Surface enhanced fluorescence of anti-tumoral drug emodin adsorbed on silver nanoparticles and loaded on porous silicon
© Hernandez et al.; licensee Springer. 2012
Received: 20 April 2012
Accepted: 12 June 2012
Published: 2 July 2012
Fluorescence spectra of anti-tumoral drug emodin loaded on nanostructured porous silicon have been recorded. The use of colloidal nanoparticles allowed embedding of the drug without previous porous silicon functionalization and leads to the observation of an enhancement of fluorescence of the drug. Mean pore size of porous silicon matrices was 60 nm, while silver nanoparticles mean diameter was 50 nm. Atmospheric and vacuum conditions at room temperature were used to infiltrate emodin-silver nanoparticles complexes into porous silicon matrices. The drug was loaded after adsorption on metal surface, alone, and bound to bovine serum albumin. Methanol and water were used as solvents. Spectra with 1 μm spatial resolution of cross-section of porous silicon layers were recorded to observe the penetration of the drug. A maximum fluorescence enhancement factor of 24 was obtained when protein was loaded bound to albumin, and atmospheric conditions of inclusion were used. A better penetration was obtained using methanol as solvent when comparing with water. Complexes of emodin remain loaded for 30 days after preparation without an apparent degradation of the drug, although a decrease in the enhancement factor is observed. The study reported here constitutes the basis for designing a new drug delivery system with future applications in medicine and pharmacy.
KeywordsSurface-enhanced fluorescence Porous Silicon Emodin Drug delivery
Porous silicon (PSi) is a mesoporous material which has been proposed for an increased number of drug delivery applications in the last few years [1, 2]. PSi, as well as mesoporous silica materials [3, 4], shows biodegradability and biocompatibility; both of them being fundamental requirements for the development of controlled-release drug delivery system. PSi materials are termed “top-down” materials as opposed to the synthesized mesoporous molecular sieves, which are so called “bottom-up” silica materials that refer to the self-assembly of silicon oxide by means of polymeric templates determining the structure obtained. Besides, the efficient visible photoluminescence of PSi, as first reported by Canham  in 1990, can be used as a sensing signal of the carried drug, once it has been duly immobilized onto the PSi surface which sometimes requires its adequate functionalization [6–8]. PSi can also incorporate metal nanoparticles (NPs) which are furthermore useful as nanocarriers and imaging agents [9, 10]. In particular, noble metal NPs, due to their localized surface plasmon resonances (LSPRs), enhance both Raman (surface-enhanced Raman scattering, SERS) and fluorescence (surface-enhanced fluorescence, SEF) signals, being possible to use such spectroscopic techniques as high sensitivity detection routes for molecular sensing of the loaded drugs even after releasing from the PSi matrix. SERS substrates based on silver/PSi [11, 12] systems or silver/Si nanowires  have been reported. Regarding SEF using silver/Si nanostructures, to our knowledge, only two papers report some results in solution: one for praxeodimium ions (Pr3+)  and the other for lanthanide ions , after adding the Ag supported on Si to the sample solutions. In both cases, the authors reported larger fluorescence enhancement factors (EF) in the range from 10 to 200 with such Ag/Si materials than that caused by unsupported silver NPs.
Emodin is a natural anthraquinone dye with anti-tumoral activity , as well as laxative, anti-inflammatory, anti-aggregation, and anti-ulcer effects; whose SERS and SEF characterizations in Ag colloid suspensions, as well as its interaction with bovine serum albumin (BSA) at different pHs values, have been thoroughly carried out in our group [17–21]. With all this previous knowledge and taking into account the interest in using PSi as a drug carrier, emodin/AgNPs (Em/Ag) and emodin-BSA/AgNPs (Em-BSA/Ag) complexes were loaded in PSi matrices and used as a model system for other drugs, followed by the detection of emodin through the corresponding SEF spectra. In order to optimize the experimental variables, PSi layers with different pore sizes were tested in different impregnation conditions and the drug penetration in the PSi channels was detected. In all cases, the spatial resolution was 1 μm. Besides, the variation of the emodin SEF signal with time was monitored, from a freshly prepared sample until 30 days, in order to evaluate the possible temporal degradation. After verifying that the drug molecules did not remain included into PSi channels, the use of AgNPs allowed loading the drug without any functionalization. The SEF measurements used in this work can discriminate between emodin monomer and their aggregates. Only the monomer form of emodin was detected, thus, avoiding possible adverse effects due to the presence of drug agglomerates. Enhancement factor obtained for the fluorescence signal of emodin in the samples studied by SEF is in the range from 10 to 24.
