Calcium carbonate end-capped,folate-mediated Fe 3 O 4 @mSiO 2 core-shell nanocarriers as targeted controlled-release drug delivery system

General materials and reagents

Iron (III) chloride hexahydrate (FeCl₃.6H₂O), iron (II) chloride tetrahydrate (FeCl₂·4H₂O), and ammonia solution (25 − 28%) were purchased from Damao reagent company (Tianjing, China). Tetraethyl orthosilicate (TEOS) and 3-aminopropyltriethoxysilane (APTES) (reagent grade, >98%) were purchased from Sigma-Aldrich. Ammonia aqueous solution (Chempur, purep.a., 25%) was used to increase pH of the reaction system (acceleration of silanes hydrolysis). 3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Prussian blue, and N,N-dimethyl-4-nitrosoaniline (RNO) were obtained from Sigma-Aldrich. DMEM medium, fetal bovine serum (FBS), trypsin-EDTA solution and penicillin–streptomycin solution were purchased from Gibco Life Technologies. 4′-6-diamidino-2-phenylindole (DAPI) was obtained from Beyotime Biotech (China). All the other chemicals were of analytical grade, and Millipore water (18.2 MΩ) was used throughout the experiments.

Instrumentation

Transmission electron microscope (TEM) analyses were performed with a JEM-2010HR TEM. UV-Vis and FTIR spectra were recorded on a Shimadzu UV-3150 spectrophotometer and an Equinox 55 Fourier transformation infrared spectrometer, respectively. Nitrogen adsorption–desorption measurements were performed on an V-Sorb X800 (Gold APP Instruments Corporation China, china) accelerated pore size analyzer at 77 K. Fluorescence microscopy of apoptosis assays was performed with an IX 70 fluorescence microscope (Olympus, Japan). MMSN drug delivery efficiency was characterized by flow cytometry using a BD FACSCalibur flow cytometer (Becton Dickinson Inc., USA). Size distribution of the particles was obtained on a Malvern Zetasizer Nano ZS90 instrument (Malvern, UK). Confocal images were obtained by confocal laser scanning microscopy (CLSM, LSM-510, ZEISS, Germany).

Preparation of Fe₃O₄@SiO₂ (MSN)

Fe₃O₄ Nps were synthesized by a coprecipitation of ferric and ferrous chlorides in alkaline medium following the method we have previously reported. ²² Fe₃O₄@SiO₂ core-shell nanocomposites were fabricated via sol-gel process of hydrolysis and condensation of TEOS. Briefly, 50 mg Fe₃O₄ compound was transferred to a beaker, dispersed in 30 ml water, assisted with ultrasounds. After that, 160 ml ethanol and 10 ml concentrated ammonia were added to the obtained aqueous colloid. The mixture was continuously stirred in ambient conditions. Afterwards, 0.5 ml TEOS was added to the above suspension under continuous mechanical stirring. After reacting for 1.5 h, the products were collected by magnetic separation and washed with ethanol for several times, and the MSN were thus prepared.

Synthesis of MMSN

MSN (30 mg) were dispersed in 1 ml chloroform, followed by addition of 20 ml aqueous solution containing 0.4 g copolymerization ratio (CTAB). ²³ After vigorously stirring, a homogeneous oil-in-water microemulsion was obtained. The microemulsion was kept at 60°C for 10 min to remove the chloroform from the solution via evaporation, forming water-dispersed nanoparticles. The resulted aqueous solution was diluted with 80 ml deionized water before 6 ml aqueous ammonia solution (25 wt %), 1 ml TEOS, and 10 ml ethyl acetate were successively added. After sonication for 30 s, the mixture was kept stirring for 6 h at 40°C with a stirring rate of 80 r/min. The resultant was collected by centrifugation and washed with water and ethanol for five times. Finally, CTAB was removed by refluxing in an ethanol solution of ammonium nitrate for 6 h, to yield MMSN. ²⁴

Preparation of MMSN-FA

About 30 mg MMSN were dispersed in a mixed solvent containing 80 ml ethanol and 15 ml acetonitrile, and then mixed with 5 ml concentrated ammonia solution (25–28 wt%) under ultrasound for 15 min. The mixture was continuously stirred in ambient conditions. Subsequently, APTES (0.5 ml) were added, and the mixture was stirred for 2.5 h. The resultant nanocomposite was collected by a permanent magnet, washed with ethanol several times, and finally dried under vacuum at room temperature for 12 h to yield MMSN-NH₂.

