Where van der Waals Force Balances the Magnetic In

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A Smart Magnetically Active Nano Vehicle for On-Demand Targeted Drug Delivery: where van der Waals force balances the magnetic interaction Sudipta Panja, Somnath Maji, Tapas K. Maiti, and Santanu Chattopadhyay ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b07706 • Publication Date (Web): 12 Oct 2015 Downloaded from http://pubs.acs.org on October 17, 2015

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ACS Applied Materials & Interfaces

A Smart Magnetically Active Nano Vehicle for On-Demand Targeted Drug Delivery: where van der Waals force balances the magnetic interaction Sudipta Panja a, Somnath Maji b, Tapas K. Maiti b and Santanu Chattopadhyaya* a b

Rubber Technology Centre, Indian Institute of Technology, Kharagpur-721302, India Department of Biotechnology, Indian Institute of Technology, Kharagpur-721302, India

ABSTRACT The magnetic field is a promising external stimulus for controlled and targeted delivery of therapeutic agents. Here, we focused on the preparation of a novel magnetically active polymeric micelle (MAPM) for magnetically targeted controlled drug delivery. To accomplish this, a number of superparamagnetic as well as biocompatible hybrid micelles were prepared by grafting four armed pentaerythretol poly(ε-caprolactone) (PE-PCL) onto the surface of Fe3O4 magnetic nanoparticles (MNPs) of two different range of sizes (~5nm and ~15 nm). PE-PCL (four armed) was synthesized by ring opening polymerization, and it has been subsequently grafted onto the surface of modified MNP through urethane (-NHCO-) linkage. Polymer-immobilized MNP (5 nm and 15 nm) showed peculiar dispersion behavior. One display uniform dispersion of MNP (5nm) while the other (15nm) reveals associated structure. This type of size dependent contradictory dispersion behaviors was realized by taking the van der Waals force as well as magnetic dipole-dipole force into consideration. The uniformly dispersed polymer immobilized MNP (5nm) was used for the preparation of MAPM. The hydrodynamic size and bulk morphology of MAPM were studied by DLS and HRTEM. The anticancer drug (DOX) was encapsulated into the MAPM. The magnetic field triggers cell uptake of MAPM micelles preferentially towards targeted cells compare to untargeted one. The cell viability of MAMP, DOX encapsulated MAPM and free DOX were studied against HeLa cell by MTT assay. In-vitro release profile displayed about 51.5% release of DOX from MAPM (just after 1h) under the influence of high frequency alternating magnetic field (HFAMF; prepared in-house device). The DOX release rate has also been tailored by on demand application of HFAMF. Keywords: Magnetic nanoparticles (MNP), van der Waal interaction, dipole-dipole interaction, on-demand drug release, and biocompatibility.

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INTRODUCTION The superparamagnetic nanoparticles (MNPs) have diverged range of applications in various facets, starting from engineering to biomedical aspects. MNPs can be used for

cell

separation, purification1, as a carrier of anti-bacterial polymer2, for magnetic targeted drug delivery (MTDD)3-6, in magnetic resonance imaging (MRI)7, in water harvesting8-9 and also in RNA fishing10. Now-a-days out of these, two most important biomedical applications, namely MTDD and MRI have gained utmost attraction in biomedical research. MNPs (Fe3O4) are extensively used for such applications because of their easy preparation (controlled size) and by virtue of their superparamagnetic nature. For MRI and MTDD, one can target the position of the MNP (or MNP tagged drug) or their magnetic moment can be manipulated by applying external magnetic field.11 These types of delivery systems can efficiently guide to reduce the side effects.12 Unlike other nanoparticles, MNPs have higher tendency to form agglomerated structures owing to their inherent cohesion due to magnetic attraction.13 The agglomeration leads to increase in an apparent size of the MNP. Increased size of MNP leads to the reduction of their circulation time in the bloodstream.14 Agglomeration may also cause the bio-elimination of MNPs by macrophages or reticuloendothelial system before it can reach the vicinity of the targeted site. To resolve this problem, MNPs are immobilized using biocompatible polymers.15-16 This technique can reduce the tendency of agglomeration of MNPs and thereby enhance the ability to carry drug molecules by the hybrid vehicles. Polymer-immobilized MNP has already been synthesized, and their dispersion behavior has also been investigated in one of our recent communication.17 Immobilization of polycaprolactone onto the surface of MNPs by click reaction improved their state of dispersion.18

Polycaprolactone immobilized MNPs synthesized by ring opening

polymerization (ROP) from ricinoleic acid modified MNP, presented successful attachments with polymer.19-20 The immobilization of caprolactone (by ROP) and 2-methoxymethacrylate (by ATRP) onto the surface of MNP exhibited core-shell morphology.21-22 The immobilization of hydrophilic poly(poly(ethylene glycol) mono methacrylate onto MNP surface resulted in non-cytotoxic and stable aqueous phase dispersed material for potential biological applications.23 Poly(ethylene glycol) methyl ether methacrylate immobilized MNP has been demonstrated to be a potential protein resistant nanoprobe for magnetic resonance contrast enhancement.24 Chitosan immobilized MNP offers excellent absorptivity for purification of the pollutants like perfluorinated compounds from water.25 MNP grafted with glycidyl methacrylate followed by dendritic PAMAM displayed fairly good dispersion 2 ACS Paragon Plus Environment

