Curcumin Delivery Using Magnetic Liposomes

Copyright © 2014 American Scientific PublishersAll rights reservedPrinted in the United States of America

ArticleJournal of

Nanopharmaceutics and Drug DeliveryVol. 1, 1–11, 2014www.aspbs.com/jnd

Curcumin Delivery Using Magnetic Liposomes

Saumya Nigam1, Asmita Kumar2, George A Thouas4, Dhirendra Bahadur3�∗, and Qizhi Chen4�∗1IITB-Monash Research Academy, IIT Bombay, Mumbai 400076, India2Padmashree Vidyapeeth, Navi Mumbai 400614, India3Indian Institute of Technology Bombay, Powai, Mumbai 400076, India4Department of Materials Engineering, Monash University, Clayton, Victoria, 3800, Australia

The objective of this work was to explore the controlled release, using magnetic liposomes (MLs), of curcumin, a drugthat selectively induces apoptosis in highly proliferative cells, as a potential cervical cancer chemotherapeutic agent.To this end, dextrin-coated iron oxide (Dx-Fe3O4) aqueous colloidal nanoparticles were synthesized using a single stepco-precipitation approach, followed by liposome encapsulation using a thin-film hydration method. Extensively chemicalanalyses demonstrated that MLs were chemically and structurally stable, with uniform dispersal in aqueous solution,and with successful surface conjugation of dextrin, lipid and curcumin. Most importantly, curcumin was found to bereleased from MLs in appreciable amounts (up to 56�3±3�9%) at elevated temperatures, following magnetic hyperthermia.Treatment of human cervical cancer derived cell lines (HeLa) in vitro demonstrated that the curcumin-loaded MLs wereeffective at inhibiting cell proliferation, with an IC50 value of 2.09 mg/ml. In conclusion, the magnetic liposomes describedin this study represent a promising system for controlled release of curcumin, as a potential chemotherapy system for thetreatment of cervical cancer.

KEYWORDS: Magnetic Nanoparticles, Liposomes, Curcumin, Drug Delivery, Cervical Cancer.

INTRODUCTIONChemotherapy involves the use of chemicals to damageDNA, RNA and proteins and to trigger cell cycle arrestor apoptosis in cancer cells, however such agents gener-ally induce apoptosis in both cancer and normal cells.1

Hence, it is highly desirable to develop a drug delivery sys-tem having a maximum chemotherapeutic efficiency andminimal side effects. An approach to achieve such drugdelivery system is to combine a chemical that selectivelyinduce apoptosis in targeted cancer cells, with a deliveryvehicle that can release the drug to the targeted tumour ina controlled manner.Curcumin (diferuloylmethane, a natural, hydrophobic

polyphenol and the primary constituent of the rhizomeof turmeric) is one of few agents that selectively induceapoptosis in highly proliferative cells. Cellular apopto-sis induced by curcumin is significantly higher in can-cer cells than in non-cancerous cells. Curcumin firstattracted attention for its antioxidant, anti-inflammatory,2

∗Authors to whom correspondence should be addressed.Emails: [email protected], [email protected]: 2 September 2013Accepted: 11 March 2014

antimicrobial3�4 and antineoplastic activities.5�6 The effi-cacy of curcumin as an therapeutic agent for cancer wasreported later.5�7�8 Curcumin has since been reported toincrease apoptotic death, control cell proliferation anddown-regulate the oncogenic phenotype by controlling thesignalling cascades involved in the cell cycle.9�10 How-ever, the low aqueous solubility, poor chemical stabilityand lipophilic nature of curcumin have limited its bioavail-ability and delivery efficiency to targeted cancer sites, andthus impaired its anticancer therapeutic potential.To enhance the chemical stability of curcumin, a few

types of polymer nanoparticles have been used for itsencapsulation,11–15 among which, poly(lactic-co-glycolic)(PLGA) is the most studied one. PLGA is a biodegrad-able polymer, with degradation products lactic and gly-colic acid, which are metabolised by cells via the Krebscycle. While PLGA nanospheres as vehicles can enhancethe delivery of curcumin compared to curcumin alone,the amphiphillic nature of the polymer results in low lev-els of encapsulation, resulting in a need to administerhigher dosages to achieve pharmaceutical activity at thetargeted site.14�16–18 In addition, PLGA nanoparticles gen-erally show an inefficient release profile, due to hydropho-bic areas on the internal surface of PLGA particles that

J. Nanopharmaceutics Drug Delivery 2014, Vol. 1, No. 4 2167-9312/2014/1/001/011 doi:10.1166/jnd.2014.1035 1

Curcumin Delivery Using Magnetic Liposomes Nigam et al.

trap curcumin. To enhance the release efficiency, saccha-ride based nanoparticle vehicles have also been used todeliver curcumin for oral administration. However, the fastwater solubility of saccharide compromises the chemicalstability of the drug system.19 In short, the above deliveryvehicles have failed to address the current issues associ-ated with the curcumin delivery.Liposomes, bi-layer vesicles of lipid molecules, have

been regarded as a promising alternative in thisregards.20–22 The amphiphillic nature of lipid moleculescauses them to form a closed vesicular structure in aque-ous solutions, with the apolar regions oriented away fromthe aqueous phase and polar regions in contact withthe water. The hydrophilic surface of liposomes rendersthem with good water solubility, while the amphiphillicnature of the lipid bilayer facilitates anchoring of bothhydrophilic and lipophilic molecules. A liposomal carriermade up of soy phosphatidylcholine and 1,1-diphenyl-2-picrylhydrazylhas been reported to enhance the antioxi-dant (therapeutic) effects of the encapsulated curcumin.23

