Targeted delivery of mesoporous silica nanoparticles loaded monastrol into cancer cells: an in vitro study
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Monastrol is a simple low molecular weight dihydropyrimidine-based kinesin Eg5 inhibitor. Its low cellular activity and its non-drug-like properties have impeded its further development. In a previous report, we have reported various topological parameters to improve the pharmacokinetic properties of monastrol. The purpose of this study is to determine the loading and release feasibility of poorly water-soluble monastrol into the synthesized mesoporous silica nanoparticles (MSNs). The synthesis of MSNs was attained by the ammonia-catalysed hydrolysis and condensation of TEOS in ethanol using polysorbate-80 as surfactant. These were characterized by BET surface area and pore size distribution analyses, SEM, XRD, UV and FTIR spectroscopy. The synthesized monastrol was successfully loaded on MSNPs and coated by hydrogels for successful controlled drug delivery. In vitro release studies are done by simple dialysis method. Monastrol-loaded MSNPs were tested on human cervical epithelial malignant carcinoma (HeLa) cell lines for studying their anticancer activity. Our presented system described a reliable method for targeted delivery of monastrol into the cancer cells in vitro.
KeywordsMesoporous silica nanoparticles Monastrol KSP Anticancer Drug delivery
The main objective in the field of nanomedicine is to acquire efficient delivery platforms for targeted delivery of therapeutic agents through proper nanomaterials. A serious interference in cancer treatment is due to the narrow range of operative biocompatible drug delivery systems. Mostly, low solubility in aqueous media is shown by the hydrophobic anticancer drugs like monastrol. Their inability to dissolve readily affects their activity in cancer treatment. Efficient drug delivery is very essential for enhancing diagnosis, reducing side effects and drug efficacy. Nanoparticles offer a capable approach towards delivering the therapeutic agents to the targeted organs and their use in cancer treatment (Nazir et al. 2014; Chen et al. 2016; Rosenholm et al. 2011; Wilczewska et al. 2012; Sahoo et al. 2007; Suri et al. 2007). Mesoporous silica nanoparticles are considered to be very promising candidate for drug delivery specifically for water-insoluble drug. Due to large surface areas and pores, these MSNPs act as reservoirs for storing hydrophobic drugs, whereas their shape and size can be altered according to the requirements (Zhang et al. 2012; Bharti et al. 2015). Several drugs with low water solubility have been explored to demonstrate this concept including ibuprofen, rifampin, etc. (Zhang et al. 2010; Mohseni et al. 2015; Ganesh and Lee 2013). Based on non-toxicity and biocompatibility, silica nanoparticles are proposed as alternative carriers for drug delivery as these can be degraded into its simple silanol units and can be removed from the body (Fent et al. 2010; Yu et al. 2009; Liu et al. 2011). The porosity and shape of MSNPs accommodate the drug molecules. Therefore, for the effective drug loading and its delivery to the targeted sites, MSNPs are selective to capture bigger bioactive molecules in substantial amount (Jong and Borm 2008; Jaganathana and Godin 2012).
Dihydropyrimidines (DHPMs) are attractive scaffold for bioactivity because they can be easily altered and designed. Monastrol being a low molecular weight compound is revealed as a cell-permeable kinesin Eg5 inhibitor (Maliga et al. 2002). New class of anticancer agents directed by monastrol showed a significant progress in cancer therapy (Prokopcova et al. 2010). As monastrol is significant in targeting a motor protein, so it is helpful in examining the problems of spindle assembly in vitro and in vivo. Monastrol is fairly soluble in DMSO and ethanol but its solubility in water is poor. Further development of monastrol was halted due to its weak inhibitory activity as kinesin Eg5 inhibitor and non-drug-like properties (Schmidt and Bastians 2007).
All the chemicals because of the highest quality were used as received. Anhydrous ethanol (99.9%), NH3 (1%), Polysorbate 80, Tetraethyl orthosilicate (TEOS), N-hydroxysuccinimide 98+% (NHS), Folic acid, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDAC), Dimethyl Sulphoxide (DMSO), Chitosan, sodium carboxymethylcellulose (CMC), Acetic acid, TPP.
Synthesis of monastrol
Yield 77%. m.p. 185 °C. 1H NMR (300 MHz, DMSO-d6): δ 1.09 (t, 3H, J = 7.2 Hz, OCH2CH3), 2.24 (s, 3H, CH3), 3.96 (q, 2H, J = 7.2 Hz, OCH2), 5.06 (d, 1H, J = 3.3 Hz, CH), 6.60 (m, 1H, Ar–H), 7.06 (t, 1H, Ar–H), 7.68 (br s, 1H, NH), 9.16 (br s,1H, NH) 9.37 (br s, 1H, OH). EIMS calculated for C14H16N2O3S (M+•) 292.4.