Emodin, BSA, and hydroxylamine hydrochloride were purchased from Sigma (Sigma-Aldrich Corporation, St. Louis, MO, USA). Pure water was obtained from a Mili-Q Integral A10 system from Millipore (Billerica, MA, USA) and methanol (MeOH) was purchased from Panreac (Barcelona, Spain).
Silver colloids were prepared using Leopold and Lendl method . Briefly, it consists of reducing an aqueous solution of AgNO3 (10−2 M) with hydroxylamine hydrochloride in basic medium. The mean diameter of silver NPs obtained and evaluated by scanning electron microscope (SEM) (images not shown) was 50 nm.
Nanostructured PSi layers were formed by electrochemical etch of boron-doped (p-type) silicon wafers (orientation, <100 > and resistivity, 0.01–0.02 Ω·cm). The low-resistivity ohmic contacts were formed by coating the backside of the Si wafers with Al and subsequently annealing at 400°C for 5 min. The electrolyte consisted of 1:2 HF (48 wt.%):ethanol (98 wt.%) solution. The wafers were galvanostatically etched under illumination from a 100 W halogen lamp. The etching current density was typically 80 mA/cm2 and the etching time was 120 seconds, leading to the formation of 5 to 7 μm-thick PSi layers with an average pore size around 60 nm. In order to fabricate PSi matrices with smaller pore size, HF concentration in the etching process was increased, leading a solution 2:1 HF (48 wt.%):ethanol (98 wt.%). Also, the applied current density was reduced to 20 mA/cm2. The resulting PSi layers were loaded by capillarity suction in two different conditions, i.e., in atmospheric conditions and in vacuum at room temperature.
Aliquots of an initial 2-mM emodin solution in MeOH were diluted to obtain different concentrations to load in PSi layers and get several samples to analyze. The sample I (Em/Ag/MeOH) contained the drug adsorbed on AgNPs surface using MeOH as solvent. Silver colloid, freshly prepared, was centrifuged and the NPs redispersed in MeOH; subsequently, aliquot of initial emodin solution was added to get 0.2 mM final concentration. Sample II (Em/Ag/H2O) carried the drug adsorbed on silver NPs using water as solvent. The final drug concentration was the same as in sample I (Em/Ag/MeOH). Sample III (Em-BSA/Ag/H2O) included the drug bound to protein albumin forming BSA-emodin complexes. A solid BSA was solved on fresh silver colloid; afterwards, aliquot of emodin initial solution was added. Final protein and drug concentrations were 0 and 0.2 mM. Lastly, sample IV (Em/MeOH) was obtained by loading a 0.2 mM solution of emodin in MeOH in absence of AgNPs. Reference samples, named sample-ref I (Ag/MeOH), sample-ref II (Ag/H2O), and sample-ref III (BSA/Ag/H2O), were prepared following the same procedure as samples I, II, and III without including emodin. Wafers were transversally cut in order to analyze the corresponding cross-sections.
Raman and fluorescence spectra were recorded in a Renishaw inVia Raman microscope (Renishaw Iberica SAU, Barcelona, Spain), using a 100x magnification objective (spectral resolution 2 cm−1). The excitation line, 532 nm, was provided by Nd:YAG laser. The output laser power was 100 mW. The acquisition time of each spectrum was 7 min. The spatial resolution was 1 μm. Raman and fluorescence measurements of one cross-section were taken in one-micron steps along the PSi layer, from crystalline Si to open air. All spectra were normalized to the Raman signal of crystalline Si in the corresponding wafer.