However, it remains to be elucidated whether the lipo-some carrier affects the anti-cancer properties of curcumin.Therefore, an objective of the present work was to inves-tigate the curcumin-encapsulation efficiency, curcumin-releasing profile, and the anticancer therapeutic effect ofcurcumin when delivered by liposomes.Another special advantage of liposomes is that they can

release encapsulated drugs under external stimuli, such aschanging pH or temperature, or ultrasound waves, allowingcontrol over the release profile and the pharmacokinet-ics of the drug molecules. Among these stimuli, chang-ing temperature is clinically the most feasible because itis virtually impossible to control pH values in the body,and ultrasound has the potential to damage the phospho-lipid membranes of healthy cells while destroying the lipo-somes. In fact, hyperthermia treatment is already used insome forms of cancer treatment, especially solid tumours,to directly kill cancer cells or to make them more sensitiveto radiation and certain anti-cancer drugs.24�25 There area number of techniques to remotely deliver heat, includ-ing infrared sources, focussed microwaves, magnetic fieldsand infusion of warmed liquids.20–22 In the case of mag-netic hyperthermia, nanoparticles can be subjected to analternating magnetic field, which induces electron flow inthe nanomaterial, thereby producing localized heat. Usingmagnetic hyperthermia to control the release of curcuminfrom liposomes comprised another objective of this work.Superparamagnetic iron oxide nanoparticles have been

employed for the hyperthermia treatment.24�26�27 and asdrug delivery vectors,28�29 such as curcumin delivery.17�18

Compared with other ferrite systems (iron, cobalt,manganese, and nickel), superparamagnetic iron oxidenanoparticles have been shown to have greater potentialdue to their better aqueous stability and biocompatibility.30

These nanomaterials also exhibit a relatively large sur-face area to volume ratio due to their small size. The

downside of superparamagnetic iron oxide nanoparticlesis that they have high surface energy leading to strongtendency to agglomerate in colloidal suspensions via vander Waals forces and magnetic dipole–dipole interactions.This agglomeration necessitates the requirement of col-loidal stability of these nanoparticles in aqueous suspen-sions for the above-mentioned biomedical applications.Surface engineering of iron oxide nanoparticles for

biomedical applications has also been performed usinga variety of organic molecules,31�32 in addition topolymers33�34 and polysaccharides.35�36 This results inbioactive surfaces that can be used to anchor differ-ent molecules of interest and thereby enhance their col-loidal and surface chemistry.32 Small organic moleculesof biological origin have been considered promisingcandidates for nanoparticle surface engineering, becausetheir biocompatible degradation products minimise therisk of toxicity to biological systems. Saccharides andoligosaccharides, for example, readily degrade into sugarmonomers, which are actively metabolised by cells. Due totheir colloidal solubility, amino acids,37 monosaccharides,35

and oligosaccharides36 are three of the most frequently usedsmall organic molecules. Oligosaccharides, such as dex-tran and �-cyclodextrin, have been reported to significantlyimprove the colloidal stability of nanoparticles.38�39 Mono-saccharides are also well known for their ease of conjuga-tion with various biomolecules, such as proteins, to formpeptidoglycans and proteoglycans, or with lipids to formglycolipids and aminoglycans. Given the above properties,dextrin was selected as the surface engineering moiety forthe iron oxide nanoparticles in the present study.After being surface engineered with small organic

molecules, magnetic nanoparticles can be further mod-ified by various macromolecules,40 polymers41 andbiomolecules42 to improve their recognition in biologi-cal systems and to lower their immunogenicity.43 In thisrespect, lipids44�45 have an advantage over other macro-molecules because they can aid in cellular uptake ofnanoparticles by facilitating transport across the phospho-lipid membranes of cells. Fabrication of magnetic lipo-somes (MLs) by coating magnetic nanoparticles with alipid bi-layer has been explored for the delivery of anumber of drugs, except for curcumin.42 In summary, theobjectives of the present work are to fabricate and charac-terize a ML formulation, to evaluate its curcumin encapsu-lation and delivery performance, and finally to assess theanti-cancer effects of the ML/curcumin formulation within a cancer cell line.

MATERIALS AND METHODSMaterialsFerric chloride, ferrous chloride, dextrin, phenanthrolinand sulphorhodamine-B were purchased from Sigma–Aldrich, USA. Hydroxylamine hydrochloride, chloroform,methanol and ammonia were purchased from Merck, India.

2 J. Nanopharmaceutics Drug Delivery 1, 1–11, 2014

Nigam et al. Curcumin Delivery Using Magnetic Liposomes

Curcumin and soy-PC were obtained from Hi-media,India, and trichloroacetic acid was obtained from LobaChemie, India. All the chemicals were of analytical gradeand were used as received without any processing.

