Thermo-Magnetoresponsive Dual Function Nanoparticles: An

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Thermo-Magneto-Responsive Dual Function Nanoparticles: An Approach for Magnetic Entrapable-Releasable Chitosan Chutamart Pitakchatwong, and Suwabun Chirachanchai ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14676 • Publication Date (Web): 03 Mar 2017 Downloaded from http://pubs.acs.org on March 10, 2017

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ACS Applied Materials & Interfaces

Thermo-Magneto-Responsive Dual Function Nanoparticles: An Approach for Magnetic Entrapable-Releasable Chitosan Chutamart Pitakchatwong,† Suwabun Chirachanchai*,†, § †

The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 103302, Thailand

§

Center for Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University,

Bangkok 10330, Thailand ∗To whom correspondence should be addressed. Tel.: 662-218-4134; Fax: 662-218-4134; E-mail address: [email protected].

ABSTRACT Magnetic polymeric nanoparticles can be used for selective binding in a magnetic field. However, as the magnetic nanoparticles (MAG) are stabilized with polymers, the separation of the MAG from the polymer chains after use is difficult. This work proposes a combination of a thermo-responsive polymer with MAG allows for the as-desired simple removal of MAG from the polymer chains. For this, chitosan (CS) was conjugated with thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) and antibody (Ab) together with the physisorbed MAG as a thermo-magneto dual functional material. The key synthesis steps are: (i) radical polymerization of NIPAM in the presence of mercaptoacetic acid so that the PNIPAM obtained contains terminal carboxylic acid groups (PNIPAM-COOH) (ii) the CSN-hydroxysuccinamide water-based system that allows conjugation of CS with PNIPAMCOOH in water at room temperature, and (iii) the weak interaction between MAG and the CS chain. As a model application, CS is conjugated with the anti-recombinant Leptospirosis Ab (rLipL32) to allow the selective binding and collection of the target antigen under the dual functions. This is the first demonstration of a simple but effective solution for MAG

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exclusion from the target molecules and will be practical for diverse applications, such as diagnosis, sensors, filtration, etc.

KEYWORDS: Magnetic

nanoparticles,

Thermo-responsive dual functions,

Poly(N-

isopropylacrylamide), Chitosan, Entrapable-releasable

1. INTRODUCTION Ferrous oxide nanoparticles or magnetic nanoparticles (MAG) have received much attention due to their unique physical properties, such as a high surface area and biocompatibility, as well as their magnetic field responsiveness.1 As the surface of MAG is the reactive oxide,2 the conjugation or coupling of MAG with specific functional molecules can generate target-specific responsive materials. In fact, as MAG tend to aggregate and precipitate, their coating with polymer chains, such as polyethylene glycol,1 polyvinyl alcohol,2 polyethylenimine,3 polylactic acid,4 alginate,5 poly (N-isopropylacrylamide) (PNIPAM)6 and chitosan (CS),7 either via weak interactions or covalent bonding, have been proposed to stabilize the MAG. The polymers on the MAG surface not only prevent particle aggregation but can also provide selective binding with target molecules through the conjugation of specific functional groups on the polymers. With respect to naturally abundant polymers for modifying the surface of MAG, CS is a good candidate. This is not only because of its bio-related properties, in terms of its biodegradability,8 biocompatibility and nontoxicity,9 but also the abundance of amino and hydroxyl side groups. The modification of the surface of MAG with CS yielded pH responsive CS- MAG,10 and a CS- MAG nanocomposite has been used for removing metal ions from water,11 while the CS oligosaccharide was shown to stabilize MAG for use in cancer treatment.12


