Design, synthesis and in vitro evaluation of anticancer and antibacterial potential of surface modified Tb(OH)₃@SiO₂ core–shell nanoparticles

Abstract

In the current study, we modified the surface of Tb(OH)₃ nanoparticles with a silica layer to enhance their solubility and biocompatibility. Transmission electron microscopy confirmed the improvements in these properties. Tb(OH)₃@SiO₂ core–shell nanoparticles (TS-CSNPs) exhibited a strong green emission peak upon irradiation with ultraviolet light, which originates from the electric-dipole transition ⁵D₄ → ⁷F₅ (543 nm) of the Tb³⁺ ion. In vitro anticancer and antimicrobial activities of the synthesized TS-CSNPs has been evaluated through their potential cytotoxicity and antibacterial activity. TS-CSNPs were shown to have more cytotoxicity against HT29 human colorectal cancer cells with a value of IC₅₀ 420.33 in an MTT assay. The alteration of the morphological features of HT29 cells was analysed using various concentrations of TS-CSNPs by inverted microscopy. Western blot analysis results of the apoptotic pathway showed that TS-CSNPs inhibited the growth of HT29 cancer cells through the induction of apoptosis, as evidenced by the down regulation of the expression of anti-apoptotic Bcl-2 and Bcl-xL gene products. Furthermore, the results of the in vitro hemolysis assay with human erythrocytes demonstrated the excellent blood biocompatibility of TS-CSNPs. Our silica coated TS-CSNPs exhibited a non-significant effect on the viability of both Gram negative and Gram positive bacterial strains up to 18 hours of exposure. These results highlight that modified TS-CSNPs can be functionalized to enhance the efficacy of cancer therapeutics due to the significant potential against cancerous cells and antibacterial activity.

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1 Introduction

Recently functionalized nano-materials have received a great deal of attention due to their potential applications in various fields of biomedical and clinical sciences such as anticancer agents. Cancer is considered one of the most dangerous diseases, however, advances in the diagnosis and therapy of cancer have been limited due to the fact that the actual theranostics rate for cancer is elusive without causing any major side effects to the health of humans and their normal microbiome, including various symbiotic bacteria.^1,2 The communities of microbes together with humans are called microbiome. Many recent studies confirmed that alterations in normal microbiota have also been connected with a number of diseases, including different type of cancers, progression of HIV and the development of various allergic diseases.^2–9 As radiation therapy and surgery may not feasible in late and advanced phases of cancer, the offered treatment is common chemotherapy without significant alterations to the normal microbiome. Among various chemotherapeutic agents, synthesized nanoparticles may be used as effective drugs in the treatment of certain cancers, including lung, head and neck breast and skin cancer,^10–13 however, the majority of the present chemotherapeutic agents lack solubility, specificity, have a small life span in the blood stream and the potential risk of many side effects. Considering these issuess, many targeted drug carriers have been formulated to upgrade the existing therapeutic efficacy and also to delay the drug from biodegradation prior to targeting the target cells, as well as to decrease the side effects.^14,15 In this situation, currently nanomaterials based on rare-earth ion doped core and core–shell nanoparticles for therapeutic and treatment applications have increased in significance in pharmaceutical industries due to their wide uses.^16–21 Furthermore, core–shell and hybrid nanoparticles containing both luminescence and magnetic properties have been applied in drug delivery, in vitro and in vivo bio-labelling, magnetic resonance imaging (MRI), and hyperthermia uses.^22–24 Drug delivery systems (DDS) based on nanomaterials with specific diameters may be preferred for their enhanced permeability and retention effect (EPR) of tumors with reduced side effects.²⁵ At present, the conjugation of luminescent/fluorescent nanomaterials such as inorganic nanoparticles, quantum dots, and metals with chitosan has given more awareness in cellular imaging, as they have made it feasible to trace the nanoparticles and their thermocokinetics through the study of fluorescence and for targeted drug delivery applications.²⁶ Among these, photo-stable luminescent lanthanides and silica coated core–shell nanoparticles are preferred to quantum dots.^27,28 In the current study, we synthesized luminescent lanthanide nanoparticles due to their ease of synthesis, specific cell targeting ability, non-photobleaching, high thermal, chemical and photo stability. We evaluated the anticancer effect of the surface of modified Tb(OH)₃@SiO₂ core–shell nanoparticles (TS-CSNPs) through the analysis of morphological changes in treated cells and the alteration in expression of Bcl-2 and Bcl-xL anti-apoptotic proteins of the apoptotic pathway. Furthermore, we also analysed the in vitro blood biocompatibility of TS-CSNPs.

