Preparation and characterization of CoFe2O4 and CoFe2O4@Albumen nanoparticles for biomedical applications

Ceramics International 45 (2019) 24971–24981

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Ceramics International journal homepage: www.elsevier.com/locate/ceramint

Preparation and characterization of CoFe2O4 and CoFe2O4@Albumen nanoparticles for biomedical applications

T

Mohd Qasim∗, Khushnuma Asghar, Dibakar Das School of Engineering Sciences and Technology, University of Hyderabad, Hyderabad, 500046, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Nanocomposites Magnetic properties Ferrites Biomedical application

Biopolymer coated magnetic nanoparticles are becoming extremely popular in the fields of biomedical research because of their enormous applications potentials. In this work, first, CoFe2O4 nanoparticles (CF NPs) of two distinct sizes have been prepared following co-precipitation method by varying the synthesis parameters and characterized. XRD, SAED and IFFT (Inverse Fast Fourier Transformed) assisted HRTEM analyses confirmed the successful formation of monodispersed spinel cubic CoFe2O4 nanoparticles. Particles size of CF NPs was found to increase with increase in the amount of reducing agent. Synthesized CF NPs was coated with an egg albumen matrix by a facile and environmental friendly method to form biocompatible CoFe2O4@Albumen nanocomposite nanoparticle (CF@Alb NP). Prepared CoFe2O4 and CoFe2O4@Albumen nanoparticles were examined for its structure, morphology, thermal stability and magnetic nature employing powder XRD, HRTEM, TGA, FTIR and VSM techniques. Low as well as high magnification TEM analyses have shown coating of amorphous albumen on crystalline CF NPs. It was observed that CF@Alb NP is composed of many smaller CF NPs engulfed in the albumen matrix forming a nano-aggregate of size ∼80–130 nm. IFFT analysis of HRTEM micrograph showed presence of crystalline CF NPs in amorphous albumen matrix. Further, TGA and FTIR results also suggested the successful coating of albumen on CF NPs. CF@Alb NP has shown a very good Dox loading ability with loading efficiency of ∼93%. A promising pH dependent Dox release behavior was observed. Dox release kinetics has also been studied using different mathematical models. Biocompatibility of the CF@Alb NP has been tested against the human monocytic cell line THP-1. This novel CF@Alb NP could have a great potential in biomedical applications, particularly in hyperthermia and targeted drug delivery.

1. Introduction Magnetic nanoparticle (MNP) and polymer based hybrid heteronanostructures or nanocomposites have gained much attention of biomedical research community in the recent past because of their extra ordinary application potential in cancer treatment [1]. Different types of magnetic nanoparticles (MNPs) are being used to develop MNP/ polymer nanocomposites and spinel ferrites based MNPs (such as Fe3O4, CoFe2O4, MnFe2O4 NPs etc) with superior magnetic properties (such as high specific magnetization) are being extensively investigated for different biomedical application [2]. Particularly, CoFe2O4 nanoparticles (CF NPs) have shown very high magnetocrystalline anisotropy, specific magnetization, outstanding physico-chemical stability, and large coercivity value that enable them for use as an appropriate agent for cancer diagnosis and therapy. Several chemical routes such as coprecipitation, thermal decomposition, and micro-emulsion are generally used to prepare MNPs [2,3]. Generally, for a biomedical

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application MNPs need to be hydrophilic, monodispersed, well crystalline and biocompatible. Coprecipitation method which can produce hydrophilic and good amount of MNPs is commonly used to prepare MNPs for biomedical fields. However, low crystallinity and broad size distribution are the drawbacks of this method that require further processing (calcinations and size sorting) of prepared samples. A variety of monodispersed and well crystalline MNPs can be prepared by thermal decomposition method but the obtained hydrophobic MNPs need further surfactant based processing [4]. Similarly, micro-emulsion based method can generate monodispersed MNPs but very less product yield is its major drawback. Thus, in spite of enormous effort in the area of synthesis of MNPs, it is still complicated and tedious to produce a MNPs of good quality (water dispersible, monodisperse, crystalline, biocompatible MNPs) in a facile way. Use of suitable chemical and adequate synthesis parameters could overcome these problems. M.Y. Rafique et al. have shown that NaBH4 has a significant effect over particle size distribution, elemental composition, as well as magnetic

Corresponding author. E-mail address: [email protected] (M. Qasim).

https://doi.org/10.1016/j.ceramint.2019.04.049 Received 30 November 2018; Received in revised form 9 March 2019; Accepted 4 April 2019 Available online 08 April 2019 0272-8842/ © 2019 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