Activation of the MMSN-NH₂ with FA was carried out via EDC/NHS chemistry according to a reported procedure with little modification. ⁴ In brief, FA (10.0 mg, 0.022 mmol) was dissolved in 30 ml anhydrous DMSO. Then, EDC (2.2 mg, 0.2875 mmol) and sulfo-NHS (115.1 mg, 1.007 mmol) were added to activate the carboxylic acid groups of FA, and the mixture was stirred gently at room temperature in a N₂ atmosphere for 4 h. Afterwards, the MMSN-NH₂ nanocomposite fully dispersed in DMSO (10 ml) was added to the activated FA solution, followed by further stirring for another 18 h at room temperature. The folate-conjugated MMSN-FA nanocomposite was separated, washed two times with DMSO and then four times with phosphate-buffered saline (PBS) (pH 7.2), and finally dried under vacuum at room temperature for 48 h. (The reaction needs to be carried out in dark conditions, folic acid needs to be protected from light.)

Preparation of DNM loaded MMSN-FA(MMSN-FA-DNM)

Drug loading was accomplished by using the solvent evaporation method with slight modifications. ²⁵ Generally, DNM was firstly dissolved in distilled water and stored at 4°C before using. For the drug loading process, 10 mg MMSN-FA were added to 10 ml 0.3 mg/ml DNM solution and the mixture was kept in a shaker for 24 h in dark conditions. Finally, the DNM-loaded MMSN-FA (MMSN-FA-DNM) were recovered by magnetic separation and washed twice with Milli-Q water to remove unbound drug molecules. The supernatant was collected to determine the drug loading content and drug encapsulation efficiency (EE) from UV-Vis absorbance at 480 nm. The amount of DNM loaded into the nanoparticles was determined from a calibration curve obtained for a series of DNM solution at different concentrations. The drug loading content and entrapment efficiency were determined by the following.

Preparation of CaCO₃-coated MMSN-FA-DNM(MMSN-FA-DNM-CaCO₃)

The MMSN-FA-DNM coating with CaCO₃ were prepared according to a method described previously. ²¹ For MMSN-FA-DNM coating with CaCO₃, empty or drug loaded MMSN-FA were dispersed in a calcium chloride solution (8 mM). After stirring for 5 min, sodium carbonate (8 mM) was added and the mixture stirred for 1 h at room temperature. The dispersion was then magnetically separated to collect the coated MMSN-FA-DNM. The supernatant was stored at 4°C to assess drug release during coating procedure through UV-vis spectroscopy, as described above.

In vitro drug release

To start the drug release, MMSN-FA-DNM-CaCO₃, MMSN-FA-DNM, MMSN-DNM-CaCO₃, and MMSN-DNM Nps were added to equal volumes of Tris buffer at pH 7.4 and 5.6, respectively, and then were thermostated at 37°C with continuously shaking. At given time intervals, each tube was collected by magnetic separation, and the released drug concentration in the supernatant was determined with UV-vis spectrophotometry. The DNM loading (%) was defined as the weight fraction of the drug in the final drug loading. ²⁶

Cell culture

HeLa and A549 cell lines were cultured in the DMEM medium containing 10% heat-activated FBS and 100 IU/ml penicillin and 100 g/ml streptomycin. They were incubated in a 37°C water-jacketed incubator equilibrated with 5% CO₂ and kept at approximately 99% relative humidity. The medium was replenished every other day until confluence was achieved. The cells were then washed with PBS and harvested with 0.125% Trypsin–EDTA solution. ²⁷

Hemolysis testing experiment

For the hemolysis testing experiment, 4 ml fresh anticoagulation human blood was diluted with 5 ml physiological saline. MMSN nanosuspension (1 ml) (concentration is 1.0 mg/ml, drug free) was incubated in the 37°C water bath for 30 min, and then 1 ml diluted blood was added to the nanosuspensions, followed by shaking for 60 min in the water bath. The mixture was then centrifuged, and the absorbance of the supernatant at 545 nm was measured. Physiological saline and distilled water were used as negative control and positive control, respectively. The hemolysis rates of materials were calculated using the following equation

Here, H% refers to the hemolysis rates of materials, A_m, A_n, A_p refer to the absorbance of the supernatant for the studied materials, the negative control and the positive control at 545 nm, respectively.