2 Materials and methods

2.1 Chemicals

Terbium oxide (99.99%, Alfa Aesar, Germany), tetraethyl-orthosilicate (TEOS, 99 wt% analytical reagent A.R.), ethanol, nitric acid, ammonium hydroxide and cetyltrimethylammonium bromide (CTAB) were used as the starting materials without any further purification. Nanopure water was used for the preparation of solutions. The ultrapure de-ionized water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals used in the preparation were of reagent grade. Fetal bovine serum (FBS), dimethyl thiazolyl tetrazolium bromide (MTT reagent), doxorubicin hydrochloride (control anti-cancerous drug), and antibodies of β-actin, Bcl2 and BclxL were procured from Sigma Aldrich (St. Louis, MO). Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI-1640 medium, 1% antibiotic–antimycotic solution, penicillin–streptomycin solution, and trypsin–EDTA solution were purchased from Life Technologies GIBCO, Grand Island, NY, USA. Human colon cancer cell lines HT29 and SW620 were obtained from American Type Cell Culture Collection (ATCC, Manassas, VA).

2.2 Synthesis of Tb(acac)₃·3H₂O metal complex

The chelate, [Tb(acac)₃·3H₂O, where acac– is the anion of acetylacetone] was synthesized by slightly modifying the standardized method.^29,30 A weighed quantity of terbium oxide (2.5 g) was dissolved in a minimum quantity of concentrated nitric acid, which was diluted with water. A solution of ammonium acetylacetonate was prepared by adding concentrated ammonium hydroxide (6 mL) together with sufficient water to an amount of acetylacetone (8 mL), which was 50% in excess of that required for the complete reaction with terbium oxide. The resulting solution of ammonium acetylacetonate was added slowly into the hot and stirred solution of terbium oxide. The pH of the reaction mixture was maintained (between 5 and 6) during the reaction. The reaction mixture was stirred for 6 hours to ensure the conversion of any basic acetylacetonate to the normal compound. The crystalline precipitate, thus formed, was filtered and washed several times with water. The crude product, thus obtained, was crystallized from chloroform.

2.3 One-pot synthesis of luminescent mesoporous Tb(OH)₃@SiO₂ core–shell nanoparticles

Luminescent TS-CSNPs were prepared via a modified W/O microemulsion process. The Tb(acac)₃·3H₂O chelating complex was prepared by a reported method prior to the preparation of the nanoparticles.³¹ In a typical procedure, firstly the microemulsion was prepared by mixing 3.54 mL of Triton X-100, 15 mL of cyclohexane, and 4.54 mL of n-hexanol under constant stirring at room temperature. Then, 2 mL of an aqueous solution of Tb(acac)₃·3H₂O chelating complex (1 M) was added drop-wise into the mixture. After that, a mixed solution containing TEOS (2 mL), H₂O (5 mL), and CTAB (50 mg) was added under vigorous stirring until a white precipitant was formed. In the presence of TEOS, a polymerization reaction was initiated by adding 1 mL of NH₄OH. After the reaction was completed, the TS-CSNPs were isolated by acetone followed by centrifuging and washing with ethanol and deionized water thoroughly (thrice) to remove any surfactant molecules.

2.4 Characterization of TS-CSNPs

The TS-CSNPs were prepared and purified through centrifugation at 22 000g for 20 min and the pellets were washed thrice carefully with ethanol and deionized water and the resulting suspension was characterized employing various available techniques.

2.4.1 Transmission electron microscopy. Field emission transmission electron microscopy (FE-TEM) was performed to determine the size and morphology of the surface modified TS-CSNPs. The samples were prepared by depositing a drop of a colloidal ethanol solution of the TS-CSNPs powder onto a carbon-coated copper grid. TEM measurements were taken with a field emission transmission electron microscope (FE-TEM, JEM-2100F, JEOL, Japan) operated at an accelerating voltage of 200 keV.

Furthermore, the energy dispersive X-ray spectroscopy (EDX) was performed to confirm the chemical stoichiometry of the silica core–shell nanoparticles. The occurrence of elemental terbium (Tb) in synthesized TS-CSNPs was verified using the EDX technique.

2.4.2 Optical absorption spectroscopy. To inspect the absorption pattern of surface modified TS-CSNPs, UV-vis absorption spectra were observed with a Perkin-Elmer Lambda-40 spectrophotometer, with the sample contained in a 1 cm³ stoppered quartz cell with a 1 cm path length, in the range 190–600 nm.

2.4.3 Photoluminescence spectroscopy. The photoluminescence properties of the TS-CSNPs were recorded using a fluorescence spectrometer at room temperature. The photoluminescence spectrum was recorded on Horiba Synapse 1024 × 256 pixels, size of the pixel: 26 microns, detection range: 300 (efficiency: 30%) to 1000 nm (efficiency: 35%). For all the experiments a slit width of 100 microns was employed, ensuring a spectral resolution better than 1 cm⁻¹.

