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Effect of sugar alcohol on colloidal stabilization of magnetic nanoparticles for hyperthermia and drug delivery applications
Accepted Manuscript Effect of sugar alcohol on colloidal stabilization of magnetic nanoparticles for hyperthermia and drug delivery applications Santosh L. Gawali, B.K. Barick, K.C. Barick, P.A. Hassan PII:
S0925-8388(17)32583-5
DOI:
10.1016/j.jallcom.2017.07.206
Reference:
JALCOM 42622
To appear in:
Journal of Alloys and Compounds
Received Date: 16 March 2017 Revised Date:
5 July 2017
Accepted Date: 20 July 2017
Please cite this article as: S.L. Gawali, B.K. Barick, K.C. Barick, P.A. Hassan, Effect of sugar alcohol on colloidal stabilization of magnetic nanoparticles for hyperthermia and drug delivery applications, Journal of Alloys and Compounds (2017), doi: 10.1016/j.jallcom.2017.07.206. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of sugar alcohol on colloidal stabilization of magnetic nanoparticles for hyperthermia and drug delivery applications Santosh L. Gawalia, b, B. K. Barickc, K. C. Baricka,b,*, P. A. Hassana,b,* a
Chemistry Division, Bhabha Atomic Research Centre, Mumbai – 400085, India b
Homi Bhabha National Institute, Anushaktinagar, Mumbai – 400094, India
c
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Dept. of Physics, Indian Institute of Technology Bombay, Powai, Mumbai – 400076, India *
Corresponding authors:- Tel: + 91 22 2559 0284, Fax: + 91 22 2550 5151;
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Emails: [email protected], (K. C. Barick), [email protected] (P. A. Hassan)
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Graphical Abstract
ACCEPTED MANUSCRIPT Effect of sugar alcohol on colloidal stabilization of magnetic nanoparticles for hyperthermia and drug delivery applications Santosh L. Gawali1, 2, B. K. Barick3, K. C. Barick1, 2* and P. A. Hassan1, 2,* 1
Chemistry Division, Bhabha Atomic Research Centre, Mumbai – 400085, India 2
Homi Bhabha National Institute, Anushaktinagar, Mumbai – 400094, India
Dept. of Physics, Indian Institute of Technology Bombay, Powai, Mumbai – 400076, India *
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3
Corresponding authors:- Tel: + 91 22 2559 0284, Fax: + 91 22 2550 5151;
Emails: [email protected], (K. C. Barick), [email protected] (P. A. Hassan)
Abstract
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We have successfully introduced sugar alcohol (mannitol) onto the surface of iron oxide magnetic nanoparticles and investigated its role on their colloidal stabilization. The mannitol
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functionalized magnetic nanoparticles (MMNPs) were prepared through co-precipitation of Fe+2 and Fe3+ ions in basic medium under N2 atmosphere followed by in-situ coating of Dmannitol. The formation of iron oxide nanoparticles is evident from XRD and TEM analysis. The coating of nanoparticles with mannitol is analyzed by FTIR, TGA, DLS and zeta-potential
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measurements. It has been observed that the presence of mannitol on the surface nanoparticles strongly affect their surface potential and colloidal stability. They show room temperature superparamagnetism with optimal magnetization of 60.5 emu/g at 20 kOe and protein resistant
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behaviour in physiological medium. These MMNPs were employed as drug delivery carrier
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using anticancer drug, doxorubicin hydrochloride (DOX). The drug molecules were loaded onto the surface of nanoparticles through electrostatic interactions between positively charged DOX and negatively charged MMNPs. A loading efficiency of 60 % has been observed at DOX to MMNPs ratio (w/w) of 1:10 and the loaded drug showed pH dependent sustained release characteristics. Further, MMNPs exhibited good self-heating ability under applied AC magnetic field, thus they can be used as efficient heating source for hyperthermia therapy.
Keywords: Magnetic nanoparticles, iron oxide, drug delivery, hyperthermia, sugar alcohol, surface functionalization. 1
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1. Introduction
Research on magnetic nanoparticles (MNPs) has increased exponentially over the past decade due to its widespread applications in various fields. Among the others, iron oxide nanoparticles have received great deal of attention in biomedical applications such as targeted
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drug delivery, hyperthermia treatment of cancer and magnetic resonance imaging contrast enhancement due to their unique physicochemical properties and low toxicity [1-7]. However, there are significant challenges involved in site-selective targeting and avoiding of their
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undesired uptake by the reticulo-endothelial system (RES). Bare particles alone are suitable for a broad spectrum of applications, but the low stability and heterogeneous size distribution in
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aqueous medium represent major setbacks. These setbacks can however be reduced or minimized through the functionalization of MNPs with various multifunctional organic and inorganic entities such as proteins, peptides, antibodies, polymers, dendrimers, silica and gold etc [8-10]. Therefore, providing proper coating and developing some effective protection
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strategies is very important as the key to the successful application of iron oxide nanoparticles lies in their surface functionalization.
Polysaccharides such as dextran, starch and carrageenan etc. [11-14] are known to
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passivate the surface of iron oxide nanoparticles and render them colloidally stable. The similarity of chemical structure of sugar alcohols with polysaccharides, it is expected that these
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additives could act as potential stabilizing and protecting agent. Among the others, mannitol (D-mannitol) is a six-carbon resistant sugar alcohol used extensively in sweets and low-calorie foods as well as a stabilizer during the freeze drying of biological macromolecules [15]. It has significant biological (biodegradable, biocompatible, bioactive) and chemical properties (due to presence of reactive groups such as -OH). It is widely used in the pharmaceutical industry as a kind of water-soluble solid dispersions of carrier materials, and can improve solubility of the drug. It has six hydroxyl groups that can easily form hydrogen bond with water as well as drug containing lone pair electrons in the atom. Mannitol is used clinically in osmotherapy and 2
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oliguric renal failure. Further, mannitol I.V. opens the blood brain barrier to hydrophilics by stretching the tight junctions between the endothelial cells [16]. However, the literature on use of this bioactive sugar alcohol on nanoparticle surfaces is not much reported. Herein, we reported the development of sugar alcohol functionalized MNPs having
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good colloidal stability and magnetic responsivity for drug delivery and hyperthermia applications. The surface modification of MNPs allows us to produce functionalised exteriors with high densities of negatively charged hydroxyl moieties for electrostatic binding of
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positively charged anticancer drug, DOX. It has been observed that the drug release increased significantly from 15 % of the adsorbed drug to about 70 % upon changing the pH of release
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medium from 7.4 to 5. Specifically, the high loading affinity for these MNPs for DOX with their sustained release profile, self-heating capacity and protein resistance characteristic makes them suitable for drug delivery as well as hyperthermia treatment of cancer. 2. Experimental Section
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All chemicals are of analytical grade and used as received. Ferrous chloride tetrahydrate (FeCl2.4H2O, ≥99%), ferric chloride hexahydrate (FeCl3.6H2O, 97%), doxorubicin hydrochloride (DOX, 98%) and bovine serum albumin (BSA) were procured from Sigma
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Aldrich, USA. D-mannitol and NH3 solution (25%, AR grade) were purchased from S. D.
