Biogenic Nanostructured Porous Silicon as a Carrier for Stabilization

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Biogenic Nanostructured Porous Silicon as a Carrier for Stabilization and Delivery of Natural Therapeutic Species Nguyen T Le, Jhansi R. Kalluri, Armando Loni, Leigh T. Canham, and Jeffery Lee Coffer Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00638 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 8, 2017

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Molecular Pharmaceutics

Biogenic Nanostructured Porous Silicon as a Carrier for Stabilization and Delivery of Natural Therapeutic Species Nguyen T. Le,a Jhansi R. Kalluri,a Armando Loni,b Leigh T. Canham,b,c Jeffery L. Coffer*a a

Department of Chemistry and Biochemistry, Texas Christian University, Fort Worth, TX, 76129, USA b

pSiMedica Ltd., Malvern Hills Science Park, Geraldine Road, Malvern, Worcestershire WR14 3SZ, UK c

Nanoscale Physics, Chemistry, and Engineering Research Laboratory, University of Birmingham, Birmingham, UK. ABSTRACT: Nanostructured mesoporous silicon (pSi) derived from the silicon-accumulator plant Tabasheer (Bambuseae) is demonstrated to serve as a potential carrier matrix for carrying and stabilizing naturally-active, but otherwise metastable, therapeutic agents. Particularly, in this study, garlic oil containing phytochemicals (namely allicin) that are capable of inhibiting Staphylococcus aureus (S. aureus) bacterial growth were incorporated into Tabasheer-derived porous silicon. Thermogravimetric analysis (TGA) indicated relatively high amounts of the extract (53.1 ± 2.2 wt%) loaded into pSi is possible by simple infiltration. Furthermore, by assessing the antibacterial activity of the samples using a combination techniques of agar disc diffusion and turbidity assays against S. aureus, we report that biogenic porous silicon can be utilized to stabilize and enhance the therapeutic effects of garlic oil for up to 4 weeks when the samples were stored under refrigerated conditions (4OC) and 1 week at room temperature (25 o C). Critically, under ultraviolet (UV) light (365 nm) irradiation for 24 hour intervals, plantderived pSi is shown to have superior performance in protecting garlic extracts over porous silica (pSiO2) derived from the same plant feedstock or extract-only controls. The mechanism for this effect has also been investigated. Keywords: porous silicon, antibacterial activity, natural products, drug delivery. 1.

Introduction

With recent advances in nanoscience, studies of new drug delivery systems based on nanostructured materials have emerged which focus on targeted delivery and controlled release of therapeutic agents to ideally reduce detrimental side effects.1,2 Retention of physicochemical stability of the active compounds to be released is also an essential element in the development of any nanoscale smart delivery system.3 This is typically achieved by inhibiting local

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environmental diminution (by pH, temperature, or the presence of other selected small molecules) of therapeutic effects of the active agents prior to reaching their targets.4 Among possible candidates for drug delivery vehicles, porous materials with large surface areas have gained a lot of attention, owing to the range of porosities and diverse surface chemistry that allows drug incorporation through various approaches.5-8 Porous materials with different pore sizes can be employed to adjust the loading of the molecules of interest.8 Such systems can be efficiently utilized in delivering therapeutic amounts of the desired therapeutic agents to the targeted disease sites, and numerous high porosity materials have been demonstrated in vitro to be capable of acting as a useful vehicle in drug delivery systems.8-12 Among possible candidates for nanoscale drug delivery carriers, porous silicon is a logical choice. Since the biocompatibility of porous silicon was first demonstrated in the mid 1990s,13 studies of biomedically-relevant applications of porous silicon have been extensively conducted, not only in the areas of drug delivery11,12, tissue engineering,14-16 and biosensing devices.17 In an aqueous environment, through an oxidative hydrolysis process, porous silicon degrades into soluble orthosilicic acid [Si(OH)4] which is a common bioavailable form of silicon in physiological systems and can be readily excreted from the body.18,19 Furthermore, the degradation rate of pSi can be harnessed by surface chemistry and porosity.20 pSi with tunable pore sizes and biodegradability have significant appeal as a novel host matrix for drug delivery. The most common method for porous silicon fabrication currently involves anodization of single crystal Si in HF-based solutions,21 with open circuit etching in acid/HF mixtures gaining some appeal.22 Both methods require the use of corrosive HF and organic solvents, and in the case of anodization, expensive Si source material.23 Alternatively, high surface area mesoporous silicon can be readily achieved through a more eco-friendly synthesis route using siliconaccumulator plants as the starting materials. Particularly, in our studies, porous silica derived from Tabasheer powder, which is extracted from the nodal joints of the Bambuseae plant, was reduced magnesiothermically to produce mesoporous silicon.24,25 Ideally porosity and pore size of pSi prepared by this route can be altered by monitoring reduction time, temperature, and the quantity and morphology of thermal regulator (NaCl). Our interest here is to assess the ability of this new biogenic porous material to act as a drug carrier to stabilize otherwise metastable therapeutics. In this work, we describe the incorporation of a natural therapeutic, the oil extract from garlic (Allium sativum), into pSi itself derived from a silicious extract obtained from the nodal joints of bamboo. Previous studies have explored the mechanism of the antibacterial and antimicrobial functions of the extract; in particular, the protection mechanism of the garlic extract against bacterial attack is triggered when the garlic tissue is damaged, from which the enzyme alliinase is readily released and catalytically converts alliin into an active antibacterial organosulfur compound known as allicin.26,27 Interestingly, and somewhat more controversial, this extract is also reported to possess a range of other protective health benefits, including hypolipidemic, hypertension-reducing, and immune-enhancing effects.27,28 Nevertheless, allicin has been


