Preparation and Characterization of Modified Polyethyleneimine Magnetic Nanoparticles for Cancer Drug Delivery

1Department of Chemistry, Faculty of Sciences, Islamic Azad University, Arak Branch, Arak, Iran 2Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14176, Iran 3Department of Pharmaceutical Biomaterials and Medical Biomaterials Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14176, Iran 4Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran


Introduction
Efficiency of many existing drugs is restricted by their poor solubility in water and toxicity against normal cells [1].Traditional chemotherapy is not commonly successful in cancer therapy due to the severe side effects of anticancer drugs and induced toxicity.However nanotechnology can reduce these side effects including dermatologic, cardiac, gastrointestinal, nervous system-related, and hematologic effects.In drug delivery, it is important to deliver a medicine to the target site of therapeutic action with a specific dose [2].For this purpose different carriers have been designed such as liposomes, hydrogels, biodegradable polymers, and magnetic nanoparticles.Polymeric nanoparticles have been widely used in drug delivery because they have small sizes that enable them to be transferred into the body from different routes [3].
The concept of drug delivery by magnetic nanoparticles was proposed by Widder et al. in 1978 [4].They coated magnetic nanoparticles with polymeric shell as suitable carriers for drug delivery due to their high stability and super paramagnetism behavior and more biocompatibility.Polymers have emerged as an efficient biocompatible coating agent with the ability of tuning the surface functionality.Yallapu et al. [5] introduced magnetic drug carriers with a pluronic polymer (F127) shell for controlled delivery of curcumin.Iron oxide is one of the nanoparticles that is used for magnetic resonance imaging and cancer treating.The polymeric nanoparticles can be prepared via two different strategies including (a) Fe 3 O 4 nanoparticles coating with a layer of polymer and (b) Fe 3 O 4 nanoparticles precipitated in the presence of a porous polymer [6].Polyethyleneimine (PEI) is a synthetic polymer that is frequently used in gene delivery systems.Amine groups can be functionalized through different methods to control its toxicity [7].
For decades there has been rapid increase in using natural products for cancer therapy.One of these natural products is curcumin.Curcumin is extracted from Curcuma longa, possessing a number of beneficial biological characteristics [8].
In this work, Fe 3 O 4 magnetic nanoparticles coated with a pH sensitive polymer and folic acid were used to increase targeting.The solubility of curcumin was increased by inclusion in the iron nanoparticles coated with modified polymer.

Synthesis of Fe 3 O 4
Nanoparticles.The coprecipitation method was used for the preparing of Fe 3 O 4 nanoparticles.This method applies ferric and ferrous ions in a 1 : 2 molar ratio [9].According to the procedure, 3.17 g of FeCl 2 ⋅4H 2 O (0.016 mol) and 7.68 g of FeCl 3 ⋅6H 2 O (0.008) were dissolved in 50 mL of deionized water.The mixture was stirred under N 2 at 85 ∘ C for 1 hr.Then 40 mL of NH 4 OH solution was added and stirred under N 2 for 1 hr and then cooled to room temperature.The precipitated particles were washed three times with deionized water and were separated by a magnet and then dried under vacuum at 65 ∘ C.

Synthesis of PEI-Sb.
Modified branched PEI network was prepared via interfacial polycondensation reaction between PEI (MW = 60,000) and sebacoyl chloride.A 2% v/v solution of Span 80 in cyclohexane (100 mL) was mixed with 30 mL of 10% wt aqueous solution of PEI (pH = 8.0).The mixture was stirred and 1.92 g (8 mmol) of sebacoyl chloride (sb) in 4 mL of cyclohexane was added.The reaction was allowed to proceed for 15 min under argon in ice bath and then it was diluted with 100 mL of cyclohexane.The resultant product in the form of beads settled within 1 day and was then separated from the liquid and rinsed several times with 100 mL of cyclohexane.The beads were transferred into the aqueous phase by dispersing the product in 100 mL of polysorbate 20 (Tween 20) (30% v/v) and 500 mL of deionized water was gradually added.The beads were then filtered and rinsed with water to eliminate traces of impurities [10].