2.5 Cytotoxicity assays

Cytotoxicity assays were performed to measure the anticancer potential of TS-CSNPs using virus-negative HT29 and SW620 human colorectal cancer cell lines and MCF-10A (a non tumorigenic epithelial cell line).

2.6 Cell culture

Human colorectal cancer cells HT29 and SW620 were purchased from the American Type Culture Collection. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with heat inactivated 10% foetal bovine serum (FBA, Invitrogen, Carlsbad, CA, USA) and 100 μg mL⁻¹ streptomycin, 100 units per mL penicillin and 2 mmol L⁻¹ L-glutamine. The cells were grown as adherent monolayers (i.e. cultured at about 70% to 80% confluence) and maintained at 37 °C in a humidified atmosphere of 5% CO₂. The cells were harvested by using 0.25% trypsin through trypsinization. The cultures (approximately 1 × 10⁴ cells) of HT29 and SW620 were seeded onto 96-well culture plates containing 100 μL of media, and incubated for 24 h at 37 °C in 5% CO₂. After the attachment of cells, the media was replaced with fresh 100 μL of media containing 0, 5, 10, 20, 40, 80, 160, 320 and 640 μg mL⁻¹ concentrations of TS-CSNPs and doxorubicin hydrochloride (control drug).

2.7 MTT assay

MTT (dimethyl thiazolyltetrazolium bromide) assay was performed to find out the synthesized TS-CSNPs quality of being cytotoxic against HT29 and SW620 colorectal cancer cells lines. The synthesized TS-CSNPs were evaluated for their cytotoxic properties against the non cancer cell line MCF-10A (non-tumorigenic epithelial cell line) using WST-1 assay. The MTT reagent was prepared freshly in PBS with a final concentration of 5 μg mL⁻¹. The MTT solution was filtered through a 0.2 μm filter and stored at 2–8 °C. The cell cultures of different lines were treated with serial concentrations of TS-CSNPs and doxorubicin hydrochloride (control drug) in separate experiments at 37 °C for 24 h in 5% CO₂. Control cells (untreated) received only 200 μL of culture medium containing 10% dimethylsulfoxide in each experiment without Co₃O₄-NPs/doxorubicin hydrochloride. After 24 hour treatments the cells were treated with freshly prepared 20 μL of MTT solutions. The cell cultures were further incubated for 3 h at 37 °C in 5% CO₂. Moreover, 100 μL of dimethyl sulfoxide (DMSO) were added in each well to dissolve the crystal of formazan, which formed in the reaction of MTT at the time of incubation. The crystals were dissolved through pipetting carefully. The absorbance of the product was measured at 590 nm using a microplate reader. The experiments were performed in the form of triplicates. The average of triplicates was used to calculate the IC₅₀ and survival percentage of the cells. The growth curves illustrate the mean and standard deviation (SD) values of a minimum of three independent experiments. The increase of inhibition was analysed using the following formula: percentage of increase inhibition = (control optical density (OD) − sample OD)/control OD × 100. The value of IC₅₀ showed the concentration of NPs that produced a 50% reduction of cell viability.

2.8 Analysis of the morphological alterations in HT29 cells

The morphological alterations in HT29 cells were analysed through inverted microscopy. Initially, cells were incubated with TS-CSNPs at different concentrations for 24 hours. The morphological changes in the cells were observed using unstained and stained cells. HT29 cells were fixed with 10% formalin for 5 minutes for staining. Staining was performed using Crystal violet (0.2%) for 30 minutes. The slides were washed twice with sterile water carefully. Images were taken at 20× using Microvisible software on Micros’ Inverted Microscope. Furthermore, western blotting was performed to examine the alteration in expression of anti-apoptotic Bcl-2 and Bcl-xL protein along with the control β-actin protein.

2.9 Western blotting

Human HT29 colorectal cancer cells were grown in DMEM (Invitrogen) containing 10% heat-inactivated fetal bovine serum, 100 μg mL⁻¹ streptomycin, 100 units per mL penicillin and 2 mmol L⁻¹ L-glutamine. The HT29 cells were treated with 10 μg mL⁻¹ concentration of TS-CSNPs for 24 hours. Whole cell lysates were prepared as described in one of our previous studies.³² Soluble proteins were analyzed by immunoblotting with anti-Bcl-2, anti-Bcl-xL (Santa Cruz Biotechnology) and anti-β-actin (Sigma). Reactivity was detected with horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (GE healthcare).