SynthesisTo synthesize the iron oxide nanoparticles, 4.44 g of ferricchloride and 1.732 g of ferrous chloride were dissolved in80 ml of Milli-Q water in a round bottom flask, and thetemperature was increased to 60 �C under nitrogen atmo-sphere with mechanical stirring at 1,000 rpm. The tem-perature was then held at 70 �C for 30 min, which wasfollowed by the addition of 30 ml of ammonia solution tothe reaction mixture and the maintenance of the tempera-ture at 70 �C for another 30 min. Finally, 10 ml of aqueoussolution of dextrin (0.07 g/ml) was added to the reac-tion mixture and the temperature was raised to 90–95 �Cunder reflux and maintained for 90 min with continu-ous stirring. A black coloured precipitate of dextrin-coatediron oxide nanoparticles (Dx-Fe3O4) was obtained and wasthoroughly washed with Milli-Q water. During each washstep, the precipitate was separated from the supernatantusing a permanent magnet.The MLs were prepared by a thin-film hydration tech-

nique that was reported previously.42 In a typical synthe-sis, 200 mg of soy-PC was dissolved in a solvent mixtureof chloroform/methanol (2:1 v/v). The solvent was evapo-rated under vacuum (120 mbar) at 35 �C in a rotary evap-orator to form a thin and uniform lipid film on the walls ofthe round bottom flask. To optimise the lipid:nanoparticleratio, the lipid film was hydrated with varying amountsof Dx-Fe3O4 nanoparticles (20, 40, 60, 80, 100, 150 and200 �g) using a water bath-type sonicator for approxi-mately 30 min. The temperature was maintained below40 �C until the lipid film was transferred to the aqueoussuspension yielding the MLs. Control liposomes were syn-thesized by hydrating the lipid film in phosphate bufferedsaline (pH 7.34). A lipid:nanoparticle ratio of 10:3 wasused for the drug delivery vesicle because it showedthe maximum encapsulation efficiency of the Dx-Fe3O4

nanoparticles and the most stable aqueous suspension. TheMLs were then stored at 4 �C until further characterisation.

Characterization of NanoparticlesThe nanoparticles fabricated above were analysed usingX-ray diffraction (XRD, Philips powder diffractometerPW3040/60) with Cu K� radiation. The particle size wasdetermined by using a high-resolution transmission elec-tron microscope, JEOL JAM 2100F (200 kV). The sur-face coatings of particles and the conjugated drug wereanalysed using a Fourier transform infrared spectrom-eter (Magna 550, Nicolet Instruments Corporation) inthe range of 4,000–400 cm−1. Thermal analyses wereperformed with a TA Instruments SDT Q600 analyserunder a nitrogen atmosphere with temperatures being

increased from room temperature to 800 �C at a rateof 10 �C/min. The hydrodynamic diameter and the zetapotential were determined with a Delsa Nano ParticleSize Analyser (Beckman Coulter Inc.) and a Zeta PALSanalyser (BI-200 Brookhaven Instruments Corp.), respec-tively. The magnetic measurements of the dried Dx-Fe3O4

powder were carried out with a vibrating sample magne-tometer (Lake Shore, Model-7410). To evaluate the spe-cific absorption rate (SAR), a radio frequency generator(Comdel CLF-5000) operating at a frequency of 425 kHzwas used. The amount of iron in the Dx-Fe3O4 sus-pension was determined by UV/Vis spectroscopy (Cecil,Model No. CE3021) using the established phenanthro-line method.46 The standard curve was prepared from astock iron solution under similar conditions (R2 = 0�998).The drug release was measured using a Varian CaryEclipse fluorescence spectrophotometer, against a stan-dard curve prepared under similar conditions (R2 = 0�996).The absorbance measurements of sulforhodamine B (SRB)assays were performed by Thermoscientific Multiskan EXmultiplate reader at wavelength of 560 nm.

Analysis of Drug Loading and ReleaseTo investigate the loading efficiency of curcumin, theabsorbance spectra of the drug was studied. In a typi-cal loading experiment, 200 mg of soy-PC was dissolvedin a solvent of chloroform/methanol (2:1 v/v) containingvarying amounts (20, 40, 60, 80, and 100 �l) of cur-cumin (stock solution-1 mg/ml in chloroform) and withthe ML:curcumin ratios of 10, 5, 3.33, 2.5 and 2. The sol-vent was evaporated under vacuum (120 mbar) at 35 �Cin a rotary evaporator to form a thin and uniform lipidfilm on the walls of the round bottom flask. The lipid filmwas then hydrated with a previously optimised amount(lipid:nanoparticle ratio = 10:3) of Dx-Fe3O4 nanoparti-cles under ultrasonication for approximately 30 min. Thetemperature was kept below 40 �C until the lipid filmwas transferred to the aqueous suspension to yield thedrug-loaded MLs. The absorbance spectrum of the super-natant (magnetic sedimentation of the curcumin-loadedMLs) was recorded with a UV-Vis spectrophotometer todetermine the amount of the drug loaded into the MLs.The curcumin-loaded MLs were re-suspended in water andstored at 4 �C until further use. The loading efficiency(w/w%) was calculated as follows:

% loading efficiency= �ACur −ASup�/ACur ×100 (1)

where ACur is the absorbance of pure curcumin and ASup isthe absorbance of the supernatant.The drug release study was carried out at temperatures

of 37 �C and 45 �C. The amount of curcumin released fromloaded MLs was quantified based on the loading efficiency.The curcumin-loaded MLs (10 mg) were suspended inphosphate-buffered saline (PBS) at pH 7.3 and placed in adialysis bag. Dialysis was performed with 200 ml of PBS

J. Nanopharmaceutics Drug Delivery 1, 1–11, 2014 3


(pH 7.3) under continuous stirring at physiological tem-perature (37 �C) and at hyperthermic temperature (45 �C).At different time intervals, aliquots were withdrawn andreplaced with fresh PBS. Fluorescence intensity of thesealiquots was analysed at �excitation = 425 nm and �emission =540 nm. Drug release was calculated from the fluorescenceintensity of the aliquots according to a standard curve pre-pared under similar conditions.The thermosensitivity of these curcumin-loaded MLs

was evaluated by the calcein release assay, which utilizesthe fluorescence property of calcein.47 At a high concentra-tion, calcein shows self-quenching resulting in a decreasein fluorescence and an increase is observed when calceinis released from the liposomal carrier into the surround-ing medium (data not shown). To establish the effect ofpH sensitivity on the release of the drug by the curcumin-loaded MLs, similar experiments were performed withsodium acetate buffer (pH 4.8) as a stimulus at 37 �C.The results showed that negligible amounts of the drugwere released from the ML system at the decreased pH,thereby establishing the independence of pH sensitivity onthe curcumin-loaded MLs.Time-dependent calorimetric measurements to evaluate

the heating ability of the Dx-Fe3O4 suspensions were per-formed using a radio frequency generator. A total of 1 ml(10 mg/ml of iron) of the Dx-Fe3O4 colloidal suspen-sion was placed in an AC magnetic field (7.64, 8.82,9.41 and 10.0 kA/m) with a fixed frequency of 425 kHzand with arrangements to minimise heat loss. The specificabsorption ratio (SAR) was calculated using the followingequation:

SAR = C× T

t× 1

mFe

(2)

where C is the specific heat of the solvent (C = Cwater =4�18 J/g 1 C), T /t is the initial slope of the time-dependent temperature curve and mFe is the mass fractionof the iron in the sample.

Evaluation of Biocompatibility and TherapeuticsThe biocompatibility of the MLs was established with amouse fibroblast cell line (L929) and a cervical cancer celllines (HeLa). Toxicity of the curcumin-loaded MLs wasevaluated with cervical cancer cell lines (HeLa) by a SRBcolorimetric assay.48 To establish the potential of thesecarriers to deliver curcumin, a dose-dependent study wasundertaken to evaluate the 50% inhibitory concentration(IC50) values of free curcumin and the curcumin-loadedMLs over 48 h. The cells were seeded in 96-well platesat a cell density of 2×104 cells per well and incubated intissue culture medium for 24 h at 37 �C in a 5% CO2 envi-ronment. After 24 h, different concentrations of the MLs(2, 1, 0.5, 0.25, 0.125, 0.0625 and 0.03125 mg/ml) andthe curcumin-loaded MLs (10, 8, 4, 2, 1, 0.5, 0.25, 0.125,0.0625 and 0.03125 mg/ml) were mixed with the growthmedia and the cells were incubated for an additional 24 h

(MLs) or 48 h (curcumin-loaded MLs). After 24 h (MLs)or 48 h (curcumin-loaded MLs), the cells were carefullywashed with PBS (pH 7.3), and an SRB assay was per-formed to determine cell viability. For the assay, cells werefixed with cold 10% trichloroacetic acid (at 4 �C) andstained with 0.4% SRB (in 1% acetic acid). After onehour of incubation in the dark, the unbound dye was thor-oughly washed with 1% acetic acid, and the cell-bounddye was later extracted with 10 mM Tris buffer (pH 10.5).Absorbance was recorded at 560 nm using a Thermo Sci-entific Multiskan EX multiplate reader. Relative cell via-bility was calculated as follows:

% relative viability

= Absorbance of treated cellsAbsorbance of control cells

×100 (3)

RESULTS AND DISCUSSIONPhysical Characterisation of the SynthesisedNanoparticles (XRD and TEM)The crystalline structure and crystallite size of the Dx-Fe3O4 nanoparticles were investigated by powder XRD(Fig. 1). The corresponding diffraction planes of theindices showed good agreement with the reported valuesfor magnetite (JCPDS Card No. 19-0629, a= 8�3967 Å).The XRD pattern revealed the formation of single-phasemagnetite, which has an inverse spinel structure with thecrystallite size of ∼ 8.48 nm, as calculated by the Scherrerformula. A high degree of crystallinity of the nanoparticleswas indicated by the presence of sharp and intense peaksof the Dx-Fe3O4 nanoparticles.Figure 2(a) shows the TEM micrograph of the Dx-Fe3O4

nanoparticles. The Dx-Fe3O4 nanoparticles were mostlyspherical in shape and were smaller than 15 nm in diame-ter. The inset in Figure 2(a) shows the electron diffractionpattern of a selected area. The pattern, which has beenindexed with the inverse spinel magnetite crystal structure,is consistent with the XRD results. Figure 2(b) shows themorphology of the MLs. The inner and outer diametersof the MLs averaged ∼ 100 and ∼ 200 nm, respectively

Figure 1. XRD pattern of Dx-Fe3O4 nanoparticles.