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properties of CoFe2O4 NPs [5]. In addition, various types of surface coating materials are being utilized to supply superb biocompatibility and hydrophilicity to the MNPs. Among them, biopolymers in particular proteins such as HSA (human serum albumin) and BSA (bovine serum albumin) are commonly used as coating materials for MNPs but they are very expensive. Till date, different kind of MNP and BSA or HSA based nanocomposites such as Fe3O4/BSA [6], MnFe2O4-BSA [7] and Fe3O4/HSA [8] have been reported. Easily available cheap egg albumen with excellent biodegradability, biocompatibility, water solubility could be a adaptable and versatile alternative shell material for different type of MNPs. CoFe2O4-Egg albumen based nanocomposites could be a potential candidate in hyperthermia and targeted drug delivery application but have not been studied and reported. In this work, first monodispersed and well crystalline CoFe2O4 nanoparticles (CF NPs) have been synthesized by NaBH4, hydrazine hydrate, NaOH and oleic acid following co-precipitation method. CF NPs of two distinct sizes are prepared by varying the synthesis parameters. In the second part of the work, synthesized CF NPs are coated with or engulfed into an egg albumen matrix by a facile and environmental friendly method to form biocompatible CoFe2O4@Albumen nanocomposite nanoparticle (CF@Alb NP). Prepared CoFe2O4 and CoFe2O4@Albumen nanoparticles were examined for its structure, morphology, thermal stability and magnetic nature employing various techniques. Biocompatibility of the CF@Alb NP has been studied by MTT assay against the human monocytic cell line THP-1. After successful formation and characterization of CF@Alb NP, its anticancer drug carrying ability also has been tested. Doxorubicin (Dox) is a well known anticancer drug with excellent anticancer activity. However, unwanted side effect (toxicity to the normal cells) of these conventional drugs compel biomedical scientific community to search for an alternative and more effective site specific targeted therapies. Loading and parceling anticancer drug in a targeted manner using MNP and pH responsive coating (albumen) could be a versatile strategy to combat the above limitations. Thus, Dox has been loaded in CF@Alb NP and its release behaviors at different pH have been investigated employing different mathematical kinetic models.

dispersed in 8 ml of water by ultrasonication. Then, 2 ml of freshly taken egg albumen was dropped into the homogeneous aqueous dispersion of CF NPs with continuous ultra-sonication. The mixture was further sonicated for 30 min and then transferred to the magnetic stirrer for 4 h. Resulted CF@Alb NP was separated magnetically, washed with water to remove free albumen and stored in the glass vial. 2.3. Dox loading and release study Dox loaded CF@Alb nanoparticle (CF@Alb-Dox NP) was obtained by stirring a fixed amount of CF@Alb NP and Dox in aqueous media. The experiment was performed in a dark environment to avoid any light induced degradation of Dox. After ∼24 h of stirring at room temperature, the resultant CF@Alb-Dox NP was separated magnetically and washed thrice to remove loosely adsorbed Dox molecules from CF@Alb-Dox NP. Obtained CF@Alb-Dox NP and supernatant were stored in dark for further studies. The amount of loaded Dox in CF@Alb-Dox NP was estimated using their respective absorbance (at λmax of Dox ∼480 nm) values from UV–Vis absorption spectra of initially taken Dox (before loading) and Dox in the supernatant. Subtraction of amount of free Dox in supernatant from the amount of taken Dox gave the amount of loaded Dox in CF@Alb-Dox NP. The loading capacity percentage (LC %) and loading efficiency percentage (LE%) were estimated from the following equations,

Amount of loaded Dox = amount of taken Dox–amount of Dox in the supernatant

(1)

LE % = amount of loaded Dox × 100 / amount of Dox taken

(2)

LC % = amount of loaded Dox × 100 / amount of loaded CF@Alb –Dox NP

(3)

FeCl3.6H2O, and CoCl2.4H2O were purchased from the Fisher Scientific. Doxorubicin (DOX, > 98%) and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) were received from Sigma. NaOH, NaBH4 and Hydrazine Hydrate were obtained from SRL India. The human monocytic cell line THP‐1 was obtained from National Centre for Cell Science (NCCS), Pune, India. Roswell Park Memorial Institute (RPMI)-1640 medium and fetal bovine serum (FBS) were procured from Gibco. Freshly extracted egg white was used as a source of albumen.

In vitro release studies of Dox from CF@Alb-Dox NP were performed by using dialysis bag method in two different buffer i.e PBS and citrate buffer of pH 7.4 and 5 respectively. For this, certain amount of CF@Alb-Dox NP was sealed in a dialysis bag of cut of 14 kDa. The bag containing CF@Alb-Dox NP was then placed into 30 ml of buffer solution (0.1 M) in a beaker. The buffer was then subjected to the continuous string at 150 rpm at 37 °C. At a regular interval of time, 1 ml released media was withdrawn and replaced with the same amount of fresh buffer. UV–Vis absorption spectra of every withdrawn released sample were recorded to estimate the amount of Dox released using their absorbance values (at λmax of Dox). A standard calibration curve of Dox was used for the estimation of the amount of Dox in the samples [9]. Obtained experimental release data were fitted with various kinetic models such as zero order, first order, Higuchi model, Korsmeyer–Peppas (K-P) model to study the involved release mechanism of Dox from CF@Alb-Dox NP.

