Colloids and Surfaces B: Biointerfaces 117 (2014) 406–413

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Doxorubicin-conjugated core–shell magnetite nanoparticles as dual-targeting carriers for anticancer drug delivery Somayeh Sadighian a,b , Kobra Rostamizadeh b,c,∗ , Hassan Hosseini-Monfared a , Mehrdad Hamidi b,d a

Faculty of Science, Department of Chemistry, University of Zanjan, Zanjan, Iran Zanjan Pharmaceutical Nanotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran c Department of Medicinal Chemistry, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran d Department of Pharmaceutics, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran b

a r t i c l e

i n f o

Article history: Received 2 October 2013 Received in revised form 8 February 2014 Accepted 2 March 2014 Available online 12 March 2014 Keywords: Dual-targeting Nanocarrier Magnetite nanoparticles Cancer targeting Doxorubicin Conjugation

a b s t r a c t The present study reports the successful synthesis of core–shell nanostructures composed of magnetite nanoparticles (Fe3 O4 -NPs) conjugated to the anticancer drug doxorubicin, intended for dual targeting of the drug to the tumor sites via a combination of the magnetic attraction and the pH-sensitive cleavage of the drug–particle linkages along with a longer circulation time and reduced side effects. To improve the carrier biocompatibility, the prepared nanocarrier was, finally coated by chitosan. FT-IR analysis confirmed the synthesis of functionalized Fe3 O4 -NPs, doxorubicin-conjugated Fe3 O4 -NPs, and chitosan-coated nanocarriers. Scanning electron microscopy (SEM) indicated the formation of spherical nanostructures with the final average particle size of around 50 nm. The vibrating sample magnetometer (VSM) analysis showed that the saturation magnetization value (Ms ) of carrier was 6 emu/g. The drug release behavior from the nanocarriers was investigated both in acidic and neutral buffered solutions (pH values of 5.3 and 7.4, respectively) and showed two-fold increase in the extent of drug release at pH 5.3 compared to pH 7.4 during 7 days. The results showed that the dual-targeting nanocarriers responded successfully to the external magnetic field and pH. From the results obtained, it can be concluded that this methodology can be used to target and improve therapeutic efficacy of the anticancer drugs. © 2014 Elsevier B.V. All rights reserved.

1. Introduction In recent years, there has been an increasing impetus for targeting therapeutic agents to specific cells of the diseased site with the aim to improve their efficiency and/or minimize the undesirable side effects [1]. Among the numerous approaches used for this purpose, targeting based on magnetic properties using magnetic nanoparticles, mainly magnetite (Fe3 O4 )-based nanoparticles, is widely considered as a promising targeted delivery system due to its distinct advantages, mainly including a well-documented biosafety, ease of preparation and handling, the possibility of controlling the characteristics of the nanocarriers, availability, affordability of the materials needed for this procedure, and more

∗ Corresponding author at: Department of Medicinal Chemistry, School of Pharmacy, Zanjan University of Medical Sciences, 45139-56184 Zanjan, Iran. Tel.: +98 241 4273635; fax: +98 241 4273639. E-mail addresses: [email protected], [email protected] (K. Rostamizadeh). http://dx.doi.org/10.1016/j.colsurfb.2014.03.001 0927-7765/© 2014 Elsevier B.V. All rights reserved.

importantly, possibility of targeting the drug(s) of interest to the desired location within the host body by using an external magnetic field. Furthermore, core–shell magnetic nanoparticles have attracted much attention due to their multifunctional properties such as small size, superparamagnetism and low toxicity [2,3]. Silica-coated Fe3 O4 –nanoparticles are one of the most extensively used Fe3 O4 –nanoparticles which possess very high specific surface with abundant Si–OH or Si–NH2 groups with ability to react with proper functional groups [4,5]. Besides the above mentioned advantages, there are a number of drawbacks against the widespread use of Fe3 O4 nanoparticles for targeted drug delivery systems. Firstly, due to the high surface-area-to-volume ratio characteristic of such nanoparticles, they tend to aggregate and form clusters with low magnetization properties. Secondly, it is shown that a major part of the naked Fe3 O4 nanoparticles are rapidly cleared from blood circulation by the reticular endothelial system (RES) before they could be able to reach the intended target site, thereby being localized in the RES organs, mainly liver [5,1]. To address these issues, one approach is to encapsulate Fe3 O4 nanoparticles within