Cell viability for biocompatibility research

The cytotoxicity of the MMSN-CaCO₃ and MMSN-FA-CaCO₃ nanocarrier against HeLa and A549 cell lines was determined using an MTT assay. The cells were seeded in a 96-well plate at a density of 5000 cells/well and incubated in Dulbecco’s modified eagle medium (DMEM) (Gibco) containing 10% FBS at 37°C in 5% CO₂ for 24 h. The medium was then removed and replaced with 100 μL medium containing nanocarrier micelles. The culture medium was exchanged and the cells were incubated with different concentrations of MMSN-CaCO₃ and MMSN-FA-CaCO₃ ranging from 12.5 to 200 μg/ml. After exposure to the nanocarrier for 4 h, the viability of cells was determined using the MTT assay at 490 nm.

Prussian blue staining

HeLa cells were seeded on 24-well plate and incubated overnight. A solution of the MMSN-FA with a concentration of 50 μg/ml was then added and the cells were cultured for 6 h. At the end of the culture period, each well was washed three times with PBS, treated with 4% paraformaldehyde solution at 4°C for 30 min to fix the cells, and washed three times with PBS again. A 1:1 mixture of 5% potassium ferrocyanide (II) trihydrate solution and 5% HCl was added to each well and the cells were incubated at room temperature for 1 h before being counterstained with neutral red. Each well was then washed three times with PBS and analyzed by light microscopy. ²⁸

Magnetic targeting study

HeLa cells were seeded in a 60 mm petri dish and incubated overnight, and then a solution of the magnetic nanospheres with a concentration of 50 μg/ml was added. For magnetic targeting study, a magnet (about 4 T) was placed beside the Petri dish. After incubation for 24 h, the Petri dish was photographed, and the cells in the position of magnet (targeting area) and the area with a much weaker magnetic field strength (control area) were observed using a light microscope.

Flow cytometry analysis of nanoparticle drug delivery efficiency

The nanoparticle drug delivery efficiency by free DNM, MMSN-FA-DNM-CaCO₃, MMSN-DNM-CaCO₃ was studied by flow cytometry. ²⁹ HeLa and A549 cells (5 × 10⁴ cells/ml) were harvested from monolayers by 0.25% (w/v) trypsine and 0.03% (w/v) EDTA solution. After 1 ml of DMEM medium was added to each well of a 12-well plate, cells in 200 ml of DMEM medium (with or without FA) were seeded in each well and were incubated for 24 h. When the cells reached 90% confluence, the medium was removed and the cells were washed twice with PBS (pH 7.4). The cells were then treated with DNM-loaded nanoparticles or free DNM (0.25 μg/ml DNM) in a humidified incubator with 5% CO₂ atmosphere at 37°C. After 4 h of incubation, the medium was discarded and the cells were washed twice with 2 ml of PBS. Cells were then detached by 0.25% (w/v) trypsin and 0.03% (w/v) EDTA solution and then dispersed in 0.2 ml of PBS for flow cytometric measurements. Nanocarrier uptake was analyzed based on the DNM fluorescence using a FACSCalibur flow cytometer. A minimum of 1 × 10⁴ cells was analyzed from each sample, with fluorescence intensity displayed on a four-decade log scale.