Figure 2. TEM micrograph of (a) Dx-Fe3O4 nanoparticles(inset shows the selected area diffraction pattern of Dx-Fe3O4

nanoparticles), (b) magnetic liposomes (inset shows magni-fied image of MLs).

with bilayer thickness of 50 nm. The MLs were stable andwell dispersed in the aqueous solution (the inset showsan image of a ML at a scale of 50 nm). The bilayerswere formed uniformly. The intact liposome has a spheri-cal shape, with varying amounts of iron oxide encapsulatedin its hydrophilic core.

Chemical Characterization of MLs (FTIR)Figure 3(a) shows the FTIR spectra of pure dextrin andthe Dx-Fe3O4 nanoparticles. The spectrum of dextrin iswell resolved and contains a few broad bands in additionto narrow bands. The very broad peak at 3297 cm−1 isthe characteristic of the aromatic sp2 C H stretch and isattributed to pyranose ring vibrations of dextrin. Character-istic peaks of the �-D-glucose units of the polysaccharidewere visible at 1203 cm−1, which occur due to the in-planeC H and O H vibrations.49 The multiple bands appear-ing in the region between 1150 and 930 cm−1 coincidewith the in-plane C H bending vibrations of the pyranosering and the C O stretching vibrations. The vibrationalbands present at 1150 and 1077 cm−1 are attributed to

Figure 3. FTIR spectra of (a) dextrin and Dx-Fe3O4 nanoparti-cles, (b) MLs and Cur-MLs.

valent vibrations of the C O C bond of the glycosidicbridge, whereas the peak at 1025 cm−1 is due to the sub-stantial chain flexibility of dextrin around the glycosidicbonds.The vibrational bands of Dx-Fe3O4 are relatively broad

and fewer compared with those of pure dextrin. Thepeaks present in the spectrum of dextrin at 1150 cm−1,1077 cm−1 and 1025 cm−1 were also present in that of Dx-Fe3O4, indicating the successful coating of dextrin on thesurface of the nanoparticles. Other peaks are interpreted asfollows. The small and narrow bands around 3000 cm−1

are due to the presence of H-bonded –OH groups fromwater molecules, which are physically adsorbed on thesurface of the Dx-Fe3O4 suspended in aqueous medium.A new peak at 3009 cm−1 is visible in the O H stretchregion. This may be the result of the formation of hydro-gen bonds between the free –OH groups on dextrin andthe oxygen from the Fe O core of the magnetite nanopar-ticles, resulting in the formation of an iron-oxy-hydroxidemonodentate coordination complex. The peaks at 2934 and2986 cm−1 present in the dextrin spectrum are retained inthe Dx-Fe3O4 spectrum, indicating that the C H bondswere unaltered during the conjugation and did not play arole in the coating process. The appearance of the peak at1660 cm−1 indicates the retention of the cyclic ring struc-ture of dextrin after its conjugation with the iron oxidenanoparticles. As seen, the peaks at 1365 and 1417 cm−1 in



dextrin shift to 1400 and 1535 cm−1 in the Dx-Fe3O4 spec-trum. This might be due to the formation of O H bondsin the iron-oxy-hydroxide complex and the overlap withC H bending vibrational bands of the dextrin molecules.The appearance of the peak at 864 cm−1 (boat conforma-tion) and the disappearance of the peak at 930 cm−1 (chairconformation) could be attributed to the loss of the chairconformation or to a conformational change to the boatconformation of the glucopyranose units after interactionwith the surface of the iron oxide. The peak at 575 cm−1

can be attributed to the stretching vibrational modes ofFe O.Figure 3(b) shows the FTIR spectra of the soy-PC, MLs,

curcumin and curcumin-loaded MLs. The spectrum of soy-PC reveals characteristic vibrational bands consistent withpreviously reported studies.50 The peak at 1739 cm−1 isdue to C O vibrations of the fatty acid chains, and thebands at 1643, 1460 and 1232 cm−1 are due to C–NH3

symmetric scissoring of the choline entity of lipid andC H scissoring and P O vibrations of the phosphatidylgroup, respectively. The shift in the vibrational bands at1739 cm−1 to 1737 cm−1 in the spectrum of the MLs isindicative of the interaction of the C O groups of soy-PC with the Dx-Fe3O4 nanoparticles by either electrostaticor van der Waals interactions. The bands at 1643 and1460 cm−1 do not exhibit any shifts, pointing to the non-participation of the –NH3 groups of the fatty acid in anytype of bond formation with the Dx-Fe3O4 nanoparticles.The band representing the P O of the choline group ofsoy-PC (1232 cm−1) exhibits a minor shift (1234 cm−1�,indicating the interaction of the group with the free –OHgroups present on the Dx-Fe3O4 nanoparticles. The overlapof soy-PC vibrations on dextrin masks the dextrin frequen-cies in the spectrum of the MLs.The FTIR spectrum of curcumin-loaded MLs shows