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characteristics.26 The hyperbranched poly(glycerol) grafted onto the surface of MNP by grafting from approach, provided a uniform dispersion of NPs.27 The polymer-immobilized MNPs have been presented as a material having antifouling surfaces and encapsulating layers.28-29 The literature is scanty on studies regarding colloidal stability and dispersion behavior of polymer-immobilized MNPs. However, a section of publication deals with dispersion characteristic of dendritic polymer grafted MNPs.26 Immobilization of polymer onto the surface of MNP is not the sole aspect of the problem (agglomeration). The change in saturation magnetization (Ms) of MNPs with variegated size scales is also to be considered with due importance. Different size of MNPs offers compatibility with different types of bioentities for instance protein (5-50 nm), the gene (2 nm wide and 10-100 nm long) and virus (20-450 nm).30 It‟s a common knowledge that MNP with high Ms leads to a higher extent of aggregation compared to others having lower Ms. For Fe3O4 based MNPs, the Ms values are largely controlled by the size factor. It has been reported that the reduction of particle size offers higher value of Ms.31 However, this trend reverses below the critical particles size (nearly10 nm).32-33 In the present work, we plan to prepare two different size range of MNPs. One of them having average size of 15 nm i.e. above critical size of superparamagnetism and other having size range of 5 nm (below the critical size). These prepared nanoparticles were immobilized by four armed PE-PCL through the urethane (-NHCO-) linkage separately. The structure of the synthesized PE-PCL was confirmed by 1H and 13C NMR spectra. The Immobilization of PE-PCL onto MNP surface was confirmed by FTIR, XRD, HRTEM, and XPS. Magnetic properties of both types of pristine MNPs and polymer immobilized MNPs were measured by using SQUID magnetometer. The size dependent dispersion behavior (from HRTEM) of both types of MNPs (15nm and 5nm) was realized by considering two fundamental forces, i.e., magnetic dipole-dipole force and van der Waals force, respectively. The thermal properties of polymer immobilized MNP were studied by DSC and TGA. The uniformly dispersed polymer immobilized MNP (5 nm) was used to prepare the MAPM. The hydrodynamic size and bulk morphology of MAPM were studied by DLS and HRTEM analysis, respectively. A model anticancer drug (DOX) was encapsulated into the MAPM. The magnetically targeted cell uptake of DOX encapsulated MAPM was studied by fluorescence microscopy. The cell viability of MAPM, DOX encapsulated MAPM and free DOX was studied against HeLa cell



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by MTT assay. The HFAMF triggered on demand release of the DOX from the MAPM was studied by UV-Visible absorption spectroscopy.

Scheme 1: Schematic representation of PE initiated four armed PE-PCL synthesis.

Scheme 2: Schematic representation of four armed PE-PCL immobilized MNP.

Scheme 3: Schematic representation of DOX-loaded MNP and DOX release under the influence of HFAMF. EXPERIMENTAL Materials N-methyl pyrrolidone, ferric chloride (FeCl3), ferrous sulphate (FeSO4), 25% aqueous NH3, tetrahydrofuran (THF), N-methyl pyrrolidon (NMP), ethanol, acetone, chloroform, hexane and methanol (analytical grades), were obtained from the Merck Chemicals Mumbai, India. 4 ACS Paragon Plus Environment

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Iron(III)acetylacetonate (Fe(acac)3), methylenebisdiphenyl diisocyanate (MDI) (mol. Wt. 250.25 g/mol), dibutyltin dilaurate (DBTDL) (mol. Wt 631.55 g/mol), pentaerythritol (PE), εcaprolactone (CL), tin(II)ethyl hexanoate (Sn(Oct)2), doxorubicin (DOX) and (3aminopropyl) tri methoxy silane (TMAS) were purchased from the Sigma-Aldrich, USA. Synthesis and modification of MNP (5 nm) MNPs were synthesized by the hydrothermal process (Scheme S1).17 Fe(acac)3 (1g, 4mmol) was dissolved in 20 mL of N-methyl pyrrolidone. The N2 gas was purged for 30 minutes to remove the dissolved O2 inside the solution. The reaction mixture was then refluxed for 1 h. Methanol (20 mL) was added to the reaction mixture as soon as the temperature dropped down to ambient temperature (25 ºC). Then the reaction mixture was centrifuged (3000 rpm for 5 minutes) to collect the precipitated particles. The precipitated particles were washed three to four times by acetone and dried in vacuum oven at 40 ºC at a reduced pressure of 400 mm Hg. Finally, the dried particles were modified by sol-gel method using TMAS as surface modifying agent. Synthesis and modification of MNP (15 nm) MNPs were prepared by the simple co-precipitation method. In a 250 mL round-bottom flask (RbF) fitted with mechanical stirrer, 0.33 g of FeCl3 and 0.28 g of FeSO4 (2:1 molar ratio) were dissolved in 50 mL deionised water. Then, after 1 h of N2 purging, the aqueous NH3 solution was added drop by drop into the solution and following this ethanol and TMAS mixture (10:1 volume ratio) added dropwise into it as shown in Scheme S1. The temperature of reaction mixture was maintained at 80 ºC and stirred at a high speed (approx. 1500 rpm). After 2 h of reaction, the heat source was removed, and stirring was continued up to next 6h. Finally, precipitated particles were washed with ethanol and acetone alternatively to remove excess TMAS. The washed particles were dried in a vacuum oven for overnight at 40 °C and then stored them in vacuum desiccators for long term use. Synthesis of four armed polycaprolactone (PE-PCL) The four armed polycaprolactone was synthesized by the simple ROP technique (scheme 1).34-35 In brief, required amount of initiator (PE) and monomer (CL) was taken into a 25 mL two neck round-bottom flask under dry N2 atmosphere. The reaction mixture was heated at 120 ºC until it formed a homogeneous mixture. The catalyst (Sn(Oct)2) was added to it. A number of the small aliquot of the reaction mixtures were taken out after certain time intervals for the checking percentage of conversion gravimetrically. The reaction was stopped after 95-97% conversion of monomer to polymer. Finally, the reaction mixture was dissolved