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biodegradable and/or biocompatible polymers [6]. Additionally, polymer-encapsulated Fe3 O4 nanoparticles can be designed in order to provide further diverse and desirable functionality to enable conjugation to the drug of interest [7]. It is believed that the use of a trigger alone as a targeting function cannot ensure complete localization of drug at the site of interest. Therefore, to enhance targeting characteristic of a carrier, it represents a well-established approach to use more than one trigger to direct carrier to the specific site. Such carriers are generally referred to as dual targeting drug delivery systems [8]. Zhang and Misra [7] synthesized a novel dual targeted magnetic carrier consisting of magnetic nanoparticles encapsulated within dextran-g-poly(Nisopropylacrylamide-co-N,N-dimethylacrylamide) co-polymer as a smart thermo-sensitive polymer showing high drug release rate for longer durations specially in acidic pH. Chitosan, a naturally-derived co-polymer of Nacetyglucosamine and d-glucosamine, has been extensively studied as a biodegradable and biocompatible polymer in drug delivery systems, gene therapy, and membranes for ultrafiltration [9,10]. It seems that chitosan-encapsulated Fe3 O4 nanoparticles would most likely improve magnetite nanoparticles characteristics in terms of biocompatibity and long circulation time. The amino groups on the chitosan structure can also be used for further functionalization with specific components, such as various drugs, targeting agents, or other functional groups. Thus, it seems to be a suitable polymer to modify the Fe3 O4 nanoparticles [11]. Feng et al. [12] synthesized monodisperse chitosan/polyacrylic acid/Fe3 O4 nanoparticles which could be used for magnetic resonance imaging (MRI). Donadel et al. [13] prepared iron oxide magnetic particles coated with chitosan intended for hyperthermia. Recently, Shen et al. [14] prepared dual-drug delivery system in which doxorubicin and verapamil were physically loaded into chitosan coated magnetite nanoparticles. The resultant nanoparticles were entrapped into the poly (lactic acid-coglycolicacid) (PLGA) nanoparticles and used as near infrared (NIR) trigger drug delivery system. The anthracycline antibiotic doxorubicin is a widely used anticancer drug in clinical practice for the treatment of a variety of cancers like leukemia, ovarian, prostate, brain cancers, especially late stage breast cancer [15]. Although doxorubicin is one of the most widely used anticancer agents, its application is still limited by its deleterious side effects, including myelosuppression, gastrointestinal toxicity and, more importantly, cardiotoxicity. Drug targeting, therefore, represents an interesting incentive to prevent side effects and increase cytotoxicity of doxorubicin [15,16]. A number of approaches including chemical conjugation and/or physical entrapment have been employed to target doxorubicin using different carriers such as dendrimers, polymeric nanoparticles, polymer–drug conjugates and micelles [17–20,16]. Herein we report the synthesis and in vitro characterization of a dual targeted drug delivery system using a core–shell magnetite nanoparticulate system conjugated with doxorubicin via acid-cleavable imine linkage. To enhance the biocompatibility of the prepared dual targeted carrier and minimize undesirable side effects of doxorubicin and Fe3 O4 nanoparticles, conjugated magnetite core–shell nanoparticles were encapsulated within chitosan.

2. Experimental 2.1. Materials and methods Ferric chloride hexahydrate (FeCl3 ·6H2 O), ferrous chloride tetrahydrate (FeCl2 ·4H2 O), tetraethylorthosilicate (TEOS), (3aminopropyl)triethoxysilane(APTES) all were purchased locally from Merck and used as received. Chitosan of molecular weight in the range of 105 –3 × 105 g/mol and degree of deacetylation

407

≥75% was provided from Sigma. Solvents of the highest grade commercially available (Merck) were purchased locally and used without further purification. Doxorubicin hydrochloride was purchased from Celonlabs (Andhra Pradesh, India). 2.2. Synthesis of spherical Fe3 O4 nanoparticles The Fe3 O4 nanoparticles were synthesized in accordance with the method of Massart [21] consisting of co-precipitation of Fe(III) and Fe(II) with ammonia in an aqueous solution. In essence, iron(II) chloride (1.0 mmol) and iron(III) chloride (2.0 mmol) were dissolved in 45 mL deionized water. Then, 3 mL aqueous ammonia (25%) was added to the solution and stirred for 1 h under the N2 flow. The black product was separated by an external magnet and washed several times with deionized water and dried at 60 ◦ C under vacuum for 12 h. 2.3. Synthesis of Fe3 O4 core–shell nanoparticles Synthesis of Fe3 O4 core–shell nanoparticles was adapted from the literature [22]. In a typical procedure, 2.0 mL TEOS was added to the suspension of Fe3 O4 nanoparticles in water and stirred at 1500 rpm for 2 h at room temperature. The nanoparticles were, then, separated by an external magnet, washed with deionized water and ethanol, each for three times, and finally were dried under vacuum at 60 ◦ C for 12 h. 2.4. Functionalization of Fe3 O4 core–shell nanoparticles For introduction of amine functional group on the surface of Fe3 O4 core–shell nanoparticles, in a three-necked flask 1.0 g of Fe3 O4 core–shell nanoparticles were dispersed in 50 mL of ethanol using an ultrasonic bath for 10 min. Then, 1.0 mL of APTES was added to the suspension, and the mixture stirred mechanically at 60 ◦ C under N2 flow for 6 h. The synthesized magnetite core–shell nanoparticles were separated using an external magnet and washed with deionized water and ethanol for several times, and finally dried at room temperature under vacuum for 12 h. 2.5. Drug conjugation to functionalized Fe3 O4 –nanoparticles Conjugation of magnetite nanoparticles with doxorubicin was accomplished by reaction between the amine group of functionalized magnetite and the carbonyl group of the drug via Schiff base chemistry. The amino-functionalized Fe3 O4 –nanoparticles (100 mg) were dispersed in 15 mL of ethanol by high speed homogenization (Heighdolph, Germany, Silent Crusher M) at 8000 rpm for 2 min in ambient temperature after adding two drops of glacial acetic acid as a catalyst. Doxorubicin (10 mg) was added to the resulting colloidal dispersion and the reaction was carried out at room temperature for 48 h, thereby leading to the chemical conjugation of doxorubicin to the nanoparticles via imine linkage. Finally, the drug-conjugated core–shell magnetic nanoparticles were separated by an external magnet, washed several times with deionized water and ethanol, and dried under vacuum at 50 ◦ C for 24 h. The drug conjugation efficiency was determined by two direct and indirect methods. Indirect method involved the analysis of the remained residual intact drug in the solution (free drug) by spectrophotometry at 480 nm. The conjugation efficiency was calculated using Eq. (1): Conjugation efficiency % =

