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Thermo-responsive copolymer coated MnFe 2 O 4 magnetic nanoparticles for hyperthermia therapy and controlled drug delivery

This study is about multifunctional magnetic nanoparticles surface-modified with bilayer oleic acid, and coated with a thermo-responsive copolymer poly(N-isopropylacrylamide-co-acrylamide) by emulsion polymerization, for controlled drug delivery and
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  Thermo-responsive copolymer coated MnFe 2 O 4  magnetic nanoparticles forhyperthermia therapy and controlled drug delivery Saqlain A. Shah a , * , M.H. Asdi b , M.U. Hashmi c , M.F. Umar d , Saif-Ullah Awan e a Department of Physics, Forman Christian College (University), Lahore, Pakistan b Faculty of Mathematics and Physics, Stuttgart University, Germany c Department of Engineering & CS, Superior University, Lahore, Pakistan d Department of Physics, University of Management & Technology, Lahore, Pakistan e Department of Physics, Quaid-i-Azam University, Islamabad, Pakistan h i g h l i g h t s < Magnetism & Magnetic Nanoparticles for Biomedical Applications. < Multifunctionality: Controlled Drug Delivery & Hyperthermia. < Drug loaded thermoresponsive polymer shell on magnetic core. < A signi fi cant absence of drug release at 37   C. < Magnetic heating causes drug release at temperature  >  LCST. a r t i c l e i n f o  Article history: Received 18 June 2012Received in revised form2 September 2012Accepted 13 September 2012 Keywords: BiomaterialsMagnetic materialsNanostructuresPolymers a b s t r a c t This study is about multifunctional magnetic nanoparticles surface-modi fi ed with bilayer oleic acid, andcoated with a thermo-responsive copolymer poly( N  -isopropylacrylamide- co -acrylamide) by emulsionpolymerization, for controlled drug delivery and magnetic hyperthermia applications. Nanoparticleswere loaded with anticancer drug doxorubicin into the copolymer chains at 25   C. Composite nano-particles (hydrated) of average diameter 45 nm were of core e shell structure having magnetic core of about 18 nm and shell was composed of organic compounds and water. Magnetic core was super-paramagnetic lacking coercive force and remanance due to the pseudo-single domain nanostructure.Lower critical solution temperature (LCST) of the thermo-responsive copolymer was observed to bearound 39   C. Below this temperature, copolymer was hydrophilic, hydrated and swelled. But above LCST,copolymer became hydrophobic, dehydrated and shrank in volume. UV visible spectrophotometer wasused to investigate the drug loading and releasing pro fi le at different temperatures as well as undermagnetic heating. There was almost absence of drug release at around 37   C (normal body temperature).Drug was released at temperatures above LCST, which is signi fi cant for controlled drug delivery. Magneticheat-generation was studied by exposing the magnetic  fl uid to alternating magnetic  fi eld of 7.2 kA m  1 having frequency 70 kHz. A simple magnetic capturing system (simulating a blood vessel) was used toanalyze the capturing of magnetic nanoparticles under various applied  fi elds for drug targeting purpose.   2012 Elsevier B.V. All rights reserved. 1. Introduction Magnetic nanoparticles (MNPs) have attracted an enormousattention for their potential use in biomedical applications likecontrolled drug delivery, cell separation, magnetic resonanceimaging and localized hyperthermia therapy of cancer etc. Ironoxide based magnetic nanoparticles are of particular signi fi cancebecauseoftheirappropriatebiocompatibilityandlowtoxicity.Suchparticles can be injected directly into the tumor sites, can bedelivered by magnetic  fi eld gradient or some other ef  fi cient drugdelivery system. Under a high frequency magnetic  fi eld, thesenanoparticles generate heat energy by hysteresis loss, Néel andBrownian relaxations dependingonthesize of theparticles. Cancercells usually perish orat least become susceptible tochemotherapyand radiotherapy around 45   C, whereas healthy body cells remainsafe at this temperature [1 e 4]. In this temperature range variouscancer damaging mechanisms may occur like apoptosis, protein *  Corresponding author. Tel.:  þ 92 3334434819. E-mail addresses:, Shah). Contents lists available at SciVerse ScienceDirect Materials Chemistry and Physics journal homepage: 0254-0584/$  e  see front matter    2012 Elsevier B.V. All rights reserved. Materials Chemistry and Physics 137 (2012) 365 e 371  denaturation and DNA