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in CHCl3 and precipitated into hexane. The precipitated polymer was dried overnight at 60 ºC under vacuum. Immobilization of PE-PCL chains (mol. Wt. = 13100 g/mol) onto modified MNP Grafting of PE-PCL onto MNPs (two different types) was performed by coupling with MDI (urethane linkage formation) as shown in Scheme 2. In a two neck round-bottom flask (RbF, 250 mL) 0.116 g of MDI was taken and dissolved in 30 mL of dry THF under N2 atmosphere. In another RBF (25 mL) 1g of PE-PCL polymer, required amount of MNP (5 nm or 15 nm) and 0.056g of DBTDL were dissolved in 10 mL of dry THF under N2 atmosphere. The above solution of MNP with polymer was sonicated for 1h. The homogeneous solution was taken into 10 mL glass syringe and added dropwise into the RbF, which already contained dissolved MDI. The temperature of the reaction mixture was maintained at 60 ºC during addition of homogeneous solution. The composition and constituents of the reaction are displayed in Table 1. The reaction was continued up to 6 h and then cooled to room temperature. Afterward, the reaction mixture was precipitated into cold methanol and collected by filtration. The filtrate was then washed with methanol (3 to 4 times). The precipitated product was then dried under vacuum at 70-80 ºC under reduced pressure. Preparation of magnetically active DOX-loaded polymer micelle The polymer immobilized MNPs were dispersed into DMSO at a concentration of 1mg/10μL. Required amount of DOX was added to the solution and stirred overnight under dark condition.36-37 100 μL of the above solution was then slowly added to 10 mL water under sonication at room temperature. The addition and sonication processes were continued for 15 minutes. The DOX molecules (hydrophobic) were entered into the hydrophobic PCL chains of the PE-PCL immobilized MNP (scheme 3). Finally, DOX-loaded magnetically active polymer nanoparticles was dialyzed (using dialysis bag of 3.5 kD cut off mol. Wt.) against water for 12 h to remove unbound DOX. The amount of DOX-loaded into the MAPM was determined by UV-Vis absorption spectra at 482 nm. A calibration curve is obtained by plotting λmax against different concentrations of DOX. The calibration curve was used later to calculate the DOX loading contents (DLC) and DOX loading efficiency (DLE) of MAPMs. The DLC and DLE were calculated by using the equations S4 and S5. Characterizations: The FTIR spectra of polymer immobilized MNP were recorded using a Perkin-Elmer FTIRspectrophotometer (model spectrum RX-I), within a range of 400-4000 cm-1 with a resolution of 4 cm-1. The structure of the synthesized polymer was established by 1H NMR (solvent: DMSO-d6) and

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C NMR (Solvent: CDCl3) by using 200 MHz Bruker Advance Instrument.


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The distributions of pristine MNPs and polymer immobilized MNPs were inspected by High Resolution (HR) TEM (JEOL 2000) operated at an accelerated voltage of 200 kV. The X-ray diffraction (XRD) analysis were performed on a Rigaku Dmax 2500 diffractometer with Co target (k = 0.179 nm) at 25 ºC. The samples were scanned from 2θ = 10–70º at the step scan mode (step size 0.038º, preset time 2 s) and the diffraction pattern was recorded using a scintillation counter detector. Differential scanning calorimetry (DSC) was carried out on a TA (DSC Q100 V8.1 Build 251) instrument at a heating rate of 20 ˚C/ minute under N2 atmosphere. In this case, the samples were heated form +25 ºC to +100 ˚C with a heating rate of 20 ºC/min then cooled down to −100 ºC with a rate 20 ºC/min and then heated again to +100 ºC with the same rate. The thermogram corresponding to the second heating cycle was used to estimate the Tm of the pristine and grafted polymers. We have calculated the percent of crystallinity by taking the sum of the areas appeared under the two melting peaks, and it was designated as „overall crystallinity. Thermogravimetric analysis (TGA) was implemented on a TA TGA Q50 V6.1 Build 181 instrument. In this case, a small amount of sample (approximately 8 mg) was heated from room temperature to 600 ˚C at a heating rate of 20 ˚C/min under N2 atmosphere. The change of weight of the samples with temperature was monitored. The magnetic measurements were accomplished on a SQUID magnetometer (MPMS; Quantum Design) under a magnetic field of 30 kOe at 27 ˚C. The composition of polymer immobilized MNP were recognized by X-ray photoelectron spectroscopy (XPS) [PHI 5000 Versa Probe II (ULVAC-PHI Inc., Japan)]. The experiments were carried out using a micro-focused (100 µm, 25W, 15 kV) monochromatic Al kα radiation of energy 1486.6 eV. DLS measurement was performed using a Malvern Nano ZS instrument. The experiment was conducted using a 4 mW He−Ne laser (λ = 632.8 nm) at 25 ˚C. In Zetasizer Nano ZS, the detector angle was fixed at 90˚. UV-Visible absorption spectra were recorded from 200 to 700 nm on the Perkin Elmer‟s Lambda 35 instrument at a slit width of 1 nm. Cytotoxicity assay The in vitro cytotoxicity of MAPM (with and without DOX loading) was investigated against HeLa cell lines by MTT assay. In MTT assay, HeLa cells were seeded in a 96 well plate at a concentration of 1×104 per well in 200 µL of MEM (with 10% FBS) complete medium. The cells were incubated in CO2 incubator and allowed to adhere for 24h. After this allotted time, used medium from each well were meticulously replaced by culture medium containing serial dilution of DOX-loaded and unloaded polymeric micelle ( in the order of µg/mL order). After next 24h of cell growth, 20 µL of MTT solution (at a concentration of 4 mg/mL in PBS) was added to each well. The added MTT was then reduced by mitochondrial 7 ACS Paragon Plus Environment


