Functionalized mesoporous silicon for targeted-drug-delivery
Graphical abstract
Highlights
► Porous silicon is functionalized for targeted-drug-delivery applications. ► Folic acid, polyethylene glycol and doxorubicin are covalently attached. ► Effect of reactant concentration and solvent of folate covalent attachment studied. ► The drug loaded particle shows higher toxicity than the free drug. ► Folate incorporation increases tremendously the cytotoxicity.
Introduction
Porous silicon (PSi) has been the focus of an ever increasing number of studies during the recent years. PSi is a versatile material whose physico-chemical characteristics like porosity, pore size, pore shape, layer dimensions and surface functional groups can be vastly tailored during the synthesis–functionalization process. Accordingly, it has been the subject of several investigations for the production of electrochemical biological sensors [1], [2], optical bio-sensors [3], bio-reactors [4], and recently, controlled-drug-delivery systems [5], [6], [7]. Concerning the last item, drugs had been incorporated into the porous matrix via adsorption [6], [8] and covalent binding [5]. Adsorption-loading suffers from the drawback of rapid drug release from the carrier. Covalent binding of drugs to PSi matrixes offers the twin advantages of slow release and possibility of triggering the process via active species [5]. The present work concerns the production of anticancer drug loaded PSi for targeted-drug-delivery applications. Published articles in this area are scarce [9].
Folate-receptors are usually over-expressed on the surface of cancer cells [10]. This phenomenon has induced researchers to design folate-conjugated drugs to accomplish a more efficient delivery of anti-cancer drugs to the malignant cells [11]. Folic acid has been successfully conjugated to gold [12], magnetite [10] and albumin [13], [14] nano-particles. The work of Zhang et al. [10] gives an informative introduction to the ‘architecture’ of folate receptor-activated functionalized nano-particles. They encapsulated magnetite nano-particles in polyethylene glycol (PEG) to avoid ‘opsonization’, i.e., to inhibit plasma coating processes when in blood. Their design suffers from the drawback that the folate-conjugated drug is just ‘adsorbed’ by Van der Waals forces on the PEG encapsulated nano-particles. Regarding silicon compounds, Rosenholm et al. report the use of folic acid for the synthesis of mesoporous silica nanoparticles for targeting of cancer cells [15]. Asadishad et al. [16] produced folate-modified polyethylene glycol-functionalized gold nano-particles for targeted delivery with doxorubicin as an anticancer drug. They synthesized the doxorubicin-loaded nanoparticles in a manner similar to the work of Zhang et al. [10]. Narayanan et al. [17] used targeted nanoparticle mediated delivery to enhance the efficacy of phytomedicines. Poly (lactide-co-glycolide) (PLGA) nanoparticles encapsulating a grape seed extract (GSE), abbreviated as nanoGSE, were prepared by a nano-precipitation technique. Folic acid was conjugated to nanoGSE as targeting ligand.
The present work actually merges the different techniques mentioned above to design an effective mesoporous silicon drug carrier for targeted delivery purposes. The PSi surface is first made hydrophilic by grafting an alkane and then, through separate treatments, the following molecules are ‘tethered’ to it: PEG, folic acid and doxorubicin (as the anticancer drug). PEG acts as an anti-opsonization ligand. Folic acid acts as a recognition element by the folate receptors, over-expressed on the tumor cells. Micro-particles of this drug carrier are sheltered from protein elements present in the blood plasma by the action of grafted PEG molecules, and can selectively be captured by the abundant folate receptors present on cancer cells (targeted delivery). The porous silicon skeleton is then susceptible to a relatively slow hydrolysis process, eventually promoted by dissolved oxygen in the blood. Silicon backbone hydrolysis products, i.e., oxyanions of orthosilisic acid, are considered of low toxicity for human body [18], [19]. Finally, the anti-cancer drug is released in the close proximity of the malignant cell, eventually retaining its original highly toxic characteristic. Wu et al. [5] attached doxorubicin to micro-particles of PSi and showed that the released drug retained its chemical characteristics. Their design did not include any folate or PEG functionalization of PSi. In other words, their composite acted merely as a controlled delivery system.