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Fine-chem Ltd., India and Thomas Baker Chemical Pvt. Ltd., India, respectively. Ferrous ammonium sulphate (AR grade) and 1, 10-phenanthroline monohydrate (Extra pure AR) were obtained from SRL Pvt. Ltd. India. Dialysis membrane-60 was procured from Himedia laboratories Pvt. Ltd., India. The acetate buffer (AB-pH-5) and phosphate buffered saline (PBS-pH-7.4) were prepared using standard protocols. MMNPs were prepared by co-precipitation of Fe+2 and Fe+3 salts at the molar ratio of 1:2 in basic medium followed by in-situ coating of mannitol [17]. In a typical synthesis of MMNPs, 0.994 g of FeCl2.4H2O and 2.703 g of FeCl3.6H2O were dissolved in 40 ml of Milli Q water in a round bottom flask. The temperature of flask was slowly raised to 70 oC under N2 3
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atmosphere with constant stirring and maintained at 70 oC for 30 min. 25% NH3 solution (15 ml) was added instantaneously to the above reaction mixture, and kept for another 30 min at that temperature. Then, 0.91 g of D-mannitol was dissolved in 3 ml Milli Q water and it was added to the round bottom flask. The temperature was slowly increased up to 90 oC and reacted
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for 60 min with continuous stirring for functionalization of MNPs with D-mannitol. The resulting black coloured nanoparticles were thoroughly washed with Milli Q water several times with the help of a permanent magnet (field strength ~2.5 kOe) and dried at 60-65°C in an
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oven. However, these dried magnetic nanoparticles were easily redispersible in water medium. The phase analysis was performed by a Phillips PW1729 diffractrometer with Cu Kα
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radiation (λ= 1.5405 nm). The crystallite size is estimated from the X-ray line broadening using Scherrer formula. The transmission electron micrograph was taken by Philips CM 200, TEM for particle size determination. The infrared spectra were recorded on a Fouriertransform infrared spectrometer (FTIR, Bomen Hartmann and Braun, MB series). The
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magnetic measurement of MMNPs (powder sample) was carried out using vibrating sample magnetometer (VSM, LakeShore, Model-7410). Thermogravimetric analysis (TGA) of samples was obtained in the range of 40 to 500 oC at scan rate of 10 oC/ min under N2
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atmosphere. Dynamic light scattering (DLS) measurements were performed using a Malvern 4800 Autosizer employing a 7132 digital correlator for the determination of hydrodynamic
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diameter. The light source of DLS instrument was He-Ne laser operated at 632.8 nm with a maximum power output of 15 mW. The zeta-potential measurements were determined by Zetasizer nanoseries, Malvern Instruments. The colloidal stability assay was investigated by measuring the absorbance of MMNPs suspensions (0.1 mg/ml) in different medium at a wavelength of 350 nm using JASCO V-650, UV-visible spectrophotometer. The concentration of Fe in MMNPs suspension was obtained by phenanthroline spectrophotometric method [18]. The heating ability of MMNPs suspensions was obtained from the calorimetric measurements using a magnetic induction heating unit. To minimize the heat loss MMNPs of 4
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different concentrations were taken in an eppendorf with suitable arrangements. The AC magnetic field of 507 Oe and frequency of 300 kHz were used to evaluate the specific absorption rate (SAR, measured in W/g) [4]. The SAR was calculated using the following equation: ΔT 1 Δt mMMNPs
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SAR = C
where, C is the specific heat of solvent (C = Cwater = 4.18 J/g oC), ∆T/∆t is the initial slope of the temperature vs. time curve and mMMNPs is mass fraction of MMNPs in solvent. However,
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the SAR value is dependent on the applied frequency and field strength. Thus, the systemindependent intrinsic loss power (ILP, measured in nHm2kg−1) was obtained using the
ILP =
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following equation [19]: SAR H2 f
where, H is the field strength and f is the frequency.
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The anticancer agent, doxorubicin hydrochloride (DOX) was used as a model drug to evaluate the loading and drug release behaviour of the MMNPs. The drug loading was carried out by incubating 0.5 ml of aqueous solution of DOX (1 mg/ml) with 2.5 ml of the aqueous
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suspension of MMNPs (5 mg) for 1h in dark (weight ratio of DOX to particle = 1:10). Then, the DOX loaded particles (DOX-MMNPs) were separated by using a permanent magnet and
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washed twice with Milli Q water to remove physically adsorbed DOX. The absorbance of supernatant at 490 nm against that of pure DOX (aqueous solution) was used to determine the loading efficiency. The loading efficiency (w/w %) was calculated using the following relation:
Loading efficiency (%) =
(A DOX − A S − A W ) A DOX
×100
5
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where, ADOX is the absorbance of pure DOX, AS the absorbance of supernatant obtained after magnetic separation of DOX-MMNPs and AW the absorbance of washed DOX (physically adsorbed DOX) For release study, DOX-MMNPs were immersed into 5 ml of release medium acetate
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buffer (AB pH-5 or of PBS pH-7.4) and then put into a dialysis bag. The dialyses were performed against 200 ml of PBS (pH-7.4) under continuous stirring at 37 oC (reservoir-sink condition). 1 ml of the external medium was withdrawn at fixed interval of time and replaced
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with fresh PBS (pH-7.4) to maintain the sink conditions. The amount of DOX released was obtained by measuring the fluorescence intensity at 585 nm (λex = 490 nm) using a micro plate
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reader (Synergy/H1 micro plate reader; BioTeK, Germany) against the standard plot prepared under similar condition. Each experiment was performed in triplicates and standard deviation was given in the plots. 3. Results and discussion
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Fig. 1 shows (a) XRD pattern and (b) TEM micrograph of MMNPs. XRD pattern displayed the six diffraction peaks corresponding to (220), (311), (400), (422), (511) and (440) lattice planes of iron oxide spinel structure. The spinel-structured magnetic iron oxide
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nanoparticles exist in two different phases such as magnetite (Fe3O4) and maghemite (γ-Fe2O3).
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Since the XRD patterns of magnetite and maghemite are very similar, it is difficult to distinguish these two phases simply from XRD patterns. Our earlier studies such as X-ray photoelectron spectroscopy and temperature dependent magnetization measurement performed on MNPs obtained by similar synthesis method primarily suggested the presence of magnetite phase [2,20,21]. However, the occurrence of magnetite partially oxidized at their surface cannot rule out as magnetite is not stable at ambient condition [22, 23]. Here, our studies mainly focused on the effect of sugar alcohol on colloidal stabilization of MNPs for hyperthermia and drug delivery applications. From X-ray line broadening, the average crystallite size was found to be around 10 nm, which is well supported by particle size 6
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observed in TEM analysis. TEM micrograph shows that MMNPs are roughly spherical in shape with an average size of 10 nm (the particle size distribution is shown in Fig. S1a). The selected area electron diffraction pattern of MMNPs (Fig. S1b) exhibits bright and distinguishable diffraction rings corresponding to the reflections of (220), (311), (400), (422),
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(511) and (440) planes of spinel structure, which is consistent with the XRD result. Further, crystal lattice fringes (inset of Fig. 1b) with the spacing of 0.29 nm correspond to the (220)
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spinel plane [24].
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(a)
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(b)
Fig. 1. (a) XRD pattern and (b) TEM micrograph of MMNPs (inset shows HRTEM micrograph revealing lattice spacing of 0.29 nm corresponding to the (220) spinel plane).
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The functionalization of mannitol with MNPs was investigated by FTIR and TGA measurements. The FTIR spectra of D-mannitol and MMNPs along with their characteristic peak assignments in the range of 4000-400 cm-1 are shown in Fig 2a. The pure mannitol shows well resolved vibration modes whereas those of MMNPs are broad and few. The FTIR
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spectrum of pure mannitol shows strong stretching vibrations peaks of –OH and C–O, C–H [25]. The –OH in plane bending and C–H bending vibrations of pure mannitol are also appeared at 1422 and 1280 cm-1. Most of these characteristic bands appeared in FTIR spectrum of MMNPs suggesting the presence of mannitol on the surface of MNPs. The highly intense
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band appeared at around 588 cm−1 in FTIR spectra of MMNPs can be ascribed the Fe-O bond
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vibration [24]. The organic modification of nanoparticles was further evident from TGA (Fig. 2b). MMNPs shows two steps thermal decomposition processes with a higher percentage of weight loss (9.4 %). The initial 3.9 % weight loss seen for MMNPs upto 180 oC can be attributed to the thermal desorption of water molecules and organic moieties physically
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adsorbed on the surface of nanoparticles. The weight loss beyond this can be associated with removal of chemisorbed organic molecules. The thermal decomposition temperature of pure D-mannitol was reported as 300 oC [26]. However, the complete removal of mannitol is not
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observed at 300 oC upon conjugated with nanoparticles. This shifting may be ascribed to the chemisorption of mannitol onto the surface of nanoparticles. Further, no more weight loss was
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observed at temperature beyond 400 oC due to the complete removal of conjugated sugar molecule. The weight loss observed by TGA was further supported by iron estimation (through phenanthroline spectrophotometric method), which showed about 10 % organic moieties are present on the surface of MMNPs.
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Fe-O vib.
