Mesoporous hemispherical silicon microparticles were fabricated by photolithography and electrochemical etching as previously described 7
. In our study, we used standard surface modification procedures developed for silicon-based materials (schematically presented in ). During the oxidation process, partial erosion of the particle surface led to an introduction of free hydroxyl groups, imparting to the particles a negative zeta potential (−31.5mV). Through silane chemistry the hydroxyl surface groups were covalently coupled to positively charged 3-Aminopropyltriethoxysilane (APTES), reversing the net surface charge of the particles to +14.73mV. APTES amine groups further served as a background for linking molecules to the particles surface. First, to estimate the range of molar ratios suitable for further conjugation of surface modifiers, we evaluated the effect of fluorescent probe concentration in the reaction medium on the fluorescence of the silicon particles. In the concentration range of 3.75–15mM of the 488-Dylight in the reaction medium, the net fluorescence intensity of the particles reached a plateau, which can be attributed to saturation of the bindings sites on the particles surface (Supplementary data, Figure 1S
). A slight reduction in the fluorescence intensity of the particles was observed at higher concentrations of the probe, which could be related to the quenching effect of the probe on the surface. This general behavior was consistent and repetitive among different experiments, though numerical values of fluorescent intensity slightly vary, due to the slightly different surface area and properties of pSi microparticles. Based on these results, a concentration of 10mM of PEG was chosen in order to obtain a saturation of the modifier on the particle surface. As in the case of the fluorescent probe, PEG molecules (MWs from 245 to 5000) were bound to the particles through APTES amine groups. No direct correlation was observed between the length of the PEG molecule and the zeta potential values (), though all PEGs and fluorescent probes bound to APTES amine groups resulted in a neutralization of the positive charges introduced by APTES thus causing a slightly negative zeta potential, which could be partially explained by the charge-shielding effect of PEG backbones.
Schematic presentation of chemical modification of pSi microparticles with APTES and PEG molecules.
Description and zeta potential values of the investigated microparticles
To evaluate the degradation rate of the particles under the simulated physiological conditions, we initially tested the degradation of small pores (10nm) and large pores (30–50nm) non-PEGylated APTES particles in phosphate buffered saline (PBS, pH 7.2) and fetal bovine serum serum (FBS) (). In agreement with the published literature, degradation kinetics of the mesoporous Si particles was strongly dependant on the pore size 13
. Particles having small pores degraded much slower than the particles with large pores. As the following step, we evaluated the influence of a modification with various PEGs on the degradation kinetics of the particles. Seven PEGs with varying molecular weights were employed: 245, 333, 509, 686, 1214, 3400 and 5000Da. shows degradation profiles of PEGylated particles with 30–50nm pores in PBS and 100% serum in vitro at 37°C. Generally, particles degraded faster in serum, and the higher was PEG’s molecular weight, the slower the degradation profile of the particles was obtained in both physiological media. The conjugation of the PEG with lowest molecular weight to the particles surface did not induce any change in the degradation kinetics in serum, but inhibited degradation and consequently the release of orthosilicic acid into buffer. When PEGs with the longer chains were evaluated, Si mass loss from the particles was slowed down, and the almost fully degraded within 18 to 24 hours in serum and within 48 hours in PBS. The most dramatic effect was observed for PEGs 3400 and 5000 which inhibited the degradation of the systems very prominently, with complete degradation achieved after four days. For these particles during the early stages of the degradation, there was a “lag” period of little or no mass loss.
Figure 2 Degradation kinetics of large pores (pores size 30–50nm) and small pores (10nm) Si microparticles as evaluated by ICP-AES. The degradation kinetic profile is expressed as a percentage of the total Si contents released to the degradation medium. (more ...)
Figure 3 Degradation kinetics of large pores PEGylated pSi microparticles as evaluated by ICP-AES. The degradation kinetic profile is expressed as a percentage of the total Si contents released to the degradation medium: (A) PBS pH 7.2; (B) Fetal Bovine Serum (more ...)