WFeed drug − WFree drug WFeed drug

× 100

(1)

where Wfeed drug and Wfree drug show the weight of drug used initially in the conjugation step and the total weight of drug found in the supernatant.

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In the direct method, doxorubicin-conjugated nanoparticles were analyzed spectrophotometrically at 480 nm and conjugation efficiency was determined using the following equation: Conjugation efficiency % =

Wconjugated drug Wfeed drug

× 100

(2)

where Wconjugated drug implies for the total weight of doxorubicin found conjugated on the magnetite nanoparticles. In order to avoid the effect of presence of Fe3 O4 nanoparticles in the absorbance change, the amount of conjugated drug was calculated from the absorbance of the dispersion after subtracting the absorbance of a drug-free nanodipersion sample in the same conditions. Besides, the results obtained by direct method were used to calculate the extent of drug content of carrier which is defined as follows (Eq. (3)): Drug content % =

Wconjugated drug Wcarrier

× 100

(3)

where Wconjugated drug and Wcarrier represent the weight of conjugated drug, and the total weight of corresponding nanoparticles, respectively. 2.6. Coating doxorubicin-conjugated Fe3 O4 core–shell nanoparticles with chitosan For coating the drug-conjugated nanoparticles with chitosan, 25 mg of the vacuum-dried drug-conjugated nanoparticles were dispersed in 15.0 mL chitosan solution, prepared as a 0.5% (w/v) solution of chitosan in acetate buffer (pH = 4.8) at room temperature, and the suspension was stirred mechanically for 1 h at room temperature. The resulting viscous suspension was centrifuged at 15,000 × g for 10 min to separate the chitosan-coated nanoparticles. 2.7. Drug release study of dual targeted carrier Fe3 O4 –doxorubicin–Chitosan The drug release behavior of the finally prepared drugconjugated Fe3 O4 core–shell nanoparticles was studied in physiological pH (7.4) as well as in acidic media with pH of 5.3. Typically, 3.0 mg of the vacuum-dried nanoparticles were placed into a dialysis bag (cut-off 12 kDa) and dialyzed against 15 mL of phosphate buffered saline (PBS) at 37 ◦ C under mild stirring (100 rpm). At predetermined time intervals, in order to determine the drug concentration, and thereby time dependent drug release profile, 1.0 mL of solution was taken out and replaced with 1.0 mL of fresh buffer solution maintained at 37 ◦ C and evaluated for drug content by UV–vis spectroscopy at wavelength of 480 nm. The percent of drug release was calculated in reference to a control sample placed at the same condition, but without nanoparticles. 2.8. In vitro characterization The crystalline structures were analyzed on a Bruker D8 Advence XRD using Cu K␣ radiation. FT-IR spectra were taken with a Matson FT-IR spectrophotomer in the range of 400–4000 cm−1 as KBr disks. Vibrating sample magnetometer (Magnetometer, Iran, Kashan) was used to measure the magnetic properties of the paramagnetite nanoparticles. Scanning electron microscopy (SEM) images were investigated on a Hitachi f4160 scanning electron microscopy using a working voltage of 30 kV. The zeta potential and particle size distribution of the prepared nanoparticles were determined by photon correlation spectroscopy (PCS) using a Nano/zetasizer (Malvern Instruments, Nano ZS, Worcestershire, UK) working on the dynamic light scattering (DLS) platform.