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Despite a number of developments and the promising applications of polymeric MAG, including CS- MAG, one important consideration is how to exclude the MAG after the target molecules have been entrapped. For MAG covalently bonded with polymer, the cleavage of the MAG is traditionally achieved via elution buffers, such as KCl-HCl (pH 1.5)13 or lysis buffer (3 M NaI; 5 M urea; 40 g/l Triton X-100; 10 nM EDTA, 25 nM TrisHCl, pH 6.5).14 If the MAG was physisorbed with polymer, the MAG removal can be achieved by changing the specific properties of the polymer, such as the net charge, pH, etc.15 However, the trace amount of MAG left after this step can potentially obstruct the analysis of the target molecules.16 Alternatively, if the polymeric MAG had an additional thermo-responsive property, the efficient detachment or release of MAG from the polymer and target molecules should be possible. From this viewpoint, PNIPAM is a good candidate as it is water soluble below the low critical solution temperature (LCST) of approximately 32 °C and precipitates above the LCST due to changes in the hydrogen bond (H-bond) network leading to the phase transition from coil to globule.17,18 Many studies have been conducted on CS/PNIPAM/MAG composites with multi-responsive properties. These include composite CS/PNIPAM/MAG based nanohydrogels for hyperthermia treatment of cancer cells,19 and the formation of a core-shell structure of CS-PNIPAM copolymer functionalized MAG via chemical bonding for drug delivery.20 For these goals, the separation of the MAG from the polymer chains was not required. In contrast, the remove of MAG after use is an important issue for further analysis in diagnostic systems, and to the best of our knowledge has not been reported yet. Recently, the application of PNIPAM with MAG to entrap and separate bovine serum albumen (BSA), as a model protein, using the temperature-dependence of PNIPAM, was demonstrated.21 Although, only BSA was separated and the MAG still remained in the



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system with the polymer, nevertheless this showed the possibility of applying thermoresponsive polymers for the entrapment and release of MAG.

Scheme 1. Overview of the thermo-magneto-responsive CS showing the synthesis steps and a model application as a biosensor, and the steps of (A) entrapment and (B) release of the MAG.

Scheme 1 summarizes our concept of dual functional thermo-magneto-responsive polymeric MAG based on an example case of a CS biosensor. Here, CS was conjugated with PNIPAM and the rLipL32 antibody (Ab) to yield CSPNIPAM-Ab, so as to gain the LCST thermo-responsiveness as well as the ability to bind with the specific (target) antigen (Ag). The surface of the MAG is stabilized with CSPNIPAM-Ab via weak interactions, especially hydrophilic-hydrophobic interactions. After the MAG CSPNIPAM-Ab was selectively bound with Ag above the LCST (step A in Scheme 1), a temperature shift below the LCST induces the coil formation of the CSPNIPAM-Ab polymer chains while separating from the MAG.


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The subsequent collection of the MAG using a strong magnetic field allows the complete exclusion of the MAG from the system (step B in Scheme 1). The point to be considered here is how to design and synthesize CS with the dual responsive functions of being thermo-magneto-responsive. That is, the CS has to be conjugated with PNIPAM to obtain the thermo-responsiveness, while CS has to remain in a favorable condition for the weak interaction (H-bond, van der Waals and hydrophilichydrophobic interactions) with the MAG. In addition, CS needs to retain the reactive sites for introducing the functional molecules, in this case the rLipL32 Ab. Moreover, considering the PNIPAM phase transition in water, the thermo-responsive CSPNIPAM, has to be water soluble, but as the application of CS in bio-systems is attractive, it is ideal that all the synthesis steps are in water to avoid residual toxic solvents. Based on the above concept, we aimed to develop thermo- and magneto-responsive CS. This report details the synthesis steps of the MAG CSPNIPAM-Ab, all of which were in water, and its detailed structural characterization. The work also demonstrates the use of the dual functional MAG CSPNIPAM-Ab as a biosensor to selectively collect and identify the target antigen (Ag), in this case recombinant Leptospirosis Ag.

