Linde Type A and nano magnetite/NaA zeolites: cytotoxicity and doxorubicin loading efficiency

2.1 Materials

Ferrous chloride (FeCl₂·4H₂O), ferric chloride hexahydrate (FeCl₃·6H₂O), sodium aluminate, sodium hydroxide, CaCl₂, and KCl were purchased from Merck and local clinoptilolite was used. SKBR3 and SCF7 human breast cancer cell lines were purchased from the National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide) and Cell Death Detection ELISAPLUS kit were purchased from Roche Diagnostics GmbH, Mannheim, Germany.

2.2 Synthesis of nano-zeolites

Nano-zeolite NaA was synthesized hydrothermally from annealed local clinoptilolite after crushing, drying, screening and washing. Clinoptilolite, sodium hydroxide, sodium aluminate and distilled water were mixed for 6h (aging). The resulting gel was treated by ultrasound for 6h, transferred to a Teflon-lined stainless steel autoclave, and heated at autogenous pressure in an air oven at 353 K. After a suitable time (the pH reached 7) the suspension was centrifuged several times and the product was dried. For the preparation of CaA (5A) and KA (6A) nano-zeolites, calcium and potassium hydroxide replaced the sodium hydroxide. The products were characterized and their adsorption properties examined.

For synthesis of nano-magnetic NaA, magnetic Fe₃O₄ was synthesized by co-precipitation from an alkaline solution of Fe²⁺ and Fe³⁺ salts and added to the carrier gel [37] after the ultrasound treatment. The next steps were same as the synthesis of nano-zeolite NaA. Nanomagnetite NaA with different Fe₃O₄ loadings (0, 2.1, 4 and 7.7 wt.%) was prepared by varying the amount of nano-Fe₃O₄ and designated as M_n4A (n=0, 2.1, 4 & 7.7).

2.3 Doxorubicin standard solutions preparation

Standard solutions of doxorubicin (5, 50, 100 mgL^–1) in deionized water were prepared quickly and kept in the dark because light immediately changes doxorubicin to a toxic material.

2.4 Preparation of drug loaded nano-zeolites

20 mg of each nano-zeolite was added to 20 mL of the standard doxorubicin solutions and stirred for 24 hours at room temperature. The white zeolite changed to the red doxorubicin color as the drug entered the zeolite pores and the loading was complete. Doxorubicin disappearance was confirmed by supernatant absorbance measurement with a Shimadzu UV-240 spectrophotometer at 485 nm. After there were no further changes in the liquid phase concentration it was assumed that the loading capacity had been reached. The reddish solid was separated by filtration, air-dried for 24h and designated DOX_n@ZA (n= 0, 5, 50, 100 & 500).

Desorption was examined at 37ºC, but the loaded nanocomposite was first washed and dried to remove non-adsorbed doxorubicin. The release profile was obtained by dispersing 100 mg of dried drug-loaded nano-zeolite in 50 mL of buffer (pH= 7.4, 5.5). As in the uptake experiments, the concentration of doxorubicin in the particle-free liquid was determined at fixed time intervals.

2.5 Measurements

A Siemens D500 diffractometer with Cu Kα radiation (λ=1.5418 Å and θ=4-80°) was used to characterize the powders. Functional group absorption bands were studied with a Bruker Tensor 27 FTIR as KBr pellets. Gold-coated particle morphology, size and elemental composition were analyzed with a Philips XL30 SEM. SEM was done at 5Kv while EDX analysis was at 15Kv.The BET surface area was measured by N₂ adsorption–desorption isotherms at liquid nitrogen temperature a using N0VA2000 (Quantachrome, USA). TEM and EDX of the samples were performed on a Zeiss LEO 912 Omega at 120 kV. TEM specimens were made by evaporating one drop of ethanolic solution on carbon-coated copper grids and blotted dry on filter paper. Magnetic properties were investigated at room temperature using a 7400 vibrating sample magnetometer.

2.6 MTT assay

The effects of concentrations of the carrier, drug and drug-loaded carrier on cell viability were investigated using identical methods.

SKBR3 and SCF7 cell lines were grown in sterile RPMI-1640 media containing 10% fetal bovine serum, streptomycin (100 μg/mL), amphotericin B (0.25 μg/mL) and penicillin (100 U/mL) at 5% CO₂ in a humidified 37°C incubator. Then 200 μL/well of supplemented medium and 12×10³ cells were incubated in 96-well plates for 24 hours at 37°C and 5% CO₂. The cells were divided into four groups in triplicate: blank, drug, carrier and drug-loaded carrier supplemented.