(a)
MMNPs Mannitol
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-CH stretch -CH2 bend -OH plane bend C-O strech
500 (b)
-OH strech
1000 1500 3000 3500 -1 Wavenumber (cm )
0
140
35
0 0. 5 1 6 12 18 24 36 48
0
-6
Time (h)
-8
-10
100
200 300 400 o Temperature ( C)
500
20 10 0
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Zeta-potential (mV)
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0 (c)
70
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Weight loss (%)
-4
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Dh (nm)
105
-2
4000
-10
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-20 2
4
6
8
10
12
pH
Fig. 2. (a) FTIR spectra of pure mannitol and MMNPs along with their characteristic peak assignments in the range of 4000-400 cm-1, (b) TGA plot of MMNPs (inset shows the variation of Dh as a function of time) and (c) variation of zeta-potential as a function of pH of the MMNPs suspension (inset shows a proposed schematic representation showing conjugation of mannitol with MNPs through some of its hydroxyl groups). 9
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Light scattering measurements were performed on the aqueous dispersion of MMNPs to explore the hydrodynamic diameter and surface charge of particles as well as their colloidal stability. The dynamic information of the particles is derived from an autocorrelation of the intensity trace recorded during DLS measurements. It has been observed that the
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autocorrelation function decays rapidly with time for MMNPs and they form aqueous colloidal suspension with intensity weighted average hydrodynamic (Dh) diameter of about 125 nm (Fig. S2) with polydispersity index of 0.18. The number-average size distributions of MMNPs were
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also obtained from the intensity-weighted hydrodynamic by using the light scattering software and found to be 37 nm. The observed higher number-average diameter by DLS than TEM is
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possibly due to the presence of hydrated adsorbed organic layers on the surface of MMNPs [27]. Both the shape irregularity and polydispersity of MMNPs could also contribute to the differences in particle size [28]. The aqueous colloidal stability of MMNPs was also assessed from the changes Dh of MMNPs (inset of Fig. 2b) as well as their normalized absorbance (Fig.
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S3) as a function of time. The insignificant change in Dh and absorbance of particle suspension (0.1 mg/ml) in water with time suggests their good colloidal stability. In present case, we believe that some of the hydroxyl groups of sugar alcohol strongly attached to iron ions on the
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nanoparticle surface to form a robust coating similar to the binding of catechol group of dopamine by forming a catecholate-iron complex [29,30]. While the uncoordinated hydroxyl
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groups extended into water medium, conferring them aqueous colloidal stability through hydrogen bonding by overcoming the attractive magnetic and van der Waals forces in water. The zeta-potential measurements (Fig. 2c) suggest that the iso-electric point (IOP) of MMNPs is around 5.3. It has been observed that the zeta-potentials of MMNPs are pH dependent and were found to flip from positive to negative while varying the pH from 2 to 12. A negative potential of about −15 mV at pH 7 implies that these MMNPs posses negative surface charge under neutral condition due to presence of surface hydroxyl group. Stiufiuc et al. [31] observed negative surface charge (-16.2 mV) on surface of PEG (MW=1500) coated gold nanoparticles. 10
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Similar results were also observed by Marchetti et al. in hydroxyl functionalized core-shell poly(styrene-co-butadiene) nanoparticles [32]. Ravikumar et al. [33] reported negligible zetapotential values for dextran coated Fe3O4 nanoparticles in the pH range of 3-11 and suggested the absence of electrostatic interaction among these particles. They have demonstrated that the
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colloidal stabilization these nanoparticles are only due to the steric repulsion raised by the nonionic dextran molecules. However, Nigam et al. [2] demonstrated that the electrostatic repulsive forces originating among the negatively charged citrate Fe3O4 coated nanoparticles
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play important role in their water stabilization. Thus, the electrostatic repulsion instigated among our mannitol coated nanoparticles also contributes to their colloidal stabilization.
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Further, the higher negative surface charge of MMNPs in 0.01 M PBS (-22.7 mV) indicate their good colloidal stability in PBS. These results demonstrated that our nanoparticles are successfully functionalized with mannitol during their synthesis process (a proposed schematic representation showing conjugation of mannitol with nanoparticles through some of its
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hydroxyl groups is shown in the inset of Fig. 2c) and provide free hydroxyl groups on surfaces
40
2 0
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Magnetization (emu/g)
60
Magnetization (emu/g)
which are ready for further conjugation and functionalization.
20
-2
-0.05
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0
0.00 0.05 Field (kOe)
(a)
(b)
-20 -40 -60
-20
-10
0 Field (kOe)
10
20
Fig. 3. Field dependent magnetization plot of MMNPs at 300 K (top inset shows expanded field dependent magnetization plot at the low-field region and bottom inset shows photographs of revealing colloidal dispersion of (a) 0.5 and (b) 2 mg/ml of MMNPs in water medium). 11
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Fig. 3 shows the field dependent magnetization plot of MMNPs at 300 K. From magnetic measurement, it has been observed that at low magnetic fields, the magnetization response is steep and approximately linear as the particles begin to align with the applied field.
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However, at higher fields, the particles are almost completely aligned with the field, and the magnetization approaches saturation. The expanded field dependent magnetization plot at the low-field region revealing negligible coercivity (2.75 Oe) and remanence (0.15 emu/g) is
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shown in top inset of Fig. 3. These results suggest that MMNPs possess superparamagnetic behaviour at room temperature. The maximum magnetization of MMNPs was found to be 60.5
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emu/g at 20 kOe, which is comparable to that reported for carboxyl decorated Fe3O4 nanoparticles (58 emu/g) by Barick et al [34]. The maximum magnetization of bare Fe3O4 nanoparticles prepared by similar method was found to be 67.6 emu/g at 20 kOe [17]. Chandra et al. prepared bare Fe3O4 MNPs by co-precipitation method and also observed similar
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magnetization value [35]. As compared to bare MNPs, about 10.5 % decreases in magnetization value are observed for MMNPs. The observed low value of magnetization of MMNPs can be attributed to the presence of non-magnetic organic coating on the surface of
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particles (as indeed suggested from TGA and Fe estimation) [36]. However, the superparamagnetic nature and good colloidal dispersion of these nanoparticles (photographs
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revealing colloidal dispersion of 0.5 and 2 mg/ml of MMNPs in water medium are shown in inset of Fig. 3) makes them suitable for various biomedical applications.
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Fig. 4. (a) temperature vs. time plots, (b) SAR values (W/g) and (c) ILP values (nHm2kg−1) of aqueous suspension of MMNPs at different concentrations of particles. Fig. 4 shows (a) temperature vs. time plots, (b) SA R values (W/g) and (c) ILP values
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(nHm2kg−1) of aqueous suspension of MMNPs at different concentrations of particles. A time dependent gradual increase in temperature of MMNPs suspension under AC magnetic field is observed from calorimetric measurements. The SAR values of MMNPs were found to be
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127.7, 103.2 and 71.3 W/g at concentration of 0.75, 1.5 and 3.0 mg/ml of particles,
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respectively. The corresponding system-independent ILP values were 0.261, 0.211 and 0.145 nHm2kg−1, respectively. Muller et al [37] developed iron oxide ferrofluids having SAR value of 87 W/g at H=11 kA/m and f=410 kHz (particle size: 13 nm, ILP: 1.75 nHm2/kg). Fortin et al. [38] reported SAR parameter of 1650 W/g at H=24.8 kA/m and f=700 kHz for iron oxide nanoparticles (particle size: 16.5 nm, ILP: 3.8 nHm2/kg). The ILP values obtained in the present study are less than these synthetic iron oxide nanoparticles. However, they in the range of those reported for commercially available ferrofluids such as Nanomag-D-spio, BNF-01808 and BNF-01708 (Manufacturer: Micromod) [39]. Further, it has been observed that SAR value decreases with increase in the concentration of particles in suspension. This could be attributed 13
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to the increase in nanoparticles aggregation and dipolar interactions with increase in concentration of particles [40-43]. Piñeiro-Redondo et al. [40] demonstrated that the heat production efficiency decreases with increasing the concentration of polyacrylic acid coated Fe3O4 nanoparticles (average particle size of 10 nm) due to the higher inter-particle dipolar
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interaction. A similar SAR dependence of the particle concentration was also observed by Linh et al. [41] and Rana et al. [42] for starch coated Fe3O4 nanoparticles (particle size: 15-17 nm) and polyaniline coated Fe3O4 nanoparticles (particle size: 10 nm), respectively. In addition to
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this, heating efficacy is also dependent on the physical properties of magnetic nanoparticles such as magnetization, particles size distribution and composition [44-46]. In general, our
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magnetic induction heating studies demonstrated that these MMNPs suspension can be used as effective heating source for hyperthermia therapy.