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dehydrogenase enzyme of cell and formed a frozen purple crystal. The frozen crystal was then dissolved by adding 200 µL DMSO per well and recorded the absorption at the wavelength of 595 nm. Scheme 4: Schematic presentation of cellular uptake study under the influence static magnetic field (average of 30 Gauss)

Cell uptake study The cell uptake study was performed by fluorescence microscopy. For fluorescence microscopy, HeLa lines were seeded into 24 well plates at a concentration of 1×105 per well with 1 mL of MEM (with 10% FBS) complete medium in a humidified incubator set to deliver 37 ºC and 5% CO2 atmosphere. The cells were cultured for 24h at 37 ºC. Afterward, culture medium were removed from each well and refilled with fresh culture medium containing free DOX, and DOX-loaded polymeric micelle at concentrations of 0.5 and 4.8 µg/mL, respectively. Then, cells were incubated at 37 ºC for predetermined time intervals of 0.5h, 2h, and 4h. To investigate the effect magnetic field on DOX-loaded MAPMs on cell uptake, one set of 24 well culture plates was placed under the influence of magnetic field (scheme 4). After the required time, the wells were washed thrice with PBS solution. The washed cells were then fixed with 4% paraformaldehyde for 20 min at room temperature followed by counterstaining the nucleus with DAPI (blue). Finally, cell images were captured by placing the well plates on Fluorescence microscope (Olympus, 1X81). Release study In order to determine the release kinetics, a 5 mL of DOX-loaded MAPM solution (at a concentration of 1 mg/mL) was placed inside the dialysis bag (cut off mol. weight 3.5 kDa). The dialysis bag was floated into a 50 mL beaker containing 25 mL PBS solution. The beaker was placed under the influence of 750 kHz frequency of alternating magnetic field at the field strength of 25 Gauss. The detailed instrumental setup is displayed in figure S5, S6, and S7. The experiment was carried out at 37 ˚C with a constant interval of magnetic field exposure time (say 5 minutes). The amount of drug release was quantified by taking the UV-Visible 8 ACS Paragon Plus Environment

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absorbance (at 488 nm) of the outer solution against time. To evaluate the concentration corresponding to the absorbance, we have used pre-determined calibration curve. RESULTS AND DISCUSSION Synthesis and modification of MNPs In situ synthesis and modification of MNPs (15 nm) was performed by the co-precipitation method. The hydrothermal method was used for the synthesis of small size MNPs (5 nm), and modification was achieved following the sol-gel method. The successful synthesis of MNPs (both 5 nm and 15 nm) was recognized by FTIR, HRTEM and XRD analysis. The FTIR spectra (figure S1) show two bands at 574 cm-1 and 3436 cm-1 that correspond to Fe-O and surface –OH stretching frequency of the unmodified MNP.

Figure 1: HRTEM photomicrographs of (a) single 15 nm MNP, (b) TMAS coated 15 nm MNP and (c) TMAS coated 5 nm MNP. TMAS modified MNPs showed a few additional bands at 1088 cm-1 and 3386 cm-1 corresponding to Si-O-Si and -NH- stretching frequency that are the characteristics of TMAS. HRTEM photomicrographs of both 15 nm and 5 nm TMAS modified MNPs are displayed in figure 1a-c. Figure 1a is the photomicrograph of a single MNP with size approximately 10 to12 nm. However, the average size of these particles has been found to be around 15 nm. Figure 1b shows that MNP particles are effectively coated with TMAS. The core-shell assembly of MNP is notified with inorganic core stabilized by organic TMAS shell. Interestingly, NPs do not appear to be very well separated from each other on the HRTEM photomicrograph at low magnification. However, by zooming the image, we get to see a clear separation of MNPs (figure 1b). Similarly, HRTEM photomicrographs of TMAS modified small size MNPs is shown in figure 1c that has an average size of 5 nm. X-ray diffraction patterns show (Figure S2) six characteristic peaks of Fe3O4 at 30.2°, 35.5°, 43.5°, 54.0°, 57.5° and 62.9° which correspond to (200), (311), (400), (422), (511) and (440) planes, respectively.38



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Synthesis of four armed PCL chains The successful synthesis of four armed poly(ε-caprolactone) was done by ROP technique. The synthesized polymer displays many signals in 1H NMR spectrum as shown in figure 2a. It shows a resonance at 4.3 ppm that corresponds to methylene group of the initiator (-CH2-). It also shows two peaks at 4.0 ppm and 2.3 ppm that are assigned to methylene protons of polymer backbone (-CH2O- and -COCH2- respectively).

13

C NMR spectrum of four armed

PCL is displayed in figure 2b. It shows a signal at 173.7 ppm corresponding to carbonyl carbon (C-3) of the ester group. It also displays some signals at 64.30 ppm, 34.27 ppm, 28.49 ppm, 25.68 ppm and 24.72 ppm corresponding to C-8, C-4, C-7, C-6 and C-5 of the repeating unit, respectively. A resonance signal at 43.7 ppm is also observed corresponding to tertiary carbon (C-1) of the initiator (PE). Some less intense peaks also appear (shown in the inset windows) which are assigned to their corresponding carbon atoms. FTIR and XRD analysis of PCL immobilized MNPs FTIR spectra of four armed PCL, MNP 1, MNP 3, MNP 5, and MNP 7 are displayed in figure 3. It shows an intense band at around 1729 cm-1 that corresponds to carbonyl stretching of the ester group of the polymer backbone. It also shows two consecutive bands at 2873 and 2940 cm-1 that are assigned to the symmetric and asymmetric stretching frequency of -CH2group. Two stretching bands appeared at 3366, and 3443 cm-1 are assigned to the -NH and OH stretching frequency of end group of the polymer. The FTIR spectra also reveal an interesting trend in the region of 3365 cm-1 (NH stretching). The intensity of –NH stretching frequency for a fixed amount of polymer gradually increase with increasing MNP content from MNP 1 to MNP 7.