2.2. Synthesis of CF NPs and CF@Alb nanocomposite

2.4. MTT assay

CF NPs were prepared by co-precipitation of Fe3+ and Co2+ (2:1 M ratio) in a reaction medium containing NaBH4, hydrazine and NaOH. Oleic acid was used as stabilizing agent. Initially, FeCl3 and CoCl2 in 2:1 M ratio were mixed in 100 ml of EtOH. Certain amount (0.5 gm and 1 gm of NaBH4 for CF1 and CF2 NPs respectively) of NaBH4 in 100 ml water was then mixed into the Fe3+ and Co2+ solution. After that, 5 ml of hydrazine, 2 gm of NaOH (dissolved in H2O), and 5 ml of oleic acid were supplied successively to the above mixture. Precipitated black color CF NPs was then separated from the mixture, rinsed with water and dehydrated at 100 °C. Following the above procedure using 0.5 gm and 1 gm of NaBH4, CF1 and CF2 NPs respectively were prepared. Albumen coated CF NP (CF@Alb NP) was fabricated by ultrasonication and stirring of the aqueous mixture of CF1 NPs and egg albumen [9]. To prepare CF@Alb NP, first 100 mg of CF1 NP was homogeneously

Biocompatibility of prepared CF@Alb NP was determined by MTT assay method against the THP-1 cells [9]. 96 well plate were first seeded with the THP-1 cells using growth medium RPMI-1640 containing 10% FBS. Then the plate was kept for 24 h incubation in 5% CO2 atmosphere and at 37 °C. After 24 h of incubation, CF@Alb NPs were added (in different dose concentrations 12.5 25, 50, 100, 200, 400 and 800 μg/ml) to the 96 wells and further incubated for 24 h. Thereafter 20 μL of MTT solution (5 mg/ml) was added in each well and allowed for further incubation of 4 h. Then, media was replaced with 100 μL DMSO to dissolve the formed formazon crystals. Finally, optical density (OD) of wells containing the culture was recorded by a microplate reader at 570 nm. The cell viability (%) of the CF@Alb NP was estimated by the following equation, Cell viability (%) = (OD of test culture/OD of control culture) × 100.

2. Experimental 2.1. Materials

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2.5. Characterizatios techniques Structural features of CF and CF@Alb nanoparticles were evaluated by a powder XRD technique (Bruker D8 Advance). Particle's size distribution, shape and morphology were investigated by Transmission Electron Microscope (FEI Tecnai T20G2). Room temperature magnetic characteristics of CF NPs and CF@Alb NP were examined up to 1.5 T range using a VSM by Lakeshore (Model 7407). Chemical structure of samples were studied using FTIR (PerkinElmer 2000). Thermogravimetric analysis (TGA) of CF NPs and CF@Alb NP were done by TGA (Mettler Toledo 851e). UV-Vis-NIR spectrometer (Jasco V670) was used for Dox loading and release studies. 3. Results and discussion 3.1. Preparation of CF, CF@Alb and CF@Alb-Dox NPs

Fig. 1. XRD patterns of CF1, CF2, CF@Alb and CF@Alb-Dox NPs.

Particles size, morphology and crystallinity of magnetic nanoparticles are dependent on their synthesis procedure, precursor concentration, calcination time etc [5]. The magnetic properties of magnetic particles are governed by particle size, crystallinity and distributions of metal ions and all these are related with the synthesis route [10]. Well crystalline and monodispersed CoFe2O4 NPs have been synthesized by a simple co-precipitation method which is well known to produce hydrophilic NPs [2,3]. Oleic acid was used as a capping agent to keep particles size smaller and to avoid aggregation during synthesis. Use of NaBH4 and hydrazine hydrate additionally with NaOH could have resulted in the formation of well crystalline monodispersed CoFe2O4 NPs at a lower temperature without any calcination. It is worthy to mention that conventional co-precipitation method requires further calcination and particles size sorting process. CoFe2O4 nanoparticles namely CF1 and CF2 were synthesized using 0.5 gm and 1 gm of NaBH4 respectively in the same reaction medium and other condition. Preparation of CF@Alb NP was carried out by environment friendly ultrasonication followed stirring method. Schematic diagram showing the formation of CF@Alb and CF@Alb-Dox NPs is shown in Scheme 1. Gelation of albumen and rapid collision during ultrasonication resulted deposition of albumen on CF NPs [9]. It is worthy to mention that ultrasonication produced very high localized heat and energy which was sufficient to initiate chemical interactions between CF NPs and albumen. Dox loaded CF@Alb nanoparticle (CF@Alb-Dox NP) was obtained by stirring the mixture of CF@Alb NP and Dox in aqueous media. Loading of Dox molecules into the CF@Alb NP can be explained in term of electrostatic interaction between the positively charged amine group of Dox and negatively charge carboxyl group of albumen matrix [11]. 3.2. XRD analysis of CF and CF@Alb NPs XRD patterns of CF1, CF2, CF@Alb and CF@Alb-Dox NPs are shown in Fig. 1. XRD pattern of CF1 and CF2 samples show presence of characteristic peaks of reflection from (220) (311) (222) (400) (422)