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reported to be highly unstable as it rapidly degrades under ambient conditions and cannot be detected once the extract is orally consumed, thus decreasing its bioavailability.28,29 Hence, in this study, we chose this metastable therapeutic species in an evaluation of pSi’s ability to stabilize the activity and deliver the active agents found in the extract against Grampositive Staphylococcus aureus (S. aureus), which is well-known for its role in deleterious infections, food poisoning, and antibiotic resistance.30 Here we specifically compare the ability of the allicin-loaded, biogenic derived nanoscale carrier with several relevant controls to inhibit bacterial growth as a function of different storage conditions. The role of composition of the nanoscale carrier itself is also evaluated by comparing plant-derived porous silica (pSiO2) with the plant-derived porous silicon product (pSi) as well, thereby providing insight regarding the role of light absorption/stabilization in protective properties of the porous drug carrier. 2. Experimental Section 2.1 Materials and Methods Tabasheer pieces were obtained from Bristol Botanicals Ltd. and ground to a powder using a ball mill. Fresh garlic bulbs were purchased from a local grocery. X-Ray Diffraction (XRD) data was obtained using a Phillips 3100 X-Ray Powder Diffractometer with Cu Kα radiation operating at 35 kV. Raman spectroscopic analysis was performed with a DeltaNu system with a laser source of 785 nm. A Seiko Instruments TG/DTA 220 was utilized to determine the weight percent of extract loaded within a given pSi sample. Field Emission Scanning Electron Microscope (FE SEM) and Energy Dispersive X-ray (EDX) analysis was conducted with a JEOL JSM-7100F operating at 15 kV. Transmission electron microscope TEM imaging was performed using a JEOL JEM-2100 Electron Microscope. Ultraviolet Light Irradiation studies were conducted on selected samples with a Black-Ray B-100 UV lamp with wavelength of 365 nm and an irradiance intensity of 8,900 µW/cm2. Differential Scanning Calorimetry (DSC) analysis was carried out using a Mettler Toledo DSC-1. Liquid Chromatography-mass spectrometry (LC-MS) was performed using Agilent Technologies 6224 TOF LC/MS system. 2.2 Synthesis of Porous Silicon Biogenic porous silicon derived from Tabasheer was prepared in a four step process. Tabasheer powder was initially washed with 10% hydrochloric acid at 100oC. The dried sample was spread evenly on a quartz boat and was calcined at 500oC under an oxygen gas flow. The resulting silica sample was then mixed thoroughly with magnesium metal powder (SiO2:Mg 1:2 w/w), and sodium chloride acting as thermal moderator was blended in the above mixture (1:1 w/w). This sample was heated in argon gas at 600oC, and the reduced powder was leached in 37% HCl to remove any residual Mg metal and MgO by-products to obtain the porous silicon. Each step described above was performed for a 2 hour duration.24,25