Figure 2: (a) 1H (in d6 DMSO) and (b) 13C NMR (in CDCl3) spectra of four armed PE-PCL


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Table 1: Composition of polymer-immobilized 5 nm and 15 nm MNP Sample code

a

5nm

15nm

MNP content (%)

MNP (5) 1

MNP (15) 1

1

MNP (5) 3

MNP (15) 3

3

MNP (5) 5

MNP (15) 5

5

MNP (5) 7

MNP (15) 7

7

PEPCL( wt. in g)

1

MDI (wt.in g)

DBTDL 5% (w.r.t total wt.)

0.116

0.056

Xc a (%) of MNP 5nm 15n m 32 37 28

34

26

30

12

27

Determined from DSC by using ∆Hº = 139.5 J/g.

On the whole, the frequency of absorption of the polymer modified MNPs appears at lower wave number compare to free -NH2 stretching (3386 cm-1, figure S1). Thus from these two consecutive observations it can be inferred that PE-PCL polymer is successfully grafted onto the surface of MNPs. However, the appearance of the two humps at 3365 cm-1 (prominent for MNP 7) confirms the presence of a few number of free amine (-NH2) groups onto the surface of the polymer immobilized MNP. The X-ray diffraction pattern reflects (figure S3) gradual increase in intensity of peaks at 2θ value of 30.2°, 35.5°, 43.5°, 57.5° and 62.9° (characteristics of Fe3O4) with increasing MNP content from 1 to 7 wt%. This observation also substantiates the fact of immobilization of polymer onto the surface of MNP.39 XPS analysis of PCL immobilized MNPs A general survey of XPS from the binding energy of 90 to 750 eV for TMAS coated MNP and four armed PCL immobilized MNP (MNP 7, 15 nm) are shown in figure 4. Silica-coated MNP shows some signals at 97, 283, 399, 529 and 709 eV that are assigned to the Si2p, C1s, N1s, O1s, and Fe2p, respectively.40

Figure 3: FTIR spectra of (a) PE-PCL, (b) MNP 7, (c) MNP 5, (d) MNP 3and (e) MNP 1. The mentioned MNPs have an average size of 15 nm. 11 ACS Paragon Plus Environment


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But the survey spectrum of polymer modified MNP only displays two prominent signals at 283 and 529 eV that are assigned to the C1s and O1s of the polymer only. The absence of N1s and Fe2p signals of MNP core in the survey spectrum of polymer immobilized MNP clearly indicates the wrapping of polymer chains onto the surface of TMAS modified MNP. For further insight into semi-qualitative estimation of surface functionalities, high-resolution XPS (HRXPS) spectrum of each detected elements as presented separately. HRXPS spectrum of Si2p, C1s, O1s and Fe 2p from TMAS modified MNP are shown in figure 5.

Figure 4: Survey XPS spectra of the polymer immobilize MNP (15 nm) scanning from the binding of 90 to 750 eV.

Figure 5: High resolution XPS spectra (a) Si 2p,(b) C 1s, (c) O 1s and (d) Fe 2p of TMAS modified MNP (15 nm).


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The high-resolution spectrum of Si2p displays two prominent deconvoluted components at 99.6 and 100.2 eV, respectively which are assigned to the Si atoms from Si-C and Si-O of TMAS. The two prominent deconvoluted components at 282.6 and 283.7eV of C1s are also observed. These two peaks are assigned to the C atoms from C-Si and C-C of TMAS, respectively. HRXPS spectrum of O1s also shows two deconvoluted components at 528.4 and 529.8 eV that are denoted for O atoms from O-Fe and O-Si of TMAS coated MNP. The two major signals at around 709, and 722.5 eV are assigned to Fe2p3/2 and Fe2p1/2 of MNP core. The HRXPS spectra of polymer immobilized MNP are shown in figure 6. HRXPS spectrum of C1s displayed three prominent deconvoluted components at 282.8, 284.3, and 286.9 eV that are designated for C atoms from C-C, -CH2O- and -COO- of PCL main backbone.41 HRXPS spectrum of O1s exhibits three deconvoluted components at 528.6, 530.2 and 531.6 eV which are assigned to the O atoms from O-Si of TMAS, -COO- of PCL backbone and -OCONH-. The „–OCONH-‟ is formed when terminal -OH groups react with the -NCO groups of MDI. The absence of Fe2p and Si2p signals in the survey and HRXPS spectra signify the fruitful immobilization of four armed PCL chains onto the surface of TMAS modified MNPs. Thermal analysis of PCL immobilized MNPs The DSC thermograms of virgin polymer and polymer immobilized MNP are displayed in figure 7. From the DSC thermograms, it is observed that overall crystallinity of polymer gradually decreases with increasing MNP content (from MNP 1 to MNP 7) as displayed in Table 1. This is schematically depicted in figure 7c and d.

Figure 6: High-resolution XPS spectra (a) C 1s and (b) O 1s of PE-PCL immobilized MNPs. The four armed polymer can stack into a plane, and the plane can further stack with one another before immobilization of polymer onto the surface of MNPs (figure 7c). But after getting immobilized onto MNPs, the polymer chains cannot be able to stack as before (figure 7d). The loss of stacking ability leads to the reduction of the overall crystallinity of the 13 ACS Paragon Plus Environment