(511) and (440) plan of cubic CoFe2O4 [10]. Thus, XRD pattern of CF1 and CF2 established the successful development of mono-phase spinel cubic CoFe2O4 nanoparticles in both cases. The appeared diffraction patterns are well matching with the standard data for CoFe2O4 (JCPDF No. 22–1086). The average crystallite sizes of CF1 and CF2 by Debye–Scherrer method were estimated to be 8 nm and 15 nm, respectively. Diffraction peaks of CF1 are broader than that of CF2 probably due to its smaller size. Crystallinity of the CF NPs was found to increase with the increase in the amount of NaBH4 in the reaction medium. The increase in the crystallite size of CF2 NP can be attributed to the increased amount of NaBH4 [5]. Upper two patterns of Fig. 1 show XRD patterns of CF@Alb and CF@Alb-Dox NPs. Both the samples show appearance of typical diffraction peaks of spinel CoFe2O4. Appearance of characteristic peaks of CF NP into the XRD pattern of CF@Alb and CF@Alb-Dox NPs established the presence of CF NPs in prepared nanocomposite. The intensities of diffraction peaks of CF@Alb NP are relatively less as compared to the pure CF NPs. It could be due to the incorporation of CF NPs in amorphous albumen matrix [12]. Due to amorphous nature of albumen, no sharp diffraction features of albumen have been observed [9]. It was also observed that the crystalline structure of CF NPs has not been affected by the albumen layer and coating process of albumen. No sharp and observable diffraction features related to Dox have been observed in XRD pattern of CF@Alb-Dox NPs and it could be due to very less amount of Dox in nanocomposite and/or its presence in amorphous state [13]. 3.3. Particles size, structure and morphology of CF NPs by TEM TEM micrographs of both CF1 and CF2 CoFe2O4 nanoparticles have been presented in Fig. 2 (a) and Fig. 2 (b) respectively with their corresponding particles size distribution (c & d) and SAED patterns (e & f). Particles sizes of CF1 and CF2 nanoparticles were estimated from the TEM micrographs and they were ∼11 and ∼15 nm respectively. The shapes of the particles are nearly spherical in nature in both cases. The

Scheme 1. Schematic diagram showing formation of CF@Alb and CF@Alb-Dox NPs. 24973

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Fig. 2. TEM micrographs of CF1 (a) and CF2 (b) NPs with corresponding Particle size distribution (c & d) and SAED pattern (e & f).

particles size distribution of CF1 is found narrower than CF2 NPs. The increase in the particles size in case of CF2 can be attributed to the increased amount of NaBH4 [5]. Uncontrolled rapid nucleation and subsequent fast particle growth rate could have caused increase in the particles size at high doses of NaBH4. This phenomenon of increment in the diameter of nanoparticles with increasing NaBH4 amount was also noticed earlier for the same ferrite NPs [5]. Polycrystalline natures of both samples CF1 and CF2 NPs were revealed by SAED analyses (Fig. 2 (e &f)) which have shown presence of characteristic dotted ring pattern corresponding to spinel cubic CoFe2O4 NPs. Appeared rings were indexed estimating their d-spacing and comparing them with standard data (JCPDF No. 22–1086). Thus, SAED observations have also corroborated the successful preparation of CF NPs. Fig. 3 (a) and (b) show HRTEM micrographs of CF1 and CF2 NPs. Appearance of well-ordered atomic planes on the surface of CF particles confirmed their well crystalline nature. In addition, appearance of atomic planes on one single particle only in one direction indicates that it is made up of a single crystallite. Closeness of particles size estimated

by TEM and crystallite size estimated by XRD, also support this observation. d-spacing of atomic plane was estimated and it has confirmed that particle was CoFe2O4 as it was well matching with d-spacing of CoFe2O4. IFFT analyses of the selected HRTEM regions were also performed. IFFT transformed HRTEM image with clear visualization of atomic planes are shown in Fig. 3 (c) and (d) respectively for marked region (in Fig. 3 (a) and (b)) of CF1 and CF2. Well arranged atomic planes can be seen in both the IFFT images having d-spacing of about 0.3 nm which correspond to (220) plane of cubic CoFe2O4. Line profiles through the atomic plans were used to estimate the d spacing and are represented in Fig. 3 (e) and (f) for CF1 and CF2 NPs. The distance between two peaks was ∼0.3 nm that corresponds to (220) plane of CF [14,15].

3.4. Particles size, structure and morphology of CF@Alb NPs by TEM Coating of magnetic nanoparticles with albumin gives hydrophilic, biocompatible and colloidally stable nanoparticles [11,16]. Presence of

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Fig. 3. HRTEM micrographs of CF1 (a) and CF2 (b) NPs. IFFT images of the marked region of (a) and (b) are given in (c) and (d). The line profiles via atomic planes of (c) and (d) are shown in (e) and (f) respectively.

magnetic nanoparticles in albumen nanocarrier will provide magnetic targeting ability to nanocarrier. Formation of CF@Alb NP and its particles size as well as morphology were studied by TEM analysis. TEM micrographs of CF@Alb NP at various magnifications are presented in Fig. 4(a–d). Particles size of CF@Alb NP was found to lie between ∼80 and 130 nm. Average particle size of CF@Alb NP was estimated to be ∼106 nm. It can be observed that CF@Alb NP is composed of many smaller CF NPs embedded and engulfed in albumen matrix. The shape of CF@Alb NP was found to vary from spherical to irregular. The covering layer of egg-albumen (light grey layer) on CF nanoparticles (embedded black smaller particles) is noticeably visible into the high magnification images (Fig. 4 (c & d)) [9]. HRTEM image of CF@Alb NP shows many crystalline CF NPs are embedded in albumen matrix. The amorphous coating of albumen and crystalline CF NPs are indicated by the arrow in Fig. 4(d). The existence of finely-ordered planes on the CF NPs suggests its well crystalline nature. IFFT image of the framed (red) region of (d) is shown in (e) which clearly showed finely ordered planes having an inter-planer spacing of ∼0.25 nm which is (311) plane of CF NPs [15]. Indexed SAED pattern of CF@Alb NP is shown in (f) and appearance of characteristic ring pattern of spinel cubic CF NPs confirmed the formation of nanocomposite or presence of CF NPs in nanocomposite. From the SAED and XRD results of CF@Alb NP, it can also be inferred that coating of albumen does not affect crystal structure of CF NPs. In addition, SAED and IFFT assisted analysis of HRTEM image confirmed the successful formation of CF@Alb NP.