2. RESULTS AND DISCUSSION: Preparation of Thermo-responsive CS and its Characterization. Thermo-responsive CS was synthesized in three steps using a modified method of the published protocol (Scheme S1).20 As CS is insoluble in organic solvents except acid, the thermo-responsive CS was prepared in known acidic conditions. For example, the conjugation of CS with carboxyl group terminated PNIPAM (PNIPAM-COOH) using conjugating agents in 2-morpholinoethane sulphonic acid (MES) was reported,22 while the Click between alkyne CS and azide terminated PNIPAM has been performed in an acidic



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buffer.23 Since the solubility of CS in water with an equimolar amount of conjugating additives, such as N-hydroxysuccinimide (NHS) and hydroxybenzyltriazole (HOBt) have been reported previously,24 this work applied this condition so that the conjugation of CS with PNIPAM-COOH could be achieved in water. Grafting PNIPAM onto CS was expected to result in different LCST values from the hydrophilic-lipophilic balance (HLB). In order to graft PNIPAM onto CS, the NIPAM monomer was primarily polymerized under a trace amount of mercaptoacetic acid with 2,2azobis(2-methylpropionitrile) (AIBN) as the initiator. In this way, the radical polymerization leads to PNIPAM-COOH, which can then be conjugated onto CS in water in the next step. From nuclear magnetic resonance (NMR) analysis, the disappearance of the protons belonging to the vinyl group (CH2=CH2) at 5.4 ppm and 6 ppm confirmed the success of PNIPAM-COOH polymerization (Figure S1B). The average molecular weight of PNIPAMCOOH, as determined by the matrix assisted laser desorption ionization-time of flight mass spectrometry analysis, was 2,967 (Figure S2). In order to identify the terminal COOH group, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR; HMBC-NMR) was applied to find the correlation between the protons at 1.9 ppm (methylene group of PNIPAM) and carbon at 172 ppm (amide group) (Figure S3). The conjugation of CS with PNIPAM-COOH was successfully performed by dissolving CS in water containing three equimolar NHS (Figure S4), which favors the CS conjugation with the functional molecules via amide and ester bonds in water.


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Figure 1. Representative (A) FTIR spectra of PNIPAM-COOH and CSPNIPAM34, (B) 1HNMR spectrum of CSPNIPAM34, and (C) HMBC 2D NMR spectrum of CSPNIPAM34.

The structural analyses of CSPNIPAM by FTIR (Figure 1A) revealed the peaks at 1640 cm-1, 1550 cm-1, 1387 cm-1 and 1367 cm-1 that correspond to C=O stretching (amide I),



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N-H (amide II) bending and two C-H bond vibrations (methyl group of PNIPAM), respectively. Note that the characteristic pyranose peaks in the 890–1100 cm-1 range became broad when the degree of substitution (%DS) of PNIPAM on CS was above 20% (Figure S5A). By changing the molar ratio of CS and PNIPAM, the %DS of PNIPAM can be varied and was quantitatively analyzed by the integral ratio between the proton at 2- position of CS at 3.1 ppm and the proton of CH2 (e position) in PNIPAM. For example, a 1:5 molar ratio of CS: PNIPAM resulted in a 34% DS (CSPNIPAM34). Here, CS with a %DS of PNIPAM of 7% (CSPNIPAM7), 28% (CSPNIPAM28) and 42% (CSPNIPAM42) were prepared (Figure S5B) as detailed in Table S1. The chain length (CL) of PNIPAM was determined by 1HNMR and is shown in Table 1. The HMBC-2D NMR was applied to confirm the covalent bond between CS and PNIPAM using the correlation between the protons at 3.8 ppm, corresponding to the proton of PNIPAM (at f position), and the carbons at 70 ppm, corresponding to the carbon of pyranose ring at C3, (Figure 1C).