Confocal image analysis

For confocal laser scanning microscopy (CLSM) studies, HeLa cells cultured in DMEM medium (with or without FA) were seeded onto 35-mm round glass coverslips, placed in a 6-well plate, and grown overnight. The medium was then removed, and the cells were washed twice with PBS. Cells were treated with DNM-loaded nanoparticles or free DNM (0.25 µg/ml DNM) for 4 h at 37◦C and 5% CO₂. Cells were washed twice with PBS and fixed with 1.5% formaldehyde. Coverslips were placed onto the glass microscope slides, and sample uptake was visualized at excitation and emission wavelengths of 535 and 590 nm, respectively. Digital monochromatic images were acquired using Leica Confocal Software. ²⁹

DAPI staining for nuclear morphology study

For visualization of HeLa cells, the nuclei of both the cells were stained with DAPI. The efficiency of MMSN-FA-DNM was tested through apoptosis study. For this purpose, HeLa cells were treated with MMSN-CaCO₃ (control set) and DNM, MMSN-DNM-CaCO₃, MMSN-FA-DNM-CaCO₃ at different DNM concentrations (0.25, 0.5, and 1 μg/ml) for 24 h at 37°C. Then, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100, and stained with 1 mg/ml DAPI for 10 min. The cells were then rinsed with PBS and examined under fluorescence microscopy. ³⁰

In vitro cytotoxicity assay

For cytotoxicity assays, HeLa and A549 cells were seeded for 48 h in standard 96-well plates at 1 × 10⁴ cells per well. Then, free DNM, MMSN-DNM-CaCO₃, or MMSN-FA-DNM-CaCO₃ spheres were added to the medium at different DNM concentrations (0.03–0.5 μg/ml), the cells were incubated in 5% CO₂ at 37°C for 48 h, and then the cells were washed carefully with PBS to remove DNM, MMSN-DNM-CaCO₃, or MMSN-FA-DNM-CaCO₃. The cells were rinsed thrice with Hank’s Buffered Salt Solution (HBSS, pH 7.4) and incubated for 4 h in 150 μl of medium containing 0.5 g/l of MTT. Absorbance was measured at 490 nm using a multiwell plate reader

ODdrug is the absorbance of cells incubated with the different concentrations of DNM, MMSN-DNM-CaCO₃, or MMSN-FA-DNM-CaCO₃ spheres, ODcontrol is the absorbance of cells incubated with culture medium, and ODblank is the absorbance of culture medium.

Statistical analysis

All of the data were expressed as the mean ± SD. Differences between two groups were analyzed by a two-tailed Student's t test. Differences with *P < 0.05 were considered statistically significant.

Preparation and characterization of MMSN-FA-DNM-CaCO3 nanoparticles

The schematic preparation process of MMSN-FA-DNM-CaCO₃ is illustrated in Scheme 1. The core-shell structure of the mesoporous silica is taken as the base drug carrier with the superparamagnetic nanoparticles as the nucleus. The MMSN microspheres were thus obtained by TEOS condensation around the CTAB template using a sol-gel method. Folic acid (for cancer-specific targeting) was linked with the amine groups on MMSN-NH₂ through formation of amide bond. ³¹ Then, after the successful loading of DNM, nanoparticles were coated with calcium carbonate after their formation by the Stober method. ³² The present approach demonstrates a relatively simple method for the formation of porous and amine functionalized silica nanoparticles as compared with the multistep modification procedure reported earlier.^33,34

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Scheme 1.

Schematic illustration of the steps for the fabrication of MMSN-FA-DNM-CaCO₃.

Figure 1 shows TEM images of the nanomaterials synthesized, namely MMSN-FA and MMSN-FA-CaCO₃, respectively. It was found that these well-dispersed nanospheres have uniform roughly spherical shapes with sizes of approximately 30–50 nm. Meanwhile, after CaCO₃ coating, the dispersion of these nanospheres slightly decreased, with PDI increasing from 0.312 to 0.592. This may result in the weakening of repulsive force between the nanoparticles.

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Figure 1.

TEM image of MMSN–FA (a, b) and MMSN-FA-CaCO3(c, d).

