Pluronic stabilized Fe₃O₄ magnetic nanoparticles for intracellular delivery of curcumin

Abstract

Pluronic stabilized Fe₃O₄ magnetic nanoparticles (PSMNPs) were developed by introducing amphiphilic tri-block co-polymer, Pluronic P123 onto the surface of hydrophobic magnetic nanoparticles (HMNPs) and investigated their efficacy for delivery of hydrophobic anti-tumor agent, curcumin (CUR). XRD and TEM analysis revealed the formation of highly crystalline Fe₃O₄ nanoparticles of size ∼7 nm. The functionalization of nanoparticles with P123 was evident from FTIR, TGA, DLS, UV-visible and zeta-potential measurements. The addition of Pluronic layer not only provides aqueous colloidal stability and biocompatibility to the particles but also promotes the encapsulation of curcumin into the interface of hydrophobic layers between Fe₃O₄ nanoparticles and Pluronic coating. The drug loading efficiency of about 98% was observed at drug to particles ratio of 1 : 2 and curcumin loaded PSMNPS (CUR–PSMNPs) showed pH dependent release behaviour. The CUR and CUR–PSMNPs showed significant reduction in proliferation of MCF-7 cells with half maximal inhibitory concentration (IC₅₀) values of be 25.1 and 18.4 μM, respectively. The higher toxicity of CUR–PSMNPs was further confirmed by cellular uptake and cellular imaging studies. These results suggested that CUR–PSMNPs formulation is superior than pure curcumin in causing tumor cytotoxicity, which is possibly due to the increase in the bioavailability of drug to the targeted site.

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Introduction

Curcumin, a natural polyphenol component isolated from the rhizomes of the herb Curcuma longa (turmeric), has gained considerable attention due to its potential pharmacological activities, including anti-inflammatory, antioxidant, antiviral and anti-microbial activities.^1,2 Curcumin has also been considered as an anticancer agent.³ However, like many other compelling therapeutic agents, its clinical application in tumor therapy is greatly hindered by its poor water solubility, instability and thus low bioavailability.^4–6 The main reasons for the low plasma and tissue levels of curcumin appear to be due to poor absorption, high rate of metabolism, and rapid systemic elimination.⁷ In order to improve its bioavailability and water solubility, researchers across the globe are actively involved in development of various nanoparticle based curcumin formulations.^8–12 For instance, Yin et al. reported the higher antitumor activity of curcumin loaded nanoparticles prepared with methoxy poly(ethylene glycol)–polycaprolactone (mPEG–PCL) block co-polymers over pure curcumin due to their enhanced in bioavailability.⁸ Yallapu et al. demonstrated that curcumin loaded cellulose nanoparticles formulation and β-cyclodextrin (CD)–curcumin nano-assembly enhanced curcumin delivery in prostate cancer cells compared to free curcumin.^9,10 Gangwar et al. prepared curcumin conjugated silica coated nanoparticles, and observed improved bioavailability and anticancer properties.¹¹ Manju et al. have reported the synthesis of water-soluble gold nanoparticles attached to curcumin–polymer conjugates and explored their targeted delivery onto cancer cells.¹² Even though many investigations have focused on improving the water solubility and bioavailability of curcumin, still challenges lie in achieving its high therapeutic efficacy at the tumor site.^5–7,11,13