cross linking etc [5 e 9]. Effectiveness of localized hyperthermia depends on various factors like particlesconcentration, duration of exposure to alternating magnetic  fi eldand tissue characteristics etc [4,9,10].Tailoring multifunctional characteristics of MNP is the mostdemanding aspect in this arena. For this purpose, core shell nano-structure is of utmost importance where MNP serves as core. Theirsurfaces are functionalized by biocompatible and surfactant layersand/or functional polymers which could also prevent agglomera-tion and opsonization [11 e 13]. Such layers become shells overmagnetic cores. Suitable drug molecules can be attached to thesesurfaces which are to be delivered at the cancer sites [9,14]. Suchmultifunctional core e shell nanoparticles serve two purposes inone therapy: localized hyperthermia of the cancerous cells andcontrolled drug delivery at speci fi c locations.Functionalized compounds that reactto external stimuli such astemperature and pH etc have attracted a huge attention inbiomedical applications. One such compound is a well knownthermo-responsive polymer poly- n -isopropylacrylamide (PNI-PAm). Its characteristic is  ‘ lower critical solution temperature ’ (LCST)belowwhichitactsashydrophilic,absorbswaterandswells.Above LCST it becomes hydrophobic and undergoes reversiblevolume shrinkage due to the expulsion of water from its chains[15 e 19]. Drug release can be controlled by manipulating thetemperature of the polymer shell resulting in higher release ratesabove LCST and vice versa [15,20 e 22]. The LCST of PNIPAm can betuned by incorporating co-monomer units such as acrylamide [23].Current study is about multifunctional magnetic MnFe 2 O 4 nanoparticles surface-modi fi ed with bilayer oleic acid and coatedwith a thermo-responsive copolymer PNIPAm-co-Am by emulsionpolymerization, for controlled drug delivery and hyperthermiaapplications. Molecules of anticancer drug doxorubicin (DOX) wereincorporated into the copolymer chains at 25   C. XRD, TEM, TGAand FTIR were used to study the morphology and composition of nanoparticles. UV visible spectrophotometer was used to investi-gate the drug loading and releasing pro fi le at different tempera-turesaswellasundermagneticheating.Asimplemagneticcapturesystem (simulating a blood vessel) was used to analyze thecapturing of magnetic nanoparticles under various applied  fi eldsfor drug targeting purpose. Fig.1 shows the schematic overview of the processes involved in this study. 2. Experimental  2.1. Synthesis of bilayer oleic acid-modi  fi ed MnFe  2 O 4  nanoparticles Single layer oleic acid-modi fi ed MnFe 2 O 4  nanoparticles wereprepared byco-precipitation method.1.47 g MnCl 2 $ 4H 2 O (98%) and4 g FeCl 3 $ 6H 2 O (98%) were dissolved in 150 ml distilled H 2 O. 5 g of NaOH (99%) was added into the solution rapidly with vigorousstirring at 80   C. Solution was stirred for 15 min under nitrogenatmosphere. Subsequently, 0.5 ml oleic acid (95%) was added intothe suspension and vigorous stirring took place for another 30 minunder the same conditions. To form a bilayer structure of oleic acid,another amount of 0.5 ml oleic acid was added into the suspensionand stirred for 30 min. Bilayer oleic acid-modi fi ed magneticnanoparticles (OA-MNPs) were separated by magnetic decantationand washed several times with distilled water  fi rst and thenrepeatedly washed with ethanol to remove the extra surfactant.  2.2. Preparation of PNIPAm-co-Am coated OA-MNP  Oleic acid-modi fi ed MnFe 2 O 4  nanoparticles were coated withpoly( N  -isopropylacrylamide- co -acrylamide) (PNIPAm- co -Am) byemulsionpolymerization [24].Initially,PNIPAm- co -Amcopolymerswere prepared. The total concentration of monomers was put to0.02mol l  1 and the molar ratio of acrylamide & PNIPAm (98%) was1:15 in the feed. 5 wt%  N  , N  -methylene bisacrylamide (based onmonomer weight) (98%) was included into the monomer mixtureas a cross linker and stirred for 10 min. OA-MNPs were then addedto the mixture. The solution was heated at 70   C along withvigorous stirring under nitrogen atmosphere. Subsequently, 3 wt%redox-initiator ammonium persulphate (98.5%) was added. After2 h of reaction, the composite nanoparticles were separated bymagnetic decantation and washed repeatedly with distilled water.  