∼580 cm−1 are due to the vibrations from octahedral and tetrahedral sites in the CoFe2O4, respectively [16]. FTIR spectra of CF@Alb NP showed the presence of specific bands of both CF NPs and albumen confirming the successful fabrication of CF@Alb NP. For better understanding, IR spectra of pure albumen is also shown in bottom of Fig. 5 [9]. It can be observed that most of the characteristic bands of pure albumen also appeared in the IR spectra of CF@Alb NP, suggesting coating of albumen on CF NPs. The specific bands of CF@Alb NP at 1642 (and 1659) and 1525 cm−1 can be assigned to the Amide I & Amide II bonds respectively of albumen [6,17]. Band at 1642 cm−1 with a small shoulder at 1659 cm−1 suggest presence of more random secondary structure of albumen with less α helix structure. Interaction with nanoparticles and reorientation of albumen chains due to high ultrasonic energy could be the reason for the more randomness in coated protein secondary structure. Other bands in IR spectra of CF@Alb NP at 1390 cm−1 arose from carboxylate C−O bending vibration, at 1227 cm−1 arose from C−O−H stretching, at 1072 cm−1 arose from C−O stretching vibration of albumen [9]. The feature at ∼2920 and 2852 cm−1 are due to the –CH and –SH stretching. The bands at 3400 and 3059 cm−1 can be assigned to the -OH with contribution from N-H stretching vibration in secondary amide of albumen coating [9,17,18]. Thus, FTIR analysis also confirmed the successful coating of albumen on the CF NP.

3.5. FTIR analysis of CF and CF@Alb NPs

Thermal stability of CF NPs and CF@Alb NP were studied by TGA analysis. The TGA results of bare CF NPs and CF@Alb NP are shown in Fig. 6. CF NPs does not show much degradation up to 600 °C. Little weight loss in case of pure CF NPs could be due to the loss of physically adsorbed water molecules and remanent oleic acid. Thus, CF NPs was found to be stable in the tested temperature rang. CF@Alb NP was found to be stable up to 200 °C with very little weight loss which was

Chemical structure and formation of CF NPs and CF@Alb NP were confirmed by FTIR analysis. FTIR spectra of bare CF NPs and CF@Alb NP and pure albumen are presented in Fig. 5. In FTIR spectra of CF NP, appearance of specific bands of spinel cubic CoFe2O4 at 420 and 580 cm−1 confirmed its successful formation. These bands at ∼420 and

3.6. TGA analysis of CF and CF@Alb NPs

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Fig. 6. TGA results of bare CF NPs and CF@Alb NP.

assigned to the elimination of water molecules. Fast degradation of albumen took place at around 300 °C that is where the major weight loss was observed [9]. The total weight loss in case CF@Alb NP was found to be ∼42%. Thus, TGA analysis also suggested the presence of albumen in the nanocomposite. 3.7. Magnetic properties of CF and CF@Alb NPs

Fig. 4. TEM images of CF@Alb NP with increasing magnifications (a–c). HRTEM image of CF@Alb NP showing crystalline CF NPs engulfed in amorphous albumen (d). IFFT image of the framed (red) region of (d) is shown in (e). SAED ring pattern of CF@Alb NP indexed with corresponding planes (f). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5. FTIR spectra of bare CF NPs, CF@Alb NP and pure Albumen.

RT M − H curve of bare CF NPs and CF@Alb NP are shown in Fig. 7. Bulk CoFe2O4 is well known for its very high magnetization value and coercivity. It was observed that both the samples are having very high magnetization value which reflects its good magnetic controllability. The Ms values of CF NPs and CF@Alb NP were found to be ∼88 and ∼64 emu/gm. Enlarge M − H of CF and CF@Alb NP are shown in the fourth quadrant of Fig. 7. The coercivity and remnant magnetization of both the samples were found to be in between 230 and 240 Oe and 11–14 emu/gm respectively (inset in fourth quadrant of Fig. 7). It is worthy to mention that (large coercivity and remanent magnetization)

Fig. 7. Room temperature M − H curves of bare CF NPs and CF@Alb NP. Enlarged M − H of CF@Alb NP is shown in fourth quadrant. Digital photographs of aqueous dispersion of CF@Alb NP without magnet (a) and near magnet (b) are shown in second quadrant. 24976

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Fig. 8. (a) UV–Vis absorption spectra of taken Dox before loading and free Dox in supernatant. (b) UV–Vis absorption spectra of CF@Alb-Dox NP and blank CF@Alb NP. (c) FTIR spectra of CF@Alb-Dox NP and blank CF@Alb NP.