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A combination of techniques, XRD, Raman Spectroscopy, FE SEM, TEM and DSC, was utilized to characterize the porous silicon produced in these experiments. 2.3 Identification of Allicin in Garlic Extract In order to confirm the presence of the active component, allicin, in the garlic oil, the chemical constituents of the extract were analyzed by LC-MS. For sample preparation, 1.2 g of the crushed garlic was mixed in 50 ml of ethanol for 10 minutes in order to extract the garlic oil. Once any garlic bulb debris was removed by vacuum filtration, 1 ml of the filtrate was further diluted with 1 ml of Ethanol. For analysis, a clear sample was obtained by further filtering the solution through glass wool. The solution was then analyzed using LC-MS. 2.4 Loading Garlic extract into Porous Silicon and Porous Silica Garlic bulbs were mechanically crushed to isolate the oil, which was then mixed with the pre-weighed pSi powder (6:1 w/w) for 30 minutes at room temperature. The loaded sample was then dried in vacuo overnight. For sample characterization, the dried product was then ground into a fine powder. For UV irradiation studies, freshly-extracted garlic oil was loaded into pSi and pSiO2 in the same manner as described above, with an oil : matrix ratio adjusted to 4:1 w/w. 2.5 Sample Storage For shelf-life assessment, the extract-loaded pSi and garlic oil samples were stored in the dark under ambient air (23oC, 60% RH) and under refrigerated conditions (4oC, 72% RH, dark) for periods of 1, 2 and 4 weeks. 2.6 Antibacterial Assays The antibacterial activity of the extract-loaded pSi was evaluated by two different methods: agar disc diffusion and turbidity assays against S. aureus. For the garlic oil-only controls, the amount of the free extract dosed in the experiment is comparable to the theoretical amount of garlic oil loaded into pSi. For the agar disc diffusion method, the efficacy of the samples against S. aureus was established based on inhibition zones of bacterial growth. In a typical experiment, 8 mg of garlic oil-loaded pSi was added onto sterilized filter disks (d = 6 mm) placed on top of a Lysogeny broth (LB) agar disc containing 100 µl of S. aureus (107 CFU/ml), and then the plates were incubated at 37oC for 24 h. The activity of the samples was quantitatively determined from inhibition zones in terms of mass-normalized diameter. In order to evaluate the antibacterial activity of the samples in an aqueous environment, turbidity assays, from which the extent of bacterial growth correlated to the turbidity of the sample-containing solution, were employed. For this method, 2 ml of LB was added into a 15-ml sterile centrifuge tube containing 10 mg of garlic oil-loaded pSi followed by 20 µL of S. aureus


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(107 CFU/ml). The samples were then incubated in a shaker incubator at 37oC for 4 h. After 4 h incubation, the supernatant was withdrawn from the test tube, and the turbidity of the solution was determined by measuring the absorbance at 600 nm.

2.7 Ultraviolet Light Irradiation Studies After the first 24 h period of antibacterial testing, equal amounts of oil-loaded pSi and pSiO2, along with free garlic extract, were irradiated under UV light at 365 nm, and another separate set of samples were stored in the dark for 24 h intervals. The antibacterial activity of the samples was evaluated in the same manner as described above.

3.

Results and Discussion 3.1 Characterization of Tabasheer-derived pSi

Initial experiments were performed with washing the feedstock Tabasheer powder thoroughly in 10% HCl to remove any metallic residues contaminating the sample during the cultivation process. Due to significant levels of plant remnants in the material, the plant powder was further calcined, thereby, burning off the organic components of the plant. The white appearance of the calcined sample is consistent with the formation of porous silica product. This material was subsequently reduced to elemental Si in the presence of Mg (Eq. 1); the addition of thermal moderator NaCl was to avoid overheating that can cause a sintering effect. Under these conditions, it should be noted that there is a negligible change in measured particle size (via SEM) between the original Tabasheer powder and derived pSi product (raw Tabasheer particles: 20.0 ± 10.0 µm; Tabasheer-derived pSi: 21.0 ± 7.5 µm). SiO2 (s) + 2 Mg (s) → Si (s) + 2 MgO (s)

(1)

As expected, the final product was dark brown in color, consistent with a porous silicon composition; significant silicon content is also observed in an EDX analysis (FE SEM) of this material (Fig. 1S). Highly crystalline Tabasheer-derived pSi was evident from TEM imaging along with the XRD pattern showing the three characteristic peaks (111), (220), and (311) associated with the cubic phase of Si (Figs. 2S and 3S). The Raman spectrum of this Tabasheerderived pSi, with a peak at 520 cm-1 also reflects its crystallinity (Fig. 4S). FE SEM images show a morphology best described as rough, debris-laden microparticles of pSi with a dendritic surface and some nanopores evident (Fig. 1). More detailed characterization of the porous nature of this plant-derived pSi can be obtained from DSC thermoporometry measurements,31 with the estimated pore volume and the pore size were determined to be 0.53 cm3/g and 12.9 nm,



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respectively. Such structural features suggest that the incorporation of compounds of interest into this carrier matrix is quite feasible (Fig. 5S). Moreover, these mesopores can also ideally play a protective role in minimizing exposure of the loaded active species to the surrounding environment, thereby stabilizing metastable antibacterial agents in this case.