Among the others, magnetic nanoparticles (MNPs) offer exciting opportunities towards the development of novel drug carrier.^14–16 The drug targeting to tumor sites is essential as most of the anticancer agents show non-specific toxicities, which affects their therapeutic gain. With MNPs as carrier, it is possible to localize therapeutic agent to the site of interest by an external magnetic field.^17,18 However, magnetic nanocarrier must address the issues related to their aqueous colloidal stability, particle size distribution, high payload, sustained release profile, retention of optimal magnetic properties and biocompatibility.^19,20 Thus, the surface modification of MNPs with suitable organic or polymer moieties is extremely important to their design and subsequent application in drug delivery.^21,22 The coatings must improve biocompatibility, and afford colloidal and chemical stability in aqueous and physiological mediums while retaining other characteristics. One of the most promising coating materials, polyethylene glycol (PEG) a water soluble, biodegradable polymer has widely been used for PEGylation of MNPs.^23,24 Owens and Peppas demonstrated that grafting of PEG and PEG-containing co-polymers to the surface of nanoparticles create a hydrophilic protective layer around the nanoparticles evading the opsonization and reticuloendothelial system (RES).²⁵ The PEG based Pluronic triblock co-polymers are amphiphilic in nature and composed of a hydrophobic central segment of poly(propylene oxide) (PPO) flanked by two hydrophilic segments of poly(ethylene oxide) (PEO). These polymers are widely used as a coating/stabilizing agent for the surface passivation of MNPs for various biomedical applications.^26,27 A few MNPs based formulations were also prepared using β-CD and polymer coating for delivery of hydrophobic drug.^28,29 For instance, Salem et al. developed a multifunctional β-CD and amine-terminated poly(propylene glycol) coated magnetite system capable of targeted curcumin delivery.²⁸ Yallapu et al. also reported the use of curcumin-loaded MNPs formulation composed of Fe₃O₄ nanoparticle coated with β-CD and Pluronic F68 polymer for breast cancer therapeutics and imaging applications.²⁹ In these systems, the hydrophobic cavity of β-CD mainly controls the loading affinity of drug molecules. Further, MNPs used in these formulations were prepared in aqueous medium at low temperature which triggers the aggregation of nanoparticles during their synthesis.

Herein, novel Pluronic stabilized Fe₃O₄ MNPs (PSMNPs) were developed by introducing amphiphilic Pluronic P123 molecules onto surface of monodispersed lauric acid coated hydrophobic MNPs (HMNPs) and investigated their efficacy for delivery of hydrophobic anticancer drug, curcumin. The addition of Pluronic layer not only promotes encapsulation of higher amount of hydrophobic drug, curcumin (encapsulation efficiency: 98%, drug loading content: 49%) into the interface of hydrophobic layer between HMNPs and Pluronic coating, but also provides aqueous colloidal stability and biocompatibility to the formulation. It has been observed that our curcumin–nanoparticle formulation is superior than pure curcumin in inhibiting the proliferation of tumor cells, which is possibly due to the increase in bioavailability of drug to the targeted site. Specifically, the present approach formed an aqueous stable drug–nanoparticle formulation having excellent payload of hydrophobic anticancer agent with higher therapeutic efficacy.

Experimental

Materials

Iron(III) acetylacetonate (Fe(acac)₃, >99%), diphenyl ether, Pluronic P123, 1,2-dodecanediol (>90%) and bovine serum albumin (BSA) were purchased from Sigma Aldrich, USA. Lauric acid (>99%) and laurylamine (>99%) were obtained from Otto Kemi, India. Curcumin (>99%) was received as a gift from Win Herbal Care, India. Dimethyl sulfoxide (DMSO, >99.9%) and Tween-80 were obtained from Alfa Aesar, Canada and Merck, India, respectively. Dulbecco's Modified Eagle Medium (DMEM), antibiotic antimycotic solution (100× liquid), sodium dodecyl sulphate (SDS, 98%), fetal bovine serum (FBS), MTT reagent (thiazolyl blue tetrazolium bromide) and dialysis membrane-60 were procured from Himedia Laboratories Pvt. Ltd., India. 0.5% trypsin–EDTA (10×) and 1,10-phenanthroline monohydrate were obtained from Gibco, Life Technologies, USA and Sisco Research Laboratories Pvt. Ltd., India, respectively. MCF-7 cells were purchased from National Centre for Cell Science (NCCS), Pune, India. All the chemicals were of analytical grade and used as such without further treatment. All the aqueous solutions were prepared using nanopure water from a Millipore-MilliQ system (resistivity ∼ 18 MΩ cm). The acetate buffer (AB) pH 5 and phosphate buffered saline (PBS) pH 7.4 were prepared using standard protocols.

