x317mm (150 x 150 DPI)


âThunderstruckâ: Plasma-Polymer-Coated Porous Silicon

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