The physical stability of nanocarrier was evaluated by monitoring the size and zeta potential of the nanodispersion prepared, immediately after preparation and throughout 7 days following preparation, while kept in ambient temperature using the method described above. Thermogravimetric analysis was carried out to monitor the mass loss of a known amount of chitosan-coated Fe3 O4 nanoparticles under N2 atmosphere on a NETZSCH STA 409 PC/PG (Selb, Germany) instrument at a heating rate of 20 ◦ C/min. For determination of iron content of the magnetic nanoparticles, an aliquot of the nanodispersion was dissolved in concentrated sulfuric acid and the iron concentration was determined by atomic absorption using a spectrophotometer (WFX-130, China, Mainland). 2.9. Statistical analysis The data were analyzed using the statistics software SPSS (version 22, USA). The results are presented as the mean ± SD, and the data were evaluated by one-way analysis of variance (ANOVA). Pvalues less than 0.05 were considered statistically significant. 3. Results and discussion In order to enhance the therapeutic efficacy of doxorubicin, while minimizing its life-threatening side effects, a dual targeting drug delivery system was designed and prepared. The carrier composed of magnetite nanoparticles in order to direct the drug toward tumor sites with the aid of an external magnetic field. On the other hand, the drug was conjugated via acid-cleavable linker to the nanostructures with the ability to release the conjugated drug in the low-physiologic pH environments (pH 5–5.5), thereby targeting the lower-than-normal pH of the tumor sites as well as the media present in the endosomes of the cancer cells. To achieve this goal, firstly Fe3 O4 –nanoparticles were functionalized and subsequently drug conjugation was accomplished via imine linkage. 3.1. Preparation and characterization of magnetic nanoparticles Magnetite nanoparticles were prepared using the coprecipitation of ferric and ferrous salts in alkaline medium (Eqs. (4) and (5)): NH4 OH ↔ NH4 + + OH− 2+

Fe

+ 2Fe

3+

−

+ 8OH → Fe3 O4 + 3H2 O

(4) (5)

The characteristic peak of Fe O stretching band at 587 cm−1 could evidence formation of Fe3 O4 (Fig. 1a). The band at 3300–3600 cm−1 for nanoparticles was ascribed to the O H vibration. It is believed that Fe3 O4 –nanoparticles synthesized by this method are prone to aggregate. To reduce the aggregation of particles, a silica-based shell was introduced onto the surface of Fe3 O4 –nanoparticles. The synthesized nanoparticles were modified by TEOS which forms a covalent Fe O Si bond between the hydroxyl groups present on the surface of the Fe3 O4 –nanoparticles and the ethoxy groups of TEOS (Fe3 O4 –core–shell nanoparticles). The surface properties of magnetite nanoparticles were studied using FT-IR (Fig. 1b). The characteristic peak of Si O bond as a relatively broad band appeared at 1055 cm−1 awardable to the corresponding stretching vibration demonstrates the presence of silica on the surface of Fe3 O4 –nanoparticles [23]. It is of great importance to functionalize the surface of Fe3 O4 –core–shell nanoparticles via introducing amine functional groups in order to facilitate the drug conjugation. Amine groups ( NH2 ) were created on the surface of Fe3 O4 –core–shell nanoparticles by chemical bonding of APTES to the nanostructures via its 3-aminopropyl group. In fact, the 3-aminopropyl functional group

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Fig. 2. UV/vis spectra recorded in water for free doxorubicin (a); Fe3 O4 –doxorubicin–Chitosan (b) and drug-free functionalized Fe3 O4 –core–shell nanoparticles (c). Fig. 1. FT-IR spectra of (a) Fe3 O4 –nanoparticles, (b) Fe3 O4 –core–shell nanoparticles, (c) functionalized Fe3 O4 –core–shell nanoparticles, (d) Fe3 O4 -doxorubicin and (e) Fe3 O4 -doxorubicin -Chitosan.

was attached to Fe3 O4 core–shell nanoparticles surface by condensation of silanol and ethoxy groups. The drug was, then, attached to the functionalized Fe3 O4 –core–shell nanoparticles through the amine groups by Schiff base condensation. FT-IR spectrum of the functionalized Fe3 O4 –core–shell nanoparticles is shown in Fig. 1c. The band at 803 cm−1 is assignable to s (Si O Si) modes. The small shoulder band at 912 cm−1 is assignable to  (Si OH). The bending vibration band of O Si O unit was found at 447 cm−1 [24]. The bands at 1030 and 1121 cm−1 were awardable to the Si O and Si O Si groups and the two bands at 3430 and 1561 cm−1 can be referred to the N H stretching vibration and bending mode of free NH2 groups, respectively. The presence of the anchored propyl group was confirmed by C H stretching vibrations that appeared at 2920 cm−1 [25]. The spectra conceived that Fe3 O4 –core–shell nanoparticles with amine-enriched surface were obtained. The amine functional groups permitted further modification of the surface with molecules such as doxorubicin. To evaluate the potential application of functionalized Fe3 O4 –core–shell nanoparticles as a tumor dual-targeting drug carrier, doxorubicin was used as a representative anticancer drug to be conjugated onto the surface of functionalized Fe3 O4 –core–shell nanoparticles via acid-labile linkers with the ability to increase the rate of drug release in an environment with low pH value (e.g. pH 5), as presented in the endosome of cancerous cells. Fig. 1d shows the FT-IR spectra of doxorubicin conjugated to the Fe3 O4 nanostructures, where the characteristic absorption peaks of 803 and 1220 cm−1 ( C O CH3 ) can be attributed to the drug [7]. The peak at 1621 cm−1 was ascribed to the characteristic absorption of C N bonds resulting from the reaction of amino group of the functionalized Fe3 O4 –nanoparticles with the carbonyl group of doxorubicin. The above observations imply that doxorubicinis chemically conjugated onto the surface of the functionalized magnetite nanoparticles. Free doxorubicin can penetrate the nuclear envelope, localize in nucleus and exert its cytotoxicity through its primary mechanism of action. Doxorubicin as conjugated to functionalized Fe3 O4 nanoparticles, however, cannot access the nucleus and, subsequently, its cytotoxic efficiency is decreased. To further minimize the side effect of the drug while increasing the circulation time of the prepared dual-targeting nanocarriers, they were encapsulated in chitosan as a naturally-derived biocompatible polycation. Fig. 1e shows the FT-IR spectra of chitosan-encapsulated, drug-conjugated