2.10 Hemolysis assay for biocompatibility of TS-CSNPs

In vitro hemolysis assay was carried out with 5 mL of fresh blood. The blood samples were collected in sterile EDTA coating blood collection tube glasses from a healthy volunteer. Whole blood was used for the separation of erythrocytes/red blood cells (RBCs) by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded containing platelets and plasma proteins. The pellets of RBCs were washed thrice carefully with an equal volume of sterile phosphate buffered saline (PBS). After that, the pellets of RBCs (2 mL) were suspended in 6 mL of PBS. Then, the suspension of RBCs (100 μL) was added to 500 μL of the TS-CSNPs suspension in PBS with their inhibitory concentration of 420.33 μg mL⁻¹. 100 μL of RBC suspension was added to 500 μL of sterile water in a tube as the positive control and 100 μL of RBC suspension in 500 μL of PBS was used as the negative control. The test samples, positive and negative suspensions were briefly vortexed and incubated at 37 °C for 4 hours under static conditions. After incubation, the samples with positive and negative controls were carefully vortexed again and centrifuged at 5000 rpm for 10 min. The above aqueous layer was collected and used for the measurement of the absorbance value of hemoglobin at 575 nm using a Perkin-Elmer Lambda 2 UV-vis. The experiments were carried out in triplicate. The average of the triplicates was used to calculate the percentage of hemolysis using the formula:

% of hemolysis = [(sample absorbance − negative control absorbance)/(positive control absorbance − negative control absorbance)] × 100.^33,34

2.11 Antibacterial activity of TS-CSNPs

The antibacterial activity of the synthesized TS-CSNPs was measured by the disk diffusion method.³⁵

2.11.1 Bacterial strains. Nine strains of bacteria, including four Gram-positive bacteria (Escherichia coli ATCC-35218, Escherichia coli ATCC-25922, Enterococcus faecalis ATCC-29212, and Bacillus subtilis NCTC-10400) and five Gram-negative bacteria (Staphylococcus aureus ATCC-29213, Pseudomonas aeruginosa ATCC-27853, Shigella sonnei ATCC-11060, Salmonella typhimurium ATCC-13311, and Proteus vulgaris ATCC-6380) were used for the determination of antibacterial activity.

Pure cultures of the bacteria were sub-cultured in Mueller-Hinton Broth overnight at 37 °C. The turbidity of the bacterial culture was adjusted to 0.5 McFerland standard. Each bacterial strain was swabbed uniformly onto separate agar plates using sterile cotton swabs. Sterile paper disks were placed on the agar plates, and 10 μL of 250 μg mL⁻¹ (w/v) concentration of TS-CSNPs were applied to the disks. The MIC values of TS-CSNPs and control drugs was determined through the standard CLSI protocol.³⁶

2.11.2 Control drugs. The standard cefotaxime MIC test strips (Liofilchem) and ampicillin (bioMérieux) were used to assess the activity of standards against nine micro-organisms. The strip of ampicillin and cefotaxime was used as the control drug in the experiments. All the plates were incubated at 37 °C for 18 hours. The tests were repeated three times. The zone of inhibition, which appeared as a clear area around the disks, was measured and compared with standards.

3 Results and discussion

3.1 Synthesis of luminescent mesoporous Tb(OH)₃@SiO₂ core–shell nanoparticles

The luminescent mesoporous Tb(OH)₃@SiO₂ core–shell nanoparticles (TS-CSNPs) were prepared by hydrolysis of TEOS with aqueous ammonia in a W/O micro-emulsion containing an aqueous solution of the Tb(acac)₃·3H₂O chelate, surfactant, co-surfactant, and an oil phase.²¹ We analysed the solubility of TS-CSNPs through the dispersion of core–shell nanoparticles in different polar and non-polar solvents. These silica-coated core–shell nanoparticles showed excellent dispersibility in polar solvents (water, ethanol) as well in a non-polar solvent (cyclohexane).

3.2 Characterization of TS-CSNPs

The following characterization methods were used to confirm the synthesis of the precise surface modified TS-CSNPs.

3.2.1 Transmission electron microscopy. The size and morphology of the as-prepared TS-CSNPs were analyzed by TEM. The result of TEM images are illustrated in Fig. 1, and are typical images of an as-prepared powder. The low resolution TEM micrograph shows that the nanoparticles are non spherical, narrowly distributed and highly aggregated (Fig. 1A and B). However, the core–shell nanostructure of the surface modified nanoparticles was not illustrated clearly due to the deposition of a thick silica layer (light grey spheres) around the surface of Tb(OH)₃ nanoparticles, which increased the dispersion of the nanoparticles in polar and nonpolar solvents. Furthermore, we used micro-emulsion process for silica surface deposition and co-precipitation process for hydrolysis of terbium tris-acetylacetonate in this case little amount of co-surfactants may be attached around the surface of particles causing the particles are aggregated. The luminescent cores are dark and silica shell is light grey color. It confirms that the irregular shape and high aggregation nature of the nanoparticles resulted from a co-precipitation process. In addition, hygroscopic nature due to surface silanol groups of the material is responsible for high aggregation of the nanoparticles. The luminescent cores (Tb(OH)₃) are dark and have an irregular shape with an average size of about 200–250 nm. The high degree dispersibility and aggregation of the core–shell nanoparticles in polar and non-polar solvent may indicate that the surface of the nuclei is covered by CTAB medium right after formation, which limits the growth of particles and stabilizes them against agglomeration.