The present work introduces an innovative multi-step procedure which uses sequential organic and aqueous media for an optimal chemical modification of the PSi surface. The PSi layer is first functionalized with undecylenic acid (UD) [20], activated with N,N′-dicyclohexylcarbodiimide (DCC)/N-hydroxysuccimide (NHS) solutions using dimethylsulfoxide (DMSO) solvent, reacted with folic acid in aqueous buffer medium, reacted with amine-terminated PEG and finally covalently bonded with doxorubicin. It should be noted that a systematic study of the amidation of silicon surface using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/NHS mixtures has already been reported in the recent years [1], [21]. Schwartz et al. [22] have reported a concise study on the attachment of PEG to PSi, although using methylene chloride as solvent. DCC and EDC are common coupling agents used for amide bond formation. Generally, DCC is considered much more efficient compared to EDC. DCC is used in organic solutions due to its insolubility in water and therefore does not suffer from the hydrolysis reaction of the EDC-formed esters [23]. Its main drawback is the possible production of dicyclohexylurea byproduct which is insoluble also in organic solvents. This fact has limited the use of DCC in the formation of peptide bonds on solid supports like resins. In the present work, a systematic approach has been followed to obtain the optimal composition of the DCC/NHS in DMSO solution for the surface activation process to take place with minimal undesired byproduct formation.
We stress the point that the incorporation of PEG is not the main point of this study and its amount will not be quantified either. Its incorporation method and its qualitative identification through FTIR analysis has been included just to provide a successful method for producing functionalized PSi for in vivo studies in the future.
The kinetics of drug release has been investigated in a broad range of pH covering acidic, neutral and basic conditions. For this means micro-particles with a size ca. 5 μm have been used. The toxicity/cellular viability characteristics of the folate/doxorubicin conjugate covalently attached to UD grafted PSi (along with PEG), doxorubicin covalently attached to UD grafted PSi (along with PEG), the empty PSi particles and free doxorubicin have been studied using KB cells and (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT) as toxicity testing agent.
Section snippets
Porous silicon synthesis
Mesoporous silicon was produced by the electrochemical etching of highly boron doped silicon wafers ((1 0 0) oriented, 0.002 Ω cm−1 resistivity) for 12 min using a current density of 76 mA cm−2 using a CVTM V1.00 galvanostat (Iranian instrument). Analytical grade HF (40 wt.% aqueous, BDH) and ethanol (96 wt.%, Merck) were used for the preparation of a 1:1 (v/v) electrolyte solution. In the case of drug release experiments, the PSi layers were subjected to grinding and ultrasound treatment to produce
Characterization of PSi and PSUD
Fig. 1 shows the FTIR pattern of the as-synthesized (PSi) and UD treated porous silicon layers (PSUD). It should be noted that Fig. 1, Fig. 2, Fig. 3, Fig. 4 are offset for clarity. The FTIR spectrum of a non-washed UD layer has also been added. The as-synthesized PSi layer shows a high concentration of SixHy groups in the wave number range of 2000–2300 cm−1. After treating with UD, the relative peak intensity due to SixHy groups decreases significantly. New peaks appear and are assigned as
Conclusions
An optimal procedure for producing folate/PEG/doxorubicin grafted PSUD layers using DCC/NHS activation has been developed. It was found that folic acid reaction with grafted NHS ester is not favored using DMSO as solvent. Amine terminated PEG was found to be a very reactive reagent for chemical grafting with NHS ester active PSi layers. Based on the FTIR measurements, it was deduced that the doxorubicin molecules are attached to the functionalized PSi layer through covalent bonds.
With a drug
Acknowledgment
Prof. A. Sahebghadam Lotfi from NIGEB is gratefully acknowledged for his kind collaboration in providing the MTT assay facility.
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