the bulk CoFe2O4 is not suitable for biomedical application as it may result in the aggregation of particles due to magnetic attraction which may cause blockage in biological system. Small size (size of CF NPs ∼11 nm, size of CF@Alb NP ∼ 80–130 nm), suitable value of coercivity and high Ms values of CF@Alb NP make them promising and suitable agent for hyperthermia application where magnetically generated heat energy is used to burn tumor cells [19–21]. In detail, CoFe2O4 is known to have large coercivity, very high magnetocrystalline anisotropy and specific magnetization which are known to influence the heat generation processes when subjected to alternating magnetic field [10,22]. Thus, CoFe2O4 NPs have been getting increasing attention in the field of magnetic hyperthermia and recently they have been proposed as excellent agents for the same by many researchers [10,23,24]. In a magnetic hyperthermia process, heat is generated by a combination of processes such as hysteresis loss, Neel relaxation and Brownian relaxation [10,25]. Heat generation by Neel and Brownian relaxations occur in very small size nanoparticles particularly in the superparamagnetic regime. In bigger size multi-domain particles dominant loss mechanism is through hysteresis loss [10,25]. Magnetic nanoparticles having size just above the superparamagnetic range but below the critical size of single domain show heat generation by combination of hysteresis loss, Neel relaxation and Brownian relaxation [19]. Further, superparamagnetic nanoparticles with some single domain nonsuperparamagnetic size nanoparticles are also expected to show heat generation by combination of hysteresis loss, Neel relaxation and

Brownian relaxation mechanisms. CF@Alb NP which is composed of very small size (∼7–15 nm, average size ∼11 nm) CF NPs showed presence of small coercivity in the M − H curve making them suitable for heat generation (during hyperthermia) through a combination of hysteresis loss, Neel relaxation and Brownian relaxation unlike pure superparamagnetic nanoparticles in which only Neel and Brownian relaxations take place [10,19–21]. Further, Ms values of magnetic nanoparticles is also directly related to its heat generation ability and high Ms values of CF@Alb NP also make them very promising and suitable agent for hyperthermia application [22,26]. Recently, some researchers have reported that CF NPs of size ∼12–13 nm are promising agent for hyperthermia applications [10,20]. Digital photographs of aqueous dispersion of CF@Alb NP taken without magnet (a) and near magnet (b) are shown in second quadrant of Fig. 7. It was observed that CF@Alb NP was well stable in aqueous media and upon bringing near to the permanent magnet, CF@Alb NP get rapidly (within minutes) attracted toward magnet bar which showed its excellent magnetic targeting ability. Good aqueous dispersibility and stability of CF@Alb NP without any observable agglomeration or clustering of CF@Alb NP have been shown in the digital image (a) placed in the inset (2nd quadrant) of Fig. 7. Hydrophilic nature of albumen coating, electrostatic repulsion between CF@Alb NP, and steric forces due to polymeric albumen coating could have facilitated the colloidal stable nature of CF@Alb NP [9] Detailed insight about stability of colloidal nanoparticles could further be studied employing dynamic light scattering (DLS) technique

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and pH dependent zeta potentials measurements [9,27]. In our previous study on a similar system (NZF and albumen based nanoparticles) good stability of NPs has been observed [9]. Thus, this biocompatible, hydrophilic, and magnetically targetable CF@Alb NP could be very useful for hyperthermia and targeted drug delivery based cancer treatment due to its suitable magnetic nature and physico-chemical properties [10,23,24]. 3.8. Doxorubicin (Dox) loading study In this work, Dox a well known and clinically accepted anticancer drug was used to study the drug loading and release ability of prepared CF@Alb NP [11]. Outstanding anticancer activity, positive charge (at pH 7.4, pK~8.3) and hydrophilic nature of Dox make it a suitable and compatible drug to be loaded in albumen (negatively charged at pH 7.4, isoelectric point ∼5) based nanocarriers [9]. Dox loaded CF@Alb NP was obtained by stirring the mixture of Dox and CF@Alb NP in dark at room temperature. Fig. 8(a) shows UV–Vis absorption spectra of taken Dox and supernatant (free Dox). Drastic decrease in the absorbance value in case of supernatant suggested a high Dox loading ability of CF@Alb NP. Digital photographs of taken Dox and supernatant are also shown in the inset of Fig. 8(a) which showed drastic reduction in the color intensity in case of supernatant. This suggested that very less amount of Dox is left in the supernatant. The loading efficiency and loading capacity were estimated to be 93.3% and 4.45% respectively. The excellent loading behavior of Dox in CF@Alb NP can be attributed to the good electrostatic interaction between positively charged Dox (protonated amine group of Dox) and negatively charged albumen (-COO-) at pH 7.4 [11]. UV–Vis absorption spectra of Dox loaded CF@Alb NP (CF@Alb-Dox NP) and blank CF@Alb NP were analyzed to confirm the loading of Dox in CF@Alb NP and are shown in Fig. 8(b). Blank CF@Alb NP did not show any characteristic absorption peak in the measured wavelength range whereas in case of CF@Alb-Dox NP, characteristic peak of Dox at ∼485 nm (in range of 450–550 nm) was observed. Appearance of the characteristic peak of Dox in the UV–Vis absorption spectra of CF@Alb-Dox NP suggested loading of Dox in CF@Alb NP. Further, loading of Dox in CF@Alb NP was also confirmed by FTIR analysis of CF@Alb-Dox NP and comparing it with that of blank CF@Alb NP. FTIR spectra of CF@Alb-Dox NP and blank CF@Alb NP in range of 1800–700 cm−1 are shown in Fig. 8(c). IR spectra of CF@Alb NP showed presence of characteristic band at 1659, 1642, 1525, 1390,1227, and 1072 cm−1 which were related to the albumen of CF@Alb NP [9]. Details about the origin of these bands are discussed above. CF@Alb-Dox NP has shown presence of all characteristic band of CF@Alb NP with additional new characteristic bands of Dox (at 1738, 1619, 1582, 1567, 1285, 1212, 1199, 975, 865 and 805 cm−1) [28,29]. The broader band in between 1600 and 1660 cm−1 with many shoulders in the IR spectra of CF@Alb-Dox NP are resultant of coupling of bands of Dox (1619 and1640 cm−1) and albumen (1642, and 1659 cm−1). The bands of Dox in IR spectra of CF@Alb-Dox are marked with star (*) symbol. The bands at 1738 cm−1 was attributed to the stretching of carbonyl groups at 13-keto position in Dox [28]. The band at 1619 cm−1 and 1582-1567 cm−1 were due to the bending vibration of N-H of Dox [29]. The bands at 1285, 1212–1199, and 975 cm−1 could be attributed to the skeleton vibration of Dox molecule, asymmetric stretching of C−O−C and stretching vibration of C−O in Dox respectively [9,28]. The bands at 865 and 805 cm−1 were related to the N-H wag of Dox [30]. Appearance of characteristic bands of Dox in IR spectra of CF@Alb-Dox confirmed the presence of Dox in CF@Alb-Dox NP. 3.9. In vitro Dox release study