Ninhydrin Test.
The content of primary amino groups in PEI-Sb was determined using ninhydrin colorimetry test [11].Polyethyleneimine was used as standard to prepare the calibration curve (40, 55, 120, and 150 ppm).Absorbance measurements were determined at 570 nm wavelength. 2 mL of PEI in mentioned concentrations and PEI-seb solution (150 mg/mL) were prepared and then 1 mL of ninhydrin (0.005 g/L) reagent was added to every solution.The samples were subsequently heated in a boiling water bath for exactly 15 minutes and cooled to 20 ∘ C for 20 minutes.Afterwards 5 mL of diluting solution was added and mixed and the absorbance was measured at 570 nm by a UV-Vis spectrophotometer.

Preparing of Folate-Conjugated Polymer (Folate-PEI-Sb).
Folic acid was initially activated by NHS [12].In summary, folic acid (1 g) was dissolved in anhydrous DMSO (30 mL) and then NHS (0.9 g) and DCC (0.5 g) were added to the solution and the reaction was completed overnight by stirring at room temperature and under argon.1,3-Dicyclohexylurea (DCU) was removed by filtration.To 5 mL of polymer solution containing PEI-Sb (0.4 g), triethylamine (0.05 mL) and then the filtered activated folate solution (3 mL) was added.The reaction proceeded at room temperature for 10 h under argon.Subsequently, the obtained solution was centrifuged and then filtered.After purification and rinsing, the obtained polymer was freeze-dried.The conversion of NH 2 -PEI-Sb into folate-PEI-Sb was evaluated by dissolving the polymer in DMSO.The absorbance was measured at 363 nm by Cecil UV-Vis spectrophotometer to confirm the presence of folate in the sample [13].

Preparation of Core-Shell Magnetic Nanoparticles.
To prepare core-shell FA-PEI-Sb-MNP, 5 mL MNP (10 mg/mL) was dispersed in water helping ultrasonication.Then, FA-PEI-Sb solution in DMSO (5.5 mg/mL) was mixed with the MNP dispersion.The suspension was stirred for 12 h at room temperature [14].MNPs covered by polymer were collected by a permanent magnet and washed three times by deionized water and dried under vacuum desiccator.

Characterization of Magnetic
Nanoparticles.Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), and thermal gravimetric analysis (TGA) were taken to characterize the magnetic nanoparticles.The magnetic properties measurements were carried out by vibrating sample magnetometry (VSM) at room temperature.Fourier transform infrared spectra (FTIR) were recorded in Nicolet 870 by a KBr pellet.XRD spectra were taken by a Bruker axs D8 advance diffractometer utilizing the X-ray beam from Cu K radiation.TGA results were obtained using L70/2003 thermal analyzer.The temperature range was raised up to 800 ∘ , at a ramp of 10 ∘ C/min with argon as inert gas.The VSM was applied to measure the magnetic properties of nanoparticles.
2.8.Drug Loading. 10 mg curcumin was dissolved in 1 mL acetonitrile and added dropwise to 10 mL of an aqueous dispersion of PEI-Sb-FA-MNPs (10 mg).Afterwards, the suspension was stirred overnight to allow penetration of curcumin into polymeric layer in core-shell nanoparticles at 12000 rpm.The obtained nanoparticles were washed three times in water to remove unloaded curcumin.The loaded curcumin was estimated at 450 nm wavelength by a UV-Vis spectrophotometer (Cecil 9000) [15].
2.9.In Vitro Release Experiments.Curcumin release from core-shell polymeric magnetic nanoparticles was accomplished in PBS at different pH levels (pH 4.5 and pH 7.4).Briefly, 10 g of MNPs containing curcumin was dispersed in 50 mL PBS solution in a tube.The sample was divided into centrifuged tubes and then placed in a shaking water bath with 120 rpm at 37 ∘ C. Because free curcumin is not soluble in water, the released curcumin in buffer at predetermined time intervals was centrifuged at 5000 rpm for 15 minutes to remove the released curcumin and redissolved in 1 mL of DMSO and the absorbance of solutions was measured at 450 nm using a UV-Vis spectrophotometer.Concentration of the solutions was determined using a calibration curve obtained from curcumin solutions (0-10 g/mL) [13,16].