Synthesis of hydrophobic Fe₃O₄ MNPs

Hydrophobic Fe₃O₄ MNPs (HMNPs) were prepared by a modified high temperature thermal decomposition approach.²⁷ In a typical reaction, thermal decomposition of iron(III) acetylacetonate (2 mmol) was carried out in presence of lauric acid (6 mmol), laurylamine (6 mmol), and 1,2-dodecanediol (10 mmol) surfactants in diphenyl ether (20 ml) medium. The reaction mixture was heated to 230 °C for 2 h under the flow of nitrogen gas to prevent oxidation and then raised to 260 °C for 1 h. The magnetic nanoparticles were separated out from the solvent by using a permanent magnet (field strength ∼ 0.25 kOe) and thoroughly rinsed with water to remove solvent and surfactants.

Synthesis of Pluronic stabilized Fe₃O₄ MNPs

Pluronic stabilized Fe₃O₄ MNPs (PSMNPs) were prepared by introducing tri block co-polymer, Pluronic P123 onto the surface of nanoparticles through self-assembly of amphiphilic molecules. Typically 3 ml aqueous solution of Pluronic P123 (20 wt% of particles) was added into the hexane dispersion of HMNPs (28 mg particles dissolved in 3 ml hexane). The resulting solution was stirred under shaking for 24 h, and then particles were separated through magnetic separation and washed thoroughly with water.

Characterizations

X-ray diffraction (XRD) study was done on a Philips PW1729 diffractometer with Cu Kα radiation (λ = 1.5405 nm). The crystallite size of PSMNPs is estimated from the X-ray line broadening using Scherrer formula:
where, λ is the X-ray wavelength used, β is the angular line width at half maximum intensity and θ be the Bragg's angle. The transmission electron micrographs (TEM) were taken by FEG TEM (JEOL JEM-2100F) for particle size determination. The infrared spectra were recorded in the range 4000–400 cm⁻¹ on a Fourier-transform infrared spectrometer (FTIR, Bomen Hartmann and Braun, MB series). Thermogravimetric analysis (TGA) of samples was carried out in the range of 40 to 500 °C at scan rate 10 °C min⁻¹ under N₂ atmosphere. Dynamic light scattering (DLS) measurement was performed using a Malvern 4800 Autosizer employing a 7132 digital correlator for the determination of hydrodynamic diameter. The zeta-potential measurements were determined by Zetasizer nanoseries, Malvern Instruments. The colloidal stability assay was investigated by measuring the absorbance of PSMNPs suspensions (0.1 mg ml⁻¹) in water medium at a wavelength of 350 nm using JASCO V-650, UV-visible spectrophotometer. The magnetic measurement was carried out using vibrating sample magnetometer (VSM, LakeShore, Model-7410).

Measurements of curcumin loading

The anticancer agent, curcumin (CUR) was used as a model drug to estimate the drug loading and release behaviour of the PSMNPs. For encapsulation of curcumin into PSMNPs, 1 ml methanolic solution of curcumin (5 mg ml⁻¹) was added to the 2 ml aqueous solution of PSMNPs (5 mg ml⁻¹) and solution was kept for stirring on vortex for 16 h. After 16 h, curcumin loaded PSMNPs (CUR–PSMNPs) were separated from solution by permanent magnet. The absorbance intensity of supernatant at 427 nm (washed drug molecules were also taken into consideration for calculations) against that of pure CUR prepared in methanol–water (1 : 2) was used to determine the encapsulation efficiency. The encapsulation efficiency (w/w%) was calculated using the following relation:
where, A_CUR is the absorbance of pure CUR solution, A_S the absorbance of supernatant and A_W the absorbance of washed CUR (surface adsorbed drug). The drug loading content was determined as follows:

Curcumin release studies

For release study, CUR–PSMNPs were immersed into 5 ml of release medium (AB pH 5/PBS pH 7.4) and then put into a dialysis bag. The dialysis was performed against 200 ml of buffer having same pH of release medium under continuous stirring at 37 °C (reservoir-sink condition). 0.1% Tween-80 was added into the buffer solutions to improve the dissolution of curcumin due to its hydrophobic nature. 1 ml of the external medium was withdrawn at fixed interval of time and replaced with fresh PBS and Tween-80 mixture to maintain the sink conditions. The amount of drug released was determined by measuring the fluorescence intensity at 510 nm (λ_ex = 420 nm) using a plate reader (SYNERGY/H1 microplate reader, BioTeK, Germany) against the standard plot prepared under similar condition (prepared in PBS 7.4–0.1% Tween-80). Each experiment was performed in triplicates and standard deviation was given in the plots.