Pure DOX Supernatant solution
0.6
0.4 0.3 0.2
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Absorbance (a.u.)
0.5
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0.1 0.0
AC C
300
350
400
450 500 550 600 Wavenumber (nm)
650
700
Fig. 5. UV-visible absorbance plots of pure DOX in water and supernatant solution obtained after magnetic separation of DOX-MMNPs (for absorbance study of pure DOX, the concentration of DOX was kept equivalent to the amount of DOX used in drug loading experiments, i.e., 0.5 mg DOX in 3 ml water).
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We have also investigated the drug loading and drug release behaviour of these MMNPs using anticancer drug, doxorubicin hydrochloride (DOX) as a model drug. The interaction of DOX with MMNPs was observed from zeta-potential and UV-visible measurements. From zeta-potential measurement, an increase in surface charge from -15 to -
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1.0 mV is observed upon reacting 10 µl DOX (1 mg/ml) with 1 ml MMNPs suspension (0.1 mg/ml). This increase in zeta-potential suggested the strong interaction between drug molecules and nanoparticles. The loading efficiency was determined from UV-visible
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spectroscopic studies and found to be 60% at DOX to particle ratio (w/w) of 1:10. In the present study, the observed drug loading can be attributed to the electrostatic interactions
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between positively charged DOX (protonated primary amine of DOX induces a positive charge) and negatively charged MMNPs. However, the loading efficiency of DOX onto the surface of MMNPs is much lower than that reported for citrate (90 %) and phosphate (82 %) coated Fe3O4 nanoparticles [2,47]. This may be due to the lower surface charge of MMNPs as
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compared to citrate and phosphate coated Fe3O4 nanoparticles.
Pure DOX at reservoir pH 5 DOX-MMNPs at reservoir pH 5 DOX-MMNPs at reservoir pH 7.4
80
AC C
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Drug release (%)
100
60 40 20
0 0
10
20 30 Time (h)
40
50
Fig. 6. Release profile of DOX from DOX-MMNPs at 37 °C under different reservoir-sink conditions.
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The release of DOX from DOX-MMNPs at reservoir pH 5 follows time dependent release behaviour. It has been observed that DOX molecules release from DOX-MMNPs over a period of 48 h. The initial stage is characterized by a rapid release of drug, followed by a slow, steady and controlled release of drug. About 70 % of loaded DOX molecules were
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released from the DOX-MMNPs system at reservoir pH 5. The pure DOX shows rapid release behaviour of DOX with t1/2 (the time need for 50 % release of the drug) about 45 min at same pH. While DOX-MMNPs show sustained release profile with t1/2 about 5 h.
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The complete release of DOX is not observed from DOX-MMNPs over the experimental period of time. However, the amount of electrostatically bound drug released from DOX-
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MMNPs at reservoir pH 5 is higher than that reported for DOX loaded citrate-stabilized Fe3O4 nanoparticles (60 %) [2], and much less than the DOX loaded phosphate anchored Fe3O4 nanoparticles (90 %) and DOX loaded folate conjugated bifunctional Fe3O4 nanoparticles (83 %) at same pH [47,48]. The release of DOX could be attributed to the weakening of the
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electrostatic interactions between DOX and partially neutralized hydroxyl groups on the surface of MMNPs at pH 5. The sustained release of DOX from DOX-MMNPs was possible since the weakening of electrostatic interactions is a slower process. It is noteworthy to
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mention that only 15 % DOX release was observed from DOX-MMNPs at reservoir pH 7.4. This is desirable for cancer therapy as the relatively low pH in tumours will stimulate the DOX
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release at the target site. Recently, Venturelli et al. reported that sugar (glucose) shell of MNPs plays a major role in recognition of cancer cells through metabolic-based assays [49]. Furthermore, the interactions of these MMNPs with bovine serum albumin (BSA) protein in physiological medium (0.01 M PBS, pH 7.4) were investigated by zeta-potential measurements (Table S1) and UV-Visible spectroscopy (Fig. S4). The MMNPs do not show any significant change in zeta-potential and absorbance values even after incubation with BSA for 2 h, revealing their protein resistance characteristic at physiological medium. Specifically, our study demonstrated the effect of sugar alcohol, mannitol on colloidal stabilization of 16
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superparamagnetic nanoparticles, and investigated the potential applications of the developed new pH-responsive nanocarriers in drug delivery and magnetic hyperthermia. 4. Conclusions Mannitol functionalized iron oxide nanoparticles were prepared through co-
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precipitation of Fe+2 and Fe3+ ions in basic medium followed by in-situ coating of D-mannitol. XRD and TEM analysis confirmed the formation of crystalline spinel nanostructure with average particles size of 10 nm. The surface passivation of these nanoparticles with D-
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mannitol was evident from FTIR, TGA and zeta-potential measurements. Light scattering measurements indicate that these particles render aqueous colloidal suspension with mean
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hydrodynamic diameters of 125 nm, and they possess pH dependent charge conversal behaviour and protein resistant characteristics in physiological medium. MMNPs show good self-heating ability under AC magnetic field, thus they can be used as effective heating source for magnetic hyperthermia. Further, these sugar alcohol functionalised nanoparticles showed
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high loading affinity for DOX and their pH dependent sustained release, which makes them suitable for the drug delivery. In addition, the functionalized exterior of MMNPs having free hydroxyl groups can provide available site for conjugation of various bioactive molecules for a
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variety of biomedical applications.
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Acknowledgements
The authors thank Dr. K. I. Priyadarsini, Head, Chemistry Division, BARC for the
encouragement and support. Authors also acknowledge Prof. D. Bahadur, Indian Institute of Technology Bombay, India for his encouragement and facilitating the use of VSM.
17
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Figure Captions:
Fig. 1. (a) XRD pattern of MMNPs, and (b) TEM micrograph of MMNPs (inset shows HRTEM micrograph revealing lattice spacing of 0.29 nm corresponding to the (220) spinel plane).
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Fig. 2. (a) FTIR spectra of pure mannitol and MMNPs along with their characteristic peak assignments in the range of 4000-400 cm-1, (b) TGA plot of MMNPs (inset shows the variation of Dh as a function of time) and (c) variation of zeta-potential as a function of pH of the
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MMNPs suspension (inset shows a proposed schematic representation showing conjugation of mannitol with MNPs through some of its hydroxyl groups).
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Fig. 3. Field dependent magnetization plot of MMNPs at 300 K (top inset shows expanded field dependent magnetization plot at the low-field region and bottom inset shows photographs revealing colloidal dispersion of (a) 0.5 and (b) 2 mg/ml of MMNPs in water medium). Fig. 4. (a) temperature vs. time plots, (b) SAR values (W/g) and (c) ILP values (nHm2kg−1) of
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aqueous suspension of MMNPs at different concentrations of particles. Fig. 5. UV-visible absorbance plots of pure DOX in water and supernatant solution obtained after magnetic separation of DOX-MMNPs (for absorbance study of pure DOX, the
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concentration of DOX was kept equivalent to the amount of DOX used in drug loading experiments, i.e., 0.5 mg DOX in 3 ml water).
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Fig. 6. Release profile of DOX from DOX-MMNPs at 37 °C under different reservoir-sink conditions.