Results and Discussion
The ninhydrin assay confirms that 54.63% of amine groups have been functionalized by carboxylic groups.
The XRD pattern shows magnetite peaks for iron oxide nanoparticles.The seven characteristic diffraction peaks (20. 1, 30.1, 35.6, 43.1, 53.8, 57.2, and 62.8) are indicating that the crystalline iron oxide was formed completely.The crystalline size was approximately 28 nm calculated by Scherrer's equation.Finally, the results indicate preservation of crystalline structure of Fe 3 O 4 for the functionalized Fe 3 O 4 NPs (Figure 1).
Thermal gravimetric analysis was performed to evaluate the thermal stability of the nanoparticles.This analysis was recorded from ambient temperature to 800 ∘ C. Figure 2 shows the TGA curves for magnetic nanoparticles in nitrogen atmosphere at 20 ∘ C/min ascending temperature.The weightloss areas were observed at 104.2 ∘ C, 232.2 ∘ C, 330.3 ∘ C, and 451.5 ∘ C, respectively.The first weight loss at the range of room temperature to 232 ∘ C could be attributed to the absorbed solvent evaporation.The second weight loss until around 451 ∘ C could be due to the polymer degradation on the surface of magnetic nanoparticles.Based on weight loss of about 10 mg at the range of 232-451 ∘ C, the coating amount of modified PEI on Fe 3 O 4 was measured to be about 10%.From Figures 1 and 2, we can realize that the nanoparticles have core-shell structure.
Magnetic hysteresis curves of Fe 3 O 4 and modified PEI Fe 3 O 4 NPs at room temperature are shown in Figure 3.
The nanoparticles display saturation magnetization equal to 57.09 and 51.4 for Fe 3 O 4 and modified PEI-Fe 3 O 4 emu/g.Saturation magnetization of FA-PEI-Sb-MNPs was lower than that of Fe 3 O 4 that could be attributed to the modified polymer on the surface of nanoparticles.Paramagnetic behavior can use for carrier targeting and make them suitable for use in the pharmaceutical field.
The SEM micrograph of Fe 3 O 4 -PEI nanoparticles is shown in Figure 4.The SEM images exhibit that the mean particle size is 17 nm.These images show that magnetic nanoparticles have spherical structure and enough monodispersity.The calibration curve was used to calculate the drug loading in nanoparticles (Figure 7).The drug loading content of obtained magnetic nanoparticles showed a value equal 28.2%.The presence of Sb moiety conjugated with PEI-NH 2 produce amphiphilic character for the polymer which can control drug loading and drug release due to the pH sensitivity of the polymer as well as high affinity to cell penetration due to the positive charge of the carrier surface interaction with.
The release study of curcumin showed that release of curcumin in acidic medium was more sensitive than neutral  medium and that is in accordance with cancer cell conditions (Figure 8).

Conclusion
Briefly, we synthesized a modified PEI coated magnetic in which PEI was modified by sebacoyl chloride and targeted by folic acid.Particles diameter can be controlled by the relative amount of polymer.Nanoparticles were characterized by FTIR, XRD, VSM, SEM, and TGA.The results adapted each other.Curcumin was loaded into nanoparticles and release behavior was investigated.

Figure 4 :
Figure 4: The SEM micrographs of FA-PEI-Sb-MNP nanoparticles and hydrodynamic size of them.