In vitro toxicity studies

Cytotoxicity study for pure curcumin, PSMNPs and CUR–PSMNPs were carried out by MTT assay using MCF-7 cell line (NCCS, Pune, India). Cells (5 × 10³) were seeded overnight in 96 well plate containing DMEM supplemented with 10% FBS and antibiotic antimycotic solution in a humidified atmosphere of 5% CO₂ at 37 °C. 13.3 mM stock solutions of CUR and CUR–PSMNPs (having equivalent amount of drug) were prepared in DMSO and diluted with the culture medium to get the desired concentration. Then different concentrations of CUR and CUR–PSMNPs were added to the cells (concentration of DMSO was <1%, which is within the permissible limits of toxicity) and these were incubated for another 48 h in culture conditions. Following this, cells were incubated with MTT reagent (5 mg ml⁻¹ in PBS) for 4 h at 37 °C. The formazan metabolites formed from the reduction of MTT by the living cells were extracted using solubilisation solution (10% SDS in 0.01 M HCl) and optical density at 570 nm was recorded using microplate reader. The percentage (%) cytotoxicity was calculated from the decrease in absorbance of treated samples as compared to that of control cells. The cellular uptake of CUR and CUR–PSMNPs were estimated by spectrophotometric method.³⁰ The cells (1 × 10⁶) were incubated with CUR and CUR–PSMNPs (at a drug concentration of 25 μM) for 4 and 7 h in culture conditions followed by washing with PBS and harvesting the cells by trypsinization. Then, pallet down at 1000 rpm in centrifuge for 5 min and washed thrice with PBS. The pellet was dried and suspended in to 1 ml of methanol and sonicated till curcumin is completely extracted into the methanol fraction. The lysate was centrifuged at 10 000 rpm for 5 min, absorption spectra of supernatant containing methanolic curcumin were recorded at 420 nm using a plate reader. The cellular uptake was determined against the standard plot prepared under similar condition. The cellular uptake of curcumin is expressed in nmol per million cells.

Results and discussion

Fig. 1 shows the (a) XRD pattern, (b) TEM micrograph, (c) HRTEM micrograph and (d) field dependent magnetization plot of HMNPs. The XRD pattern reveals the formation of single-phase inverse spinel magnetite, Fe₃O₄ with lattice constant, a = ∼8.378 Å, which is very close to the reported value (JCPDS card no. 88-0315, a = 8.375 Å). From X-ray line broadening, the crystallite size was found to be around 7 nm. TEM micrograph clearly shows the formation of highly monodisperse spherical nanoparticles of average size 7 nm (particle size distribution shown in inset of Fig. 1b). The average interfringe distance of HMNPs was measured to be ∼0.30 nm, which corresponds to (220) plane of Fe₃O₄.²⁷ Furthermore, the selected area electron diffraction pattern (inset of Fig. 1c) can be indexed to the highly crystalline reflections of Fe₃O₄, which is consistent with XRD result.

Fig. 1 (a) XRD pattern, (b) TEM micrograph (inset: particle size distribution), (c) HRTEM micrograph (inset: selected area electron diffraction pattern) and (d) field dependent magnetization plot of HMNPs.

The field-dependent magnetization (M vs. H) plot exhibits superparamagnetic behavior of HMNPs with maximum magnetic moment of 58 emu g⁻¹ at a field of 20 kOe. The low value of magnetic moment of Fe₃O₄ MNPs as compared to 92 emu g⁻¹ of bulk Fe₃O₄ can be ascribed to the combined effect of nano-sized particles and non-magnetic coating of organic molecules on their surface.³¹ However, HMNPs prepared in presence of hydrophobic surfactants (coated with lauric acid) are hydrophobic in nature.^27,32 In order to make them hydrophilic and biocompatible, surface passivating agent, P123 coating was introduced onto the surface of HMNPs. The schematic representation of P123 coating on the surface of HMNPs and photographs revealing their phase change from hydrophobic to hydrophilic is shown in Fig. 2. It is noteworthy to mention that the Pluronic modification retain the nanocrystalline nature and magnetic response of Fe₃O₄ particles. The XRD pattern, TEM/HRTEM micrographs and magnetic field responsivity of Pluronic stabilized Fe₃O₄ magnetic nanoparticles (PSMNPs) are shown in Fig. S1 (ESI†).