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polymer. The DSC thermogram displays two melting peaks, one at the higher temperature and other at the lower temperature. The first one is attributed to the assembly of end groups that can be realized as an intra-molecular organization (figure 7b). Second one is attributed to the main chain crystallization that can be termed as „intermolecular crystallization‟ (figure 7c).42 The melting peaks are shifted to lower temperature region because of immobilization of polymer chains onto the surface of MNPs, leading to the decrease in overall crystallinity of the polymer. From the TGA thermogram (Figure 8), it is noticed that thermal stability of virgin PE-PCL is higher (Tmax = 400 0C) compared to PE-PCL immobilized MNP. Usually thermal stability of the polymer increases by adding filler particles into the polymer matrix. As we immobilize the polymer onto the surface of the MNP, the thermal stability of polymer immobilized MNP decreases compared to that of the virgin polymer. The decrease in thermal stability can be rationally explained by considering the resultant decrease in overall crystallinity (Table 1) of the polymer during immobilization onto the surface of MNP. The above phenomenon leads to an increase in the number of amorphous chains those are susceptible to degradation at even lower temperature by allowing easier diffusion of degradation products.43 The amount of polymer immobilized onto the surface of MNP (i.e. grafting density) is calculated from the weight loss (Figure 9) in between the temperature range of 160 to 500 ˚C. The calculated grafting densities (from Equation S3) for both 15 and 5 nm MNPs are displayed in Table 2. The grafting density of both the particles gradually decreases with increasing MNP content from MNP 1 to MNP 7 caused by decreasing the number of polymer chains available per particle.

Figure 7: (a) DSC thermogram of pure PE-PCL and PE-PCL immobilized MNP. (b) Intra and (c) intermolecular crystallization represented schematically.(d) Schematic presentation of PE-PCL immobilized MNP. The mentioned MNP has an average size of 15 nm. 14 ACS Paragon Plus Environment

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It is observed that the grafting density on the surface of MNPs having an average size of 15 nm is higher compared to those having an average size of 5 nm for all cases. As grafting density on the 15 nm particles is higher compare to 5 nm particles, the colloidal stability of later is supposed to be lower. But in practice, 15 nm MNPs forms agglomerated structures. This agglomeration is due to the strong dipole-dipole interaction energy (105.85 kT) that subdues the steric effects. Table 2: TGA weight loss and determination of grafting density Sample

MNP (%)

MNP 1

1

96.4

96.4

28.6

17.5

MNP 3

3

95.4

95.7

22.2

14.6

MNP 5

5

93.6

94.3

15.5

10.8

MNP 7

7

89.2

92.1

8.7

7.5

a

Weight loss (%)a 15 nm 5 nm

Grafting density ( µ mol m-2) 15 nm 5 nm

Weight loss in between the temperature range of 160 to 500 ˚C

Figure 8: TGA thermogram of virgin PE-PCL and PE-PCL immobilized MNP. The mentioned MNP has an average size of 5 nm. HRTEM photomicrographs and intra-particles force of interaction The superparamagnetic nanoparticles have an inherent magnetic moment that assists in forming agglomerates. The extent of aggregation among the MNPs can be explained by the balance of two types of the intermolecular force of interaction namely, van der Waals and magnetic dipole-dipole (gravitational force is ignored, for simplicity). The aggregation tendency can be reduced by decorating the MNP with four armed polycaprolactone. Although 15 ACS Paragon Plus Environment


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large size (15 nm) MNPs are grafted more by the branched polycaprolactone chains, yet it shows some fairly higher extent of aggregation (figure 9 lower part). The aggregation comes into prominence with increasing MNP content (for a given weight percent of polymer). At higher MNP content, a few numbers of small size particles forms nano-colony that leads to the increase of effective particle size. Thus, by comparing the different dispersion behaviour, it can be inferred that polymer immobilized large size (15 nm) MNP shows prevalent aggregation (especially, at higher MNP content) compared to small size MNP (5 nm). The phenomena can be made clear by calculating van der Waals (Equation S1) and dipole-dipole interaction (Equation S2) energy for both sizes MNPs separately. Since, dipole-dipole interaction is more sensitive towards the radius of MNP compared to van der Waals interaction; hence dipole-dipole interaction becomes an imperative component in explaining this type of dispersion behaviour.44-45 The individual forces of interaction as calculated (from Equations S1and S2) are presented in Table 3. For the small size MNP (5nm), there is not so much difference between van der Waals and dipole-dipole (0.174 kT and 0.170 kT respectively) interactions energy. However, the van der Waals interaction energy is to some extent higher compared to the dipole-dipole interaction that constitutes the basis for uniform dispersion of such particles.

Figure 9: HRTEM photomicrographs of 5 (upper part) and 15nm MNP (lower part).The micrographs are presented as MNP 7, MNP 5, MNP 3& MNP1 (from left to right). Unlike small size MNP, large size MNP (15nm) has a very large difference between the values of dipole-dipole interaction energy and van der Waals interaction energy (105.85 kT and 16.66 kT, respectively). The above phenomenon would certainly favor the formation of 16 ACS Paragon Plus Environment

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aggregates for large size MNPs. The superparamagnetic nature of the polymer immobilized MNP and pristine MNP (in the inset windows) are displayed in figure 10. A very high value of saturation magnetization (Ms) (169.34 emu/g) is manifested by 15 nm pristine MNP compared to 5 nm MNP (Table 4). Because of the high value of Ms, the entire polymer immobilized 15 nm MNP exhibits associated dispersion behavior. A low value of Ms is displayed by all the polymer-immobilized MNPs compared to the pristine one. Magnetic measurement The Ms values of polymer-immobilized MNPs and pristine MNP are summarized in Table 4. Each kind (5 nm and 15 nm) of polymer-immobilized MNPs pursued a decreasing trend of Ms value on moving from MNP 7 to MNP 1. The increase of the amount of polymer onto MNP surface (i.e. grafting density) is the responsible factor for decreasing Ms value from MNP 7 to MNP 1. We have measured the Ms in emu/g unit. Hence, for a fixed amount of sample, with increasing the amount of polymer onto the surface of the MNP (from MNP 7 to MNP 1) the effective MNP content decreases (in the sample). This fact leads to the decrease of saturation magnetization. Table 3. Calculated values of intra-particles interaction energy. Assumption 5 nm