2.3. Drug-loading with doxorubicin Anticancer drug doxorubicin (DOX) was loaded onto thecopolymer-coated nanoparticles by incubating 300 mg of thedehydratedparticlesin5mlofDOX-watersolutionfor24hat25  C.DOX concentration in distilled water was 1 mg ml  1 . After drugloading, the hydrated composite nanoparticles (CNP) were sepa-rated by magnetic decantation. The post-loading DOX concentra-tion of the aqueous solution was determined using UV  e visiblespectrophotometer. The drug loading of CNP was calculated by thereduction in the amount of DOX in solution during the incubationprocess. 3. Characterization MnFe 2 O 4  crystalline phases were identi fi ed using x-raydiffraction (XRD) and mean particle size was calculated usingScherrer ’ s formula [4]. Morphology and core e shell nanostructureof composite nanoparticles were observed using transmissionelectron microscope (TEM) (JEOL JM-2100). Fourier transforminfrared (FTIR) (Bruker tensor 27) analysis was performed on thecomposite particles in the wavenumber range 500 e 3500 cm  1 tostudy the inter-atomic nature of bonds and to con fi rm the coatingof copolymer. Thermo gravimetric analysis (TGA) (SDT Q600) wasperformed to determine the amount of organic substance coatedonto the magnetic particles. Magnetic properties of OA-MNP andCNP were studied using vibrating sample magnetometer (VSM)(Lakeshore 7407) operating within 6 kOe  fi eld. Lower criticalsolution temperature (LCST) of thermo-responsive copolymer andquantity of loaded & released drug were studied using UV  e visiblespectrophotometer (UVS) (DR-2800). Fig. 1.  Schematic overview of the processes involved in this study. S.A. Shah et al. / Materials Chemistry and Physics 137 (2012) 365 e  371 366   3.1. Temperature triggered DOX release 200 mg of DOX-loaded CNP were dispersed in 7 ml of distilledwater at 25   C in test tubes. The test tubes were placed at 25   C,35   C & 45   C for 24 h. UV visible spectrophotometer was used todetermine the amount of DOX released after every 4 h.  3.2. Heat generation under high frequency magnetic   fi eld Heat generation capability of the CNP was determined usingmagnetic heateroperating at 7.2 kA m  1 magnetic fi eld and 70 kHzfrequency. 30 mg CNP and OA-MNP (separate observations) weredispersed in 1 ml distilled water at 25   C in a thermally insulatingglass container and placed within the heating coil. Magnetic heaterwas operated for 1 h and temperature variations were noted afterevery 10 min.  3.3. Magnetic-heating triggered DOX release 200 mg of DOX-loaded CNP were dispersed in 7 ml of distilledwater at 25   C in thermally insulating glass container and placedwithin the coil of magnetic heater. Magnetic heater was operatedfor 1 h and UV visible spectrophotometer was used to determinethe amount of DOX release after every 10 min.  3.4. Magnetic capturing test  A simple magnetic capturing system (simulating a blood vessel)[25]wasusedtoanalyzethecapturerateofmagneticnanoparticlesunder an applied  fi eld, for drug targeting purpose. The apparatusconsisted of an injection pump, a silicon tube of inner diameter2 mm, a permanent NdFeB magnet (max.  fl ux density 0.1 T) anda 20-mL syringe as shown in Fig. 2. OA-MNP and CNP magneticsuspensions of concentration 10 mg mL   1 were prepared for thecapture tests. 20 ml of the magnetic suspension was placed in thesyringe and pushed through the tube. The tube size at the pole wasmuch larger than the arterioles (0.07 mm) found in the humanbody. The fl owratewas controlled bythe syringepump, whichwasset5mLmin  1 correspondingtoa fl owvelocityof10mms  1 insidea2-mmtube.Thedispersedsuspensionswerepumpedthroughthemagnetic  fi eld. The un-captured samples were collected in a bottlelocated outsideof themagneticsystem.The percentage ofcapturedparticles in the magnetic capture system was determined by theweight difference using UV  e visible spectrophotometer. 4. Results and discussions Magnetic nanoparticles were synthesized by co-precipitationmethod and surface-modi fi ed by oleic acid (OA). This surfacetreatment was necessary for two reasons: its role as an adhesive toprovide af  fi nity to thermo-responsive copolymer to attach withMNP and its role as a surfactant to reduce particles agglomerationwhich may occur due to dipole e dipole interactions andhydrophobic nature of MnFe 2 O 4 . Magnetic nanoparticles have highsurface to volume ratio and so they tend to reduce their surfaceenergy by forming clusters due to dipole e dipole interactions andvander-wall ’ s forces [26,27]. Stability and dispersion of particlescan be enhanced by coating the particles with surfactantslike oleic acid (OA), which is done in the current study. OAwas chemisorbed onto the surface of MnFe 2 O 4  nanoparticlesforming the  fi rst layer of surfactant. Afterward, a