Fe3 O4 nanoparticles. The bands around 1044 cm−1 and 1621 cm−1 verified the C N and N H groups of chitosan, respectively. These results revealed that drug-conjugated Fe3 O4 nanoparticles encapsulated successfully with chitosan. UV-spectrophotometry was applied to further confirm the presence of doxorubicin in the conjugated Fe3 O4 –nanoparticles. The Fe3 O4 –nanoparticles were evaluated by UV absorption spectra before and after conjugation with doxorubicin as shown in Fig. 2. As it is clear, the UV spectrum in Fig. 2b corresponding to the dual-targeting carrier showed absorption maximum at 488 nm assigned to the ␲ → ␲* band of the drug, while those of functionalized Fe3 O4 –core–shell nanoparticles did not show any absorbance signal at the same wavelength (Fig. 2c). Compared with pure doxorubicin (maximum absorption evident at 485 nm), the conjugate showed a maximum absorption shift of 4–5 nm, which may be explained by the drug conjugation on the magnetite nanoparticles. It means that the drug was covalently conjugated to the Fe3 O4 –nanoparticles surfaces. The X-ray diffraction patterns of the naked Fe3 O4 –nanoparticles, functionalized Fe3 O4 –core–shell nanoparticles and Fe3 O4 –doxorubicin–Chitosan samples are shown in Fig. 3.

Fig. 3. XRD patterns of the naked Fe3 O4 –nanoparticles (a) functionalized Fe3 O4 –core–shell nanoparticles (b) and Fe3 O4 –doxorubicin–Chitosan (c).

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Fig. 4. Simultaneous thermal analysis of chitosan-coated Fe3 O4 .

A series of characteristic peaks which corresponds to (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0) Bragg reflections were observed for Fe3 O4 –nanoparticles (Fig. 3a) and functionalized Fe3 O4 –core–shell nanoparticles (Fig 3b). The intensity of Fe3 O4 –nanoparticles diffraction peaks for functionalized Fe3 O4 core–shell nanoparticles were weaker than those of the naked Fe3 O4 –nanoparticles, which indicates the presence of amorphous materials (silica) on the surface of Fe3 O4 –nanoparticles. As expected, in case of Fe3 O4 –doxorubicin–Chitosan, all peaks were broadened because of presence of amorphous chitosan on the surface of nanoparticles. The chitosan content of chitosan-coated Fe3 O4 nanoparticles was measured by STA under nitrogen atmosphere condition. Fig. 4 illustrates the STA curve, depicting the variation of residual mass of the sample with temperature. As it is clear, the first weight loss was about 0.99 wt% at 25–103 ◦ C which can be ascribed to the evaporation of water molecules in the polymer matrix. The second weight loss was about 3.18 wt% in the range of 103–243 ◦ C, which may be awardable to scission of the ether linkage in the chitosan backbone. In the third stage, the weight loss was about 6.34 wt% in the range of 243–536 ◦ C, which corresponds to the thermal decomposition of glucosamine residues of chitosan [26], thus indicating the chitosan content of the nanocarrier. In order to assay the iron content of the nanocarrier, atomic absorption spectroscopy was used. The results revealed that the iron content of chitosan coated nanoparticles was 72 ± 2% (w/w). It is obvious that high saturation magnetization is one of the prerequisites to maximize the targeting efficiency of nanoparticles using an external magnetic field. The magnetic properties of nanoparticles were evaluated by vibrating sample magnetometer (VSM). Fig. 5 shows the magnetization curves of naked Fe3 O4 –nanoparticles, functionalized Fe3 O4 –core–shell nanoparticles, and Fe3 O4 –doxorubicin–Chitosan. The results showed that all samples exhibited superparamagnetic properties. The saturation magnetization values (Ms ) of the Fe3 O4 –nanoparticles, functionalized Fe3 O4 –core–shell nanoparticles and Fe3 O4 –doxorubicin–Chitosan nanoparticles were 56.8, 47 and 6 emu/g at 8500 Oe, respectively. Magnetic properties of Fe3 O4 –nanoparticles coated by chitosan exhibited a lower level of magnetization in comparison to the naked Fe3 O4 –nanoparticles. Indeed, such a significant decrease in magnetization can be related to the presence of chitosan shell on the surface of magnetite nanoparticles which is consistent with many studies reporting the reduction in magnetization by surface modification of Fe3 O4 –nanoparticles [11]. This observation can also be explained by the overall reduction in the total magnetic content in the composite nanoparticles related to the total mass of naked