and 7.4 (pH of blood plasma pH) at 37 °C. Fig. 9 (a) and (b) show UV–Vis absorption spectra of Dox's release media at pH 5 and 7.4 respectively, with respect to time [11,31]. In both the case increase in the absorption intensity (at around 480–490 nm) of Dox were observed which suggested increase in the cumulative concentration of Dox in the released media with incubation time. Fig. 9 (c) shows time dependent Dox release profile from CF@Alb-Dox NP at pH 5 and 7.4. Both the pH have shown sustained as well as controlled Dox release profile and a pH dependent Dox release was observed. Dox release % at pH 5 was significantly higher than that of pH 7.4. Up to 24 h of incubation around 22% and 9% Dox release were observed at pH 5 and 7.4 respectively. Thereafter the release rate becomes relatively slower and ∼33.4 and 24.4% Dox release were observed for pH 5 and 7.4 up to 72 h. Thus the release data clearly indicated a pH dependent Dox release behavior and suggested pH responsive nature of CF@Alb-Dox NP. The obtained sustained release profile of CF@Alb-Dox NP (with ∼22% Dox release at pH 5 in 24 h) is better than that of reported similar nanocarriers (∼40–45% Dox release in 24 h) [31]. Both the observed feature (controlled sustain release and pH dependent release) are very promising and desirable for targeted drug delivery of anticancer drug. It is worthy to mention that pH responsive and magnetic nature of CF@Alb-Dox NP will enable delivery of Dox preferably at tumor site which has slightly acidic micro-environment and thus will reduce systemic toxicity. Sustained release of Dox from CF@Alb-Dox NP could be associated to its loading inside the albumen matrix. The increased release of Dox from CF@Alb-Dox NP at lower pH (pH 5) could be explained in term of increased solubility of Dox at lower pH as well as reduced electrostatic interaction between Dox and albumen matrix at lower pH. At pH 7.4 or above, the negatively charged carboxyl group of albumen could bind electro-statically with the positively charged amine group of Dox (pKa ∼8.3) [11]. The isoelectric point of albumen is ∼5 and thus remains negatively charged at pH 7.4 [32]. When the CF@Alb-Dox NP was subjected to the lower pH 5, possibly the protonations of albumen molecules could have resulted in the reduced electrostatic interaction between Dox and albumen resulting in increased Dox release at pH 5 than 7.4 [9]. In order to understand the involved release kinetics and mechanism for the release of Dox from CF@Alb-Dox NP at pH 5 and 7.4, the obtained release data were fitted with different mathematical models such as zero order model, first order model, Higuchi model, and KorsmeyerPeppas model [1]. Best correlation coefficient (R2) was used to identify the best suited model for the release data. Release profile data fitted with above mentioned models are shown in Fig. 10(a–d). Different kinetic parameters such as R2, K0 (zero order rate constant), K1 (first order rate constant), KH (Higuchi rate constant) and n (release exponent) were noted and shown in Table 1. It was observed that the release profile data at pH 5 best fitted with Higuchi model with highest correlation coefficient (R2 ∼0.98) whereas it does not fitted well with other studied model. Well fitting with Higuchi model of release profile data at pH 5 suggests that the release of Dox take place by diffusion controlled process according to Fick's law. The release data at pH 7.4 equally well fitted with zero and first model with R2 ∼0.96, whereas it does not well fitted with Higuchi (R2 ∼0.92) and K-P (R2 ∼0.91) model. It was also observed that release rate constant (K0, K1,and KH) at pH 5 were relatively higher (1.56–1.66 times) than those at pH 7.4 (Table 1). Increased release rate at pH 5 could be due to the reduced electrostatic interaction between Dox and albumen. Reduced electrostatic interaction between Dox and albumen at pH 5 could also be the reason for the diffusion controlled Dox release [9,11]. Further study of Dox release behaviors from CF@Alb-Dox NP into release medium having composition and concentration similar to human body plasma such as simulated body fluid (SBF) will provide a better and deep insight about the applicability of CF@Alb-Dox NP for drug delivery applications [33].