Investigation of the Thermo-responsive Properties. The LCST of the copolymer was determined as a function of the temperature from the midpoint of the increasing hydrodynamic diameter, as measured by dynamic light scattering (DLS), and is shown in Figure 2A. The DLS measurements were conducted from 15 °C to 55 °C. A series of CSPNIPAM with different %DS of PNIPAM were prepared and their LCSTs were investigated. The CSPNIPAM7 showed a narrow phase transition that was hard to recognize as the LCST, but the size changed from 40 to 300 nm. However, in the cases of CSPNIPAM28 and CSPNIPAM34, the LCSTs were confirmed at 32 °C and 34 °C, respectively, where their sizes dramatically changed from 30 nm and 40 nm to 1.5 µm and 4.5 µm, respectively. Note that CSPNIPAM42 had a LCST of 32 °C, which is lower than 8 ACS Paragon Plus Environment

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those for CSPNIPAM34 and CSPNIPAM28 (Table 1), and leads to the question of what are the factors required to fine tune the LCST?

Figure 2. (A) Log scale of the hydrodynamic diameter of CSPNIPAM7, CSPNIPAM28, CSPNIPAM34 and CSPNIPAM42 (prepared in 6 mg/mL aqueous solution) as a function of the temperature (CL of PNIPAM 1,000–2,000) and the appearance of the suspension. (B) Representative TEM images of the CSPNIPAM28 obtained at (a) 4 °C and (c) 50 °C. Scale bars are (a) 200 nm and (c) 5 µm. Histogram of the anhydrous particle size distribution of CSPNIPAM28, as obtained from TEM images, at (b) 4 °C and (d) 50 °C.



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Table 1 shows that the %DS and CL of PNIPAM are related to the LCST values. Here, the HLB, as determined by the Griffen equation25 shown in Eq. (1), was applied. In this way, both the %DS and CL were taken into consideration as factors to control the LCST. ×

=

%

,

(1)

where Mh is the molecular mass of the hydrophilic portion of the molecule, and M is molecular mass of the whole molecule.

Table 1. Chain length (CL) of PNIPAM, HLB and the LCST of CSPNIPAM LCST CSPNIPAM DS

PNIPAM CLa

HLBb (°C)c

a

CSPNIPAM7

8

3.6

30.5

CSPNIPAM28

17

6.2

32

CSPNIPAM34

28

6.8

34

CSPNIPAM42

11

6.1

32

CSPNIPAM25d

15

5.9

31

CSPNIPAM25d

56

7.0

34

CL was calculated from the 1H-NMR spectra in D2O; bHLB was calculated using the Griffen

equation; cLCST was measured from the midpoint of the increasing hydrodynamic diameter measured by DLS. dThe structural confirmation is shown in Figure S6.

For CSPNIPAM, the hydrophilicity was based on the amino and hydroxyl groups of CS in addition to the acrylamide group of PNIPAM, whereas the lipophilicity was based on the isopropyl units of PNIPAM.26 The HLB values are summarized in Table 1, and their calculation is shown in Table S1. It is clear that the higher the HLB value, the higher was the 10 ACS Paragon Plus Environment

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LCST of CSPNIPAM. A high HLB implies a high level of hydrophilicity and so the hydrophilic interactions are dominant due to the strong interactions with water molecules. The transmission electron microscopy (TEM) images were also used to observe the size and the morphology of the anhydrous particles. For example, in the case of CSPNIPAM28, spherical shaped particles with an average diameter of about 50 nm were observed at 4 °C (below LCST) (Figure 2B (a)) while they became as large as 1.5 µm at 50 °C (above the LCST) (Figure 2B (c)). Obviously, self-aggregation due to the likely hydrophobic interactions leads to the larger spherical inclusions. This indicates that at this level of %DS of PNIPAM, the PNIPAM is in a favorable condition to initiate the non-aggregation and aggregation of CSPNIPAM depending on the LCST, respectively. Figure 2B (b, d) shows the size distribution of CSPNIPAM28 under each condition. The narrow size distribution at 4 °C implied a good interaction with water molecules. The distribution observed here is also in an agreement with that obtained from the DLS analysis (SD = ± 10). In contrast, above the LCST the size distribution was broad, implying a random aggregation of CSPNIPAM28.