Fig. 5. Magnetization curves of Fe3 O4 –nanoparticles (a), functionalized Fe3 O4 –core–shell nanoparticles (b), and Fe3 O4 –doxorubicin–Chitosan (c) at room temperature.

nanoparticles. As it can be seen, there was no magnetic remanence for the magnetite nanoparticles and the initial slope of the magnetization curve was quite steep, which are characteristics of superparamagnetic behavior with a high magnetic susceptibility. If the magnetic susceptibility is zero it means that the material does not respond with any magnetization. The value of magnetic susceptibility can be obtained from the initial slope of the magnetization curve shown in Fig. 5. The magnetic susceptibility of Fe3 O4 –nanoparticles, functionalized Fe3 O4 –core–shell nanoparticles and Fe3 O4 –doxorubicin–Chitosan nanoparticles were determined to be 0.08, 0.14, and 0.008 emu/g Oe, respectively. The low magnetic susceptibility of chitosan-coated nanoparticles may be explained by the presence of the high concentration of functional groups in the chitosan backbone which allows for spinpinning of the iron oxide surfaces [27]. Considering all findings, the results indicate that all variants of nanoparticles possess sufficient magnetic properties to exhibit high enough magnetic response in order to direct the particles to a specific location in body. The size of Fe3 O4 –nanoparticles before and after each step of surface modification was investigated by DLS analysis. In all samples, particle size distribution curves exhibited only one peak with a relatively high polydispersity index, probably indicating the Fe3 O4 –nanoparticles aggregation in solutions. Table 1 presents the hydrodynamic diameters of Fe3 O4 –nanoparticles, SiO2 - and APTES-modified Fe3 O4 –nanoparticles, Fe3 O4 -doxorubicin, and Fe3 O4 –doxorubicin–Chitosan nanoparticles. From the results shown in Table 1, a trend toward the increase of diameter after each modification step was observed. The final product (Fe3 O4 –doxorubicin–Chitosan) showed an average diameter of 136 nm with a polydispersity index of 0.493. Fig. 6 shows the typical SEM image and corresponding histogram for Fe3 O4 –doxorubicin–Chitosan nanoparticles. Although the sample preparation for SEM observation may induce some aggregation of the magnetic particles during deposition on a grid, comparing the SEM image of Fe3 O4 –nanoparticles in the absence of chitosan, the results indicate that the aggregation degree of the chitosan coated magnetic particles is far smaller than that of the naked Fe3 O4 –nanoparticles. The corresponding histogram of SEM image also shows that the average size of nanoparticles are about 50 nm which is much smaller that the results obtained by DLS technique. Indeed, such a difference can be explained by the fact that in spite of SEM image, DLS results imply to the hydrodynamic diameter of nanoparticles.

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Table 1 Average size and zeta potential of Fe3 O4 –nanoparticles. Each data represents the mean ± S.D. (n = 3).

Size (nm) Zeta potential (mV)

Fe3 O4 –nanoparticles

Fe3 O4 core–shell nanoparticles

functionalized Fe3 O4 core–shell nanoparticles

Fe3 O4 -doxorubicin

Fe3 O4 –doxorubicin–Chitosan

186 ± 5.2 −31.2 ± 1.1

97 ± 3.2 +8.9 ± 0.63

125 ± 2.5 +14.1 ± 0.962

126 ± 1.2 +18.5 ± 1.02

136 ±2.3 +25 ±2.13

The zeta potential of naked Fe3 O4 –nanoparticles in aqueous dispersion was determined to be about −31.2 mV. The zeta potential of Fe3 O4 –nanoparticles was fairly increased to +8.9 mV and it was further moved toward positive charge (+25 mV) by coating with chitosan, thereby indicating the presence of abundant amino groups of chitosan on the surface of dual-targeting carriers. This trend can lead to the conclusion that chitosan not only can be considered to enhance the biocompatibility of the nanocarrier, but also owing such high zeta potential, it can play a critical role in improvement of the stability of the carrier. 3.2. Stability of nanoparticles The stability in biological milieu is of significant importance when choosing an appropriate drug carrier. The particle size and zeta potential of modified Fe3 O4 –nanoparticles were measured to investigate the stability of the nanoparticles under the physiological pH. Fig. 7 shows the variation of zeta potential and particle size of nanoparticles through one week in phosphate-buffered saline (PBS; pH 7.4). As shown in Fig. 7, in case of Fe3 O4 –core–shell nanoparticles, functionalized Fe3 O4 –core–shell nanoparticles, Fe3 O4 -doxorubicin conjugate and Fe3 O4 –doxorubicin–Chitosan nanoparticles, the particle size and zeta potential of the nanoparticles did not show significant change during the time of investigation, but naked Fe3 O4 –nanoparticles showed slight change which can be attributed, in part, to the higher magnetic properties of naked Fe3 O4 –nanoparticles. From these results, it is evident that the functionalized Fe3 O4 –nanoparticles probably have good stability in biological milieu and may form a stable suspension in biological fluids, at least in terms of pH and tonicity, which is a highly desirable characteristic for a drug nanocarrier.

