Rapid Synthesis of Drug-Encapsulated Films by Evaporation-Induced Self-Assembly for Highly-Controlled Drug Release from Biomaterial Surfaces

Abstract

Infection at the surgical site for dental implants results in failed procedures, patient pain, burdensome economic impact, and the over-prescription of prophylactic antibiotics. Mesoporous silica films as coatings for implants may provide an ideal antimicrobial drug storage and local release vector to the site of infection, however traditional drug loading techniques result in insufficient drug load and short-term release kinetics. In this work, we have applied a method to use a surfactant-antimicrobial drug octenidine dihydrochloride (OCT) as a template for mesostructured silica, to demonstrate silica-OCT composite films. The films are synthesized by evaporation induced self-assembly (EISA) and we explore the effects of synthesis parameters on porous film structure, OCT incorporation, and OCT drug release rates. Drug micelle incorporation into the silica mesostructure was highly dependent on silica precursor pre-reaction to form silica oligomers before film spin-casting. The OCT drug concentration of the synthesis solution dictated the time required for effective incorporation (without phase separation), with total loading in the film of up to 90 % by mass. The OCT content in the films was found to directly determine the timescale of drug release, from 2 to 8 h for a single layer film. The total release timescale was increased by the addition of multiple layers of OCT-silica films to nearly 2 weeks. Drug release from films completely inhibited Streptococcus mutans (UA159) growth, while drug-free porous silica films showed no increase in bacterial growth over non-porous control. These OCT-silica films have a significant potential to store and release antimicrobial drugs from dental implant surfaces.

1. Introduction

Concerns over implantable medical device-related bacterial infection have been well documented.¹ Non-degrading permanent and temporary implant materials, such as the titanium used in orthopedic and dental implants, provide an ideal surface for the attachment of pathogenic biofilms, allowing bacteria to proliferate and remain resistant to traditional systemic antibiotic therapies.^2–5 Dental implants are used to replace missing teeth with more than 5 million procedures annually in North America.⁶ Post-placement infections may result in pain, tenderness, swelling, delayed recovery and implant loss.^7–9 Local prevention of infection at the implant site has been the subject of intense research.^{7, 10–12} Prevention of bacterial colonization and proliferation at the interface between dental implant, gingiva and oral cavity, at the implant neck and abutment and below the crown (false tooth), may prevent infection during the first two weeks of wound healing.

Several methods have been proposed to impart infection control mechanisms to the surfaces of implants, including anti-attachment surfaces, surfaces with bactericidal moieties permanently attached or released by bacterial activity,^13–16 and more traditional antimicrobial drug eluting surfaces.^{12, 17} Direct mixing of insoluble drugs with monomers prior to polymerization, such as in commercially available antibiotic-eluting bone cements, can produce very high loads of drug (30% wt.) within the material, and are some of the only drug-eluting implant products in wide use.¹⁸ However, these drug molecules tend to aggregate, the release rate is low and declines rapidly (for example 24 to 2.4 μg cm⁻² d⁻¹ over 1 week¹⁹), or are permanently trapped in the matrix, which limits their timeframe of efficacy.^{18, 20, 21}

Mesoporous silica coatings, with their ordered array of pores, can provide a large reservoir of antimicrobial agent while controlling rate of release via pore size and tortuosity.^22–24 These coatings may be synthesized via evaporation induced self-assembly (EISA)²⁵ onto Ti implants. The resultant surfactant micelles (in ordered arrays) within the silica matrix may be removed to create pores, into which drugs can be loaded. In principle, this can create a drug release reservoir (of 50–60 vol%), with further modification possible using gate-keeper molecules for triggered release.²⁶ However, a major limitation to this approach is the limited internal drug loading that is typically achieved (generally < 1% wt.), due to low diffusion of drug into pores.),^27–32 with most of the drug just adsorbed on the surface.^33–36