Fig. 1 Typical FE-TEM images of the synthesized and surface modified TS-CSNPs at a 50 nm scale: (A) micrograph of the mesoporous core–shell nanoparticle TS-CSNPs; (B) outer layer of mesoporous core–shell nanoparticle TS-CSNPs at a higher magnification; (C) EDX image of the synthesized luminescent mesoporous TS-CSNPs.

These surface modified silica-coated core–shell nanoparticles exhibit excellent dispersibility in polar solvents such as water, ethanol and non-polar solvent including cyclohexane. Similarly to the N,N,N¹,N¹-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)-phenylpyridine]tetrakis(acetate), Tb³⁺-doped silica nanoparticles,³⁷ the dark dots embedded inside the silica network that can be observed in the TEM images show that the Tb(III) chelate molecules in the nanoparticles exist by physically interacting with the silica network. Due to the silica-surface modification and small presence of surfactant and co-surfactant, the nanoparticles are not well separated (aggregated) in organic solution (ethanol) media as evident from the TEM micrographs. These silica coated core–shell nanoparticles with small pore sizes are advantageous and favourable for drug delivery applications. The EDX images confirmed the presence of terbium in surface modified TS-CSNPs (Fig. 1). The strongest Si peaks are clearly indicated together with Tb and O peaks. It should be noted that the origin of strong Cu peaks that appear in the EDX spectra originated from the copper micrometer grids. The C peak was also generated from the carbon-coated Cu-TEM grid. No other impurities are evident in the figure, implying that the resulting TS-CSNPs are pure in chemical composition (Fig. 1C).

3.2.2 Optical absorption spectroscopy. Optical absorption spectroscopy was employed to characterize the optical features of the synthesized TS-CSNPs. The suspension of TS-CSNPs was dispersed thoroughly in an ultrasonic bath prior to observing the absorption spectra. The optical absorption spectra show that the surface modified TS-CSNPs dissolved in de-ionized ethanol (Fig. 2). The absorption spectra of TS-CSNPs display two broad absorption bands centred at 225 and 295 nm. The absorption band peaking at 225 nm originates from the silica surface, which also agrees with the spectra of previous literature reports.²⁰ The absorption band at 295 likely originates from the 4f–4f transition within the Tb(III) ion.³⁸ The appearance of this band in the visible region clearly indicates the successful formation of the bifunctional nanocomposite. As the silica shell began to cover its surface, this peak shifted to longer wavelengths due to changes of the dielectric constant of the environment near the Tb(III) surface. The FTIR spectrum of TS-CSNPs shows all the prominent peaks of silica as well as the asymmetric and symmetric stretching vibrational modes of hydroxyl groups at ∼1088, 956, 783 cm⁻¹ (Si–O–Si) and 3454 and 1636 (O–H) and 470 cm⁻¹ (Tb–O), respectively.²¹

Fig. 2 UV-vis absorption spectra of surface modified TS-CSNPs in ethanol and inset shows the FTIR spectra of TS-CSNPs.

3.2.3 Photoluminescence spectroscopy. The properties of photoluminescence in TS-CSNPs were examined under the excitation of 325 nm by fluorescence spectrometer at room temperature. As shown in Fig. 3, the spectral transition measured in the range from 350 to 750 nm is associated with the transitions from the excited ⁵D₄ level to ⁷F_J (J = 1, 2, 3, 4, 5 and 6) levels of Tb³⁺ activators; the most intense hypersensitive emission is the ⁵D₄ → ⁷F₅ transition located in the range of 555–535 nm, corresponding to the green emission (543/548 nm), in good accordance with the Judd–Ofelt theory.^39–41 This hypersensitive emission transition is the true fingerprint of the characteristic emission lines corresponding to 4fⁿ–4fⁿ transitions of terbium ions, which can give information about the chemical environment of the Tb(III) ion. A broadening and splitting of the spectral lines are also observed and are induced by the change in the chemical environment of Tb³⁺ ions during the formation of new chemical bond between silica and terbium metal ions. It is observed that the emission spectrum is uplifted due to the existence of a high quantity silica surface around the TS-CSNPs, which makes it a suitable energy acceptor/quencher in FRET-based assays.

Fig. 3 Photoluminescence spectrum of surface modified TS-CSNPs.