sharp, intense characteristic peaks of soy-PC due tothe high ratio of ML:curcumin. Characteristic peaks ofcurcumin are also present in the FTIR spectrum ofcurcumin-loaded MLs, which are in good agreement withthe vibration spectrum of curcumin reported by Mohanet al.51 According to their interpretations, the band at3502 cm−1 is due to phenolic –OH stretching vibrations,the peak at 1427 cm−1 is attributed to olefinic bendingvibration of C H bound to the benzene ring of curcumin,the peak at 1272 cm−1 due to an enol vibration (C O),the band at 958 cm−1 due to benzoate trans –CH vibra-tions, and the vibrations at 709 cm−1 due to cis-CH ofan aromatic ring. The above results further indicate thesuccessful loading of curcumin into the MLs.The successful conjugation of curcumin to the MLs was

indicated by the shift of some peaks in the FTIR spectrumof curcumin-loaded MLs, as discussed as follows. A shiftobserved in the band at 1737 cm−1 in MLs to 1741 cm−1 inthe curcumin-loaded MLs is attributed to the attachment ofthe drug to the –COO groups of the fatty acid. Other bands

shifted from 1643 to 1651 cm−1, 1460 to 1457 cm−1, 1234and 1232 to 1238 cm−1 and 1056 to 1067 cm−1 in thespectrum of the curcumin-loaded MLs. The shift in thesebands indicates that the –CH3 side chains and the free –OHgroups may be involved in the conjugation of curcuminto the MLs, confirming the drug loading into the MLs.The above results confirm the surface conjugation of dex-trin onto the Dx-Fe3O4 nanoparticles, encapsulation by thelipid bilayer and the subsequent conjugation of curcumin.

Organic Mass Percentage of MLs(TGA-DTA Analysis)TGA-DTA analysis was conducted to determine thedecomposition profile of formulations and relative mass ofdextrin (Fig. 4(a)) and liposome (Fig. 4(b)) coatings. Thethermal profile of the Dx-Fe3O4 nanoparticles revealed thedegradation of the nanoparticles occurred in three primarysteps. Initially, a weight loss of 3.45% occurred, whichcorresponded to the endothermic DTA peak at 80 �C.This loss could be attributed to water molecules surfaceadsorbed by the superficial dextrin molecules. A gradual5.11% loss in weight of nanoparticles was then observedin the temperature range of 100–430 �C correspondingto two DTA peaks at 285 and 400 �C respectively. Dex-trin molecules underwent a decomposition step at 320 �Cand thus this observed decrease in weight could be dueto the surface conjugated dextrin molecules.52 The thirdweight loss step of 0.39% was observed at 570 �C having acorresponding sharp exothermic DTA peak. This decreaseoccurs due to the loss of FeO molecules during degrada-tion of magnetite to maghemite.The thermogram of MLs indicated that weight loss

occurred in three distinct steps. In the first step, an initialweight loss of approximately 3.71% occurred, correspond-ing to a small endothermic DTA peak at 60 �C, due to theremoval of physically absorbed water. In the second step,a steep decline in the curve was observed. This drop inthe TGA plot represents a weight loss of approximately9.0% and has a corresponding broad exothermic DTA peakat 280 �C, again due to the removal of the organic dex-trin and lipid molecules by thermal decomposition fromthe surface of the iron oxide nanoparticles. This large and

Figure 4. TGA-DTA plots of magnetic liposomes.



well-defined weight loss may be due to the larger surfaceconcentration and high molecular weight of the moleculesmade up of carbon, oxygen, hydrogen and phosphorus.In the third step, a negligible weight loss of approximately0.29% occurs. There is a corresponding exothermic DTApeak at 600 �C due to the removal of iron (II) oxide (FeO)during a phase transformation of magnetite to maghemite.Hence, the total weight loss percentage was approximately13.7%. This mass loss represents the weight percentage oforganic coatings on the surface of the iron oxide nanopar-ticles and the encapsulating liposome bilayer.

Aqueous Stability and Dispersion of theMLs and the pH SensitivityThe colloidal stability and dispersion of the nanoparticlesis associated with the electric charge of the particle sur-face. Figure 5 shows the zeta potential of the Dx-Fe3O4

and MLs at different pH values, indicating that the con-jugation of the dextrin molecules onto the surface of theiron oxide nanoparticles creates a highly negative surfacecharge with an isoelectric point at pH 5. These zeta poten-tial values confirm the presence of negatively charged dex-trin groups on the surface of the iron oxide nanoparticles.The addition of the lipid bilayer to the Dx-Fe3O4 loweredthe surface charge of the MLs and shifted the isoelectricpoint to pH 4.5. At pH 7.0, the MLs showed a negativesurface charge of approximately − 30 mV, which is highlydesirable, as it ensures high aqueous stability of the MLsin a physiological environment before reaching targetedcancer cells. The dynamic light scattering (DLS) measure-ments on the MLs (Fig. 5(b)) showed that the MLs hada mean hydrodynamic diameter of 1.2 �m, which can beattributed to the presence of the water associated with thehydrophilic heads of the lipid layer.