We discovered that the surfactant-like self-assembly of certain antimicrobials can themselves direct the mesostructure formation to produce ‘drug-templated’ mesoporous silica, and avoiding post-synthesis drug loading altogether. Although this limits the potential range of applications to ones where these specific drugs are useful, many of the drugs are powerful antiseptics effective against oral pathogens. We have previously optimized this process to produce antimicrobial octenidine-silica co-assembled particles, through a precipitation synthesis.³⁷ Octenidine dihydrochloride (OCT) is a commercially-produced antimicrobial, common to mouthwash, wound cleansers, and mucous membrane antiseptic rinses. We have shown that these drug-loaded silica materials demonstrate extended-release rates compared to conventionally drug-loaded mesoporous silica. These templating OCT molecules are highly loaded (30–50 % wt.) and exhibit slowed release (t_1/2 = 21 days) due to the high amount of interaction between templating molecules and the pore wall, as well as the depth of loading within the silica to extend the drug release rates.³⁸

In this study we aim to synthesise this OCT-silica material as a uniform, scalable, antimicrobial surface coating through a 1-step EISA process (summary schematic in Figure 1). We hypothesize that increasing order of the templating molecule in the silica structure from disordered pockets to a pore network will extend the effective release of templating OCT, providing a new technology with potential for post-operative infection control applications on implants. We have investigated the effect of synthesis parameters on the structure and release properties of silica-OCT composite films to demonstrate control over antimicrobial drug loading and release rate, two critical factors in the use of long-term release vectors for dental implant infection prevention.

Figure 1:

A schematic comparing the drug-loading procedures for traditionally synthesized mesoporous silica and for drug-templated mesoporous silica on a dental implant neck and abutment. The drug templated process is ready for use immediately after the formation of mesoporous silica, while a traditional approach requires many additional steps to remove the templating surfactant and load drug. Furthermore, this drug loading typically causes an abundance of drug on the coating surface and a minimal amount is deposited within pores.

2. Experimental

2.1. Materials

All chemicals were used without further purification. Type 1 ultra-pure water was used at 18.2 MΩ cm produced in our laboratory (Direct-Q 3 UV, Millipore, USA). Octenidine dihydrochloride (OCT) was purchased from TCI America (USA), with purity confirmed by LC-MS (Xevo G2-XS QTof, Waters, USA). Tetramethyl orthosilicate (TMOS) was purchased from Sigma-Aldrich (Oakville, ON, Canada). Hydrochloric acid (6.0 N), sodium chloride, potassium chloride, disodium phosphate, and monopotassium phosphate were purchased from Bioshop Canada Inc. (Burlington, ON, Canada). Anhydrous ethanol was purchased through the University of Toronto MedStore (house brand, Toronto, ON, Canada).

2.2. Film synthesis

Synthesis solution molar ratios are made according to Table 1. OCT is dissolved in water or water and ethanol by sonication, before adding HCl and TMOS and mixing at 750 RPM for 30 min. Solutions are then aged according to experimental protocol at room temperature. Substrate Si wafers ([100] orientation, Wafer World, FL, US) or glass slides (VWR, ON, CA) are first cleaned with ethanol followed by oxygen plasma cleaning for 10 s (Harrick Plasma, NY, USA), then spin-coated with the synthesis solution at varying RPM. All wafers are coated with 90 μL of solution and allowed to spin for an additional 10 s after all the solution has been dropped. The samples are then left at 70°C for 24 h. The process was repeated for multiple layers, allowing 24 h between coatings.

Table 1:

OCT-templated mesoporous silica film synthesis molar ratios

Label	H2O	HCl [x103]	TMOS	OCT	EtOH
Et1	8.4	1.4	1	0.112	2.3
Et2 a)	8.4	1.4	1	0.112	2.9
Et3	8.4	1.4	1	0.112	3.5
Et4	8.4	1.4	1	0.112	4.1
Et5	8.4	1.4	1	0.112	4.6
H1	10.0	1.5	1	0.112	5.8
H2	10.0	10.0	1	0.112	5.8
H3	10.0	32.5	1	0.112	5.8
H4	10.0	65.1	1	0.112	5.8
H5	10.0	97.6	1	0.112	5.8
O1	15.7	1.4	1	0.062	2.9
O2	15.7	1.4	1	0.075	2.9
O3	15.7	1.4	1	0.087	2.9
O4	15.7	1.4	1	0.100	2.9
O5	15.7	1.4	1	0.124	2.9