second layerof OA was adsorbed onto the  fi rst layer forming bilayerOA-modi fi ed magnetic nanoparticles (OA-MNP). Consequently,thermo-responsive copolymer PNIPAm-co-Am was precipitatedonto the OA-MNP by emulsion polymerization forming core e shellnanostructure. Ultimately, molecules of anticancer drugdoxorubicin (DOX) were loaded to copolymer, forming hydratedcomposite nanoparticles (CNP).XRD patterns of co-precipitated but uncoated magnetic nano-particles revealed the crystallization of MnFe 2 O 4  having averageparticlesize to bearound20nmascalculated byScherrer ’ sformula[4]. Fig. 3 shows the TEM morphology of the (DOX-loaded/ hydrated) CNP. Particles are of the spherical core e shell nano-structure.TheaveragediameterofCNPisabout45nm,outofwhichabout 18 nm corresponds to the magnetic core, which is in agree-ment with the particle size of OA-MNP calculated by Scherrer ’ smethod.Particlesappeartobewellseparatedasawhole.Sizeoftheparticles is signi fi cant for biomedical applications. The most suit-able particles size for cancer-cells penetration is below 10 nm, butthe particles of this size are rapidly cleared by the renal in fi ltrationand extravasations. Particles exceeding the size of 50 nm are fi ltered out by the kupffer cells of liver [18,28]. Therefore, the Fig. 2.  Magnetic capturing system (simulating a blood vessel) [25]. Fig. 3.  TEM image of (drug-loaded/hydrated) composite nanoparticles having core e shell structure. S.A. Shah et al. / Materials Chemistry and Physics 137 (2012) 365 e  371  367  appropriate size range of drug-loaded magnetic nanoparticles isabout 10 e 50 nm for magnetic hyperthermia applications [15], andthe composite particles in this work lie in this range. The presenceof organic compounds onto the MNP surface was further analyzedusing TGA operating in the temperature range of 25 e 500   C. Fig. 4shows that about 60% of the weight of composite particles was lostin this temperature range. The initial weight loss in the range 95 e 135   C may correspond to the evaporation of water that wasexpelled outof the copolymerchains above LCST. Weightreductionlater in the graph may be attributed to the elimination of organiccompounds DOX, OA and PNIPAm- co -Am. This suggests thataround 40% (wt) of the (hydrated) CNP was composed of magneticcore and the rest consisted of organic compounds & water.Fig. 5 shows the FTIR patterns of CNP. Absorption bands at1551 cm  1 and 1653 cm  1 may correspond to the amide-II C ] Ostretchbondsofthethermo-responsivecopolymerPNIPAm- co -Am. e CH 3  asymmetric stretch mode at 2970 cm  1 and amide-II N e Hstretch vibrations at 3292 cm  1 also may correspond to thecoated copolymer indicating its due presence. Absorption bands at588 cm  1 ,610cm  1 and630 cm  1 maybe assignedtoFe e Ostretchmode of tetrahedral and octahedral sites in MnFe 2 O 4  particles.Absorption bands at 1520 cm  1 and 1618 cm  1 may correspond toN e H bending and those at 3330 cm  1 may be assigned to H e Ostretching, all corresponding to doxorubicin.Thermo-responsive polymers are known for absorbingwater and swelling due to hydration below certain temperatureand releasing water to shrink above that temperature. Thistemperature is called  ‘ lower critical solution temperature ’  (LCST),below which polymer is hydrophilic but it becomes hydrophobicabove this temperature. LCST of PNIPAm- co -Am was estimated byheating the composite particles from 25   C to 50   C and studyingthe UV  e visible spectrophotometer data at various temperatures(Fig. 6). From 25   C to about 38   C, the plot is almost horizontalundergoing least absorption, indicating the copolymer to behydrophilic in this temperature range. A sudden rise in absorptionoccurs at 39   C which is supposed to be the LCST. Copolymerbecomes hydrophobic at this temperature and expels water bydehydrating. Due to the shrinkage of copolymer volume, itsporosity decreases and thus UV absorption increases for alltemperatures above LCST.Anticancer drug doxorubicin (DOX) was loaded onto thecomposite nanoparticles by incubating the particles in water-DOXsolution for 24 h at 25   C (below LCST) and UVS data of thesolution was taken after every 4 h. Fig. 7 shows the DOX-loadingvariation with time. Slope of the curve is maximum for the initial4 h suggesting that about 33% of the drug was loaded within the fi rst4h.AfterwardDOXloadingwaspassiveandalmostsmoothforlater hours. Fig. 4.  TGA graph of (drug-loaded/hydrated) composite nanoparticles. Fig. 5.  FTIR plot of composite nanoparticles. Fig. 6.  