Fig. 2 Schematic representation showing Pluronic P123 coating on surface of HMNPs along with photographs revealing the change of HMNPs from hydrophobic to hydrophilic.

In order to investigate the Pluronic coating onto the surface of HMNPs, we have performed FTIR, TGA, DLS, UV-visible and zeta-potential measurements. Fig. 3 shows (a) FTIR spectra and (b) TGA plots of HMNPs and PSMNPs. FTIR spectra (Fig. 3a) of HMNPs and PSMNPs retained a strong absorption band at 588 cm⁻¹ attributed to the Fe–O stretching vibration.¹⁶ The bands appeared at ∼2920, 2850 and 1380 cm⁻¹ can be ascribed to asymmetric stretching, symmetric stretching and bending vibrations of C–H, respectively.^10,33 The bands appeared in the range of 1150–1025 cm⁻¹ corresponds to C–O bond vibrations. Compare to the FTIR spectrum of HMNPs, PSMNPs showed an intense band at around 1108 cm⁻¹ owing to the typical C–O–C vibration of Pluronic.³⁴ The appearance of intense C–O–C vibrational mode as well as the increase in intensity of C–H stretching and bending vibrations in FTIR spectrum of PSMNPs suggests the successful introduction of P123 onto the surface of HMNPs. This was further confirmed from the TGA (Fig. 3b). Both HMNPs and PSMNPs showed three steps thermal decomposition with a total weight loss of about 9.15 and 15.5%, respectively. The first step loss demonstrated the removal of physically adsorbed water and organic molecules, whereas the second and third step losses were related to the removal of chemically adsorbed organic molecules like lauric acid and P123.^35,36 The higher weight loss in case of PSMNPs indicated the coating of P123 on surface of HMNPs. The higher weight loss observed in PSMNPs was further supported by iron estimation through phenanthroline spectrophotometric method which showed that about 17% organic moieties are present on the surface of particle.³⁷

Fig. 3 (a) FTIR spectra and (b) TGA plots of HMNPs and PSMNPs.

The colloidal stability of nanoparticles is important for biological applications. Fig. 4 shows (a) size distribution of PSMNPs in aqueous medium as obtained from DLS analysis and (b) pH dependent zeta-potential plot of aqueous suspension of PSMNPs. The DLS measurements (Fig. 4a) indicate that PSMNPs sample renders aqueous colloidal suspension with z-average intensity-weighted diameter of about 175 nm. However, the z-average size of HMNPs (in hexane medium) was found to be around 150 nm (Fig. S2, ESI†). This is consistent with the formation of single layer of Pluronic shell around each Fe₃O₄ nanoparticle, as the expected diameter of hydrophobic particles covered with block co-polymer shell of thickness 10 nm is 170 nm. This negates any possibility of encapsulating multiple particles in the block co-polymer assembly. The colloidal stability of the PSMNPs was also assessed from the changes in absorbance of their aqueous suspensions using UV-visible spectrophotometer (Fig. S3, ESI†). The insignificant change in absorbance of PSMNPs suspension (0.1 mg ml⁻¹) in water with time (even after 24 h) indicates their good colloidal stability. We believed that the hydrophobic part of Pluronic (PPO) forms robust coating around HMNPs though hydrophobic–hydrophobic interaction, while the hydrophilic part of Pluronic (PEO) extends into water medium conferring a high degree of aqueous stability to MNPs. Further from the zeta-potential studies (Fig. 4b), it was observed that PSMNPs possess a high negative surface charge at pH 6 and above. The Pluronic P123 is non-ionic molecule; however it possessed a higher hydrophilic–lipophilic balances (HLB) value (ethylene oxide, EO = 39 units) and hence has a higher affinity for water. The increased association of water (negatively charged hydroxyl groups) results the observed increase in zeta-potential value (negative) at higher pH.³⁸ Thus, the electrostatic repulsive force originating on particles surface also provides stability to them in water medium. It is worth mentioning that the negative surface charge of PSMNPs at physiological medium could decrease the possibility of its interaction with haemoglobin, which would play a vital role in improving the stability and blood compatibility. We have also addressed the interaction of PSMNPs with BSA protein. The insignificant change in the zeta-potential (Table S1†) of PSMNPs even after incubation with BSA for 2 h, revealed their protein resistance characteristics in physiological medium.