2.3. Film characterization

Field emission scanning electron microscopy was performed at 1 kV accelerating voltage (SU8230, Hitachi, Japan) without further sample preparation, after fracturing films and substrates. Image processing software was used to analyse sample thickness from micrographs (ImageJ, NIH, USA). Thickness was measured at 3 points per image, three images per sample, and averaged between samples in the same group. X-ray diffraction (XRD) was carried out from 1 to 6 ° using a beam energy of 30 kV and current of 10 mA from a Cu K α radiation source with sample rotation (MiniFlex 600, Rigaku, Japan). Sample opacity was analysed using glass-slide substrates mounted in a custom absorptivity jig at λ_abs = 400 nm (Cary 60 UV-Vis spectrophotometer, Agilent, USA). Opacity samples were imaged using a stereomicroscope under identical conditions (Trinocular Stereo Microscope with 3 MP camera, AMScope, USA).

2.4. Drug release analysis

Film samples on silicon were submerged in 10 mL PBS with gentle orbital shaking. PBS was prepared via the Cold Spring Harbor protocol and adjusted to a pH of 7.2 via HCl. OCT concentration was monitored in 1 mL aliquots diluted in PBS as necessary at λ_max = 281 nm (Cary 60 UV-Vis spectrophotometer, Agilent, USA). Aliquots were replaced with equal volumes of PBS in the sample.

2.5. Antimicrobial analysis

2 cm² samples on silicon wafers were prepared using recipe Et2 (Table 1) after selection based on previous results in the study. Drug-free sample was prepared by calcining samples at 600°C for 6 hours. S. mutans UA 159 (ATCC/Cedar Lane Labs, Burlington, ON, Canada) was grown to mid-log phase at 37°C and 5% CO₂ in Todd Hewitt broth supplemented with 10% yeast extract (THYE, BD Biosciences, Mississauga, ON, Canada).³⁹ Samples were submerged in S. mutans in THYE supplemented with 10% sucrose. Specimens were incubated for 30 hours at 37°C and 5% CO₂, before media was aspirated and specimens were rinsed with PBS. Specimens were stained using a LIVE/DEAD BacLight Bacterial Viability Kit (Life Technologies, Thermo Fisher Scientific, USA) according to manufacturer’s instructions. Imaging was performed via fluorescence microscopy (BX63, Olympus, Japan).

2.6. Statistical Analysis

All statistical analyses were carried out with analysis of variance (ANOVA) and Tukey’s HSD post-hoc analysis via JMP 13 (SAS Institute, USA) with a minimum sample size of 3 per group and significance defined as p<0.05. Details are presented alongside each set of results.

3. Results and discussion

3.1. Synthesis and fabrication of films

We deposited films by spin-coating on either silicon wafers for XRD and SEM analysis and drug release studies, or glass slides for optical studies. Although these substrates may not be representative of the materials and geometries found clinically, they provide a consistent substrate that has minimal effect on the film properties, and thus have allowed for a more independent study of the films. Using the molar ratios of our previous particulate OCT-mesoporous silica synthesis as a starting point, an array of recipes were formulated with one reactant molar ratio varied in each series (Table 1, variations of Et: ethanol, H: hydrochloric acid, O: octenidine dihydrochloride). Solution Et2 was studied to assess the feasibility of spin coating silica-OCT solutions as it formed a homogenous solution and film. A range of spin-coat speeds were assessed.

In this film synthesis, the tetramethyl orthosilicate (TMOS) is a silica precursor, and the antimicrobial drug OCT acts as both a structure-directing template agent, and an encapsulated antimicrobial. Films are deposited at low pH to catalyse the reaction, while ethanol acts as a volatile solvent to drive ingredient concentration and subsequent self-assembly.²⁵ As the solution spreads across the surface of the spinning substrate, its thickness decreases and the volatile ethanol and water solvent mixture quickly evaporates and reactants are concentrated. In the ideal case, this results in the well understood evaporation-induced self-assembly (EISA) process,⁴⁰ where TMOS is locally concentrated at the outer surface of OCT micelles. At this locally high concentration, TMOS undergoes an acid-catalysed hydrolysis and condensation, polymerizing in the remaining space between micelles. This reaction proceeds until a sol-gel film interlaced with octenidine has formed on the surface.