if one can develop targeting carriers for this drug in the way to avoid its side effects during intravenous injection to the patients. To achieve this goal, doxorubicin molecule was chemically conjugated onto the surface of functionalized magnetite nanoparticles synthesized in this study. The conjugation efficiency (%) was calculated indirectly to be 54%, while by direct method, the value of conjugation efficiency (%) was found to be 42.4%. Such difference can be related to the presence of some loosely adsorbed drugs, which are being removed through washing steps or possibility of drug degradation through conjugation procedure. Drug content (%) of carrier was also calculated to be about 13.2%. Considering all these results, it seems that the resultant doxorubicin carrier system is highly promising carrier considering the goals of this study. 3.4. Drug release from dual targeting carrier The cumulative release of doxorubicin from the prepared dual-targeting carrier was investigated in two different pH values of 5.3 and 7.4 at the physiological temperature of 37 ◦ C,

3.3. Conjugation efficiency The use of doxorubicin is somehow limited in clinical practice, because of its harmful side effects, mainly the life-threatening cardiotoxicity. Therefore it is highly attractive for the chemotherapists

Fig. 6. Scanning electron microscopy Fe3 O4 –doxorubicin–Chitosan.

(SEM)

image

of

the

synthesized

Fig. 7. (a) Zeta potentials and (b) hydrodynamic diameters of different variants of the magnetite nanoparticles in phosphate-buffered saline (PBS; pH 7.4). Each data point represents the mean ± S.D. (n = 3).

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S. Sadighian et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 406–413 Table 2 Dissimilaroty (f1 ) and similarity (f2 ) factors of the carriers at different condition. The data were evaluated by one-way analysis of variance (ANOVA). P-values less than 0.05 were considered significant.

Fig. 8. Release profiles of doxorubicin from Fe3 O4 -doxorubicin (A in pH = 7.4, B in pH = 5.3) and Fe3 O4 –doxorubicin–Chitosan (C in pH = 7.4, D in pH = 5.3) nanoparticles while incubated in phosphate-buffered saline (PBS; pH 7.4) at 37 ◦ C (n = 3). Each data point represents the mean ± S.D. (n = 3).

to investigate the pH-based tissue targeting of this system, considering the acidic nature of the tumor tissue compared to the healthy tissues. The cumulative drug release was expressed as the percent of drug delivered to the surrounding aqueous media from the dialysis sacs containing the nanocarriers as a function of time, taking into account the overall drug content of nanocarrier. Fig. 8 shows the cumulative release of doxorubicin from Fe3 O4 –doxorubicin–Chitosan nanoparticles and Fe3 O4 -doxorubicin nanoparticles. Of particular note was that both variants showed a considerable sustained drug release behavior with no significant initial burst release (Fig. 8). The reason behind this observation probably lies in the fact that the release of drug is considerably controlled by the cleavage of chemical bond between magnetite and doxorubicin rather than simple diffusion from carrier. Moreover, the coating of carrier by chitosan resulted in decreasing, to some extent, rate and extent of drug release (Fig. 8C and D compared to Fig. 8A and B). In order to evaluate the differences between the drug release profiles of various formulations, the model independent mathematical approach proposed by Moore and Flanner and implemented by the US Food and Drug Administration (FDA) was considered [28]. The equations are represented as follows:

n

f1 =

i=1

|ti − ri |

2

i=1

f2 = 50 × log

|ri |

⎧ ⎨ ⎩

1+

× 100

(6)

n

(ti − ri ) i=1 n

2

−0.5

⎫ ⎬

× 100

⎭

Formulation

f1

f2

Fe3 O4 -doxorubicin (pH 7.4) vs. Fe3 O4 -doxorubicin (pH = 5.3) Fe3 O4 -doxorubicin (pH 7.4) vs. Fe3 O4 –doxorubicin–Chitosan (pH 7.4) Fe3 O4 -doxorubicin (pH = 7.4) vs. Fe3 O4 –doxorubicin–Chitosan (pH 5.3) Fe3 O4 -doxorubicin (pH = 5.3) vs. Fe3 O4 –doxorubicin–Chitosan (pH 7.4) Fe3 O4 -doxorubicin (pH = 5. 3) vs. Fe3 O4 –doxorubicin–Chitosan (pH 5.3) Fe3 O4 –doxorubicin–Chitosan (pH = 7.4) vs. Fe3 O4 –doxorubicin–Chitosan (pH 5.3)