3.3 Cytotoxicity assays

To present inclusive results concerning the potential uses of synthesized TS-CSNPs for non-persistent purposes, it is necessary to evaluate the cytotoxicity of the synthesized nanoparticles. The effect of TS-CSNPs was screened for measuring its cytotoxicity through MTT assay. The HT29, SW620 cells and normal cells MCF-10A (non tumorigenic epithelial cell line) were treated with a range of concentrations of TS-CSNPs (0, 5, 10, 20, 40, 80, 160, 320 and 640 μg mL⁻¹) and doxorubicin hydrochloride for 24 h to determine the IC₅₀ values. The untreated cancerous cell line and normal cells did not show any significant cytotoxicity. Cancerous cells HT29 and SW620 demonstrated the potential cytotoxicity of TS-CSNPs with respect to untreated cells where the viability was assumed to be 1 (i.e. 100%). The results demonstrate that TS-CSNPs induced a potentially cytotoxic response (Fig. 4 and 5). The treatment of HT29 and SW620 cells with TS-CSNPs for 24 h at a concentration of 5 μg mL⁻¹ showed a slight alteration in cell viability against HT29 cells, however the cytotoxic effect of TS-CSNPs in the SW620 cell line was negligible. It may be due to the resistant nature of SW620 against the various ranges of concentrations of TS-CSNPs.

Fig. 4 Cytotoxicity (in terms of survival percentage of cells) of TS-CSNPs against HT29 cells determined by MTT assay after 24 h of treatment.

Fig. 5 Cytotoxicity (in terms of survival percentage of cells) of TS-CSNPs nanoparticles against SW620 cells determined by MTT assay after 24 h of treatment.

The HT29 cells treated with increasing concentrations (0, 5, 10, 20, 40, 80, 160, 320 and 640 μg mL⁻¹) of TS-CSNPs for 24 h illustrate a noticeable dose-dependent reduction in cell viability during experiments. The results of the MTT assay demonstrate that TS-CSNPs have a profound effect on human colorectal cancer cells HT29 but not on SW620 with IC₅₀ (inhibition of 50% viable cells) values of 420.33 μg mL⁻¹ and 2704 μg mL⁻¹, respectively. Also, the outcome of the MTT assay with serial concentrations of control drug doxorubicin demonstrated that doxorubicin exerts a more significant effect on HT29 cells with a 11 μg mL⁻¹ value of IC₅₀ (inhibition of 50% viable cells). The results showed that doxorubicin also induced a significant cytotoxic response (Fig. 6). However, cancerous cells SW620 did not show significant cytotoxicity against doxorubicin. The maximum concentration (640 μg mL⁻¹) of synthesized TS-CSNPs and the control drug did not shown significant cytotoxicity against MCF-10A cells (non tumorigenic epithelial cell line). These outcomes of cytotoxicity experiments confirmed the non-toxic nature of TS-CSNPs in the in vitro model. The results of the present study are in agreement with the fact that the most employed biocompatible material for the preparation of nanoparticles is the terbium oxide. Interestingly, TS-CSNPs have non-significant toxic effect (about 80% viability at the highest concentration) on a normal cell line, whereas HT29 cancer cells demonstrated a sufficient cytotoxic effect against the same TS-CSNPs. Thus, it is advocated that the severe cytotoxicity mainly is initiated from the cellular internalization of TS-CSNPs instead of the physical injury of the cell membrane. In addition, various studies have confirmed that nanoparticles may enter the cytoplasm through diverse routes and a variety of mechanisms.^42–44

Fig. 6 Cytotoxicity (in terms of survival percentage of cells) of doxorubicin against HT29 cells determined by MTT assay after 24 h of treatment.

One of the studies showed that differently sized gold nanoparticles accumulate in different compartments of the cells and exert different levels of cytotoxicity. GNPs of 3 nm and 10 nm sizes entered the nucleus, while 25 nm and 50 nm particles accumulated around the nucleus.^45,46 The 3 nm GNPs demonstrated the highest toxicity whereas large sized GNPs exhibited a smaller cytotoxic effect in HEp-2 cells using MTT assay.⁴⁵ In the Trojan horse mechanism, once the cells have been penetrated by the metal present in the particle, metal ions can escape from the existing particle and produce reactive oxygen species (ROS) in the internal environment of the cell, causing oxidative stress to the cells.^47,48 Our results of cytotoxicity of TS-CSNPs were inconsistent against SW620 cells. In order to investigate the morphological changes and mechanism of cytotoxicity in cancerous cells, Crystal violet staining and western blotting analysis were performed with apoptotic marker genes.