A Cell Death Detection ELISAPLUS kit was used according to the manufacturer’s protocol to induce cell apoptosis and necrosis. Briefly, supernatants and cell lysates were prepared and incubated in microtiter plates coated with an antihistone antibody then analyzed at 405 nm.

After incubation the used media were discarded and the wells washed with pH 7.4 phosphate-buffered saline. 50 μL of 2 mg/mL MTT solution and 150 μL culture medium were added to each well. The cells were incubated at 37°C and 5% CO₂ for 24 hours; then the media was removed and 200 μL of dimethyl sulfoxide and 25μL Sorenson solubilizer buffer added to each well. Finally, an ELISA plate reader (BioTeck, Bad Friedrichshall, Germany) was used to read the absorbance at 405 nm. All results were analyzed relative to the untreated cells then normalized.

3.1 Characterization of DOX@ZA

Figure 1A shows the pXRD of zeolite 4A before and after DOX loading. All characteristic peaks of zeolite 4A are shown. In Figures 1A(b-d), 4.7ppm, 42.5ppm and 80ppm DOX were loaded. XRD pattern similarity before and after drug loading indicates that the framework did not change. However, overall reduction in the peaks’ intensity indicated a slight decrease in zeolite crystallinity following drug adsorption which is more obvious at higher drug loadings. The XRD patterns of the other zeolites are similar.

Figure 1

XRD patterns of: A) a: zeolite 4A, b: DOX_4.7 @ 4A, c: DOX_42.5 @ 4A, d: DOX₈₀ @ 4A; B) a: M₄4A, b: DOX_42.5 @ M₄4A, c: DOX₈₀ @M₄4A.

The XRD show only characteristic 4A zeolite and Fe₃O₄ peaks; no other structures or changes in cell parameters were observed (Figure 1B). The XRD of the DOX_n@M₄4A (n = 42.5ppm and 80ppm; Figures 1B (a-c)) were similar to the M₄4A XRD with reduced peak intensities.

TEM and SEM examined nano-zeolite morphology before and after loading. The DOX@ZA with the lowest DOX concentration (5 ppm) is shown in Figure 2a. The morphology and structure were unchanged after drug loading. As DOX is larger than the nano-zeolite pores, it is adsorbed on the outer surface. Drug and zeolite -OH and –NH₂ groups hydrogen bond causing aggregation. The average particle size of the starting 4A was about 50-100 nm (Figure 2b) and 80-120 nm for M4A (Figure 3a).

Figure 2

SEM images of: a) DOX₅@ 4A, b) TEM image of 4A.

Figure 3

a)SEM image of M₄4A, b)EDX of M₄4A.

3.2 Loading efficiency and release

4A zeolite (94% of DOX₅@ZA) has significantly higher loading efficiency than 3A and 5A zeolites (86% and 79%) as shown in Figure4a. The higher amounts of DOX in the 4A zeolite suggest higher drug loadings in the pores due to different pore sizes. The initial red of the DOX solution became colorless after a few minutes of zeolites contact; respectively, 92%, 84% and 74% of the initial DOX was adsorbed in 30 min by 4A, 3A and 5A (Figure 4a). Adsorption of the remaining DOX was slower and saturation was complete in approximately 120 min. DOX solution UV spectral changes after contact with zeolites were not significant, indicating that their interaction is only physical

Figure 4

Drug loading efficiency of a) 3A, 4A &5A; b) A type zeolites with different amounts of DOX; c) 4A and M_n4A for different amounts of DOX.

Figure 5

Release profiles of a) DOX₅₀ @ ZA(3A, 4A, 5A); b) DOX₅₀ @ 4A and DOX₅₀ @ M₄4A.

Hydrogen bonding of the DOX ‒NH₂ (δ⁺) groups to the zeolite ‒OH (δ^‒) groups causes drug loading.

At higher DOX concentrations (DOX₅₀@ZA and DOX₁₀₀@ ZA), loading efficiency was lower (Figure 4b). The mean pore size of the A zeolites is 0.87 nm which is much smaller than the DOX molecule, so adsorption occurred only on the external surface as a poremouth phenomenon [34]. The 4A BET external specific surface area of 5.27 m²/g means that although the nanocomposite has a considerable DOX storage capacity, the composite surface area (≈ 220-255 m²g^–1) will not be fully occupied. Its loading may come from physical attachment rather than encapsulation.. Substrate surface electronic structure and charge transfer dynamics can strongly influence interactions with the adsorbate.

The DOX concentration in DOX₅@ZA is very low; after 24 h approximately 60% of the loaded drug (4.7 ppm from 5 ppm) way released.