Magnetic Response of the Synthesised MLsFigure 6 shows the field-dependent magnetization(M vs. H ) plot of the Dx-Fe3O4 nanoparticles and the MLsat room temperature. Both exhibited superparamagneticbehaviour, i.e., zero magnetic hysteresis and remanence.

Figure 5. Zeta potential of Dx-Fe3O4 and MLs as a functionof pH. Inset shows the hydrodynamic diameter of the MLsobtained from DLS measurements.

Figure 6. Field-dependent magnetization (M vs. H) plot of Dx-Fe3O4 and MLs at room temperature.

The maximum magnetisation of the Dx-Fe3O4 and theMLs was 54.18 emu/g and 24.29 emu/g, respectively, at afield of 20 kOe. The observed magnetisation of Dx-Fe3O4

is comparable to that (approximately 60 emu/g) of bareiron oxide nanoparticles reported in previous work.53 Dex-trin chains with more than 10 glucose units tend to adopt ahelical structure in aqueous solution, which contain cavi-ties that easily form an inclusion complex with low molec-ular weight compounds such as iron oxidenanoparticlesand ions.54 These helical cavities may interfere with thedomain alignment of the iron oxide nanoparticles with theapplied magnetic field, thus lowering the value of its mag-netisation in comparison to bare nanoparticles. The strongmagnetic response of these aqueous-stable nanoparticlescould be exploited for various applications such as mag-netic targeting, hyperthermia treatment, bio-sensing andmagnetic resonance imaging. The substantial drop in themagnetization values of the MLs is attributed to the highmolecular weight of the non-magnetic soy-PC bi-layer.This bilayer masks the domain alignment and restrictsthe response of the Dx-Fe3O4 particles trapped within thebilayers to the applied magnetic field.Figure 7 shows the time-dependent SAR of the Dx-

Fe3O4 nanoparticles in response to the application ofvarying AC magnetic fields. The calorimetric measurementwas used to determine the heating rate of the Dx-Fe3O4

Figure 7. Time-dependent specific absorption measurementsof Dx-Fe3O4 nanoparticles.



suspension. The SAR values of the nanoparticles were 9.51,19.50, 22.03 and 32.90 W/g of iron with an applied field(H ) of 7.64, 8.82, 9.41 and 10 kA/m, respectively. Figure 7illustrates that with the increment of the applied field, thetime required to reach a temperature of 45 �C from an ini-tial ambient temperature was reduced, which is consistentwith the relationship between the heat generation and theapplied AC magnetic field (the inset of Fig. 7).

Encapsulation (Loading) of CurcuminThe absorbance spectra of the pure curcumin andsupernatant (magnetic sedimentation) obtained from theMLs are given in Figure 8, which shows that theabsorbance of the supernatant decreased with an increasein the ML:curcumin ratio. The decrease in absorbance isattributed to the conjugation of the curcumin moleculeswith the phosphatidyl choline groups, due to the increasein the concentration of the latter relative to that of the for-mer. In other words, the decrease of absorbance indicatesan increased encapsulation of curcumin. The hydropho-bic curcumin and the lipophilic ends of soy-PC in anaqueous environment interact with each other in a waythat facilitates the encapsulation of the drug within thelipid bilayer. This been reported to be strongly dependenton the ratio of phosphatidyl choline to curcumin duringthe thin-film formation step because a single moleculeof curcumin binds with six molecules of phosphatidylcholine.55�56 In the present work, a maximum encapsu-lation efficiency of 97.6% was achieved when the ratioof phosphatidyl choline to curcumin was 1:0.6, with thecurcumin-loaded MLs showing a high degree of homo-geneity and stability in aqueous suspensions. The encap-sulation efficacy observed was higher than values reportedfor similar nano-carrier systems.14�57

Release of Curcumin at Elevated TemperaturesFigure 9 shows the release profile of the curcumin-loadedMLs under physiological (37 �C) and elevated (45 �C)temperature in reservoir-sink conditions. Sink (PBS) wasspiked with 0.1% (v/v) chloroform to facilitate the diffu-sion of hydrophobic curcumin in an aqueous environment.

Figure 8. Absorbance spectra of curcumin-loaded MLsagainst pure curcumin.

Figure 9. Drug release profiles of curcumin-MLs at physio-logical and hyperthermic temperatures.

Under normal physiological conditions, curcumin releasewas lower than 6%, which is negligible. In contrast, it wasreleased rapidly under hyperthermia temperatures follow-ing an initial short period (25 min) of slow release. At45 �C, curcumin release reached a plateau at 150 min andhad a total release percentage of ∼ 56%. The release ofthe curcumin molecules could be attributed to the disrup-tion of the heat sensitive lipid bilayer at elevated temper-atures, which may have weakened and thus disrupted thehydrophobic interactions between the curcumin and thephosphatidyl choline groups in the bilayer, thereby expos-ing curcumin to the surrounding environment. The phasetransition temperature is an important parameter in deter-mining the fluidity of the bi-layer. The fluidity, in turn,affects the release of curcumin from the MLs. Since physi-ological temperature is below the transition temperature ofsoy-PC, the release of curcumin in physiological environ-ments is inhibited. In contrast, a hyperthermic temperatureof 45 �C is above the transition temperature and fluidisesthe bi-layer, thus enhancing the release of the curcumin.58