3.4 Analysis of morphological alterations in HT29 cells

The results of Crystal violet stained HT29 cells clearly showed a reduction in cells numbers with different concentration of TS-CSNPs compared to the untreated control (Fig. 7A–D). Morphologically cells became circular, an indication of apoptotic cells. Also, the loss of membrane integrity, inhibition of cell growth and cytoplasmic condensation were observed with TS-CSNPs treatment in unstained HT29 cells compared to the untreated control (Fig. 8A–D). These results indicate that TS-CSNPs inhibit cellular proliferation by inducing cell death.

Fig. 7 (A–D) HT29 cells were treated with increasing concentrations of TS-CSNPs. Crystal violet staining was done and images were taken using Microvisible software. Morphological changes and the reduction in the number of cells indicates the activity of TS-CSNPs compared to untreated control cells.

Fig. 8 (A–D) Unstained HT29 cells were also treated with increasing concentrations of TS-CSNPs. Morphological alterations and the reduction in the number of cells indicates the activity of TS-CSNPs compared to untreated control cells. Phase contrast microscopy images were capture at 20× using Microvisible software.

3.5 Western blotting

The effect of TS-CSNPs was studied on the expression of the anti-apoptotic Bcl family protein in the human colorectal cancer cell line HT-29. Western blot analysis was performed to analyze the expression levels of the anti-apoptotic protein (Bcl-2 and Bcl-xL) in HT29 cells. The expression level of β-actin was used as a control in our experiments. The HT-29 cells were treated with TS-CSNPs at a concentration of 420 μg mL⁻¹ for 24 hours. The results showed that the TS-CSNPs down regulate the expression of anti-apoptotic Bcl-2 and Bcl-xL gene products and inhibit the proliferation of HT29 cells compared to the untreated control and β-actin as the loading control (Fig. 9A and B). Our study is in agreement with the recently published work of Jeyaraj et al. (2015), which showed Bax/Bcl2 and caspase–cascade mediated dysfunction in mitochondria.⁴⁹ The results indicated that no significant change was observed in the expression of the untreated control and loading control β-actin (Fig. 9C). Although, the results of the MTT assay showed a significant toxic effect of TS-CSNPs against HT29 cells, the mechanistic approach needs confirmation of these result. Various studies show that the over expression of Bcl-2 and Bcl-xL significantly inhibits programmed cell death.^50,51 In other words, the down regulation of the expression of Bcl-2 and Bcl-xL accelerates the process of apoptosis through by controlling the activation of caspase proteases.⁵¹ Our results indicated that TS-CSNPs inhibited cellular proliferation through the down regulation of the expression of anti-apoptotic Bcl-2 and Bcl-xL proteins. Therefore, the down regulation in the expression of anti-apoptotic markers (Bcl-2 and Bcl-xL) validates our MTT assay results through western blotting.

Fig. 9 Western blot analysis of the expression profile of anti-apoptotic proteins Bcl-2, Bcl-xL, and control β-actin of the apoptotic pathway in HT29 cells treated with 500 μg mL⁻¹ TS-CSNPs, with untreated HT29 cells as the control (C) for 24 hours. At the end of the treatment, cells were lysed in buffer and the cell extract subjected to western blots antibodies: anti-Bcl-2, anti-Bcl-xL and anti-β-actin. The images of the immunoblot show the expression levels of anti-apoptotic proteins. (A) Down regulation of the expression of Bcl-2 showed in the immunoblot. (B) Down regulation of the expression of Bcl-xL showed in the immunoblot. (C) The unchanged expression level of β-actin in the immune-blot as the control.

3.6 Hemolysis assay for biocompatibility of TS-CSNPs

The result of various pathological conditions is hemolysis, which may potentially cause anaemia. To measure the biocompatibility of TS-CSNPs, an in vitro hemolysis assay was performed to evaluate the possible toxic nature of the synthesized TS-CSNPs as described in the literature.^52,53 The hemolytic activity of TS-CSNPs nanoparticles on human RBCs is shown in Fig. 10. The results showed that the hemolytic activity of TS-CSNPs was less considerable with respect to the positive control, which demonstrates the harmless nature of TS-CSNPs in applications. Our results showed the better biocompatibility of the synthesized TS-CSNPs in vitro. The acceptable level of hemolysis of biological based materials is 5%, as per the guidelines of the International Organization for Standardization/Technical Report 7406. The results of the synthesized TS-CSNPs demonstrate inadequate levels of hemolysis, showing their biocompatibility and suitability in biomedical sciences.^33,54

Fig. 10 In vitro hemolysis assay demonstrates the biocompatibility of TS-CSNPs compared to positive and negative controls.