FTIR spectra of MMSN, MMSN-FA, MMSN-NH₂ and free FA are shown in Figure 2. In the IR spectrum of MMSN, a pair of bands at 595 cm⁻¹ and 620 cm⁻¹, and another pair of bands at 953 cm⁻¹, and 1087 cm⁻¹ were attributed to the characteristic Fe–O and Si–O–Si stretching. In the spectrum of MMSN-NH₂, peaks at 1087 cm⁻¹, 1458 cm⁻¹, 1647 cm⁻¹ could be attributed to Si–O–Si, C–N stretching vibrations and N-H bending, respectively, which suggests a successful grafting of APTES in a single, in situ approach. Additionally, two peaks at 2960 cm⁻¹ (asymmetric CH₃ stretching) and 2925 cm⁻¹ (symmetric CH₂ stretching) were observed (Figure 2(c)), which may be attributed to the presence of APTES on the nanoparticles. The IR spectrum of MMSN-FA show peaks at 1450, 1530, 2960 and 3400 cm⁻¹, which correspond to O=C-NH stretching, N−H bending, -CH₂ bond stretching, and C-H bond vibration, respectively, demonstrating the successful FA modification on the MMSN-NH₂ nanoparticles. ³⁵

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Figure 2.

FTIR spectra of (a) MMSN, (b) MMSN-FA, (c) MMSN-NH2 and (d) free FA.

Figure 3(a) shows the UV/Vis spectra of FA, MMSN-NH₂ spheres, and MMSN-FA spheres. The characteristic UV/Vis absorption peaks of FA at 295 nm and 370 nm were observed on the spectrum of MMSN-FA spheres, which confirmed that FA ligands have been grafted by the amide reaction. However, compared with free FA, a bathochromic shift could be noticed, which revealed the modification in the environment of the FA between the free and grafted states. The result is similar to the previous reported. ³⁶ Figure 3(b) shows zeta potentials of FA, MMSN-NH₂, and MMSN-FA spheres, which varies from −17.97 ± 0.377 to 6.63 ± 0.350, and −15.5 ± 0.499, respectively.

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Figure 3.

UV/Vis spectra (a) and (b) Zeta potentials of FA, MMSN-NH2, and MMSN-FA nanospheres.

The total surface area and the average pore diameter of the coated MMSN were evaluated by nitrogen adsorption/desorption isotherm analysis. As shown in Figure 4, the isotherm of MMSN-FA nanoparticles displayed a typical type IV feature, which indicated that MMSN-FA possess a defined mesoporous structure. The BET surface area for MMSN was found to be 106.6 m²/g (Figure 4(a)). BJH pore distributions (Figure 4(b)) for MMSN show unequal pore size distribution in the range 2 nm–50 nm, with the maximum around 2–6 nm. The average pore volume and pore diameter of MMSN were found to be 0.27 cm³/g and 3.2 nm, respectively. ³⁷

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Figure 4.

(a) N₂ adsorption-desorption isotherm of MMSN-FA hollow mesoporous spheres and (b) corresponding pore size distribution.

Drug loading and releasing on MMSN-FA-CaCO₃ nanoparticles

We next investigated the influence of DNM feeding concentration on the loading capacity. MMSN solutions were mixed with DNM at serial concentration ranged from 0.14 to 0.86 mg/ml. It was found that the loading capacity increased as the DNM feeding concentration increased (Figure 5). However, the influence of DNM concentration on the loading capacity became less obvious when the concentration fell in the region from 0.65 mg/ml to 0.86 mg/ml. The concentration of 0.65 mg/ml was thus used for drug loading.

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Figure 5.

DNM loading profile at different DNM concentrations (in water).

The in vitro release of DNM from CaCO₃-coated MMSN nanospheres was then studied in Tris buffer pH 7.40 (to simulate physiological conditions) and 5.6 (to simulate the tumor microenvironment and lysosomal compartments) at 37°C, respectively. The release was monitored by UV, and the release ratio results are given in Figure 6. The release of DNM from the non-coated particles presents a step-wise profile at pH 7.4 and 5.6. The cumulative release of MMSN-FA-DNM spheres at 120 h was 74.98%, indicating that most of the drug can be released in pH 5.6. However, the release at pH 7.40, which is the condition of blood plasma, is relatively slow and sustained. This indicates that MMSN-FA-DNM-CaCO₃ spheres are pH sensitive and have pH-controlled releasing property. ³⁸ This study clearly indicated the pH-sensitive release property of MMSN-FA-DNM-CaCO₃ spheres, which was expected to minimize premature release of the cytotoxic drug during blood circulation and facilitate the active drug release at the acid target issues, such as the microenvironments of extracellular tissues of tumors and the intracellular lysosomes and endosomes.