Films of Et2 were analysed by SEM (Figure 2). Films were smooth and uniform across the substrate, with no visible agglomerates (Figure 2 A). Spin coat speed unsurprisingly had an inverse effect on film thickness of Et2 samples, but as speed was increased beyond 3000 RPM a minimum thickness of approximately 3.5 μm was observed (Figure 2 D). A spin-speed of 3000 RPM was used for all subsequent studies.

Figure 2:

Initial results using the Et2 synthesis parameters successfully produced smooth, porous films. An SEM micrograph in A) shows a large smooth surface with no cracking or major features. Increasing spin coating speed decreases the thickness of films (D), but there are diminishing returns after 3000 RPM (lettering signifies p<0.05 Tukey’s HSD post-hoc analysis, N=3 per group, error bars represent ± 1 SD).

3.2. XRD analysis of film structure

Mesopores present in silica produce XRD peaks indicative of the average pore-pore spacing in the particles.^41–44 This peak may be broad and low in the case of a disordered structure with loose control over average pore-pore spacing, or very sharp repeating peaks in line with well understood crystal structures.

In all O-series recipes, very sharp peaks at 2.68 and 5.12 degrees were seen that correspond to peaks seen in powdered pure OCT alone, along with a low, broad baseline diffraction peak (Figure 3 A). This indicated an abundance of free OCT in its original crystal form poorly incorporated into the film. This poorly incorporated OCT, whether on the film surface or in large pockets within the film, may be quickly solvated and released, and may result in structurally compromising voids in the film, and is thus undesirable for long-term release applications.

Figure 3:

XRD analysis of various film recipes demonstrate the effect of ingredient molar ratio on drug incorporation and order of a pore mesostructure. In A), XRD analysis of O series is displayed (O1-light red, to O5-dark red). Large diffraction peaks at 2.68 and 5.12 degrees are caused by crystalline OCT drug that are not incorporated in a mesostructure. B) shows the effect of aging a synthesis solution of Et2 (light orange-30 minutes, to dark orange-3 days), showing the elimination of crystalline drug diffraction peaks and an increasing, broad diffraction peak at 3.6 degrees. The effect of 1-day aging on the O series from A) is shown in C), with the elimination of all crystalline drug peaks. 3-day aging of Et samples (Et1-light blue to Et5-dark blue) shown in D) demonstrating the strongest mesopore peak from Et 2 and 3. 3-day aging of H samples (H1-light green to H5-dark green) shown in E) demonstrating the strongest mesopore peak from low HCl concentrations. All y axis are in relative arbitrary units.

It is well understood that dilute mesoporous EISA synthesis solutions still experience some hydrolysis and condensation of silica precursor, resulting in branched silica oligomers in solution.⁴⁵ Therefore, solutions of Et2 were aged for D0–3 (days) and cast, before analyzing by XRD. D0 samples exhibited strong unincorporated OCT peaks as expected (Figure 3 B). However, after aging to “pre-react” the synthesis solutions prior to casting, Et2 samples displayed no OCT peaks, and showed a broad low peak at 3.6 degrees that grew stronger with increasing solution aging. This peak matched that seen for OCT-mesoporous silica nanoparticles.³⁷ The formation of silica precursor oligomers is important, and affects the species’ mobility, charge density, solution pH, and ability to integrate micelles of OCT under the more rapid reaction conditions during deposition, where instantaneous evaporation of volatile solvents increases the concentration of all solution components and drives self-assembly.⁴⁵ Acid catalysed hydrolysis of TMOS will result in positively charged silica precursor, which may be electrostatically repulsed by the positively charged OCT micelles in solution, leading to agglomeration of OCT.⁴⁶ With pre-deposition hydrolysis and condensation of TMOS species, the resultant silica oligomers should have a lower charge-per-silica atom and allow for increased OCT micelle incorporation in the film. O-series samples re-cast after aging for 1 day did not display OCT and contained a corresponding mesophase peak that increased in intensity with increasing OCT concentration (Figure 3 C).