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Effect of sugar alcohol on colloidal stabilization of magnetic nanoparticles for hyperthermia and drug delivery applications Santosh L. Gawalia, b, B. K. Barickc, K. C. Baricka,b,*, P. A. Hassana,b,* a
Chemistry Division, Bhabha Atomic Research Centre, Mumbai – 400085, India b
Homi Bhabha National Institute, Anushaktinagar, Mumbai – 400094, India
c
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Dept. of Physics, Indian Institute of Technology Bombay, Powai, Mumbai – 400076, India *
Corresponding authors:- Tel: + 91 22 2559 0284, Fax: + 91 22 2550 5151;
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Emails: [email protected], (K. C. Barick), [email protected] (P. A. Hassan)
Highlights
Preparation of sugar alcohol functionalized water-dispersible MNPs
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Presence of mannitol on MNPs surface provides colloidal stability to particles Good heating ability under AC magnetic field shows its use in hyperthermia therapy High payload of positively charged anticancer drug and their pH triggered release
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Hydroxyl groups on surface of MNPs can be explored for conjugation of biomolecules
S0925-8388(17)32583-5
DOI:
10.1016/j.jallcom.2017.07.206
Reference:
JALCOM 42622
To appear in:
Journal of Alloys and Compounds
Received Date: 16 March 2017 Revised Date:
5 July 2017
Accepted Date: 20 July 2017
Please cite this article as: S.L. Gawali, B.K. Barick, K.C. Barick, P.A. Hassan, Effect of sugar alcohol on colloidal stabilization of magnetic nanoparticles for hyperthermia and drug delivery applications, Journal of Alloys and Compounds (2017), doi: 10.1016/j.jallcom.2017.07.206. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of sugar alcohol on colloidal stabilization of magnetic nanoparticles for hyperthermia and drug delivery applications Santosh L. Gawalia, b, B. K. Barickc, K. C. Baricka,b,*, P. A. Hassana,b,* a
Chemistry Division, Bhabha Atomic Research Centre, Mumbai – 400085, India b
Homi Bhabha National Institute, Anushaktinagar, Mumbai – 400094, India
c
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Dept. of Physics, Indian Institute of Technology Bombay, Powai, Mumbai – 400076, India *
Corresponding authors:- Tel: + 91 22 2559 0284, Fax: + 91 22 2550 5151;
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Emails: [email protected], (K. C. Barick), [email protected] (P. A. Hassan)
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Graphical Abstract
ACCEPTED MANUSCRIPT Effect of sugar alcohol on colloidal stabilization of magnetic nanoparticles for hyperthermia and drug delivery applications Santosh L. Gawali1, 2, B. K. Barick3, K. C. Barick1, 2* and P. A. Hassan1, 2,* 1
Chemistry Division, Bhabha Atomic Research Centre, Mumbai – 400085, India 2
Homi Bhabha National Institute, Anushaktinagar, Mumbai – 400094, India
Dept. of Physics, Indian Institute of Technology Bombay, Powai, Mumbai – 400076, India *
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3
Corresponding authors:- Tel: + 91 22 2559 0284, Fax: + 91 22 2550 5151;
Emails: [email protected], (K. C. Barick), [email protected] (P. A. Hassan)
Abstract
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We have successfully introduced sugar alcohol (mannitol) onto the surface of iron oxide magnetic nanoparticles and investigated its role on their colloidal stabilization. The mannitol
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functionalized magnetic nanoparticles (MMNPs) were prepared through co-precipitation of Fe+2 and Fe3+ ions in basic medium under N2 atmosphere followed by in-situ coating of Dmannitol. The formation of iron oxide nanoparticles is evident from XRD and TEM analysis. The coating of nanoparticles with mannitol is analyzed by FTIR, TGA, DLS and zeta-potential
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measurements. It has been observed that the presence of mannitol on the surface nanoparticles strongly affect their surface potential and colloidal stability. They show room temperature superparamagnetism with optimal magnetization of 60.5 emu/g at 20 kOe and protein resistant
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behaviour in physiological medium. These MMNPs were employed as drug delivery carrier
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using anticancer drug, doxorubicin hydrochloride (DOX). The drug molecules were loaded onto the surface of nanoparticles through electrostatic interactions between positively charged DOX and negatively charged MMNPs. A loading efficiency of 60 % has been observed at DOX to MMNPs ratio (w/w) of 1:10 and the loaded drug showed pH dependent sustained release characteristics. Further, MMNPs exhibited good self-heating ability under applied AC magnetic field, thus they can be used as efficient heating source for hyperthermia therapy.
Keywords: Magnetic nanoparticles, iron oxide, drug delivery, hyperthermia, sugar alcohol, surface functionalization. 1
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1. Introduction
Research on magnetic nanoparticles (MNPs) has increased exponentially over the past decade due to its widespread applications in various fields. Among the others, iron oxide nanoparticles have received great deal of attention in biomedical applications such as targeted
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drug delivery, hyperthermia treatment of cancer and magnetic resonance imaging contrast enhancement due to their unique physicochemical properties and low toxicity [1-7]. However, there are significant challenges involved in site-selective targeting and avoiding of their
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undesired uptake by the reticulo-endothelial system (RES). Bare particles alone are suitable for a broad spectrum of applications, but the low stability and heterogeneous size distribution in
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aqueous medium represent major setbacks. These setbacks can however be reduced or minimized through the functionalization of MNPs with various multifunctional organic and inorganic entities such as proteins, peptides, antibodies, polymers, dendrimers, silica and gold etc [8-10]. Therefore, providing proper coating and developing some effective protection
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strategies is very important as the key to the successful application of iron oxide nanoparticles lies in their surface functionalization.
Polysaccharides such as dextran, starch and carrageenan etc. [11-14] are known to
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passivate the surface of iron oxide nanoparticles and render them colloidally stable. The similarity of chemical structure of sugar alcohols with polysaccharides, it is expected that these
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additives could act as potential stabilizing and protecting agent. Among the others, mannitol (D-mannitol) is a six-carbon resistant sugar alcohol used extensively in sweets and low-calorie foods as well as a stabilizer during the freeze drying of biological macromolecules [15]. It has significant biological (biodegradable, biocompatible, bioactive) and chemical properties (due to presence of reactive groups such as -OH). It is widely used in the pharmaceutical industry as a kind of water-soluble solid dispersions of carrier materials, and can improve solubility of the drug. It has six hydroxyl groups that can easily form hydrogen bond with water as well as drug containing lone pair electrons in the atom. Mannitol is used clinically in osmotherapy and 2
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oliguric renal failure. Further, mannitol I.V. opens the blood brain barrier to hydrophilics by stretching the tight junctions between the endothelial cells [16]. However, the literature on use of this bioactive sugar alcohol on nanoparticle surfaces is not much reported. Herein, we reported the development of sugar alcohol functionalized MNPs having
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good colloidal stability and magnetic responsivity for drug delivery and hyperthermia applications. The surface modification of MNPs allows us to produce functionalised exteriors with high densities of negatively charged hydroxyl moieties for electrostatic binding of
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positively charged anticancer drug, DOX. It has been observed that the drug release increased significantly from 15 % of the adsorbed drug to about 70 % upon changing the pH of release
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medium from 7.4 to 5. Specifically, the high loading affinity for these MNPs for DOX with their sustained release profile, self-heating capacity and protein resistance characteristic makes them suitable for drug delivery as well as hyperthermia treatment of cancer. 2. Experimental Section
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All chemicals are of analytical grade and used as received. Ferrous chloride tetrahydrate (FeCl2.4H2O, ≥99%), ferric chloride hexahydrate (FeCl3.6H2O, 97%), doxorubicin hydrochloride (DOX, 98%) and bovine serum albumin (BSA) were procured from Sigma
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Aldrich, USA. D-mannitol and NH3 solution (25%, AR grade) were purchased from S. D.