Optimal CSPNIPAM: MAG ratio for the Entrapment and Release of MAG. As CSPNIPAM was designed to entrap MAG by weak interactions, the optimization of the ratio of MAG and CSPNIPAM was attempted. For example, in the case of CSPNIPAM28, quantification of the optimal conditions was performed as follows. The MAG and CSPNIPAM28 were mixed and stirred for 5 min. The MAG were collected with a strong magnet leaving the residual solution that was then heated to 40 °C (above the LCST) and the turbidity of the solution was determined by measuring the absorbance at 650 nm (A650) in comparison to the calibration curve (Figure S7) to obtain the amount of residual CSPNIPAM28 and hence the efficiency of MAG entrapment.



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Figure 3. Amount of residual CSPNIPAM28 in the solution with (A) different CSPNIPAM28: MAG (w/w) ratios at a temperature (●) above the LCST and (○) below the LCST, and (B) MAG collected from CSPNIPAM28 at 6 °C and 40 °C for four cycles. Total concentration of CSPNIPAM28 was 2.8 mg/mL. Data were obtained from three independent experiments and are presented as the mean (± SD). When the ratio of CSPNIPAM28 to MAG was 1:10 (w/w), the solution after MAG collection was transparent. This means that almost no CSPNIPAM28 remained in the solution (∼0 mg/mL), as shown in Figure 3A. Thus, at this ratio all of the added CSPNIPAM28 interacted with the MAG and there was no excess CSPNIPAM28. In contrast, for a CSPNIPAM28: MAG ratio of 1:5 (w/w), a certain amount of free CSPNIPAM28 remained, as seen from the turbidity. It should be emphasized that the MAG were expected to 12 ACS Paragon Plus Environment

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be entrapped in CSPNIPAM under weak interactions, especially H-bonds and hydrophilic/hydrophobic interactions. The determination of the amount of MAG released from CSPNIPAM is also important and was evaluated as follows. The CSPNIPAM28 and MAG at a 1:10 (w/w) ratio obtained at 40 °C were collected by the strong magnet, yielding the MAG CSPNIPAM28. Deionized-water (DW) was then added to the MAG CSPNIPAM28 and the suspension was cooled to 6 °C and left for 20 min before separating the MAG with the strong magnet. The aqueous phase was collected and heated to 40 °C for 5 min prior to measuring the A650 to evaluate the amount of CSPNIPAM28 in the solution. Here, if the solution is turbid, it means that there are free CSPNIPAM28 (as a consequence of releasing MAG) in the solution. As expected, for a 1:10 (w/w) ratio of CSPNIPAM28: MAG, the solution had almost 100% of CSPNIPAM28 present (Figure 3A), indicating that the CSPNIPAM28 was completely released from the MAG. Thus, a 1:10 (w/w) ratio of CSPNIPAM28: MAG was optimal for entrapping and releasing the MAG completely. Note that CSPNIPAM28: MAG ratios above or below 1:10 did not show a complete capture and release. For example, at a 1:1 ratio of CSPNIPAM28: MAG, the CSPNIPAM28 entrapped MAG for only 20% but released almost 70%, while increasing the ratio to 1:20 led to almost 100% entrapment but an incomplete MAG release (Figure 3A). This might be due to the fact that those high ratios initiated the aggregation of MAG in CSPNIPAM28 leading to difficulty in the separation of MAG from CSPNIPAM28 at temperatures below the LCST. All the subsequent studies on CS as a biosensor (shown in the next section) were performed at this optimal 1:10 (w/w) ratio of CSPNIPAM28: MAG. The entrapment/release process was repeated over four cycles and was found to be reproducible (Figure 3B).