The degradation process as a function of time as shown in , can be separated into two phases: phase I
, up to about 24 hours; and phase II
, from 24 hours onward. The percentage of Si released (Mt
) in solution over time can be described quite accurately in both phases employing a general power law αtβ
with different scaling coefficients. Regarding the phase I
, the APTES modified surface and short PEG chains (PEG245) behave similarly with Mt
growing with time following a square root relationship (
) with α=23.10 and 23.48 (R2=0.965 and 0.984
as from ), respectively. For coating made with longer PEG chains, the exponent β
grows with the length of the polymer as listed in Tab. 2S, with β
ranging from 0.7 to 1.5; whereas α decreases leading to longer degradation times. Higher-order degradation laws with Mt
have been observed for PEG3400 and PEG5000 with α =0.0047 and α=0.0020, respectively with R2=
0.999 in both cases as from (, ). For phase II
, only particles coated with PEG3400 and PEG5000 exhibit a significant degradation, whereas APTES modified and particles with short PEG chains (up to PEG1214) have almost fully degraded after 18 hours. For PEG3400 and PEG5000, the degradation law can be again described through a general power law of the type αtβ
) and α=
Power law coefficients and R2 values for the first degradation phase.
Comparison between the experimental data (dots) and the best fitting power laws αtβ for the first phase of the degradation process according to scaling low Mt =α·tβ
Interestingly, for APTES modified and PEG245 coated particles, the degradation laws exhibit a square root behavior which is possibly associated with a diffusive release of silicic acid from the porous silicon matrix into the surrounding solution. As the length of the PEG chains attached on the particle surface increases, the diffusion of the silicic acid from the pores, where most of the degradation occurs, to the surrounding media is more and more hindered possibly by surface steric interactions with the polymer chains. Notably, a similar behavior is observed for PEG3400 and PEG5000 during phase II, with degradation laws exhibiting an exponent β=0.6, which is very close to that associated with pure diffusion (β=0.5). This would suggest that, during phase II, most of the PEG chains decorating the particle surface have been removed and released in the surrounding medium because of the degradation of the first porous layers.
The deterioration of the pSi microparticle surface morphology over time was evaluated by Scanning Electron Microscopy (SEM). presents SEM micrographs of the particles during the degradation process. The rate of deterioration of the microparticles was associated with the rate of Si chemical degradation, and microparticles conjugated to higher molecular weight PEGs exhibited surface deterioration at a much slower rate. It can be seen that the degradation of the APTES modified (non-PEGylated) particles over time occurred by means of erosion of the particles surface as well as of the pores. As the study progressed, the pore sizes became wider and the surface of the particle less smooth and more irregular. This degradation profile is in general agreement with the published reports on degradation of porous silicon structures [10
]. With intermediate PEG (MW 861), the appearance of the particles during the in vitro degradation process changed. The most prominent erosion is seen in the pores in comparison to the particle’s outer surface. This different degradation pattern could be attributed to the steric hindrance of the hydrophilic polymer molecules which probably cover particle surface more efficiently outside the pores, thus preventing penetration of water and other components, which play an important role in the degradation process. In the case of high MW PEG (5000) almost no degradation is seen within the first 48 hours, which confirms the data obtained by ICP-AES analysis.
SEM images of the pSi particles during the degradation process in PBS pH 7.2. Systems shown: a) APTES particles; b) Particles modified with PEG 861; c) Particles modified with PEG 5000. Timepoints: 2, 8, 18 and 48 hours.
To evaluate the kinetics of surface degradation of the particles, APTES and PEG3400 particles were labeled with the Dylight 488 fluorescent probe. The release kinetics of the probe from particles surface into the degradation media was followed by fluorescence intensity and FACS. Based on the fluoremetric analysis, for non-PEGylated particles, the fluorescent probe conjugated to the surface was released into the degradation medium within 8–16 hours depending on the degradation medium. For PEGylated particles the surface erosion rate was significantly extended and the fluorescent probe was released from the particle surface only after 24–48 hours () as was also confirmed by FACS analysis (data not shown). The obtained profiles were in agreement with the data on degradation kinetics of the particles surface as evaluated by ICP-AES and SEM.
Figure 6 Erosion of fluorescent PEG vs low MW probe from the pSi particle surface as followed up by fluorimetry in the degradation medium in PBS and FBS. The degradation kinetic profile is expressed as a percentage of the total fluorescence released to the degradation (more ...)