Concentration of ethanol and hydrochloric acid were also altered (Figure 3 D and E respectively). Ethanol concentration will directly affect synthesis solution component concentration, the rate of pre-deposition reaction of silica during aging, and the viscosity of the cast solution. HCl acts to lower pH as a catalyst for the hydrolysis and condensation of TMOS, and therefore also affects the electrostatic interactions between templating OCT molecules, their micelles, and silica. Mid-range ratios of ethanol (Et2 and Et3) resulted in the most defined mesophase peak, while low concentrations of HCl yielded the most defined peak. The presence and strong definition of a peak indicates the presence of a porous structure, with increasing intensity and definition the result of an increasing mesophase signal. An extremely sharp peak would indicate a highly ordered mesostructured following a crystal arrangement.

Across all materials containing a mesophase peak, this peak is low and broad, in contrast to some mesoporous materials that exhibit sharp peaks indicative of a wide range of crystal structures.⁴² These broad, singular peaks are similar to those seen by other researchers producing disordered mesophase materials,^{47, 48} and differ significantly from the peaks observed when an ordered repeating pore structure is present. This disordered structure indicated by XRD will impact the path OCT must diffuse through during release, and therefore significantly affect release rate and the material’s biological applications.

3.3. Effect of drug concentration and aging on drug incorporation in films

Mesoporous silica films with discrete and continuous mesophases tend to be optically clear due to the nano length scale of the micelle and pore structure.²⁵ When templating material is instead present in large crystalized agglomerates, these scatter light and cause the film to appear cloudy. These poorly incorporated pockets of OCT may compromise the film integrity and lead to uncontrolled burst release of drug, and therefore cloudy-films may indicate unsuitable synthesis parameters for implant coating. We have used transparency to analyse silica-OCT composite films on glass slides through light absorbance to determine variations in OCT incorporation in the film mesophase.

New octenidine-silica-series synthesis solutions (abbreviated OctSi) were prepared according to Table 2 and aged until the solution reacted to the point of forming a solid gel (aging time denoted by “D[time in days]”). Films were cast every day until gelation, imaged via light microscope (Figure 4 A), and analysed for light absorbance at λ_abs = 400 nm (maximum absorbance, Figure 4 B). Si-film (zero OCT) and Oct-film (zero TMOS) were cast as controls. From Figure 4 A, several general trends may be observed. First, increasing the concentration of OCT in synthesis solutions allows for increased aging time without the complete reaction of silica and gelation of the solution. In fact, the experiment was stopped before solutions OctSi9 and OctSi10 gelled. This is in line with previous studies that have shown that some synthesis solutions react at an extremely slow rate over a course of months.⁴⁵ This may be due to OCT micelles sequestering TMOS or decreasing the mobility of silica oligomers, both limiting their rate of reaction.

Table 2:

Molar ratios of OCT- and aging-variation films

Label	H2O	HCl [x103]	TMOS	OCT	EtOH
OctSi1	8.4	1.4	1	0.02	10.0
OctSi 2	8.4	1.4	1	0.04	10.0
OctSi 3	8.4	1.4	1	0.10	10.0
OctSi 4	8.4	1.4	1	0.20	10.0
OctSi 5	8.4	1.4	1	0.30	10.0
OctSi 6	8.4	1.4	1	0.36	10.0
OctSi 7	8.4	1.4	1	0.42	10.0
OctSi 8	8.4	1.4	1	0.48	10.0
OctSi 9	8.4	1.4	1	0.54	10.0
OctSi 10	8.4	1.4	1	0.60	10.0
Oct-film	8.4	1.4	0	0.30	10.0
Si-film	8.4	1.4	1	0	10.0

Figure 4:

Drug concentration and aging of synthesis solution both affect the opacity of resultant films. Films from the B series of synthesis parameters aged for various times and deposited on glass, photographed over a 5 mm University of Toronto crest to demonstrate cloudiness with increased drug concentration, and increased clarity with aging (A). Absorbance of these films at λ_{400 nm} is shown in B), quantifying the trend observed in A). N=3 per group, error bars represent ± 1 SD. Cross-sections of OctSi-series samples (Table 2) were also analysed by SEM for thickness and structure (Figure 5). Sample OctSi8 D0 appears to have a large amount of rough deposited material on the surface of the film (Figure 5 C), but does not appear on samples aged for 4 d (Figure 5 D). The poorly incorporated OCT micelles are deposited on the surface of the film due to separation of the silica and OCT during deposition, resulting in the cloudy appearance seen in Figure 4 A, and the large XRD peaks indicative of crystalline OCT from Figure 3. No external material is seen in other clear films (OctSi5 D0 and OctSi10 D6, Figure 5 B and E respectively). In general, increasing drug content within the films results in increasing film thickness (Figure 5 F). This is especially interesting when comparing OctSi8 D0 and D4, where increased aging and incorporation of OCT in the film results in a thicker silica-OCT composite layer. Increased OCT mesophase content and a similar amount of deposited silica likely result in an increase in the overall thickness of the film. Increased film thickness may in turn increase the signal and peak height in XRD analysis.