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Figure 6.

DNM release profile at different conditions from MMSN-FA-DNM (a, c) and MMSN-FA-DNM-CaCO₃ (b, d).

Biocompatibility of MMSN

The biocompatibility of the MMSN was evaluated by hemolysis rate and the cell toxicity. The hemolysis rate for the MMSN nanospheres at a high concentration of 125 µg ml⁻¹ was calculated to be 1.97%, which is far lower than the maximum hemolysis rate (5%) for the medical devise requested by FDA. ³⁹ This result suggests that the studied material is biocompatible for the circulating blood.

The study on cytotoxicity of drug delivery vehicles is very important for drug delivery systems. Although mesoporous silica and FA ligands have been reported to be suitable for drug delivery, it is necessary to investigate the cytotoxicity of MMSN-CaCO₃ and MMSN-FA-CaCO₃ nanospheres, because FA conjugation on MMSN spheres in this study was conducted under organic solution conditions. Figure 7(a) and (b) shows the effects of MMSN-CaCO₃ and MMSN-FA-CaCO₃ nanospheres on the viabilities of HeLa and A549 cells measured by MTT assay. It could be found that MMSN-CaCO₃ spheres and MMSN-FA-CaCO₃ spheres exhibit similar biocompatibility, and both have negligible cytotoxicity to HeLa cells at the concentrations up to 150 µg/ml. Meanwhile, these two kinds of nanospheres exhibit good biocompatibility and little cytotoxicity to A549 cells at the concentration lower than 200 µg/ml (Figure 7(c) and (d)). Therefore, CaCO₃ capping and FA modification did not decrease the biocompatibility of the system, and the MMSN-FA-CaCO₃ nanospheres could be used as potential drug delivery vehicles. ²⁴

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Figure 7.

Evaluation of the biocompatibility profile of MMSN-CaCO₃ and MMSN-FA-CaCO₃ in Hela cells (a, b) and A549 cells (c, d) at 48 h, respectively.

Cellular uptake of the magnetic nanospheres

Since it is well known that the biochemical processes that control the complexity of life occur in the cell cytosol and different subcellular organelles, the cellular uptake efficiency of the magnetic nanospheres was evaluated in order to assess its potential for efficient delivery of drug molecules into cells. ⁴⁰ The cellular uptake study for these magnetic nanospheres was conducted using HeLa cells.

To detect the presence of the magnetic nanospheres in HeLa cells, intracellular “Fe” was detected by the Prussian blue staining method. In the absence of MMSN-FA nanospheres, we did not observe any blue staining of the control cells (Figure 8(a)). However, as shown in Figure 8(b), after the incubation with the MMSN-FA nanospheres, substantial blue spots were observed in most of the cells. The majority of the blue spots seem to be localized around the cell membrane and throughout the cytoplasm, indicating high cellular uptake of the magnetic nanospheres.

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Figure 8.

(a) Prussian blue staining of HeLa cells treated in the absence and (b) the presence of the MMSN-FA magnetic nanosphere (50 μg/ml) for 4 h.

Magnetic targeting study of the magnetic nanospheres

In vitro magnetic targeting experiment was carried out to examine the magnetic targeting property of the magnetic nanospheres. A magnet (about 4 T) was placed beside the Petri dish. The cells closest to the magnet were referred to as the targeting area (orange circle) and the cells far from the magnet were referred to as control area (blue circle). After incubating with MMSN-FA-DNM-CaCO₃ nanospheres, the cells were photographed and compared (Figure 9).

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Figure 9.

Optical micrographs of HeLa cells at the targeting area (orange circle) and the control area (blue circle) after incubating with the magnetic nanospheres (50 μg/ml) for 24 h under an external magnetic field (about 4 T).