15 nm

Interactions energy (kT)

5 nm particles

15 nm particles

r = (5+3)=8

r = (15+1)=16

van der Waals

0.1745

16.66

& D=5

& D = 15

Magnetic dipole-dipole

0.170

105.85

D = particles size and r = Centre to centre separation distance between two particles Characterization of magnetically active polymer micelles (MAPM) Polymer-immobilized MNP 5 (5 nm) was mainly used for the preparation of MAPM due to its favorable dispersion characteristics, according to the procedure as illustrated earlier in the experimental section. Bulk morphology of polymer micelle was inspected by HRTEM analysis (fig. 11). Figure 11 shows a uniform appearance of MAPMs (as the uniform array).The black spots inside the MAPM are the signature of MNPs presents inside the core of polymer MAPMs. The average particles size of the MAPM is around 100 nm that is very close to the hydrodynamic radius (116 nm) of MAPMs as determined from DLS measurement (figure 11b). The MAPM shows the zeta-potential around -28 mV. The negative zeta potential signifies exposure of unused amine group (present onto the surface of the polymer immobilized MNP, discussed in FTIR section) from core to the outer surface 17 ACS Paragon Plus Environment


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during the preparation of MAPM. The MAPM gets stabilized by posting these amine groups towards the aqueous phase. The magnetic responses of MAPMs are shown in figure 11d. Figure 11c indicates the formation of a stable water dispersed MAPMs. However, MAPAMs are spontaneously attracted towards the magnetic fields from stable aqueous dispersions. The nanoscale MAPMs are found to be totally separated from the water within 3 h in presence of a bar magnet bar (figure 11d).

Figure 10: Magnetic measurement of (a) 15 nm and (b) 5 nm polymer immobilized MNP and pristine MNP (in the inset windows). 18 ACS Paragon Plus Environment

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Table 4: Saturation magnetization (Ms) of pristine MNP and polymer immobilized MNPs. Sample name Pristine MNP

Saturation magnetization (emu g-1) 15 nm 5nm 169.34 65.02

MNP 7

11.75

6.25

MNP 5

6.82

2.65

MNP 3

4.19

1.32

MNP 1

3.20

0.77

Cytotoxicity assay (MTT) The cell viability of free DOX, MAPM and DOX-loaded MAPM were checked by the MTT assay against the HeLa cells. The MTT assay was performed by incubating different concentrations of earlier mention components with the HeLa cells for 24h. Figure 12 displays the cell viability results of all these components together. MAPMs (prepared from MNP 1 and MNP 3) are showing a high degree of cell viability (about 90 %) even at higher concentration (100µg/mL). Both free DOX and DOX-loaded MAPM (prepared from MNP 1 and MNP 3) are showing high cytotoxicity i.e. low cell viability compare to the blank MAPM. Between free DOX and DOX-loaded MAPM, free DOX shows higher cytotoxicity compare to DOX-loaded MAPM.

Figure 11: (a) HRTEM photomicrographs of MAPM, (b) DLS and schematic presentation of MAPM, (c) water dispersed MAPMs at 0h and (d) after 3h in presence of external magnet. 19 ACS Paragon Plus Environment


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Figure 12(a) MTT assay of the above mention components shows in column chart model (b) MTT assay in line symbol format. Quick incorporation of free DOX into the cell as well as in the nucleus is the responsible factor for its high cytotoxicity. For DOX-loaded MAPM, DOX molecules are present in the micelle and take some extra time to diffuse from it. This time dependent movement of DOX molecules (present in the polymeric micelle) is the answerable reason for low cytotoxicity i.e. high cell viability of DOX-loaded micelles compare to free DOX one. The dose required for 50 % cell growth inhibition i.e. the IC50 values of DOX-loaded micelle, and free DOX are 4.47 and 0.54 µg/mL, respectively. These results manifest that DOX-loaded MAPMs are ready to incorporate into the cell revealing their expected biological action. Magnetic targeted cell uptake study To investigate the enhanced cell uptake study of DOX-loaded MAPM, one set of DOXpolymeric micelle treated cultured plate was placed directly under the influence of magnetic field (~average field of 30 Gauss) and the other set was kept as control (without magnetic field). Because of self-fluorescence (red color) property, intercellular distribution of DOX molecule can be prominently detected by fluorescence microscopy. Study was conduct by incubating both free DOX and DOX-loaded polymeric micelle with HeLa cell line in presence and absences of static magnetic field for 0.5 h, 2 h and 4 h, respectively as discussed in experimental section. In the absence of magnetic field, fluorescence microscopic image (figure 13) displays a very small intercellular uptake of DOX-loaded MAPM even after 4 h of incubation. While in the presence of magnetic field, DOX-loaded MAPM shows (figure 14) an effective intercellular uptake just after 0.5 h of incubation.


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Figure 13: Fluorescence microscopic images of HeLa cell treated with DOX-loaded MAPM in absence of static magnetic field where nucleus was stained by DAPI (blue). The mention scale bar is 40 μm. The intensity of red fluorescence implying the signature of DOX-loaded MAPM is gradually increased with time up to 4 h. The red fluorescence of DOX-loaded MAPM is significantly observed mainly in the cytoplasm but not in the nucleus. Appearance of DOX fluorescence mainly in the cytoplasm indicating the uptake of DOX-loaded polymeric micelle might be happening through endocytic pathway.46 In contrast, incubation of free DOX with the cell shows (Figure S4) significant red fluorescence in the nucleus just after 0.5h of incubation. This phenomenon indicates rapid internalization of free DOX molecule into the nucleus through passive diffusion mechanism.47 The MAPM with loaded DOX that is present in the cell media attracted strongly towards the magnetic field. The experience of strong magnetic attraction provides a quick contact of MAPM with the cytoplasmic membrane of the cell which then gets internalized into the cell through the endocytic mechanism. In the absence of magnetic field, the MAPM is present in suspended state in culture medium and required more time to get in contact with the cell membrane. Hence, shows very low internalization of DOX-loaded MAPM even after 4 h of incubation. Release study The anticancer drug, DOX was encapsulated into the MAPM according to the procedure as described in the experimental section. The amount of drug loaded into the MAPM was calculated by the Equation S4. The DLC of the MAPM prepared from the 5 nm MNP 1, MNP 3, MNP 5 and MNP 7 samples are 32%, 28%, 18% and 12%, respectively. 21 ACS Paragon Plus Environment


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Figure 14: Cell uptake study of DOX-loaded MAPM on HeLa cell in presence of static magnetic field where nucleus was stained by DAPI (blue) and scale bar is 40µm.