The second trend is that film cloudiness is generally linked with increasing OCT concentration in the synthesis solution, and low aging time, further corroborating the XRD results presented in Figure 3. Silica-OCT composite films with low amounts of OCT in the synthesis solution show high optical transparency regardless of aging time, suggesting that low amounts of OCT micelles are easily incorporated into the silica as a mesostructure. However, at high OCT concentrations in synthesis solution, silica pre-reaction is necessary to optimize micelle incorporation. Finally, solution aging appears to have an optimal point to produce optically clear films (that have well-incorporated mesophases of OCT), as seen in the increasing cloudiness of OctSi9 and OctSi10 D5+ samples. The increasing viscosity of these samples as silica oligomer size increases may limit the mobility of OCT micelles and silica, slowing the rapid organization and reaction during deposition, resulting in agglomeration of both silica and OCT in the final film.

Therefore, films with application potential appear to be synthesized at low concentrations of OCT in the synthesis solution, with increasing synthesis OCT requiring aging to allow micelle incorporation in the final films.

3.4. Film drug content and release

The effect of drug templating on drug load and release was analyzed (Figure 6 A). With a film of deposited OCT alone as a control (Oct-film), samples submerged in neutral buffer exhibited a range of release rates. OctSi5 D0 exhibited the fastest release kinetics of the mesoporous films, with total release under 2 h. Furthermore, the total release is less than that of the pure OCT drug film. This is unsurprising given the film’s similar thickness to the pure drug film, but lower ratio of drug. The fast release of OCT from OctSi5 D0 films is likely due to the lack of extensive synthesis solution aging, resulting in a poorly developed mesostructure and excess OCT deposited in pockets or on the film surface. OctSi8 D0 films are significantly thicker than OctSi5 D0, resulting in a higher total drug release and longer release times (6 h). Aging OctSi8 synthesis solutions (OctSi8 D4) to allow full mesostructure development and drug incorporation extended release to approximately 8 h. OctSi10 D6, despite being slightly thinner than OctSi8 D4, showed a similar total amount of OCT release and similar kinetics, suggesting that there is a uniform mesoporous structure through the film. Spacing of mesopores as indicated by XRD peak location remained constant with OCT concentration changes and therefore the density of mesopore packing is unlikely a factor (Figure 3 A, C).

Figure 6:

Aging samples may extend release duration but does not appear to effect drug encapsulation efficiency within films. Cumulative release from B series films is shown in A), with Oct-film (x), OctSi5 D0 (▲), OctSi8 D0 (♦), OctSi8 D4 (●) and OctSi10 D6 (■). The presence of silica prolongs drug release compared to a cast drug solution alone, and increasing drug content results in increased release. Aging synthesis solution prolongs release further. Encapsulation efficiency (B), or the amount of deposited drug that should theoretically be contained within films as determined by thickness and silica-drug ratio, is greater at lower concentrations of drug, but does not appear to change between higher drug concentrations or with aging (asterisk p<0.05 utilizing Tukey’s HSD post-hoc analysis, N=3 per group, error bars represent ± 1 SD).

Drug encapsulation/incorporation efficiency (EE), as measured by drug to silica ratio in the film versus the synthesis solution, is inversely related to the amount of drug in the synthesis solution (Figure 6 B). Values are calculated from total OCT release, film thickness, and the relative densities of OCT and silica.³⁷ With lower initial concentrations of drug, OctSi5 D0 samples obtain slightly higher efficiencies than B8 D0, OctSi8 D4 and OctSi10 D6 samples due to the relatively constant pore spacing between samples and therefore constant drug density within mesoporous silica. Less-than 100% EE can be attributed to the separation of solution components on the substrate surface, or the evaporation and spreading of solvent during film carrying away excess unincorporated drug. In general, OCT concentration in synthesis solution must be a balance between achieving an optimal mesostructure for release kinetics, a high load, and the amount of excess wasted drug.