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Fine-chem Ltd., India and Thomas Baker Chemical Pvt. Ltd., India, respectively. Ferrous ammonium sulphate (AR grade) and 1, 10-phenanthroline monohydrate (Extra pure AR) were obtained from SRL Pvt. Ltd. India. Dialysis membrane-60 was procured from Himedia laboratories Pvt. Ltd., India. The acetate buffer (AB-pH-5) and phosphate buffered saline (PBS-pH-7.4) were prepared using standard protocols. MMNPs were prepared by co-precipitation of Fe+2 and Fe+3 salts at the molar ratio of 1:2 in basic medium followed by in-situ coating of mannitol [17]. In a typical synthesis of MMNPs, 0.994 g of FeCl2.4H2O and 2.703 g of FeCl3.6H2O were dissolved in 40 ml of Milli Q water in a round bottom flask. The temperature of flask was slowly raised to 70 oC under N2 3
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atmosphere with constant stirring and maintained at 70 oC for 30 min. 25% NH3 solution (15 ml) was added instantaneously to the above reaction mixture, and kept for another 30 min at that temperature. Then, 0.91 g of D-mannitol was dissolved in 3 ml Milli Q water and it was added to the round bottom flask. The temperature was slowly increased up to 90 oC and reacted
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for 60 min with continuous stirring for functionalization of MNPs with D-mannitol. The resulting black coloured nanoparticles were thoroughly washed with Milli Q water several times with the help of a permanent magnet (field strength ~2.5 kOe) and dried at 60-65°C in an
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oven. However, these dried magnetic nanoparticles were easily redispersible in water medium. The phase analysis was performed by a Phillips PW1729 diffractrometer with Cu Kα
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radiation (λ= 1.5405 nm). The crystallite size is estimated from the X-ray line broadening using Scherrer formula. The transmission electron micrograph was taken by Philips CM 200, TEM for particle size determination. The infrared spectra were recorded on a Fouriertransform infrared spectrometer (FTIR, Bomen Hartmann and Braun, MB series). The
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magnetic measurement of MMNPs (powder sample) was carried out using vibrating sample magnetometer (VSM, LakeShore, Model-7410). Thermogravimetric analysis (TGA) of samples was obtained in the range of 40 to 500 oC at scan rate of 10 oC/ min under N2
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atmosphere. Dynamic light scattering (DLS) measurements were performed using a Malvern 4800 Autosizer employing a 7132 digital correlator for the determination of hydrodynamic
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diameter. The light source of DLS instrument was He-Ne laser operated at 632.8 nm with a maximum power output of 15 mW. The zeta-potential measurements were determined by Zetasizer nanoseries, Malvern Instruments. The colloidal stability assay was investigated by measuring the absorbance of MMNPs suspensions (0.1 mg/ml) in different medium at a wavelength of 350 nm using JASCO V-650, UV-visible spectrophotometer. The concentration of Fe in MMNPs suspension was obtained by phenanthroline spectrophotometric method [18]. The heating ability of MMNPs suspensions was obtained from the calorimetric measurements using a magnetic induction heating unit. To minimize the heat loss MMNPs of 4
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different concentrations were taken in an eppendorf with suitable arrangements. The AC magnetic field of 507 Oe and frequency of 300 kHz were used to evaluate the specific absorption rate (SAR, measured in W/g) [4]. The SAR was calculated using the following equation: ΔT 1 Δt mMMNPs
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SAR = C
where, C is the specific heat of solvent (C = Cwater = 4.18 J/g oC), ∆T/∆t is the initial slope of the temperature vs. time curve and mMMNPs is mass fraction of MMNPs in solvent. However,
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the SAR value is dependent on the applied frequency and field strength. Thus, the systemindependent intrinsic loss power (ILP, measured in nHm2kg−1) was obtained using the
ILP =
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following equation [19]: SAR H2 f
where, H is the field strength and f is the frequency.
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The anticancer agent, doxorubicin hydrochloride (DOX) was used as a model drug to evaluate the loading and drug release behaviour of the MMNPs. The drug loading was carried out by incubating 0.5 ml of aqueous solution of DOX (1 mg/ml) with 2.5 ml of the aqueous
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suspension of MMNPs (5 mg) for 1h in dark (weight ratio of DOX to particle = 1:10). Then, the DOX loaded particles (DOX-MMNPs) were separated by using a permanent magnet and
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washed twice with Milli Q water to remove physically adsorbed DOX. The absorbance of supernatant at 490 nm against that of pure DOX (aqueous solution) was used to determine the loading efficiency. The loading efficiency (w/w %) was calculated using the following relation:
Loading efficiency (%) =
(A DOX − A S − A W ) A DOX
×100
5
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where, ADOX is the absorbance of pure DOX, AS the absorbance of supernatant obtained after magnetic separation of DOX-MMNPs and AW the absorbance of washed DOX (physically adsorbed DOX) For release study, DOX-MMNPs were immersed into 5 ml of release medium acetate
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buffer (AB pH-5 or of PBS pH-7.4) and then put into a dialysis bag. The dialyses were performed against 200 ml of PBS (pH-7.4) under continuous stirring at 37 oC (reservoir-sink condition). 1 ml of the external medium was withdrawn at fixed interval of time and replaced
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with fresh PBS (pH-7.4) to maintain the sink conditions. The amount of DOX released was obtained by measuring the fluorescence intensity at 585 nm (λex = 490 nm) using a micro plate
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reader (Synergy/H1 micro plate reader; BioTeK, Germany) against the standard plot prepared under similar condition. Each experiment was performed in triplicates and standard deviation was given in the plots. 3. Results and discussion
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Fig. 1 shows (a) XRD pattern and (b) TEM micrograph of MMNPs. XRD pattern displayed the six diffraction peaks corresponding to (220), (311), (400), (422), (511) and (440) lattice planes of iron oxide spinel structure. The spinel-structured magnetic iron oxide
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nanoparticles exist in two different phases such as magnetite (Fe3O4) and maghemite (γ-Fe2O3).
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Since the XRD patterns of magnetite and maghemite are very similar, it is difficult to distinguish these two phases simply from XRD patterns. Our earlier studies such as X-ray photoelectron spectroscopy and temperature dependent magnetization measurement performed on MNPs obtained by similar synthesis method primarily suggested the presence of magnetite phase [2,20,21]. However, the occurrence of magnetite partially oxidized at their surface cannot rule out as magnetite is not stable at ambient condition [22, 23]. Here, our studies mainly focused on the effect of sugar alcohol on colloidal stabilization of MNPs for hyperthermia and drug delivery applications. From X-ray line broadening, the average crystallite size was found to be around 10 nm, which is well supported by particle size 6
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observed in TEM analysis. TEM micrograph shows that MMNPs are roughly spherical in shape with an average size of 10 nm (the particle size distribution is shown in Fig. S1a). The selected area electron diffraction pattern of MMNPs (Fig. S1b) exhibits bright and distinguishable diffraction rings corresponding to the reflections of (220), (311), (400), (422),
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(511) and (440) planes of spinel structure, which is consistent with the XRD result. Further, crystal lattice fringes (inset of Fig. 1b) with the spacing of 0.29 nm correspond to the (220)
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spinel plane [24].
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(a)
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(b)
Fig. 1. (a) XRD pattern and (b) TEM micrograph of MMNPs (inset shows HRTEM micrograph revealing lattice spacing of 0.29 nm corresponding to the (220) spinel plane).
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The functionalization of mannitol with MNPs was investigated by FTIR and TGA measurements. The FTIR spectra of D-mannitol and MMNPs along with their characteristic peak assignments in the range of 4000-400 cm-1 are shown in Fig 2a. The pure mannitol shows well resolved vibration modes whereas those of MMNPs are broad and few. The FTIR
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spectrum of pure mannitol shows strong stretching vibrations peaks of –OH and C–O, C–H [25]. The –OH in plane bending and C–H bending vibrations of pure mannitol are also appeared at 1422 and 1280 cm-1. Most of these characteristic bands appeared in FTIR spectrum of MMNPs suggesting the presence of mannitol on the surface of MNPs. The highly intense
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band appeared at around 588 cm−1 in FTIR spectra of MMNPs can be ascribed the Fe-O bond
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vibration [24]. The organic modification of nanoparticles was further evident from TGA (Fig. 2b). MMNPs shows two steps thermal decomposition processes with a higher percentage of weight loss (9.4 %). The initial 3.9 % weight loss seen for MMNPs upto 180 oC can be attributed to the thermal desorption of water molecules and organic moieties physically
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adsorbed on the surface of nanoparticles. The weight loss beyond this can be associated with removal of chemisorbed organic molecules. The thermal decomposition temperature of pure D-mannitol was reported as 300 oC [26]. However, the complete removal of mannitol is not
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observed at 300 oC upon conjugated with nanoparticles. This shifting may be ascribed to the chemisorption of mannitol onto the surface of nanoparticles. Further, no more weight loss was
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observed at temperature beyond 400 oC due to the complete removal of conjugated sugar molecule. The weight loss observed by TGA was further supported by iron estimation (through phenanthroline spectrophotometric method), which showed about 10 % organic moieties are present on the surface of MMNPs.
8
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Fe-O vib.