Fig. 1. FE SEM images of Tabasheer-derived pSi, (A,B) reflecting the relative size of the particles, 21.0 ± 7.5 µm. A rough, highly porous surface morphology of the particles is demonstrated in higher magnification images (C, D).

For the garlic extract, the isolated oil was viscous with a density of 1.15 ± 0.12 g/ml. LC-MS analysis confirms the presence of the desired allicin (m/z = 163) component in the extract (Fig. 6S). In this study, due to instability of garlic extract in water and other organic solvents (e.g. ethanol), simple drug loading involved blending pure garlic oil with pSi, and relatively high loadings of garlic oil was obtained. In a typical Tabasheer-derived pSi sample, quantitative analysis of TGA indicates the amount of garlic oil in the loaded sample is 53.1 (± 2.2) wt % (Fig. 2). However, the presence of some residual surface oil in these samples cannot be excluded, as a mesopore volume of 0.533 cm3/g and oil density of 1.15 g/cm3 leads to a maximum internal loading (100% pore filling) of approximately 37 wt %. Variations in the amount of extract loading can be readily obtained by adjusting the ratio of the extract to pSi (w/w). A typical TGA trace shows two significant losses at around 194oC and 260oC as evidence for two main components in the extract loaded into the pSi. Since the boiling point of diallyl disulfide, which is one of the main components of garlic extract, is 180-190oC, it is suggested that the first temperature may correspond to that chemical compound.32 Moreover, the latter temperature is also consistent with the decomposition point of allicin (262oC), confirming the presence of this active component in the oil.33


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105 95

320

85 Mass%

Temperature oC

420

75 220

65

120

55

Temperature

45

Mass%

20

35 0

20

Time (min)

40

60

Fig. 2. Typical Thermogravimetric Analysis (TGA) measurement for garlic extract-loaded pSi. 3.2 Antibacterial Activity Studies Agar disc diffusion assays allow assessment of diffusion of the therapeutic agent(s) of interest from the porous matrix to the surrounding environment, as indicated by the inhibition zones where no bacterial growth is detected. In terms of inhibition zone per mg of extract, a comparison of garlic oil-loaded into pSi with unstabilized extract reveals that regardless of stabilization conditions, the oil-loaded pSi samples exhibit a significantly higher activity against S. aureus (Fig. 3A and Table 1). For example, within the first 24 hrs of exposure to the bacteria (at 37oC), there is a nearly two-fold enhancement of antibacterial activity brought about by the garlic extract-loaded porous Si carrier relative to the unstabilized extract. This is amplified upon extended storage. After one week at room temperature, the garlic extract loaded into pSi has retained ~50% of its mass normalized inhibition, while the unstablized garlic extract is completely inactive toward S. aureus under these conditions. Storage at lower temperatures permits retention of antibacterial activity in both species for longer periods. After 1 month storage at 4oC, >90% activity is still in place for the pSi-stabilized extract, with more than 2.5 times greater activity for this material relative to the unstabilized garlic under these conditions (albeit with a slightly larger standard deviation for this type of sample).

A

B

5 4

Garlic oil

1.2 Garlic oil

1

Garlic oil-loaded pSi

Garlic oil-loaded pSi

0.8 3

OD600

Mass-normalized diameter (mm/mg)

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2

0.6 0.4

1

0.2

0

0 Day 1

Week 1

Week 2

Week 4

Day 1

Week 1

Week 2

Week 4

Fig. 3. Graphical representation of: A) Mass- normalized inhibition zones (mm/mg extract) of free extract and garlic oil-loaded pSi obtained from agar disc diffusion assays versus S. aureus as a function of overnight exposure at 37oC and extended storage at 4oC; B. Optical Density



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measurements at 600 nm (OD600) of the supernatant solutions obtained from the extract only and extract-loaded Tabasheer-derived pSi Incubated in S. aureus under the same conditions as part A. Table 1. Mass-normalized inhibition zones of the free and Tabasheer-derived pSi loaded garlic extract before and after the samples were stored for various time periods at 4oC. Mass-normalized Inhibition Zone (mm/mg) Extract-loaded pSi