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Dual Functions of Thermo-Magneto responsive CSPNIPAM. Figure 4 allows visualization of the thermo- and magnetic-responsive CSPNIPAM28, through the studies on entrapable-releasable MAG of MAG CSPNIPAM28 at the optimal 1:10 (w/w) ratio. The MAG CSPNIPAM28 were left in phosphate buffered saline (PBS) at 40 °C for 5 min (Figure 4A (a-1)) before the strong magnet was attached aside (Figure 4A (a-2)). The solution remained clear after the MAG were entrapped (Figure 4A (a-3)), and after subsequent treatment above the LCST at 40 °C (Figure 4A (a-4)). Since CSPNIPAM28 is colloidal at a temperature at its LCST, this implies there was no residual CSPNIPAM28 in the solution and so the entrapment of MAG was complete (See Movie S1). The well mixed suspension of MAG with CSPNIPAM28 at 40 oC, as in Figure 4A (a1) above, was cooled down to 6 °C (Figure 4B (b-1)) and the MAG were removed by the strong magnet (Figure 4B (b-2)). The residual solution was collected (Figure 4B (b-3)) and heated to 40 °C, whereupon the solution became turbid (Figure 4B (b-4)) confirming the existence of CSPNIPAM28 in the solution and so the CSPNIPAM28 had released the MAG from their matrices (see Movie S2). Figure 4C demonstrates the possible mechanisms of the dual function MAG CSPNIPAM28. When the temperature was above the LCST, CSPNIPAM28 became hydrophobic and aggregated. At the same time, CSPNIPAM interacted with MAG via a Hbond network (Figure 4C (c-1)). By simply shifting the temperature below the LCST, CSPNIPAM28 became hydrophilic and interacted with water molecules becoming water soluble (Figure 4C (c-2)) and excluding the MAG from the CSPNIPAM28 assemblies. The TEM images of the solid phases, as shown in Figure 4D, support this mechanism. The samples in Figure 4D (d-1), and Figure 4D (d-2) were prepared by collecting the solid phase from Figure 4A (a-3) and 4B (b-3), respectively. It is clear that above the LCST, the solid phase showed well-separated particles, and the formation of MAG embedded inside the 14 ACS Paragon Plus Environment

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CSPNIPAM28 matrix. The black small particles represent the MAG surrounded by the grey area of CSPNIPAM28 (Figure 4D (d-1)). In contrast, the TEM image of Figure 4D (d-2) shows the small black particles without any area of polymer.

Figure 4. Entrapment-release of MAG CSPNIPAM28. (A) (a-1) Suspension of MAG and CSPNIPAM28 at 40 °C in water, (a-2) harvesting the MAG by a strong magnet, (a-3) collecting the water phase from a-2 and transferring to another vial and (a-4) the appearance of water from a-3 at 40 °C. (B) (b-1) The suspension of MAG CSPNIPAM28 after cooling from 40 °C to 6 oC in water, (b-2) harvesting the MAG by a strong magnet at 6 °C, (b-3) collecting the water phase from b-2 and transferring to another vial and (b-4) the appearance of water from b-3 at 40 °C. (C) Schematic representation of the reversible temperaturetriggered entrapment-release of MAG CSPNIPAM28, and (D) representative TEM images of the solid phase from (d-1) a-3 and (d-2) b-3.