UVS absorption showing LCST of thermo-responsive copolymer. Fig. 7.  Drug loading in thermo-responsive copolymer in 24 h. S.A. Shah et al. / Materials Chemistry and Physics 137 (2012) 365 e  371 368  DOX release pro fi le was studied by incubating the compositenanoparticles for 24 h at three different temperatures, i.e., 25   C,35   C & 45   C. Fig. 8 shows the UVS plots of drug release at thesetemperatures. At 25   C (below LCST), maximum drug released after24hwas13%only,outofwhich7%wasreleasedwithinthe fi rst4h.At 35   C (below LCST), maximum drug released after 24 h was 22%,out of which 12% was released within the  fi rst 4 h. At bothtemperatures below LCST, DOX may have come out of the hydro-philic polymer due to the simple diffusion process. However, at45  C(aboveLCST),DOXreleasedafter24hwasabout70%,and60%of that drug was released within the initial 4 h due to the hydro-phobic dehydration of thermo-responsive copolymer. Afterward,the rate of drug release was passive and smooth probably due toadditional diffusion process.Fig.9showstheVSMplotsofOA-MNPandCNPinthemaximummagnetic  fi eld of 6 kOe. Saturation magnetization (Ms) of OA-MNPis about 60 emu/g, whereas that of CNP is about 24 emu/g. 6 kOeappears to be insuf  fi cient magnetic  fi eld to saturate all themagnetic moments in its direction. Saturation magnetization of CNP is even less than half of that of OA-MNP probably becauseamount of magnetic substance per gram of CNP is almost less thanhalf of magnetic substance per gram of OA-MNP. As discussedearlier, TEM data shows that average particle size of CNP is about45nm outofwhich magnetic core isof about 18 nmin diameter. Sothe values of Ms are in accordance with the amount of magneticsubstance in each material. Further, VSM plots don ’ t show anycoercive force (Hc) and remanance magnetization (Mr). Absence of Hc & Mr indicates that the magnetic particles are super-paramagnetic having single domain structure. MnFe 2 O 4  hasa mixed spinel structure represented by the structural formulaMn 1  d 2 þ Fe d 3 þ [Mn d 2 þ Fe 2  d 3 þ ]O 42  , where  d  is the inversion parameter[29].About20%oftheMn 2 þ occupyoctahedral(B)sitesandtherestoccupy tetrahedral (A) sites. The remaining tetrahedral and octa-hedral sites are occupied by Fe 3 þ . Strong A e B interactions give riseto ferrimagnetism.Magnetic heat-generation characteristics of OA-MNP & CNPwerestudiedbydispersingnanoparticlesinwaterandsubjectingtoalternating magnetic  fi eld of 7.2 kA m  1 and frequency 70 kHz for1 h. Fig.10 shows the temperature variation with time of magnetictreatment. Initial temperatures were 25   C for both observations.Temperature of magnetic  fl uid containing OA-MNP increased toabout 70   C in 60 min, whereas CNP caused a temperature increaseof about 50   C in the same time. Temperature of the CNP  fl uid roseto 45   C in about 27 min, which is an effective and appropriatetemperature for the localized hyperthermia treatment of cancer. Atthis temperature, various cellular damaging mechanisms mayoccurlikeapoptosis,proteindenaturationandDNAcrosslinkingetc[5 e 9]. Magnetic heat-generation can be further tailored by settingmore appropriate magnetic parameters like  fi eld strength,frequency of applied  fi eld and concentration of magnetic particles.As mentioned earlier, size of magnetic portion of nanoparticles isabout 18 e 20 nm forming a pseudo single domain structure. Due tothe absence of coercive force and remanance magnetization,particles exhibit superparamagnetism and heating occurs due toNéel and Brownian relaxations. Néel relaxation arises due to therapidly changing direction of the magnetic moments along theapplied  fi eld within the crystal lattice (internal dynamics).Anisotropyenergy that tries to orient the magnetic moments alonga particular axis within the crystal lattice actually hinders therotation of moments under alternating magnetic  fi eld. So the Néelheating occurs due to the energy dissipation in the reversibleprocess of relaxation of moments to their equilibrium positionsafter the removal of applied  fi eld. Brownian heating occurs due tothe physical rotation of the magnetic particles within a  fl uidmedium when alternating magnetic  fi eld is applied (external Fig. 8.  Drug release pro fi le at different temperatures. Fig. 9.  Magnetic pro fi le of OA-MNP and CNP. Fig. 10.  Heat generation of OA-MNP and CNP under alternating magnetic  fi eld. S.A. Shah et al. / Materials Chemistry and Physics 137 (2012) 365 e  371  369
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