49.52

2.38

36.23

2.80

18.93

4.23

57.35

1.41

45.78

1.75

27.12

6.10

chitosan-coated dual-targeting carrier after 12 days, while the maximum drug release attainable after 12 days was about 60% of the whole loaded doxorubicin in the absence of chitosan. It is possible to explain this behavior as a consequence of an additional barrier made by chitosan, which prevents the water from penetration into the core segment of nanostructure, thereby slowing down the rate of hydrolytic cleavage of drug–particle bonds and, finally the drug release rate out of the carrier. Lower than physiologic pH environment (pH 5–5.5) present in the endosomes of the cancer cells is widely used as a promising approach to develop different targeted delivery systems. To examine the validity of this approach, the release behavior of the drug from our dual-targeting carriers was examined in buffer solutions with two different pH values (5.3, 7.4). As Fig. 8 and Table 2 show it was found that a significantly higher and different drug release occurs in acidic dissolution medium (P < 0.05, final time points). For instance, the percent drug release was about 60% at pH 5.3 after 12 days, while it was less than 50% at pH 7.4 at the same time. In fact, the released fraction of doxorubicin from both the chitosancoated and uncoated Fe3 O4 core–shell nanoparticles seems to be higher in lower pH (corresponding to that of the cancer cells) than the physiologic pH throughout the time period tested. This clear pH-dependency in drug release rate from the nanocarriers can be attributed to the presence of acid-labile linker used for drug conjugation to the particles. The results are consistent with the other researcher’s report [29] which demonstrates that the weak acidic condition could accelerate cleavage of the imine bonds linker. Thus, briefly, it can be concluded that under our experimental condition, the drug release from the drug-conjugated core–shell magnetic nanostructures is influenced by the triggered drug release mechanism (chemical bond cleavage vs. simple diffusion) and pH of the surrounding medium, both being beneficial for the purpose of this study.

(7)

where f1 represents dissimilarity factor, and f2 indicates similarity factor of drug release throughout a definite time interval, ri and ti are drug release percent from the reference and test formulation, respectively, at time t, and n indicates the total number of sampling time points. Generally, values between 0–15 for f1 and 50–100 for f2 indicate similarity or equivalence of the two drug release curves. Table 2 shows the results of f1 and f2 for the different carriers at various conditions. It is obvious that for all comparisons f1 was up to 15 and f2 was smaller than 5 which verify the significant difference between drug release profiles from the corresponding formulations. Fig. 8 clearly illustrates these differences, where only about 35% of the overall amount of conjugated doxorubicin was released from

3.5. Drug release kinetics To understand the factors that control the drug release from Fe3 O4 –doxorubicin–Chitosan, the release data were fitted with mathematical model. Drug release kinetics were analyzed by plotting the cumulative release data vs. time by fitting to an exponential equation using the equation proposed by Korsmeyer and Peppas as represented below (Eq. (8)). Mt = kt n M∞

(8)

here Mt /M∞ represents the fractional drug release at time t, k is a constant characteristic of the drug delivery system and n is an empirical parameter characterizing the release mechanism. Using the least squares method, we estimated the values of n and k.

S. Sadighian et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 406–413

The results showed that the equation is not valid for all the drug release stages, in particular the later release stage. However, the experimental data satisfy the linear fitting up to 60% doxorubicin release from carriers. The value of n, and K were determined to be 0.68 and 101.2 (R2 = 0.96), respectively. Since n was in the range between 0.5 and 1.0, it can be concluded that for the majority of drug release from carrier, the water penetration into the chitosan shell of carrier and release of drug via diffusion mechanism occurs simultaneously. However, the partial deviation of the fitting parameters from the Fickian kinetic can be attributed to the involvement of the cleavage of drug from conjugated carrier in the release condition. 4. Conclusion Synthesis of dual-targeting core–shell nanostructures composed of magnetite (Fe3 O4 ) nanoparticles conjugated with doxorubicin via pH sensitive linkage with characteristics of substantially longer drug release time in circulation compared to the similar physically-entrapped drug, as well as capability to deliver the drug specifically to the tumor-bearing tissues based on both the external magnetic localization and pH-sensitive drug cleavage from the nanoparticles was achieved in this study. To improve carrier biocompatibility and also to reduce the nonspecific drug release rate from the carriers, the dual-targeting carrier was coated by chitosan. FT-IR confirmed the synthesis of functionalized Fe3 O4 –nanoparticles, doxorubicin-conjugated Fe3 O4 –nanoparticles, and chitosan-coated magnetite nanoparticles. Scanning electron microscopy (SEM) and particle size analysis confirmed the formation of spherical nanostructures with the final average particle size of 50 nm. The VSM analysis showed a saturation magnetization value (Ms ) of about 6 emu/g for nanocarrier. The drug release behavior from the prepared dual-targeted carriers was investigated in acidic and neutral buffer solutions (pH: 5.3 and 7.4). The release study of doxorubicin revealed that the extent of drug release at pH = 5.3 was promisingly more than drug release at pH = 7.4. From the results it can be concluded that the dual-targeting carrier with possibility to respond to the external magnetic field and pH was successfully prepared and can be used to improve therapeutic efficacy of anticancer drugs.

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