(a)
MMNPs Mannitol
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-CH stretch -CH2 bend -OH plane bend C-O strech
500 (b)
-OH strech
1000 1500 3000 3500 -1 Wavenumber (cm )
0
140
35
0 0. 5 1 6 12 18 24 36 48
0
-6
Time (h)
-8
-10
100
200 300 400 o Temperature ( C)
500
20 10 0
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Zeta-potential (mV)
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0 (c)
70
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Weight loss (%)
-4
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Dh (nm)
105
-2
4000
-10
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-20 2
4
6
8
10
12
pH
Fig. 2. (a) FTIR spectra of pure mannitol and MMNPs along with their characteristic peak assignments in the range of 4000-400 cm-1, (b) TGA plot of MMNPs (inset shows the variation of Dh as a function of time) and (c) variation of zeta-potential as a function of pH of the MMNPs suspension (inset shows a proposed schematic representation showing conjugation of mannitol with MNPs through some of its hydroxyl groups). 9
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Light scattering measurements were performed on the aqueous dispersion of MMNPs to explore the hydrodynamic diameter and surface charge of particles as well as their colloidal stability. The dynamic information of the particles is derived from an autocorrelation of the intensity trace recorded during DLS measurements. It has been observed that the
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autocorrelation function decays rapidly with time for MMNPs and they form aqueous colloidal suspension with intensity weighted average hydrodynamic (Dh) diameter of about 125 nm (Fig. S2) with polydispersity index of 0.18. The number-average size distributions of MMNPs were
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also obtained from the intensity-weighted hydrodynamic by using the light scattering software and found to be 37 nm. The observed higher number-average diameter by DLS than TEM is
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possibly due to the presence of hydrated adsorbed organic layers on the surface of MMNPs [27]. Both the shape irregularity and polydispersity of MMNPs could also contribute to the differences in particle size [28]. The aqueous colloidal stability of MMNPs was also assessed from the changes Dh of MMNPs (inset of Fig. 2b) as well as their normalized absorbance (Fig.
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S3) as a function of time. The insignificant change in Dh and absorbance of particle suspension (0.1 mg/ml) in water with time suggests their good colloidal stability. In present case, we believe that some of the hydroxyl groups of sugar alcohol strongly attached to iron ions on the
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nanoparticle surface to form a robust coating similar to the binding of catechol group of dopamine by forming a catecholate-iron complex [29,30]. While the uncoordinated hydroxyl
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groups extended into water medium, conferring them aqueous colloidal stability through hydrogen bonding by overcoming the attractive magnetic and van der Waals forces in water. The zeta-potential measurements (Fig. 2c) suggest that the iso-electric point (IOP) of MMNPs is around 5.3. It has been observed that the zeta-potentials of MMNPs are pH dependent and were found to flip from positive to negative while varying the pH from 2 to 12. A negative potential of about −15 mV at pH 7 implies that these MMNPs posses negative surface charge under neutral condition due to presence of surface hydroxyl group. Stiufiuc et al. [31] observed negative surface charge (-16.2 mV) on surface of PEG (MW=1500) coated gold nanoparticles. 10
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Similar results were also observed by Marchetti et al. in hydroxyl functionalized core-shell poly(styrene-co-butadiene) nanoparticles [32]. Ravikumar et al. [33] reported negligible zetapotential values for dextran coated Fe3O4 nanoparticles in the pH range of 3-11 and suggested the absence of electrostatic interaction among these particles. They have demonstrated that the
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colloidal stabilization these nanoparticles are only due to the steric repulsion raised by the nonionic dextran molecules. However, Nigam et al. [2] demonstrated that the electrostatic repulsive forces originating among the negatively charged citrate Fe3O4 coated nanoparticles
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play important role in their water stabilization. Thus, the electrostatic repulsion instigated among our mannitol coated nanoparticles also contributes to their colloidal stabilization.
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Further, the higher negative surface charge of MMNPs in 0.01 M PBS (-22.7 mV) indicate their good colloidal stability in PBS. These results demonstrated that our nanoparticles are successfully functionalized with mannitol during their synthesis process (a proposed schematic representation showing conjugation of mannitol with nanoparticles through some of its
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hydroxyl groups is shown in the inset of Fig. 2c) and provide free hydroxyl groups on surfaces
40
2 0
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Magnetization (emu/g)
60
Magnetization (emu/g)
which are ready for further conjugation and functionalization.
20
-2
-0.05
AC C
0
0.00 0.05 Field (kOe)
(a)
(b)
-20 -40 -60
-20
-10
0 Field (kOe)
10
20
Fig. 3. Field dependent magnetization plot of MMNPs at 300 K (top inset shows expanded field dependent magnetization plot at the low-field region and bottom inset shows photographs of revealing colloidal dispersion of (a) 0.5 and (b) 2 mg/ml of MMNPs in water medium). 11
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Fig. 3 shows the field dependent magnetization plot of MMNPs at 300 K. From magnetic measurement, it has been observed that at low magnetic fields, the magnetization response is steep and approximately linear as the particles begin to align with the applied field.
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However, at higher fields, the particles are almost completely aligned with the field, and the magnetization approaches saturation. The expanded field dependent magnetization plot at the low-field region revealing negligible coercivity (2.75 Oe) and remanence (0.15 emu/g) is
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shown in top inset of Fig. 3. These results suggest that MMNPs possess superparamagnetic behaviour at room temperature. The maximum magnetization of MMNPs was found to be 60.5
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emu/g at 20 kOe, which is comparable to that reported for carboxyl decorated Fe3O4 nanoparticles (58 emu/g) by Barick et al [34]. The maximum magnetization of bare Fe3O4 nanoparticles prepared by similar method was found to be 67.6 emu/g at 20 kOe [17]. Chandra et al. prepared bare Fe3O4 MNPs by co-precipitation method and also observed similar
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magnetization value [35]. As compared to bare MNPs, about 10.5 % decreases in magnetization value are observed for MMNPs. The observed low value of magnetization of MMNPs can be attributed to the presence of non-magnetic organic coating on the surface of
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particles (as indeed suggested from TGA and Fe estimation) [36]. However, the superparamagnetic nature and good colloidal dispersion of these nanoparticles (photographs
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revealing colloidal dispersion of 0.5 and 2 mg/ml of MMNPs in water medium are shown in inset of Fig. 3) makes them suitable for various biomedical applications.
12
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Fig. 4. (a) temperature vs. time plots, (b) SAR values (W/g) and (c) ILP values (nHm2kg−1) of aqueous suspension of MMNPs at different concentrations of particles. Fig. 4 shows (a) temperature vs. time plots, (b) SA R values (W/g) and (c) ILP values
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(nHm2kg−1) of aqueous suspension of MMNPs at different concentrations of particles. A time dependent gradual increase in temperature of MMNPs suspension under AC magnetic field is observed from calorimetric measurements. The SAR values of MMNPs were found to be
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127.7, 103.2 and 71.3 W/g at concentration of 0.75, 1.5 and 3.0 mg/ml of particles,
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respectively. The corresponding system-independent ILP values were 0.261, 0.211 and 0.145 nHm2kg−1, respectively. Muller et al [37] developed iron oxide ferrofluids having SAR value of 87 W/g at H=11 kA/m and f=410 kHz (particle size: 13 nm, ILP: 1.75 nHm2/kg). Fortin et al. [38] reported SAR parameter of 1650 W/g at H=24.8 kA/m and f=700 kHz for iron oxide nanoparticles (particle size: 16.5 nm, ILP: 3.8 nHm2/kg). The ILP values obtained in the present study are less than these synthetic iron oxide nanoparticles. However, they in the range of those reported for commercially available ferrofluids such as Nanomag-D-spio, BNF-01808 and BNF-01708 (Manufacturer: Micromod) [39]. Further, it has been observed that SAR value decreases with increase in the concentration of particles in suspension. This could be attributed 13
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to the increase in nanoparticles aggregation and dipolar interactions with increase in concentration of particles [40-43]. Piñeiro-Redondo et al. [40] demonstrated that the heat production efficiency decreases with increasing the concentration of polyacrylic acid coated Fe3O4 nanoparticles (average particle size of 10 nm) due to the higher inter-particle dipolar
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interaction. A similar SAR dependence of the particle concentration was also observed by Linh et al. [41] and Rana et al. [42] for starch coated Fe3O4 nanoparticles (particle size: 15-17 nm) and polyaniline coated Fe3O4 nanoparticles (particle size: 10 nm), respectively. In addition to
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this, heating efficacy is also dependent on the physical properties of magnetic nanoparticles such as magnetization, particles size distribution and composition [44-46]. In general, our
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magnetic induction heating studies demonstrated that these MMNPs suspension can be used as effective heating source for hyperthermia therapy.