The field emission scanning electron microscopy (FE-SEM) images were taken with Hitachi SU8000 (Hitachi High-Technologies Corporation, Tokyo, Japan); SEM images were obtained using a JEOL JEM2000Fx (JEOL Ltd., Tokyo, Japan).
Results and discussion
Hence, AgNPs present two principal advantages: firstly, they act as a "linker" between the PSi surface and the drug; secondly, they produce an enhancement of the fluorescence of the drug signal due to the localized surface plasmon resonance (LSPR) (SEF effect) which is clearly perceptible. This enhancement of the fluorescence varies with the solvent used in the loaded process and decreases in all cases after 30 days of preparation.
Fluorescence enhancement factor of samples studied
Air, 30 days
Sample I (Em/Ag/MeOH)
Sample II (Em/Ag/H2O)
Sample III (Em-BSA/Ag/H2O)
As concentration of emodin in sample III (Em-BSA/Ag) was ten times lower than that of other samples, the EF estimated must be considered as a low threshold. Experiments recorded with higher concentrations were not possible because of solubility problems. Two simultaneous phenomena were present in these systems that are absent in sample I (Em/Ag/MeOH) or sample II (Em/Ag/H2O) and make fluorescence emission intensity not proportional to concentration. The first one is the inner filter effect , which is minimized on the geometry used here, and affects all the samples studied. The second one is the resonance energy transfer between the protein, BSA, acting as a donor, and the emodin, acting as an acceptor . This effect is present only in sample III (Em-BSA/Ag/H2O) and is derived from the overlap between the protein emission and emodin excitation spectra. Quantification of independent contributions is difficult and is not necessary for an estimation of the global EF presented in Table 1.
No SERS was observed in any of the samples. This is a consequence of two factors; the first one is the high intensity of the fluorescence, and second one is the absence of aggregating agent in the preparation of the silver colloid, thus allowing SEF but hindering SERS . Only isolated NPs with smaller size than that of PSi channels are able to penetrate in the pores. On the contrary, AgNPs aggregates produced by emodin, which also aggregates in these conditions  and is responsible for the SERS effect, remain on the surface of the PSi layers, and are subsequently eliminated after washing of the preparation of the samples. This was confirmed as unwashed samples showed SERS spectra on the PSi-air interface.
PSi layers were fabricated and loaded with emodin by capillarity suction in atmospheric conditions and in vacuum. The emission fluorescence of emodin and PSi overlap, and signals are almost identical with no possibility of discrimination. This problem has been overcome using silver colloid as drug carrier. Emodin was adsorbed on AgNPs using either MeOH or H2O as solvent. The drug has also been loaded bound to BSA, forming the transporter complex. The presence of the AgNPs has been responsible of the observation of SEF effect due to the existence of LSPR. The differences in fluorescence drug intensity allowed monitoring of its location in the PSi layer at different distances of the air-PSi interface.
The presence of protein gives the highest enhancement factor of the drug fluorescence signal. In all cases molecules, which are loaded, go 5 to 6 μm inside the pores. Fluorescence signal decreases noticeably after 30 days of preparation. Inclusion in atmospheric conditions gives better results than in vacuum ones. In all cases emodin is detected as monomer and no aggregate is observed. The system presented allows the detection of low concentrations of drugs and could constitute the basis for new drug delivery systems used in medicine and pharmacy.
localized surface plasmon resonances
surface-enhanced Raman scattering
bovine serum albumin
emodin-silver nanoparticles complex
emodin-bovine serum albumin-silver nanoparticles complex
The Secretaria de Estado de Investigacion, Desarrollo e Innovacion (MINECO) (Project FIS2010-15405: Plasmonics: Enhanced Molecular Sensing on Metal Nanostructures (POEMS), Comunidad de Madrid (MICROSERES Project, S2009TIC-1476) and grupo investigación 950247 of the UCM are gratefully acknowledged for their financial support. David Gomez (characterization service from the ICTP-CSIC) is also acknowledged for the FE-SEM images.
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