Fig. 4 (a) Size distribution of PSMNPs in aqueous medium as obtained from DLS analysis and (b) pH dependent zeta-potential plot of aqueous suspension of PSMNPs.

In order to explore the use of PSMNPs as efficient carriers for delivery of hydrophobic drug, we have investigated drug loading and release behaviour using CUR as a model drug. The loading of curcumin into PSMNPs was apparent from the decrease in absorbance intensity of the supernatant liquid after removal of CUR–PSMNPs through magnetic separation. The encapsulation efficiency of CUR into PSMNPs was found to be around 98% with drug to particle ratio of 1 : 2 from standard plots of curcumin (Fig. S4, ESI†). The drug loading content was determined to be 49%. The high drug payload on a delivery system is necessary to achieve a good therapeutic dose at the site of interest. The loading of CUR into PSMNPs was also investigated by FTIR spectroscopic analysis (Fig. 5). The FTIR spectrum of CUR shows one sharp peak at 3510 cm⁻¹ indicating the presence of OH (phenol group of curcumin). The band at 1627 cm⁻¹ has a predominantly mixed (C=C) and (C=O) characteristics.³⁹ The vibrational bands appeared at 2839 and 2926 cm⁻¹ can be ascribed to the symmetric and asymmetric stretching modes of aliphatic C–H, respectively.³³ CUR also depicted absorption bands at 1587, 1512, 1276 and 1026 cm⁻¹ corresponding to the stretching vibrations of benzene ring (C=C vibrations), keto C=O stretching, enol C–O and C–O–C stretching modes, respectively.^39,40 The presence of most of these characteristic functional groups of curcumin in FTIR spectrum of CUR–PSMNPs confirmed their encapsulation into PSMNPs. However, no observable new characteristic peaks were found in the FTIR spectrum of CUR–PSMNPs. Thus, there was no change in the chemical composition of the samples before and after loading of curcumin. In present case, the hydrophobic interface between HMNPs and Pluronic layer provides site for encapsulation of drug without any changes in chemical composition (inset of Fig. 5). Furthermore, the z-average intensity-weighted diameter of CUR–PSMNPs was found to be 190 nm, which suggest that PSMNPs assembly structure was not perturbed during the curcumin loading (Fig. S5, ESI†).

Fig. 5 FTIR spectra of CUR and CUR–PSMNPs (inset shows the schematic representation of CUR loading into PSMNPs at the hydrophobic interface between HMNPs and Pluronic layer).

The drug release profiles of CUR–PSMNPs were investigated under reservoir-sink condition at 37 °C. The release of drug molecules from CUR–PSMNPs (Fig. 6) follows a time dependent release profile (gradually over a period of time). The loaded drug probably diffuses out from the hydrophobic–hydrophobic interface under the influence of concentration gradient. The release of curcumin was observed even with naked eyes from the conversion of colourless solution of sink medium to a yellow coloured solution after drug release experiment. The photo-graphs depicting the release of curcumin from CUR–PSMNPs at reservoir pH 5 are shown in Fig. S6 (ESI†). Further, about 65% of CUR was released from the CUR–PSMNPs system at pH 5.0, while only 7% of CUR was released at pH 7.4 after 50 h. This data revealed the higher release of curcumin in acidic conditions as compared to physiological medium which favors its targeted delivery to the tumor cells. The higher release of curcumin in acidic conditions may be attributed to the conformational changes of hydrophilic PEO chains of Pluronic assembled on surface of nanoparticles. It is reported that when the concentration of the protons is increased, the conformation of the hydrophilic PEO chains becomes more stretched.⁴¹ Thus, the contact of Pluronic polymer with the surface of HMNPs decreases, which allows the higher release of drug molecules.