Pure DOX Supernatant solution
0.6
0.4 0.3 0.2
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Absorbance (a.u.)
0.5
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0.1 0.0
AC C
300
350
400
450 500 550 600 Wavenumber (nm)
650
700
Fig. 5. UV-visible absorbance plots of pure DOX in water and supernatant solution obtained after magnetic separation of DOX-MMNPs (for absorbance study of pure DOX, the concentration of DOX was kept equivalent to the amount of DOX used in drug loading experiments, i.e., 0.5 mg DOX in 3 ml water).
14
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We have also investigated the drug loading and drug release behaviour of these MMNPs using anticancer drug, doxorubicin hydrochloride (DOX) as a model drug. The interaction of DOX with MMNPs was observed from zeta-potential and UV-visible measurements. From zeta-potential measurement, an increase in surface charge from -15 to -
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1.0 mV is observed upon reacting 10 µl DOX (1 mg/ml) with 1 ml MMNPs suspension (0.1 mg/ml). This increase in zeta-potential suggested the strong interaction between drug molecules and nanoparticles. The loading efficiency was determined from UV-visible
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spectroscopic studies and found to be 60% at DOX to particle ratio (w/w) of 1:10. In the present study, the observed drug loading can be attributed to the electrostatic interactions
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between positively charged DOX (protonated primary amine of DOX induces a positive charge) and negatively charged MMNPs. However, the loading efficiency of DOX onto the surface of MMNPs is much lower than that reported for citrate (90 %) and phosphate (82 %) coated Fe3O4 nanoparticles [2,47]. This may be due to the lower surface charge of MMNPs as
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compared to citrate and phosphate coated Fe3O4 nanoparticles.
Pure DOX at reservoir pH 5 DOX-MMNPs at reservoir pH 5 DOX-MMNPs at reservoir pH 7.4
80
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Drug release (%)
100
60 40 20
0 0
10
20 30 Time (h)
40
50
Fig. 6. Release profile of DOX from DOX-MMNPs at 37 °C under different reservoir-sink conditions.
15
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The release of DOX from DOX-MMNPs at reservoir pH 5 follows time dependent release behaviour. It has been observed that DOX molecules release from DOX-MMNPs over a period of 48 h. The initial stage is characterized by a rapid release of drug, followed by a slow, steady and controlled release of drug. About 70 % of loaded DOX molecules were
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released from the DOX-MMNPs system at reservoir pH 5. The pure DOX shows rapid release behaviour of DOX with t1/2 (the time need for 50 % release of the drug) about 45 min at same pH. While DOX-MMNPs show sustained release profile with t1/2 about 5 h.
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The complete release of DOX is not observed from DOX-MMNPs over the experimental period of time. However, the amount of electrostatically bound drug released from DOX-
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MMNPs at reservoir pH 5 is higher than that reported for DOX loaded citrate-stabilized Fe3O4 nanoparticles (60 %) [2], and much less than the DOX loaded phosphate anchored Fe3O4 nanoparticles (90 %) and DOX loaded folate conjugated bifunctional Fe3O4 nanoparticles (83 %) at same pH [47,48]. The release of DOX could be attributed to the weakening of the
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electrostatic interactions between DOX and partially neutralized hydroxyl groups on the surface of MMNPs at pH 5. The sustained release of DOX from DOX-MMNPs was possible since the weakening of electrostatic interactions is a slower process. It is noteworthy to
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mention that only 15 % DOX release was observed from DOX-MMNPs at reservoir pH 7.4. This is desirable for cancer therapy as the relatively low pH in tumours will stimulate the DOX
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release at the target site. Recently, Venturelli et al. reported that sugar (glucose) shell of MNPs plays a major role in recognition of cancer cells through metabolic-based assays [49]. Furthermore, the interactions of these MMNPs with bovine serum albumin (BSA) protein in physiological medium (0.01 M PBS, pH 7.4) were investigated by zeta-potential measurements (Table S1) and UV-Visible spectroscopy (Fig. S4). The MMNPs do not show any significant change in zeta-potential and absorbance values even after incubation with BSA for 2 h, revealing their protein resistance characteristic at physiological medium. Specifically, our study demonstrated the effect of sugar alcohol, mannitol on colloidal stabilization of 16
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superparamagnetic nanoparticles, and investigated the potential applications of the developed new pH-responsive nanocarriers in drug delivery and magnetic hyperthermia. 4. Conclusions Mannitol functionalized iron oxide nanoparticles were prepared through co-
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precipitation of Fe+2 and Fe3+ ions in basic medium followed by in-situ coating of D-mannitol. XRD and TEM analysis confirmed the formation of crystalline spinel nanostructure with average particles size of 10 nm. The surface passivation of these nanoparticles with D-
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mannitol was evident from FTIR, TGA and zeta-potential measurements. Light scattering measurements indicate that these particles render aqueous colloidal suspension with mean
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hydrodynamic diameters of 125 nm, and they possess pH dependent charge conversal behaviour and protein resistant characteristics in physiological medium. MMNPs show good self-heating ability under AC magnetic field, thus they can be used as effective heating source for magnetic hyperthermia. Further, these sugar alcohol functionalised nanoparticles showed
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high loading affinity for DOX and their pH dependent sustained release, which makes them suitable for the drug delivery. In addition, the functionalized exterior of MMNPs having free hydroxyl groups can provide available site for conjugation of various bioactive molecules for a
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variety of biomedical applications.
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Acknowledgements
The authors thank Dr. K. I. Priyadarsini, Head, Chemistry Division, BARC for the
encouragement and support. Authors also acknowledge Prof. D. Bahadur, Indian Institute of Technology Bombay, India for his encouragement and facilitating the use of VSM.
17
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Figure Captions:
Fig. 1. (a) XRD pattern of MMNPs, and (b) TEM micrograph of MMNPs (inset shows HRTEM micrograph revealing lattice spacing of 0.29 nm corresponding to the (220) spinel plane).
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Fig. 2. (a) FTIR spectra of pure mannitol and MMNPs along with their characteristic peak assignments in the range of 4000-400 cm-1, (b) TGA plot of MMNPs (inset shows the variation of Dh as a function of time) and (c) variation of zeta-potential as a function of pH of the
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MMNPs suspension (inset shows a proposed schematic representation showing conjugation of mannitol with MNPs through some of its hydroxyl groups).
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Fig. 3. Field dependent magnetization plot of MMNPs at 300 K (top inset shows expanded field dependent magnetization plot at the low-field region and bottom inset shows photographs revealing colloidal dispersion of (a) 0.5 and (b) 2 mg/ml of MMNPs in water medium). Fig. 4. (a) temperature vs. time plots, (b) SAR values (W/g) and (c) ILP values (nHm2kg−1) of
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aqueous suspension of MMNPs at different concentrations of particles. Fig. 5. UV-visible absorbance plots of pure DOX in water and supernatant solution obtained after magnetic separation of DOX-MMNPs (for absorbance study of pure DOX, the
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concentration of DOX was kept equivalent to the amount of DOX used in drug loading experiments, i.e., 0.5 mg DOX in 3 ml water).
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Fig. 6. Release profile of DOX from DOX-MMNPs at 37 °C under different reservoir-sink conditions.
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Effect of sugar alcohol on colloidal stabilization of magnetic nanoparticles for hyperthermia and drug delivery applications Santosh L. Gawalia, b, B. K. Barickc, K. C. Baricka,b,*, P. A. Hassana,b,* a
Chemistry Division, Bhabha Atomic Research Centre, Mumbai – 400085, India b
Homi Bhabha National Institute, Anushaktinagar, Mumbai – 400094, India
c
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Dept. of Physics, Indian Institute of Technology Bombay, Powai, Mumbai – 400076, India *
Corresponding authors:- Tel: + 91 22 2559 0284, Fax: + 91 22 2550 5151;
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Emails: [email protected], (K. C. Barick), [email protected] (P. A. Hassan)
Highlights
Preparation of sugar alcohol functionalized water-dispersible MNPs
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Presence of mannitol on MNPs surface provides colloidal stability to particles Good heating ability under AC magnetic field shows its use in hyperthermia therapy High payload of positively charged anticancer drug and their pH triggered release
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Hydroxyl groups on surface of MNPs can be explored for conjugation of biomolecules