The ability to control the release of drug and imaging agents from pharmaceutical systems is critical for many clinical applications. In the case of the multistage delivery carrier 7,16–17
comprised of 1st
stage microparticles bearing 2nd
stage nanoparticles within the pores of pSi, the release of the second stage nanoparticles from the 1st
stage pSi microparticles will depend on several mechanisms, including their diffusion outside the pores, as well as on the simultaneous Si erosion and degradation of the matrix. The mechanism of degradation and drug release from biodegradable controlled release systems can generally be described in terms of three basic parameters. First, the type of the hydrolytically unstable linkage in the system and its position. Second, the way the system biodegrades, either at the surface or uniformly throughout the matrix, affects device performance substantially. The third significant factor is the design of the drug delivery system encountering for system geometry and morphology as well as for the mechanism of loading of therapeutic agents. For example, the active agent may be covalently attached to the particle matrix and released as the bond between drug and polymer cleaves.
The size and number of pores in pSi affects its physiochemical properties, and as a consequence different types of mesoporous Si particles degrade in aqueous solutions and biological fluids at different rates, which could be directly translated to release of free drugs or second stage microparticles from the matrix. The pores of the particles could be considered as a void fraction, being in constant contact with the degradation fluids and presumably originating the orthosilicic acid - the degradation product of porous silicon. Orthosilicic acid, Si(OH)4, is the biologically relevant water soluble form of silicon (Si), recently proven to be play a significant role in bone and collagen growth. Porous Si films release Si(OH)4 (silicic acid) in aqueous solutions in the physiological pH range through hydrolysis of the Si-O bonds, 18
which is harmlessly excreted in the urine through the kidneys 19
. In this study we addressed the question how the surface modification of pSi surface with PEG affect the degradation kinetics. APTES particles are a subject of homogenous surface degradation, where the erosion occurs homogeneously throughout the whole surface of the particle as well as the pores. In the case of PEGylated Si particles, the obtained degradation profile could be defined as heterogeneous erosion which besides the surface area, geometry and morphology of the particles is also defined by the length of the polymer chains covering the particle surface. PEGylation in this case appears to be the factor which controls penetration of solutes into the Si matrix of the particles. Derivatization can dramatically affect light-emitting properties of pSi and is degradation. For example, Buriak and co-workers have demonstrated that hydrosilyation with 1-dodecyne may sufficiently stabilize the material to render it corrosion-resistant 20
. It is also noteworthy that the higher degradation rates are observed for the particles incubated with serum as compared to PBS, indicating the stronger ionic strength or involvement of some biological processes such as enzymatic degradation.
Events that follow the administration of foreign material into the body could provoke acute or chronic inflammation, while the last one is characterized by the presence of macrophages and release of inflammatory cytokines. Injectable biomaterials are expected to be biocompatible in terms of lack of immunogenic and inflammatory responses. Though silicon has been recognized as an essential trace element in the body which participates in connective tissue, especially cartilage and bone formation 19
, some forms of crystalline silicon dioxide are known as a cytotoxic agent in macrophages 21–23
. Thus, it was important to assess the effect of pSi microparticles with various surface modifications on human immune cells. Keeping this in mind, we evaluated the biocompatibility of the systems with human monocyte derived differentiated cultured macrophages. Data clearly demonstrate that the tested systems did not induce release of proinflammatory cytokines IL-6 and IL-8 over 48 hours period time in THP-1 macrophages (). On contrary, when the cells were incubated with zymosan particles, a positive control, a very prominent increase in the cytokines release was observed. Phagocytic receptors on macrophages bind zymosan, stimulate particles engulfment and cytokines release. This agent is well known to induce inflammatory signals in macrophages through toll-like receptors TLR2 and TLR6. These results are in agreement with previously published studies on zymosan and other Si based systems. As an example, silica based Sol-gel glass was reported not to induce a significant inflammatory response by polymorphonuclear leukocytes 24
and oxidized nanoporous silicon was found to be compatible with primary hepatocytes 25–26
. In our recent study, we have also shown that vascular endothelial cells, following internalization of silicon microparticles, maintain cellular integrity, viability and normal mitotic division 17
. More explicit biocompatibility studies of the pSi microparticles for biomedical applications are currently underway in various in vitro and in vivo models in our laboratory.
Release of proinflammatry cytokines IL-6 and IL-8 by human cultured THP-1 macrophages following incubation with pSi particles with various surface modifications.