Synthesis of mesoporous silica nanoparticles (MSNPs)
The synthesis of hollow mesoporous silica nanoparticles was attained by the ammonia-catalysed hydrolysis and condensation of TEOS in ethanol using Polysorbate 80 as a surfactant. Typically, 1% solution of TEOS, 50 mL ethanol and 5 mM tween 80 were mixed and stirred for half an hour and then 1% ammonia solution was added. The reaction mixture was kept overnight under stirring. The reaction mixture then turned turbid and the product thus formed was filtered after settling down the solution. MSNPs were obtained after calcination at 200 °C for 6 h and then at 400 °C for 4 h.
Loading of monastrol on silica nanoparticles
Monastrol was dissolved in ethanol to which powdered MSNPs were added under constant magnetic stirring for 24 h at 37 °C. The ratio of drug to nanoparticles was selected as 2:1 (w/w). After 24 h of continuous stirring at 200 rpm, the particles were allowed to settle, centrifuge and wash with ethanol. Finally, the powdered monastrol-loaded MSNPs were recovered after drying in open air at 37 °C.
Formation of hydrogel
For creating a CMC and chitosan hydrogel, NPD conjugate solution (in ethanol) was mixed in CMC solution (0.01 g in 20 mL water) to which chitosan solution was added under constant magnetic stirring (200 rpm). The pH of the reaction mixture was maintained in the range 4–5 and then again CMC solution was added in drop-wise manner. For covalent linkages, TPP solution was added followed by constant agitation for further 30 min. The appearance of turbidity thus confirms the formation of hydrogel. Finally, the solution was centrifuged to get gel encapsulated drug-loaded silica particles.
Coating of folic acid
For coating, 0.25 g folic acid was dissolved in 30 mL DMSO and sonicated for 15 min till a clear solution was obtained. Then, 0.225 g NHS and 0.125 g EDAC were added to folic acid solution and stirred for 2 h. In this way, folic acid was activated. Afterward, 0.9 g gel was dissolved in 9 mL of ethanol to which 4 mL activated folic acid added and stirred for 4 h. Finally, the gel products were centrifuged and air dried.
Human cervical epithelial malignant carcinoma (HeLa) cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and grown in RPMI 1640, supplemented with l-glutamine, 10% heat-inactivated Fetal Bovine Serum (FBS), 50 U/mL penicillin and 50 mg/mL streptomycin (Mediatech, Manassas, VA). The cells were maintained in a humidified incubator at 37 °C in an atmosphere of 5% carbon dioxide. Monolayer cultures were seeded in 96-well plate (103 cells/well) for 24 h before treatment with the nanoconstructs. Increasing concentrations of nanoconstructs were incubated with the cells for 24 h at 37 °C. The cell viabilities were measured using MTT assay.
MTT viability assay was prepared in a physiologically balanced solvent and cells were incubated with 100 µL of MTT solution for 3 h after removing the media. MTT-containing media was replaced with 100 µL of detergent and absorbance was measured on microplate reader (AMP PLATOS R-496) at the wavelength of 570 nm. Active metabolic action in the viable cells converts MTT into a purple-coloured formazan product while the dead cells lose this ability, thus colour formation with an absorbance maximum near 570 nm serves as a suitable and useful marker of viable cells.
Results and discussion
Characterization of MSNPs
Monastrol release kinetics
In vitro innate cytotoxicity
The effect of free drug and drug nano-carrier on inhibition of cell proliferation in HeLa cells was monitored after addition of 0.34, 0.26 and 0.17 µM of Monastrol contained in 100, 75 and 50 μg/mL of NP-D and NP-D-F. The effects of Monastrol, SNPs, NP-D and NP-D-F on cell viability were determined using MTT assay. The absorbance measurement, by using this assay, reflects the total metabolic activity of a cell population and is therefore an indirect measurement of cell proliferation. Although commonly used as a viability assay, the MTT assay more specifically represents a measure of mitochondrial function.
From the above-mentioned study, it can be concluded that the mesoporous silica nanoparticles were successfully synthesized by sol–gel method. These particles were then characterized by BET surface area and pore size distribution analyses, UV, FTIR, XRD and SEM. The surface area of MSNPs was found to be 328 m2/g and the maximum pore size of silica nanoparticles was about 5.6 nm. Monastrol drug was loaded on MSNPs for controlled drug delivery, where UV and FTIR analyses were done on every important step. The cell viability was observed to decrease with NPD and decreases further after loading folic acid. MSNPs with high payloads of monastrol were effectively delivered into the cancer cells in vitro. Folic acid conjugation ensued the targeted delivery, whereas CMC-based hydrogel of MSNPs effectively packed monastrol deep into the pores of MSNPs.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
The manuscript does not contain clinical studies or patient data.
- Mohseni M, Gilani K, Mortazavi SA (2015) Preparation and characterization of rifampin loaded mesoporous silica nanoparticles as a potential system for pulmonary drug delivery. Iran J Pharm Res 14:27–34Google Scholar
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