Tumor-Targeted Drug Delivery Systems Based on Supramolecular Interactions Between Iron

Tumor-Targeted Drug Delivery Systems Based on Supramolecular Interactions between Iron oxide-Carbon Nanotubes and PAMAM-PEG-PAMAM Linear-Dendritic Copolymers

Mohsen Adeli1 and 2*, Masoumeh Ashiri1, Beheshte Khodadadi1 and Pezhman Sasanpour3

1) Department of chemistry, Faculty of Science, LorestanUniversity, Khoramabad, Iran.

2) Department of Chemistry, Sharif University of Technology, Tehran, Iran.

3) Institute for Nanoscience and Technology, Sharif University of Technology, Tehran, Iran

Experimental

Materials

MWCNT were prepared by chemical vapor deposition procedure in the presence of Co/Mo/MgO as catalyst at 900 ºC. Citric acid monohydrate (MW=210.14), poly ethylene glycol (MW=1000), CDDP, [Fe (NO3)3.9H2O] and HNO3were purchased from Merck. The cell lines (mouse tissue connective fibroblast adhesive cells (L929) were obtained from the National Cell Bank of Iran (NCBI) Pasteur institute, Tehran, Iran. MTT powder, Annexin-V FLUOS Staining Kit, was obtained from Sigma.

Characterization

Nuclear magnetic resonance (1HNMR) spectra were recorded in D2Osolution on a Bruker DRX 400 (400 MHz) apparatus with the solvent proton signal for reference. Infrared spectroscopy (IR) measurements were performed using a Nicolet 320 FT-IR. Ultraviolet (UV)spectra were recorded on a shimadzu (1650 PC) scanningspectrophotometer. Ultrasonic bath (Model: 5RS, 22 KHZ,Made in Italy) was used to disperse materials in solvents. The particle size, polydispersity and zeta potential of materials were determined using Dynamic Light Scattering (DLS) (zetasizer ZS, Malvern Instruments).

Morphology and size of materials were investigated using the Philips XL30 scanning electron microscope (SEM) with 12 and 15 A accelerating voltages.

Surface imaging studies were performed using atomic force microscopy (AFM) to estimate surface morphology and particle size distribution. The samples were imaged with the aid of Dualscope/Rasterscope C26, DME, Denmark, using DS 95-50-E scanner with vertical z-axis resolution of 0.1 nm.Raman spectra was obtained with an Almega Thermo Nicolet Dispersive Raman Spectrometer with second harmonic @532 nm of a Nd:YLF laser.

Thermogravimetric analysis (TGA) was performed on a PL-STA 1500 thermal analyzer set up under dynamic atmosphere of an inert gas (i.e. Ar) at 10 ml/min (room temperature). The Transmission electron microscopic (TEM) analyses were performed by a LEO 912AB electron microscope with accelerating voltage of 200 kV. The X-ray power diffraction pattern of products were recorded on Siemens D-500 diffractometer with Cu Kα radiation (λ= 1.54056 Ǻ) in 2θ range from 15º to 80º.

The magnetic moment (M) of the hybrid nanomaterials was measured using LakeShore model 7400 Vibrating Sample Magnetometer (VSM).

Simulation for NDDSs in the magnetic field was performed usingFinite element method COMSOL multiphysics software.

Preparation of PAMAM-PEG-PAMAM linear-dendritic copolymers

PAMAM-PEG-PAMAM linear-dendritic copolymers were prepared according to slightly modified reported procedure in literature [1-3]. Poly ethylene glycol(MW=1000) (9 gr)dissolved in 5cc distiled water containing 0.64 gr NaOH was added dropwise to a solution containing Cyanuric chloride(13 gr)dissolved in 250 cc dichloromethane and stirred at 0˚c for 1 h and then at room temperature for 1h. The mixture was then refluxed for 6 h. The resulted mixture was filtrated and the solvent was removed under vaccume. Then 250 cc diethyl eter was added and stay for 10 minutes. The mixture was decantated and the solution cooled to 0˚c and the product precipitate.

Synthesis of the amin terminated poly ethylene glycol.

Ethylene di amin(12cc, 179mmol) in 15 cc methanol was added dropwise to a solution containing tetrachloro poly ethylene glycol(5 gr, 3.85 mmol) in 10 cc methanol and stirred for 12 h at 25 ˚c. and then was stirred at 70˚c for 24 h. the solvent was evaporated under vaccume and the mixture was precipitated in di ethyl eter. A yellow viscose product with 90% yield was prepared.