After successful loading of Dox in CF@Alb NP, it's in vitro release behavior were studied at two different pH of 5 (pH inside cancer cells) 24978

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Fig. 9. UV–Vis absorption spectra of Dox release media at pH 5 (a) and pH 7.4 (b) with respect to time. Time-dependent Dox release profiles of CF@Alb-Dox NP at pH 5 and 7.4.

3.10. In vitro cyto-toxicity study by MTT assay

Table 1 Release kinetic parameters of Dox released from CF@Alb-Dox at pH 5 and 7.4.

Biocompatibility of CF@Alb NP has been studied by in vitro MTT assay against the THP-1 cells. Fig. 11 shows cell viabilities of THP1 cells treated with different concentrations of CF@Alb NPs after 24 h of incubation. Very less cell inhibition (∼4.5%) was observed up to dose of 50 μg/ml and thereafter a concentration dependent cell inhibition was observed. In other words, very good cell viability was found (95.5%) up to dose of 50 μg/ml. Thus, MTT assay result indicated good biocompatible nature of CF@Alb NPs. High cells viability (∼77%) was observed even at high dose concentration (800 μg/ml) of CF@Alb NPs which also suggested biocompatible and non-toxic nature of CF@Alb NPs at high concentration. The excellent biocompatibility of CF@Alb NPs can be attributed to the albumen coating [9]. Biocompatible nature

Dox release at

pH 5 pH 7.4

Zero-order

First-order

Higuchi

Korsemeyer- Peppas

K0

R2

K1

R2

KH

R2

n

R2

0.444 0.284

0.882 0.962

0.0054 0.0032

0.912 0.962

4.129 2.482

0.984 0.927

0.711 0.414

0.930 0.917

of CF NP and CF NP based systems has also been reported earlier [34,35]. Thus considering the above obtained suitable physico-chemical characteristic of CF@Alb NP, it is anticipated that albumen and CF NP based magnetic nanocarriers could be a potential agent for biomedical

Fig. 10. Dox release data (at pH 5 and 7.4) fitted with zero order (a), first order (b), Higuchi model (c), and Korsmeyer-Peppas model (d). 24979

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Fig. 11. Cell viabilities of THP-1 cell line treated with different concentrations of CF@Alb NPs after 24 h of incubation.

application specially targeted drug delivery (due its biocompatibility, drug loading/releasing ability and magnetic nature) and hyperthermia based cancer therapy (due to its water dispersibility, suitable size, coercivity and high saturation magnetization value) [20]. Seongtae Bae et al. have also established that CF NPs have high biocompatibility and appropriate magnetic and structural properties for a hyperthermia agent application [35]. 4. Conclusions In conclusion, two different sizes of well crystalline and monodispersed CF NPs were prepared by changing the reaction parameter and characterized. Then, novel, hydrophilic, biocompatible, and stable CF@Alb NP was successfully developed by a simple green route using cheap egg albumen. The CF@Alb NP was extensively characterized for its different physicochemical properties. The average particles size of CF@Alb NP was found to be ∼106 nm with spherical to irregular morphologies. CF@Alb NP has shown a very good Dox loading ability with loading efficiency and loading capacity of ∼93.3% and 4.45%. A promising and desirable pH dependent in vitro Dox release behavior was also observed. CF@Alb NP was found to be biocompatible against the THP-1 cells. In light of our observation, it is anticipated that this kind of inexpensive, biocompatible, hydrophilic CF and egg albumen based system could be a very useful candidate for biomedical applications in the coming years. Acknowledgments Mohd Qasim acknowledges the MANF fellowship (2012–13/MANF2012-13-MUS-UTT-15733) received from UGC, Govt. of India. The infrastructural support received from DST PURSE & FIST grants toward consumables and contingencies are gratefully acknowledged Instrumental support such as UV–Vis spectrophotometer and TEM, VSM from CIL and CFN, University of Hyderabad is truly recognized. References [1] K. Asghar, M. Qasim, G. Dharmapuri, D. Das, Investigation on a smart nanocarrier with a mesoporous magnetic core and thermo-responsive shell for co-delivery of doxorubicin and curcumin: a new approach towards combination therapy of cancer, RSC Adv. 7 (46) (2017) 28802–28818. [2] L.H. Reddy, J.L. Arias, J. Nicolas, P. Couvreur, Magnetic nanoparticles: design and characterization, toxicity and biocompatibility, pharmaceutical and biomedical applications, Chem. Rev. 112 (11) (2012) 5818–5878.

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