Figure 15: (a) The release kinetic of DOX-loaded MAPM prepared from MNP 5(5 nm) at 37 and 45 ˚C and (b) the release kinetics of MNP 3 and MNP 5(5nm) under the influence of HFAM F at 37 ˚C. The decrease in the trend of DLC from MNP 1 to MNP 7 is attributed to the decrease in the number of drug preserving branched PE-PCL chains per particles i.e. decrease in the grafting density (Table 2). The DOX-loaded MAMPs were placed under the influence of high frequency alternating magnetic field (HFAMF) according to the procedure as described in the experimental section. Under the influence of HFAMF, the superparamagnetic nanoparticles produce internal heat. Neel‟s relaxation is the cause of internal heat effect.48 The amount of heat generated is proportional to the strength and exposure time of the applied HFAMF.49 The 22 ACS Paragon Plus Environment

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amount of DOX released with time is displayed in figure 15. Figure 15a shows a cumulative release of DOX from MAPM at 37 and 45 ˚C. The release profile shows only 8.3 and 9.7 % (after 24 h) release of the DOX molecule at 37 and 45 ˚C, respectively. Therefore, the differences in release behaviors at two different temperatures are not remarkable. Thus, the Neel relaxation does not have a significant influence on drug release. A step up increase in the rate of DOX released under the influence of HFAMF is displayed in the figure 15b. After 1 h, MAPMs show about 51.5% and 29.1% release of DOX molecule from MNP 5 and MNP 3, respectively. The high Ms value of MNP 5 (compare to MNP 3, Table 4) offers the MAPM to experience more effect of HFAMF and releases high amount of DOX. The MAPM releases the DOX molecule by virtue of forming many pores on the wall of the micelle or by rupturing of the self-assembled structure by the application of HFAMF.50 The rate of the drug release (51.5% / 1 h) is significantly higher than those reported in literature.48-49 MAPM with such advantage of externally controlled on the DOX release rate is quite an advanced and superior for a patient compared to the other conventional methods used for drug delivery (diffusion, pH, thermal response, etc.). CONCLUSION The successful synthesis of four armed PE-PCL has been confirmed by 1H and

13

C NMR

spectrum. The four armed polymer chain could be efficiently immobilized onto the surface of MNP as confirmed by FTIR, XRD, HRTEM and XPS analysis. Large size (15 nm) polymer immobilized MNPs displayed associated structure because of their extremely high magnetic dipole-dipole interaction energy (105.8kT) compared to the van der Waals interaction energy (16.6 kT). In other words, small size (5 nm) polymer immobilized MNP displayed its colloidal stability because of comparable or slightly higher van der Waals interaction energy (0.174 kT) subduing the magnetic dipole-dipole interaction energy (0.170 kT). Although the amount of polymer immobilized onto the surface of 15 nm MNP (i.e. grafting density, determined from TGA) is higher compared to 5 nm MNPs, still it displayed associated structure owing to their extremely high magnetic dipole-dipole interaction energy due to their superparamagnetic nature. The decrease in overall crystallinity during immobilization is attributed to the loss of stacking ability of polymer chains. The appearance of two melting peaks (from DSC), one at higher and other at lower temperature is associated with intra and intermolecular organization of four armed PE-PCL chains, respectively. MAPM has been prepared successfully, and its magnetic activity has been realized in the presence of external bar magnet. HRTEM photomicrograph displayed a regular appearance of spherical MAPM 23 ACS Paragon Plus Environment


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having an average size of 100 nm that is at par with the hydrodynamic size (116 nm) evaluated from DLS measurement. Biocompatibility test provided information that all MAPM are showing biocompatibility above 90% even at higher concentration (100 μg/ mL). Selective and higher cellular uptake of DOX-loaded MAPM is observed for magnetically targeted HeLa cell line compared to the untargeted one. Plenty of DOX-loaded micelles enters into the cytoplasm of the magnetically targeted cell just after 0.5 h of incubation. In contrary, there is no significant uptake of loaded micelle for the untargeted HeLa cell line even after 4h of incubation. Unlike free DOX molecules, loaded micelles are mainly concentrated in cytoplasm indicating endocytic pathway that they have followed for entering into the cells. The MAPM shows a quick on-demand release of about 51.5 % DOX (just after 1h) under the influence of HFAMF. Hence, using such novel MAPM we can able to tailor the drug release rate according to the status of the patient (i.e. on demand) by switching „off‟ or „on‟ HFAMF device. ACKNOWLEDGEMENT SP is thankful to UGC, New Delhi (Ref. No. 20-06/2010 (i) EU-IV) for sponsoring the fellowship. ASSOCIATED CONTENT The supporting information contained the FTIR, XRD, calculation of inter-particle interactions and HFAMF instrumental setup. AUTHOR INFORMATION Corresponding author: Santanu Chattopadhyay (Email: [email protected]) REFERENCES 1.

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TOC


Where van der Waals Force Balances the Magnetic In

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