Day 1

Week 1

Week 2

Week 4

2.70 ± 0.40

2.71 ± 0.40

2.78 ± 0.32

2.52 ± 1.67

Garlic extract

1.43 ± 0.14

1.37 ± 0.101

1.42 ± 0.27

1.05 ± 0.02

Table 2. Optical density values at 600 nm (OD600) of the supernatant solutions obtained from the extract-only control and extract-loaded tabasheer-derived pSi Incubated in S. aureus Solution. (Note: Week 1, 2, and 4 samples are stored at 4oC prior to the assays). OD600 Extract-loaded pSi

Day 1

Week 1

Week 2

Week 4

0.30 ± 0.11

0.41 ± 0.10

0.29 ± 0.08

0.53 ± 0.27

Garlic extract

0.65 ± 0.22

0.62 ± 0.28

0.45 ± 0.24

0.92 ± 0.10

A complementary assay of relative changes in antibacterial activity is turbidity, measured by optical density of a given bacteria-containing sample at 600 nm. Taking into account the possible rapid degradation of the extracts’ chemical components in aqueous media, a 4 hour window was chosen to examine the activity of the free extract and garlic oil-loaded pSi. In these experiments, for a given exposure condition to S. aureus in LB, the extract loaded into pSi (at comparable oil concentration) yielded significantly stronger activity (Fig. 3B and Table 2). Critically, for shelf-life assessment, when being stored at 4oC for up to 4 weeks, these turbidity assays consistently affirm the ability of the garlic oil-loaded pSi to maintain the enhanced activity relative to the unprotected garlic extract controls, typically by nearly a factor of two (Fig. 7S). It should be pointed out, however, that for a given measurement these turbidity assays do show a slightly larger standard deviation in optical density (Fig. 3B) as a consequence of possible scattering from suspended pSi particles present in the LB medium. Nevertheless, these studies confirm the efficacy of pSi in acting as a protective matrix for enhancement of the activity of the garlic extract in terms of shelf-life. 3.3 Protection of the Garlic Extract under UV Irradiation by Tabasheer-derived pSi Some reports describe allicin as relatively light stable,34 while others note some degradation taking place in unprotected allicin in the presence of room light for extended periods (weeks).35 In our experiments reported here, we find that under extended UV irradiation (λ = 365 nm, 24 hours), the free extract completely loses all measurable antibacterial activity versus S.


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aureus in terms of either disk diffusion or turbidity assays. This in turn raises a fundamental question with regard to the role of composition and intrinsic light absorption in the ability of these plant-derived nanostructured materials, namely tabasheer-derived pSiO2 versus pSi, upon loading with garlic extract, to inhibit bacterial growth before and after UV irradiation. Notably, due to the instrinsic hygroscopic properties of the pSiO2, a slightly lower oil: silica ratio (w/w) was employed in the extract loading process, adjusted to 4:1 (Fig. 8S). In terms of disk diffusion assays, while the unprotected oil completely lost its activity after exposure to UV light (24 hr irradiation at 365 nm using 9 mW/cm2), only a 5% reduction of the mass-normalized inhibition zone was recorded for the extract loaded into pSi (at identical extract concentrations and irradiation conditions) (Fig 4A). In contrast, the extract-loaded into pSiO2 derived from tabasheer lost a significant amount of its activity, with a lowering of the massnormalized value by 25% (for original assay data, see Fig. 9S).

B

120 100

% Degradation in Activity

A

% Degradation in Activity

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80 60 40 20 0

120 100 80 60 40 20 0

G.Oil

pSiO2

pSi

G.Oil

pSiO2

pSi

Fig. 4. Effect of UV irradiation (24 hr at λ = 365 nm, 9 mW/cm2) on degradation of antibacterial activity versus S. aureus, as indicated by changes in: A) mass-normalized inhibition zones; and B) turbidity assays for unprotected garlic extract (G. Oil), extract loaded into porous silica derived from tabasheer (pSiO2), and extract loaded into porous silicon derived from tabasheer (pSi).