As shown in Figure 9, after incubation, in the orange circle (magnetic region), most of the adhered HeLa cells appeared to be round and bright, with floating in the cell culture media. This is the significant signal of cell death. However, for the region where the magnetic field is much weaker (blue circle), the cells suffered much fewer death than the magnetic position, indicating the magnetic nanospheres rapidly accumulated in the targeting area under a magnetic field. This result allowed the conclusion that the external magnetic field can significantly increase the local concentration of magnetic nanospheres, which implies that the magnetic nanospheres could efficiently accumulate to the targeted area under a magnetic guidance.

Drug delivery efficiency

The flow cytometry profiles of HeLa and A549 cells incubated with MMSN-FA-DNM-CaCO₃ spheres, MMSN-DNM-CaCO₃ spheres, or free DNM for 4 h in culture medium containing serum. Cells without any DNM sample treatment were used as a negative control. As demonstrated in Figure 10 (a) and (b), for HeLa cells, the MMSN were efficiently internalized into the cancer cells, with MMSN-FA-DNM-CaCO₃ presented higher mean fluorescence intensity (MFI) than MMSN-DNM-CaCO₃. This result indicates that the cellular uptake of the drug-loaded carriers is effectively enhanced by attaching folate to their surface. However, in A549 cells, no significant difference was observed in the MFI between MMSN-DNM-CaCO₃ and MMSN-FA-DNM-CaCO₃ groups (Figure 10(c) and (d)). This could be explained by the fact that the A549 are folate receptor (FR)-negative cells while the HeLa cells were FR-positive ones. Thus, MMSN-FA-DNM-CaCO₃ could selectively target the FR-positive cancerous cells by FR-mediated endocytosis.

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Figure 10.

MMSN loaded drug delivery efficiency to Hela cells and A549 cells. (a) Representative histograms of DNM in different nanospheres (b) and MFI flow cytometry analysis to HeLa cells. (c) Representative histograms of DNM in different nanospheres (d) and MFI flow cytometry analysis to A549 cells.

Cell uptake

To observe the intracellular behavior, free DNM, MMSN-DNM-CaCO₃, and MMSN-FA-DNM-CaCO₃ nanospheres were incubated with HeLa cells at the concentration of 0.5 μg/ml DNM, respectively, and imaged with CLSM. As shown in Figure 11, it was apparent that within 4 h of incubation, free DNM was internalized by the HeLa cells and localized in the nucleus. In the case of the DNM-loaded MMSN-CaCO₃, the fluorescence was distributed in whole cells after 4 h incubation. Weakened fluorescence in the nucleus and enhanced fluorescence in the cytoplasm was observed, indicating the DNM was released from MMSN particles and from lysosomes diffused to the nucleus. The fluorescence intensity of HeLa cells cultured with MMSN-FA-DNM-CaCO₃ nanospheres in FA-free medium (Figure 11(d)) was higher than that of cells cultured with MMSN-DNM-CaCO₃ spheres (Figure 11(b)). Meanwhile, more fluorescently marked cells were clearly visualized in the FA-free medium than in the FA-containing medium after incubation with MMSN-FA-DNM-CaCO₃ spheres (Figure 11(c) and (d)). MMSN-FA-DNM-CaCO₃ spheres were therefore suggested to be targeted to HeLa cells by a folate receptor-mediated endocytosis process.

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Figure 11.

Cellular uptake of (a) free DNM, (b) MMSN-DNM-CaCO₃, (c) MMSN-FA-DNM-CaCO₃, (d) MMSN-FADNM-CaCO₃ in the presence of 1 mM free FA in HeLa cells after 6 h of incubation exhibited by CLSM. Images from left to right show the cell nuclei stained by DNM fluorescence in cells (green), DAPI (blue), and merged images.

Morphological assessment of apoptosis

DAPI staining was carried out to investigate the apoptosis induction of free DNM, MMSN-DNM-CaCO₃, and MMSN-FA-DNM-CaCO₃ nanospheres on HeLa cells (Figure 12). DAPI was a blue fluorescent dye and stained the cell nucleus. When cells were treated with DAPI, live cells with uniformly light blue nuclei were observed under fluorescence microscope, while apoptotic cells exhibited bright blue because of karyopyknosis and chromatin condensation, and the nuclei of dead cells could not be stained.

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Figure 12.

Apoptosis study of HeLa cells treated with (a) DNM, (b) MMSN-DNM-CaCO₃ and (c) MMSN-FADNM-CaCO₃ at concentrations of 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, respectively.