3.7 Antibacterial activity of TS-CSNPs

The antibacterial activity of the synthesized TS-CSNPs in this study was investigated against Escherichia coli ATCC-35218, Escherichia coli ATCC-25922, Enterococcus faecalis ATCC-29212, and Bacillus subtilis NCTC-10400 (Gram-positive bacteria) and Staphylococcus aureus ATCC-29213, Pseudomonas aeruginosa ATCC-27853, Shigella sonnei ATCC-11060, Salmonella typhimurium ATCC-13311, and Proteus vulgaris ATCC-6380 (Gram-negative bacteria).

The TS-CSNPs did not show any antibacterial activity with 250 μg mL⁻¹ against all strains of bacteria used in this study (Fig. 11A–I). The control drug cefotaxime (CTX) showed higher antibacterial potential with respect to ampicillin (AM) against all bacterial strains used in this study except Enterococcus faecalis ATCC-29212 (for which both control drugs demonstrated a MIC value of 250 μg mL⁻¹). The observed MIC values of TS-CSNPs were >10000 μg mL⁻¹ with compression of the control drug, Table 1. As we know that the host and microbiome including bacteria, fungi and archaea have co-evolved into a complex form, the complicated associations benefit the host in various ways, such as through metabolism and nutrition.^55–57 The dysbiosis in microbiome such as E. coli and other symbiotic bacteria may act as a potential factor in the growth and development of many types of cancer, including colon cancer.^2,3,8 These TS-CSNPs exhibited non-significant effects on various strains of Gram positive and Gram negative bacteria, which might be more beneficial for the development of anticancer nanomedicines due to their minimum effect on the normal microbiota of the host. The overall results of the antimicrobial activity of our study suggest that TS-CSNPs can open a new door into cancer research, therapeutics and management.

Fig. 11 Synthesized TS-CSNPs showed negligible antibacterial activity against four Gram positive (Escherichia coli ATCC-35218, Escherichia coli ATCC-25922, Enterococcus faecalis ATCC-29212, and Bacillus subtilis NCTC-10400) and five Gram negative bacteria (Staphylococcus aureus ATCC-29213, Pseudomonas aeruginosa ATCC-27853, Shigella sonnei ATCC-11060, Salmonella typhimurium ATCC-13311, and Proteus vulgaris ATCC-6380) at a 250 μg mL⁻¹ (as indicated by T) concentration compared to the control drugs ampicillin (AM) and cefotaxime (CTX, indicated with C).

Table 1 The MIC values of TS-CSNPs and control drugs was determined as per the CLSI (Clinical Laboratory Standards Institute) protocol. The MIC of the control was determined through commercial E-test strips. (*All values are in μg mL⁻¹; *AM – ampicillin; **CTX: cefotaxime)

Bacterial strains	Tb(OH)3@SiO2 core–shell nanoparticles	*AM (control)	**CTX (control)
Escherichia coli ATCC-35218	>10 000	>256	0.032
Staphylococcus aureus ATCC-29213	>10 000	>256	1.5
Escherichia coli ATCC-25922	>10 000	>256	0.032
Pseudomonas aeruginosa ATCC-27853	>10 000	>256	24
Enterococcus faecalis ATCC-29212	>10 000	>256	1.5
Bacillus subtilis NCTC-10400	>10 000	>256	256
Shigella sonnei ATCC-11060	>10 000	256	0.016
Salmonella typhimurium ATCC-13311	>10 000	0.75	0.016
Proteus vulgaris ATCC-6380	>10 000	>256	24

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4 Conclusion

In this study, using human colorectal cancer (CRC) cell lines as the assay system, we performed a systematic investigation of the cellular response to TS-CSNPs exposure in both MTT and western blotting assay. The presence of a silica-shell on the surface of the Tb(OH)₃ nanoparticles is highly favourable, because it can reduce the toxic effect and enhance the biocompatibility of the material. Our data indicate that TS-CSNPs exposure could cause significant toxicity in the cancerous cell line HT29 through the down regulation of the anti apoptotic gene products Bcl-2 and Bcl-xL. Considering the potential toxicity of TS-CSNPs in hypoxic HT29 cells, the knowledge gained here will be helpful for directing the development of TS-CSNPs-related therapies in CRC patients and guaranteeing the safe applications of other nanotechnologies. The exposure of TS-CSNPs shows very low levels of hemolysis, demonstrating their appropriateness and biocompatibility for biomedical uses. Considering the chemical inertness and biocompatibility of silica, this technology may provide a new platform for nonviral gene delivery as well as anti-cancer drugs. This system provides a new dimension for the development of new diagnostic and therapeutic agents. Future investigations may provide more understanding of the relationship between surface properties and cellular uptake, translocation, metabolism, oxidative effects and other biological effects of size controlled TS-CSNPs in vivo and in vitro. Therefore, TS-CSNPs may be a potential anticancer agent in the future.

Acknowledgements

The authors are very grateful to the Deanship of Scientific Research and Research Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.

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