Turbidity assays also confirmed the efficacy of pSi over pSiO2 in stabilizing this garlic extract. In terms of percentage of degradation based on the change in the absorbance at 600 nm, pSi demonstrates significant activity against S. aureus was largely unaffected (3.8 % degradation) (Fig. 4B). The extract loaded into pSiO2 was not nearly as effective in inhibiting bacterial growth as indicated by degradation up to 51.5 %. Note this slightly higher percent degradation in the turbidity assay for pSiO2 material (and associated standard deviation) is again likely a consequence of the sensitivity of optical absorption measurements to scattering in solution by suspended Si/SiO2 particles. The observation of superior light absorbing ability of pSi suggests this plant-derived material can be utilized to protect therapeutically active compounds from degradation, relative to pSiO2, under light exposure. Measurements of the UV-Vis absorption spectra of these two siliconcontaining materials (as KBr pellets) in the 180 – 1000 nm window clearly reveals that the pSi



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absorbs 365 nm UV light more efficiently than pSiO2, consistent with the absorption coefficients of these materials in this particular region of the electromagnetic spectrum36,37 and our observations that the tabasheer-dervied pSi matrix more effectively protects the active components of the extract from light damage (for the disk diffusion data, see Fig. 10S). It has been suggested that under illumination, reactive oxygen species (ROS), such as singlet oxygen O2 and superoxide O2+, are generated from (anodized) pSi as a consequence of the interaction between excitons and oxygen molecules adsorbed onto the silicon surface.38 Thus, it was hypothesized that one possible contribution to the observed antibacterial activity in these silicon materials might be a consequence of the action of ROS, which are reported to be cytotoxic. However, from our control experiments, namely the disk diffusion assay data shown in Fig. 11S indicated no activity was detected from pSi-only samples as well as pSiO2 after 24-h UV irradiation. A similar trend emerges from turbidity assays, where comparable OD600 values of a given sample (pSi or pSiO2) stored in the dark versus those irradiated under light are obtained, suggesting no antibacterial action caused by ROS. Thus, control experiments from both types of assays exclude measurable contribution by ROS to the antibacterial activity of the samples, and rather affirm the mechanism of action arising from the diffusion of antibacterialactive garlic extract emerging from a porous carrier. 4. Conclusions The benefits of a plant-derived porous silicon drug carrier, specifically stabilization of naturally-occurring therapeutic extracts exemplified here by the case of allicin derived from garlic, are clearly demonstrated here. The specific utility of a strongly absorbing carrier such as elemental silicon to inhibit UV light induced degradation of a loaded active substance is an added benefit of this eco-friendly-derived matrix. Other potential uses of this ability to exploit porous silicon derived from plants, including additional examples as an effective drug carrier in maintaining activity and permitting sustained delivery of metastable therapeutic agents, are under investigation. AUTHOR INFORMATION Corresponding Author Email: [email protected]


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Author Contributions NTL and JLC conceived and planned the experiments; NTL prepared all porous silicon samples and carried out the antibacterial assays and related controls; JRK assisted with the electron microscopic characterization, thermal methods of sample analysis, and mass spectrometry measurements; AL prepared and characterized the Tabasheer powder; JLC and LTC provided overall supervision for the project; NTL, JLC, and LTC analyzed the data and wrote the manuscript. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xx Information includes: Additional characterization data of Tabasheer-derived pSi (EDX. XRD, TEM, Raman, DSC); mass spectral data of garlic extract; additional antibacterial inhibition zone data in the presence/absence of UV irradiation; additional TGA data; UV/Vis spectra of Tabasheer-derived pSiO2 and pSi. Funding Sources Financial support by the Robert A. Welch Foundation (Grant P-1212) and the Texas Christian University - Science and Engineering Research Center (SERC) are gratefully acknowledged. ACKNOWLEDGMENT The authors would like to thank Dr. Joakim Riikonen of the University of Eastern Finland for valued assistance with the thermoporometry method. The authors also thank Dr. Shauna McGillivray of the TCU Biology Department for assistance with the antibacterial assays.

5. References 1. Hoffman, A. S. Drug Delivery Systems. In An Introduction to Materials in Medicine, B.D. Ratner, A.S. Hoffman, F.J. Schoen, J.E. Lemons, Eds. New York: Academic Press, 2013, pp 1024-1027. 2. DeJong, W. H.; Borm, P. J. A. Drug Delivery and Nanoparticles: Applications and Hazards. Int. J. Nanomedicine, 2008, 3(2), 133–149. 3. Wang, B.; Hu, L.; Siahaan. T. J. Drug Delivery: Principles and Applications, John Wiley & Sons, Inc. Hoboken, 2016.



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TOC Graphic for Manuscript

allicin

Loaded porous Si

Staph Si

Aureus

nanocrystals


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Biogenic Nanostructured Porous Silicon as a Carrier for Stabilization

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