As given in Figure 12(d), nuclei in the control cells (cells incubated with MMSN-FA) were regular in shape. However, the cells became shrunken and dark after treatment with MMSN-FA-DNM-CaCO₃ for 24 h (Figure 12(c)) and the nuclei showed chromatin condensation and marginalization or nuclear beading. The number of apoptotic cells gradually increased as the DNM concentration increased, and at high concentrations, the cells showed morphological features typical of apoptotic cells (Figure 12(a)). The percentage of apoptotic or necrotic cells increased in a dose-dependent manner. The same phenomena are found in Figure 12 (b) and (c). After staining with DAPI, HeLa cells treated with MMSN-FA-DNM-CaCO₃ for 24 h showed an apoptotic-like abnormal morphology (condensation and frag-mentation) of nuclei. As the DNM concentration increased (from 0.25 μg/ml to 1 μg/ml), the number of apoptotic cells increased. Compared to native DNM, MMSN-DNM-CaCO₃ nanospheres showed high apoptotic rate to HeLa cells.

In vitro cytotoxicity

Dose-dependent cytotoxicity profiles for free DNM, MMSN-DNM-CaCO₃ spheres, and MMSN-FA-DNM-CaCO₃ spheres are shown in Figure 13(a). DNM alone significantly reduced viability of HeLa cells in a dose-dependent manner. The concentration killing 50% of the cells (IC₅₀) was estimated as 0.255 ± 0.013 µg/ml⁻. Both of the DNM-loaded carriers showed lower cytotoxicities than free DNM, perhaps due to the slower endocytosis process of DNM-loaded carriers. In contrast, MMSN-FA-DNM-CaCO₃ spheres exhibited a higher cytotoxicity than MMSN-DNM-CaCO₃ spheres, but similar cytotoxicity with free DNM in the folate-free medium (Figure 13(b)). This can be attributed to the higher uptake of MMSN-FA-DNM-CaCO₃ spheres than MMSN-DNM-CaCO₃ spheres by HeLa cells, resulting to the higher DNM release into the cells. Figure 13(d) shows the growth inhibition of A549 cells by MMSN-DNM spheres, MMSN-FA-DNM-CaCO₃ spheres, and free DNM in folate-containing medium. MMSN-DNM-CaCO₃ and MMSN-FA-DNM-CaCO₃ spheres exhibited similar cellular growth inhibition levels. This could be explained by the fact that A549 cells have no overexpressed surface folate receptors and MMSN-FA-DNM-CaCO₃ could only be taken up through a slow non-specific endocytosis mechanism like MMSN–DNM-CaCO₃ spheres.

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Figure 13.

(a) Viability of HeLa cells incubated with MMSN-DNM-CaCO₃, MMSN-FA-DNM-CaCO₃ or free DNM. (b) Viability of HeLa cells incubated with MMSN-DNM-CaCO₃ or MMSN-FA-DNM-CaCO₃ at different concentrations in folate-containing medium and folate-free medium. (c) Calculated IC50 values of MMSN-DNM-CaCO₃, MMSN-FA-DNM-CaCO₃, or free DNM against HeLa. (d) Viability of A549 cells incubated with MMSN-DNM-CaCO₃, MMSN-FA-DNM-CaCO₃, or free DNM.

To delineate whether folate-mediated endocytosis effectively contributes to the equipotent cytotoxicity of MMSN-FA-DNM-CaCO₃ spheres, cell viability studies were performed in the presence of 1 mM FA. Inclusion of this competitor for the folate receptor binding sites is anticipated to reduce folate-mediated endocytosis of MMSN-FA-DNM-CaCO₃ spheres. As expected, from Figure 13(c), increased IC₅₀ was observed for the MMSN-FA-DNM-CaCO₃ spheres incubated with HeLa cells which were pretreated by FA. The overexpressed surface folate receptors on the cancer cells were presaturated with free folate in the medium, thereby blocking folate receptor-mediated endocytosis. MMSN-FA-DNM-CaCO₃ spheres should only be taken up through a slow non-specific endocytosis mechanism.