Another factor that must be considered in the evalua-tion of anti-cancer drug release is the acidic microenviron-ment around a tumour. Tumours, due to their hypoxic (lowoxygen) conditions and high lactic acid secretion, tend tocause acidity in their surrounding microenvironment witha pH value below physiological (7.2–7.4). Hence, a pHvalue lower than 7.0 is typically used to evaluate the pHsensitivity of cancer drug release. In this work, the effect ofpH sensitivity on the release of the curcumin-loaded MLswas investigated with sodium acetate buffer (pH 4.8). Theresult showed that little curcumin was released from theML system at the decreased pH value, thereby establish-ing the independence of pH sensitivity on the curcumin-loaded MLs. Considering this result, this work focusedon the temperature-stimulated drug release of curcumin-loaded MLs at pH 7.0.

Biocompatibility of MLs Alone and AnticancerTherapeutic Effect of Curcumin-Loaded MLsFigure 10(a) shows cell viabilities of mouse fibroblast(L929) and cervical cancer (HeLa) cell lines incubated in



Figure 10. (a) Percentage cell viability of MLs incubated withmouse fibroblasts and cervical cancer cells for 24 h. (b) Dose-dependent evaluation of Curcumin-MLs for determination ofIC50 with HeLa cells.

growth medium containing MLs. A SRB assay was per-formed to quantify the viable cell population, thereby indi-cating the effect of the MLs on the growth and phenotypeof the L929 and HeLa cells. The results suggested that theMLs alone are biocompatible and thus they are safe forin vivo studies. The relative cell viability, calculated fromEq. (2), exceeded 100% when the concentration of thenanoparticles was low, which was attributed to the facili-tation of cell growth by the iron released by the cellulardegradation of iron oxide.59

Figure 10(b) illustrates the results of the dose-dependentstudy performed with HeLa cells to evaluate the half maxi-mal inhibitory concentration (IC50) of the curcumin-loadedMLs. The results indicate that the formulations were ableto inhibit cell proliferation by approximately 65% in HeLacells. The amount of pure curcumin that reduced the cellpopulation by half (IC50) was 125 �g/ml over a periodof 48 h. The IC50 value of curcumin has been reportedto range from 15 to 30 �M with various cancer celllines.14�60�61 The dose-response curve fitting with Origin 8software showed that the curcumin-loaded MLs hinderedcell growth and reduced the cell population by half (IC50)at a concentration of 2.09 mg/ml (R2 = 0�98154). How-ever, cell viability was decreased to 70% at a concentration∼ 62.5 �g/ml, as indicated by Figure 10(b). The IC50 value

of 2.09 mg/ml for curcumin-loaded MLs was higher thanthat (125 �g/ml) of pure curcumin, which is due to theloading of the Dx-Fe3O4 and encapsulative lipid bi-layer,as well as the ML release profile over 48 h. In a physi-ological environment, the anticancer therapeutic effect ofpure curcumin is hampered by its low aqueous solubilityand poor chemical stability, which limits its bioavailabilityat the targeted cancer site. Hence, the MLs would enhancethe therapeutic effect by increasing the delivery efficiencyof curcumin compared with pure curcumin. In short, thework has demonstrated the ability of MLs to efficientlydeliver curcumin to cancer cells and to maintain its anti-cancer activity in an aqueous environment for the treat-ment of cervical cancer.

CONCLUSIONSControlled release of curcumin using the ML system andthe therapeutic effect of this drug formula have beenexplored for the treatment of cervical cancer. The sur-face engineered magnetic nanoparticles, dextrin-coatediron oxide (Dx-Fe3O4), were fabricated using a single-stepfacile co-precipitation approach. Dx-Fe3O4 nanoparticleswere successfully encapsulated within the hydrophilic coreof liposomes, producing a magnetic liposome (ML) system.The conjugation of dextrin, lipid and curcumin moleculesonto the iron oxide nanoparticles was also achieved. TheMLs exerted little or no toxic effects on normal fibrob-last cells (L929), showing a potential to be used as a drugcarrier to deliver hydrophobic drugs in aqueous environ-ments without harming normal cells. The release of cur-cumin molecules can be controlled by varying temperature,which can be achieved by applying an AC magnetic field.The fabricated curcumin-loaded MLs can release curcuminin appreciable amounts (up to 56�3±3�9%) at 45 �C underthe control of magnetic stimulation. More significantly, thecurcumin-loaded MLs can effectively inhibit cancer cellgrowth and viability and have an IC50 value of 2.09 mg/ml.In conclusion, the curcumin-loaded MLs explored in thiswork could offer an effective treatment for cervical cancer.

Acknowledgments: The author (Saumya Nigam)acknowledges IITB-Monash Research Academy for pro-viding the fellowships. The financial support by Nanomis-sion of DST, Government of India and Nanotechnologydivision, DIT, Government of India is also gratefullyacknowledged by Saumya Nigam.

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Curcumin Delivery Using Magnetic Liposomes