Initial DOX uptake by magnetite-zeolite nanocomposites (M4A) was very fast, with 94% adsorption in 30 min. Drug saturation was completed in approximately 2h. At higher DOX concentrations drug loading was lower for all the composites (Figure 4c). As shown in Figure 4, drug loading increased with increased Fe₃O₄ percentage but at the highest percentage (7.7%) the loading decreased. At 4 wt% Fe₃O₄, maximum adsorption was achieved because the iron oxide was well dispersed into the zeolite pores, caused by strong interactions between the zeolite oxygen atoms and Fe₃O₄. Increasing the Fe₃O₄ content blocked the channels, reducing adsorption.

The dried nanocomposite contains more than 75% drug. Its release was measured by desorption and diff usion into pH 7.4 buff er; released DOX % is given by:

22% (9.24 ppm), 26% (11.06 ppm), 22% (9.02 ppm) and 21% (9.55 ppm) of the DOX on the 3A, 4A, 5A zeolites and M₄4A nanocomposite was released in 24 h. However, at the lower cancer cell pH (5.4), more desorption from DOX₅₀@ ZA occurred (70-80%). The release profiles of DOX₅@ZA are the same. There was no dependence of the (DOX@ZA) characteristics on the storage time.

Arruebo et.al reported magnetite/Y zeolite prepared by milling as a potential DOX delivery vehicle. They found 92% of DOX adsorbed in 25 h and 77% of the load was released in 12.6 h [34].

3.3 Cytotoxicity of the combination of nanozeolite with doxorubicin

For breast cancer, the required DOX dosage is 50 mg/m² times the Mosteller value for the BSA (body surface area) [38]:

For a 170 cm, 65 kg female the required DOX dosage is 87.7 mg. Her total body water (TBW) is calculated by Watson’s formula [39]:

Therefore, the DOX concentration should be 2.73 μg/mL. Based on the DOX = 5 ppm loading and release profiles, we achieve this concentration in 25 min from 4A and 20 min from M₄4A, respectively. DOX@ZA was selected for the rest of the study.

Zeolites should have small or no effect on cell viability to achieve a suitable delivery system. Investigation of the starting zeolites’ cytotoxicity was carried out with the SKBr3 & MCF-7 cell lines. Different DOX₅@ZA concentrations (0.50, 1.00, 2.50 and 5.00 mg/mL) were suspended in the culture medium (RPMI 1640) and treated with ultrasound for 2 min before use for better homogeneity.

Figure 6 illustrates the effect of increasing carrier amounts (zeolites and composites) on cell viability determined by the MTT assay (P^> 0.05). The insignificant differences between controls (no zeolite) and zeolite concentrations tested show that 3A, 4A & 5A zeolites are not cytotoxic over the 24h incubation period. All of the zeolites and composites gave similar results for a 48 h incubation. For magnetite zeolites containing 7.7% Fe₃O₄ cell viability decreased, but very mild toxicities appeared. M₄4A magnetic nanocomposites had no cytotoxicity suggesting biomedical application.

Figure 6

Effect of the different starting zeolites and magnetite form on the SKBr3 cell viability.

The effects of DOX₅@ZA concentration and zeolite type (3A, 4A, 5A & M_3.44A) on SKBr3 & MCF-7 cell viability were investigated. When the DOX₅@ZA concentrations (0.5, 1.0, and 2.5 mg/mL) increased, cell growth inhibition was observed in both cancer cell lines (Figure7). Loading doxorubicin on zeolite improves tumor inhibition over doxorubicin alone.

Figure 7

effect of DOX and DOX@4A on the SKBr3 and MCF-7 cells viability.

This increased efficacy may be due to slow release. We believe that like other systems [33,40,41,42], zeolite delivery systems increase the bioavailability and promote DOX entry into the cell, explaining this increase in potency. Upon exposure to the acidic endosomes/lysosomes environment, DOX is released intracellularly, resulting in efficient apoptotic cell death [43]. Figure 7 shows the treatment-induced apoptosis (^> 80%) and necrosis (^< 20%), obtained from ELISA analysis for the SKBR3 line.

As shown in Figure 8, the cytotoxicity of delivery with type A zeolite (1.0 mg/mL) was more pronounced for 4A and M₄4A, which have greatest loading and release capacity.

Figure 8

Effect of 1.0 mg/mL of DOX and DOX@ZA (3A, 4A, 5A & M₄4A) on the SKBr3 cell viability.

Figure 9 shows the M₄4A magnetization curve at room temperature. The nanocomposite showed superparamagnetism and the curve’s coercivity was negligible. The saturation magnetization and susceptibility values for the nanocomposite were found to be 43.4 emu/gFe and 17.65 emu/gFe kOe, respectively.

Figure 9

Magnetization versus applied field curve of M₄4A nanocomposites.