Synthesis of the linear dendritic copolymer

amin terminated poly ethylene glycol was used as the polymeric supporter and the PAMAM dendrimer was extended outward from the PEG core by repetition of Michael addition and amidation

(1) Michael Addition. First, amin terminated PEG was dissolved in methanol and added dropwise to 200 equiv of methyl acrylate kept at 37 °C for the complete reaction. After 48 h, methanol and unreacted methyl acrylate were removed under vacuum. The residue was precipitated with an excess of cold ethyl ether to remove residual methyl acrylate and dried under vaccum to remove ethyl ether, leaving a weak yellow solid, PAMAM-PEG-PAMAM G 0.5.

(2) Amidation. Second, PAMAM-PEG-PAMAM G 0.5 was dissolved in methanol and added dropwise to 400 equiv of ethylenediamine

kept at 37 °C. After 48 h, methanol and ethylenediamine were removed under vaccum. The residue was identically precipitated with an excess of ethyl ether to remove residual ethylenediamine and dried under vacuum to remove ethyl ether, leaving a weak yellow solid, PAMAM-PEG-PAMAM G 1.0.

These Michael addition and amidation reactions were performed three times repeatedly for the synthesis of the second generation of the linear dendritic copolymer.

Preparation of Fe3O4-MWCNTs hybrid materials

Fe3O4-MWCNTs hybrid materials were prepared according to reported procedures in literature [4].For the preparation of CNTs-iron oxide magnetic composites, CNTs were first refluxed with concentrated nitric acid at 140˚C for 1 h. The magnetic composites were prepared from a suspension of 1.0 g oxidized CNTs in a 300 ml solution of 0.585 g FeCl3 6H2O and 0.390 FeSO4 6H2O at 70˚C under N2 atmosphere. NaOH solution (18.7 ml, 0.5 mol l-1) was added dropwise to precipitate the iron oxides. After the addition of NaOH solution, the mixture was adjusted to pH 11.0 and stirred for 30 min. Then the mixture was aged at 70˚C for 2 h and washed 3 times with deionized water. The obtained material were dried in an oven at 45˚C.

Preparation of Fe3O4-MWCNTs/ PAMAM-PEG-PAMAM hybrid nanomaterials

Fe3O4-MWCNTs hybrid material (9.3 mg) was dispersed in 5 ml of deionised water and sonicated for 5 minutes then PAMAM-PEG-PAMAM G2 (2.3 mg) was added to the suspension and sonicated for 10 minutes again. The mixture was kept at room temperature to investigate its stability.

Preparation of DOX/Fe3O4-MWCNTs/PAMAM-PEG-PAMAM hybrid nanomaterials

A solution containing 0.04 mg DOX was added to the Fe3O4-MWCNTs/ PAMAM-PEG-PAMAM hybrid nanomaterials mixture and sonicated for 10 minutes. The mixture was kept at room temperature to investigate its stability.

Cell culture

Mouse tissue connective fibroblast adhesive cells (L929) were cultivated in RPMI-1640 medium supplemented with10% fetal bovine serum, 2mM L-glutamine, 100 U/ml of penicillin, and 100 µg/ml of streptomycin sulfate at 37ºC in a humidified incubator with 5% CO2. The cells were maintained in an exponential growth phase by periodic subcultivation.

Cytotoxicity assay

In vitro cytotoxicity of the nanomaterials was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cells (2500 cells/well) were seeded in 96-well plates. Nanomaterials (5 and 1000µg/ml) were then added to the wells in triplicates and incubated for 72 hours. After the incubation period, 20 l of MTT dye (5 mg/ml in PBS) was added to each well, and they were incubated in the dark at 37 ºC for 5 hours. Then media were removed and formazan crystals were dissolved in 200 μL dimethylsulfoxide (DMSO) and 20 l of glycine buffer. Then the absorbance of each well was measured by an ELISA reader (Statfax–2100 Awareness Technology, USA) at 570 nm.

Cell viability was calculated using the following equation:

Cell viability (%) = (Ints/Intscontrol) ×100

Where “Ints” is the colorimetric intensity of the cells incubated with the samples, and “Intscontrol” is the colorimetric intensity of the cells incubated with the Media only (positive control).

OutlierDetection

All MTT experiments were performed in triplicate or more, with the results expressed as mean ± standard deviation; standard deviation values are indicated as error bars in the MTT results plots. The results were statistically processed for outlier detection using a “T procedure” (REF) using MINITAB software (Minitab Inc., State College, PA). One-way analysis of variance (ANOVA) with p<0.05 was performed for each set of MTT assay test repeats. Outlier samples have then been excluded from the corresponding asset viability calculations.

In this method, a T-ratio is calculated as follows

Where the suspected outlier is point (normally the smallest or the largest value in a set of measurements), is the sample mean, and is the (estimated) standard deviation. If the calculated value of T is equal to or exceeds a critical value, the outlier point is removed with a significance level of 0.05. In the latter case, assuming that the data were sampled from a normal distribution, there is at least a 95% chance that the suspected point is in fact far from other points.