Fig. 6 pH dependent release of CUR from PSMNPs in reservior-sink condition at 37 °C.

Cytotoxicity studies were performed to determine the effects of CUR and CUR–PSMNPs on cell viability. From MTT assay, it has been observed that CUR and CUR–PSMNPs showed significant reduction in proliferation of MCF-7 cells (Fig. 7a). The CUR–PSMNPs was more effective as compared to that of CUR. The IC₅₀ of CUR and CUR–PSMNPs in MCF-7 cell lines was found to be 25.1 and 18.4 μM, respectively through sigmoidal dose response fitting (Fig. S7, ESI†). It is noteworthy to mention that PSMNPs are highly biocompatible in nature and cell viability was found to be >90% at a concentration of 1 mg ml⁻¹. The higher toxicity of CUR–PSMNPs was further visualized by cellular imaging (Fig. 7b–d), which confirmed higher percentage of cell death as compared to the control. Further, we have investigated the cellular uptake of CUR and CUR–PSMNPs for different times. The cellular uptake studies (Fig. 8) confirmed the time dependent uptake of curcumin by MCF-7 cells for both CUR and CUR–PSMNPs. The curcumin uptake were found to be 1.37 and 2.9 nmol per million cells for CUR at 4 and 7 h, respectively, whereas those of CUR–PSMNPs were 5.0 and 7.2 nmol per million cells at 4 and 7 h, respectively. These results suggested that our drug–nanoparticle formulation has significantly higher uptake as compared to that of pure drug. Thus, the enhanced toxicity of CUR in our drug–nanoparticle formulation is possibly due to the increase in the bioavailability of drug to the targeted site. Specifically, the present investigation demonstrated the development of nanocarrier for intracellular delivery of hydrophobic drug, curcumin and investigation of their therapeutic efficacy.

Fig. 7 Bar graph showing cytotoxicity of CUR and CUR–PSMNPs in MCF-7 cells (a), and images showing control cells (b), cells treated with 25 μM CUR (c) and cell treated with CUR–PSMNPs at 25 μM (d). The results of cytotoxicity is presented as mean ± SEM, n = 3.

Fig. 8 Cellular uptake of curcumin for CUR and CUR–PSMNPs at different times.

Conclusions

Pluronic stabilized Fe₃O₄ magnetic nanoparticles were successfully developed for intracellular delivery of hydrophobic anticancer drug, curcumin. The formation of highly crystalline Fe₃O₄ nanoparticles of size ∼7 nm is evident from XRD and TEM analysis and its Pluronic modification was confirmed from FTIR, TGA, DLS, UV-visible and zeta-potential measurements. The introduction of Pluronic layer provides aqueous colloidal stability and biocompatibility as well as creates hydrophobic interface for encapsulation of curcumin. PSMNPs showed high loading affinity for curcumin, its pH dependent release and substantial internalization in tumor cells. Specifically, our drug–nanoparticle formulation showed higher therapeutic efficacy as compared to that of pure curcumin possibly due to the increase in the bioavailability of drug to the targeted site.

Acknowledgements

The authors thank Dr B. N. Jagatap, Director, Chemistry Group, BARC for the encouragement and support. Authors also acknowledge Prof. D. Bahadur, Indian Institute of Technology Bombay, India for his encouragement and allowing us to use some of the experimental facilities.

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Footnotes

† Electronic supplementary information (ESI) available: XRD pattern, TEM micrograph, HRTEM micrograph, particle size distribution, UV-visible absorbance and sigmoidal-dose response plots, photographs depicting magnetic field responsivity and curcumin release, and protein–particles interaction (Fig. S1–S7 and Table S1). See DOI: 10.1039/c6ra21207g

‡ These authors are equally contributed to this work.

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