3.5. Multi-layer films and drug release

With seemingly limited difference in drug load, encapsulation efficiency, and release rate from increasing initial OCT concentration, the most obvious method for increasing drug load and release timeframe is the layering of multiple films to increase the total film thickness and therefore drug payload. This may increase the duration of drug dosage to a wound healing site, for example. The Et2 synthesis recipe (Table 1) aged for 3 d was chosen as a base recipe for multi-layer analysis, since it resulted in homogenous films with a strong mesophase. Each layer was deposited and allowed to dry for 24 h before depositing the next layer. Each layer was cast from a separate synthesis solution aged for 3 d each. Films were analysed by XRD and for OCT release rate (Figure 7).

Figure 7:

Layering of well-ordered drug-mesoporous silica increases total drug payload and prolongs the duration of predictable drug release. XRD of multiple layers of 3-day aged Et2 recipe in A) shows a good degree of order from 1 layer (L1, light purple) to 5 layers (L5, dark purple), with an approximate increase in intensity as layers are increased. Cumulative release (B) increases and is prolonged with increasing layers from 2 layers (■) to 5 layers (♦) to 6 layers (●). C) The release from a 6-layer sample may be fit to a t^0.5 model with good fit during the initial phases of release when drug diffusion from the mesoporous silica material follows the Higuchi model. N=3 per group, error bars represent ± 1 SD.

Increasing the number of film layers increases the intensity of the XRD diffraction peak (height difference between peak and height at 2 degrees, Figure 7 A). This is easily explained by the increasing thickness of the film resulting in a stronger diffraction intensity from an increasing number of mesopores.³⁷

Released mass and timeframe increases with additional layers of film (Figure 7 B), approaching the previously mentioned two-weeks of effect that would be desired during dental implant wound healing. However, total OCT release is not linear with increasing layers, implying that some drug is lost between films. This could be due to the solvation of the previous layer’s surface OCT and removal during the deposition of the next layer. Release timeframe is extended from approximately 2 to 10 d from 1-layer to 6-layers. The greater film thickness combined with well defined continuous mesophase results in an increase in diffusion path length within the film, both for penetrating water into the pore prior to drug solvation, and solvated drug from the pore.³⁵ Release follows the Higuchi formula,^{35, 36} (Figure 7 C) with release of 709.53 μg cm⁻² day^−0.5. The goodness-of-fit of this model suggests that release closely follows the Higuchi equation, as has been seen previously in drug-templated mesoporous silica materials.³⁷ The addition of more layers should result in the same release profile, but should follow that profile for a longer period of time for a larger cumulative release of OCT as none of the rate-controlling parameters within the film are altered (drug solubility, diffusivity, concentration of drug in the substrate, substrate porosity, and pore tortuosity factor should remain unchanged). This allows some degree of customization of the release duration and total dose to meet various application demands. In addition, application of the coating on rough or porous implant surfaces may further increase the diffusion path of the drug, extending release duration.

3.6. Antimicrobial efficacy of released drug and films

A preliminary antimicrobial study was performed to confirm retention of antimicrobial capability of OCT incorporated in the silica films, and to demonstrate the potential capability of the material to inhibit biofilm growth. Samples were prepared following Et2 molar ratios (Table 1) and a single layer of film on silicon substrates, with silicon alone and drug-free calcined samples as controls. Streptococcus mutans, an oral pathogen frequently associated with the early colonization of surfaces in the mouth and implicated in early biofilm formation, was used as a demonstrative target.^{37, 49} S. mutans is easily cultivatable, and its susceptibility to OCT is similar to that of other oral and non-oral pathogens.^{50, 51} Live/dead stained fluorescence microscopy images of sample and control groups are shown in Figure 8. Both silicon wafer (Figure 8 A) and drug-free calcined porous silica (Figure 8 B) show strong S. mutans proliferation and healthy biofilms. Mesoporous silica after its drug reservoir is exhausted did not increase bacterial proliferation. Nanoscale pores an order of magnitude larger than the ones templated by OCT (20 nm) were previously shown to increase the attachment of some bacterial species and decrease attachment of others.^{37, 52} It may therefore be important to study bacterial proliferation and biofilm formation in an orally relevant multi-species system on this material in the future, as well as bacterial and biofilm attachment strength to the surface.