Results and discussion

Figure 1 SM (a-d) shows the IR spectra of PAMAM-PEG-PAMAM, Fe3O4-MWCNTs, Fe3O4-MWCNTs/PAMAM-PEG-PAMAM, DOX/Fe3O4-MWCNTs/PAMAM-PEG-PAMAM hybrid nanomaterials. In IR spectrum of PAMAM-PEG-PAMAM linear-dendritic copolymers (Figure 1aSM), an absorbance band at 3269 cm-1 arises from the symmetric stretching of NH functional groups while a band at 2933 cm-1 shows the C-H stretching of methylene groups. C=O functional groups appeared at 1649 cm-1 and the absorbance bands at 1559 cm-1, 1114 cm-1, are assigned to bending vibration of N-H and stretching vibration of C-O functional groups respectively.

The IR spectrum of the Fe3O4-MWCNTs (Figure 1bSM), showed an absorbance band at 1558 cm-1 assigned to the carbonyl groups created on the surface and tips of carbon nanotubes. Furthermore, sharp absorbance peaks are observed at low frequency region (520 and 474 cm-1) in the IR spectrum of Fe3O4-MWCNTs, could be ascribed to the Fe–O bond vibrations in the Fe3O4 nanoparticles. The characteristic absorbance bands in Fe3O4-MWCNTs /PAMAM-PEG-PAMAM hybrid nanomaterial (Figure 1cSM) were appeared at 3490-3636, 1634, 600-800 cm-1 that are assigned to the vibration of N-H, C=O and Fe–O functional groups respectively. NH functional groups of Fe3O4-MWCNTs were appeared at higher frequency than in PAMAM-PEG-PAMAM proving noncovalent interactions between Fe3O4-MWCNTs and PAMAM-PEG-PAMAM linear-dendritic copolymers. In IR spectrum of DOX/Fe3O4-MWCNTs/PAMAM-PEG-PAMAM hybrid nanomaterial (Figure 1dSM), absorbance bands at 3440-3450, 1632, 600-800 cm-1are assigned to the vibration of N-H, C=O and Fe–O functional groups respectively. Decreasing the N-H vibration frequency in this hybrid nanomaterial in compare to that for Fe3O4-MWCNTs/PAMAM-PEG-PAMAM proves loading of DOX onto the surface of Fe3O4-MWCNTs. Appearance the absorbance bands at 600-800 cm-1 in all hybrid nanomaterial including Fe3O4-MWCNTs proves decoration of MWCNTs by Fe3O4 NPs. Non-covalent interactions between polymers and CNTs could be evaluated by 1H NMR spectroscopy, because protons of polymer that are closer to the CNT surface show broader and weaker signals than others. 1H NMR spectrum of PAMAM-PEG-PAMAM is shown in figure 3a. Signals assigned to PAMAM-PEG-PAMAM were also appeared in 1H NMR spectrum of Fe3O4-MWCNTs/PAMAM-PEG-PAMAM (Figure 2bSM) and DOX/Fe3O4-MWCNTs/PAMAM-PEG-PAMAM hybrid nanomaterials (figure 2cSM). However some of these signals were extended and some of them were disappeared. Signals belong to PAMAM-PEG-PAMAM segment in Fe3O4-MWCNTs/PAMAM-PEG-PAMAM are waked and broadened than in PAMAM-PEG-PAMAM proving interactions between Fe3O4-MWCNTs and PAMAM-PEG-PAMAM. But in DOX/Fe3O4-MWCNTs/PAMAM-PEG-PAMAMthe intensity of these signals increased again and this proves that loading of DOX on Fe3O4-MWCNTs surface affects the interaction between Fe3O4-MWCNTs and PAMAM-PEG-PAMAM.

Figure 1SM.IR spectra of (a)PAMAM-PEG-PAMAM, (b)Fe3O4-MWCNTs, (c)Fe3O4-MWCNTs/PAMAM-PEG-PAMAM, and (d)DOX/Fe3O4-MWCNTs/PAMAM-PEG-PAMAM hybrid nanomaterials.

Figure 2SM.1H NMR spectra of (a)PAMAM-PEG-PAMAM(b) Fe3O4-MWCNTs /PAMAM-PEG-PAMAM, and (c)DOX/Fe3O4-MWCNTs/PAMAM-PEG-PAMAM hybrid nanomaterials.

Figure 3SM. (a) Geometry and mesh structure of simulated structure of hybrid nanomaterials. (b) Flow chart describing simulation routine.

References

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