Figure 8:

Mesoporous silica films containing OCT drug inhibit attachment and proliferation of S. mutans. Biofilm growth was investigated using a fluorescence microscope and a live/dead stain (live-green, dead-red). S. mutans readily attaches and forms biofilms over Si substrate (A), as well as drug-mesoporous films that have been calcined to remove OCT drug (B). However, drug released from intact Et2 samples on Si (C) completely inhibited biofilm formation, killing all bacteria on the surface of the material.

Silica-OCT composite films completely inhibited bacterial proliferation and biofilm formation on sample surfaces over the short-term, likely through the inhibition of planktonic cells in the incubation medium (Figure 8 C), agreeing with previous results.^{37, 53} This confirms that the octenidine incorporated into the mesoporous structure retains its antimicrobial capability after release. The minimum inhibitory concentration of OCT against S. mutans is 2 μg mL⁻¹.³⁷ It is therefore unsurprising that growth is inhibited with the release of hundreds of micrograms per cm² per day at the material surface. These results indicate good ability to prevent colonization of a coated implant surface by released OCT in the short-term, and ability to dose the surrounding tissue to prevent infection, however further work must be done with multi-layer samples to extend length of action, and confirmation of attachment to a realistic substrate surface such as titanium. Future study in this direction may be able to correlate these longer-term release profiles with inhibition of surface colonisation by bacteria, even with levels of release that do not inhibit surrounding planktonic bacteria, as has been seen with release from dental resins containing octenidine.³⁷

4. Conclusion

We have demonstrated the use of OCT as a pore-directing agent in the synthesis of mesoporous silica films by spin-coat deposition. Through the variation of several synthesis parameters, silica-OCT composite films with OCT wholly incorporated in the silica mesostructure were obtained under specific conditions. The inclusion of OCT in the pore mesophase extended the drug release due to the increased diffusion path for drug dissolution. Alternatively, the disordered sol-gel encapsulation approaches allowed OCT to be easily solvated by penetrating media when bulk pockets of drug are exposed and quickly released in an uncontrolled and unpredictable manor. Direct pore templating by micellizing drug molecules overcame these limitations. Although the present study was limited to the investigation of OCT, other effective self-assembling drug molecules may function well in this role, however it should be noted that not all bioactive compounds will produce mesoporous films suitable for drug release.

We propose using these highly-loaded OCT/silica composite films as antimicrobial coatings for dental or orthopedic implants, to prevent post-operative infection. However, direct templating of mesoporous silica films by any suitable molecule of interest may be applied to a wide variety of long-term release applications. There is still crucial work to be done to demonstrate the synthesis and efficacy of these coatings in a more clinically representative setting and on clinically relevant geometries and materials to analyse the coatings’ physical properties, interfacing with host tissues, and adaptability to a dip-coating synthesis. Future work should also more realistically model the oral flora and rate of salivary dilution to assess the surface inhibition of bacteria. Despite the current limitations, this study provides a rational for continued investigation of drug-silica co-assembled coatings for long-term drug delivery.

Figure 5:

Drug concentration and aging of synthesis solution greatly affects film thickness and morphology. Cross-sectional SEM images of fractured OctSi series film edges on glass are shown in A) through E), with the glass substrate marked by an s when visible, the film edge marked by e, and the film top by t. Images are of A) Si-Film; B) OctSi5 D0; C) OctSi8 D0; D) OctSi8 D4; E) OctSi10 D6. Film thicknesses were measured by separate directly edge-facing images and are summarized and compared in (F), with the general trend that increasing drug concentration and aging time (D – days) increases film thickness (lettering signifies p<0.05 utilizing Tukey’s HSD post-hoc analysis, N=3 per group, error bars represent ± 1 SD).

Data Availability

The raw/processed data required to reproduce these findings will be shared upon request.