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Observation of H-Bonds by Temperature Dependent FTIR and X-ray photoelectron spectroscopy (XPS). It is clear that at a temperature above the LCST, MAG were entrapped by CSPNIPAM28 whereas at a temperature below the LCST, the MAG were released from CSPNIPAM28. Temperature dependent FTIR was applied to trace the changes in the microstructure related to the MAG entrapment and release. After MAG were mixed with CSPNIPAM28 at a temperature above the LCST for a while, the MAG were recovered with the strong magnet, dried and analyzed by FTIR. The intensities of the bands due to O-H and N-H stretching (3400–3200 cm-1), amide I (1650 cm-1) and amide II (1560 cm-1) decreased and shifted to a higher frequency, indicating the disruption and weakening of the H-bonds with increasing temperature (Figure 5A).27,28 The interaction between CSPNIPAM28 and MAG at a temperature above the LCST was also confirmed by x-ray photoelectron spectroscopy (XPS) analysis.29 Figure 5B shows the peaks of binding energy N 1s of CSPNIPAM28 at 398 eV and 399 eV, which represent the amino groups involved in the H-bonded amine (H-NH2-) and amide (NH-CO), respectively. In contrast, the MAG CSPNIPAM28 expressed a new band of N 1s at 402 eV which was shifted to the higher binding energy and was attributed to the chelation between the amino groups and iron ions (-NH2-Fe).30 In fact, the H-bond can be described as an electron delocalization from the electron donor to electron acceptor.31 The transfer caused a decrease of electrons around the N atom resulting in the shifting to higher binding energy. Oxygen signals of CSPNIPAM28 were found at 530, 531 and 532 eV, which were assigned to -C=O, C-OH and O-C-O, respectively. In the case of MAG CSPNIPAM28, the O 1s peak was observed at 535 to 529 eV. All peaks were shifted to a higher binding energy, including a new dominant peak at 531 eV belonging to Fe-O, confirming the H-bond formation between MAG and CSPNIPAM28.


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Figure 5. (A) Temperature-dependent FTIR spectra of CSPNIPAM28 and MAG CSPNIPAM28 in the range of 55–120 °C at a wavenumber range of (a) 2800–3600 and (b) 1400–1700. (B) High-resolution XPS spectra of the (a) N 1s region for CSPNIPAM28, (b) N 1s region for MAG CSPNIPAM28, (c) O 1s region for CSPNIPAM28 and (d) O 1s region for MAG CSPNIPAM28.

Entrapment/Release Evaluation by DOSY NMR. Although the interactions of MAG with CSPNIPAM28 were observed by turbidity, temperature dependent FTIR and XPS, we also 17 ACS Paragon Plus Environment


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attempted to support it by NMR. However, it is important to note that NMR cannot be used to analyze MAG due to their magnetic property. Thus, 3 nitrosalicylic acid (3-NiSA) was selected as a model molecule that can form a H-bond with CSPNIPAM in place of the MAG because its structure provides functional hydroxyl groups to interact with CSPNIPAM via Hbonds. The CSPNIPAM28 was mixed with 3-NiSA at 15 °C, which was below the LCST. Figure 6A shows two distinct diffusion coefficients (D) at 3.981 x 10-10 m2s-1 (log D =-9.4) and 1.995 x 10-11 m2s-1 (log D =-10.7), from the clearly separate free 3-NiSA and CSPNIPAM28, respectively. However, at a temperature at 40 °C (above the LCST) only one diffusion coefficient for both species, at 6.309 x 10-10 m2s-1 (log D =-9.2), was observed, confirming that CSPNIPAM28 entraps 3-NiSA when CSPNIPAM28 was above the LCST. This then provides indirect information to support the MAG entrapment and release by CSPNIPAM28.

Figure 6. Representative DOSY NMR spectra for a mixture of CSPNIPAM28 and 3-NiSA in D2O at a temperature (A) below the LCST (15 °C) and (B) above the LCST (40 °C). The horizontal axis represents chemical shifts whereas the vertical axis is the diffusion coefficient.


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Biosensor under Dual Functions of MAG-CSPNIPAM28. To emphasize the practical application of thermo-magneto-responsive MAG CSPNIPAM28, a model biosensor was studied. The monoclonal rLipL32 Ab (anti-recombinant Leptospirosis Ab) was conjugated onto

CSPNIPAM28

using

NHS/1-ethyl-3-[3-dimethylaminopropyl]

carbodiimide

hydrochloride (EDC) at 4 °C in water. The success of the conjugation was confirmed by sandwich ELISA based on the evaluation of the amount of Ab conjugated on CSPNIPAM28 using the horse radish peroxide conjugated secondary Ab, followed by 3, 3′, 5, 5′tetramethylbenzidine substrate (Figure S8 (d)). By comparing the Ab concentration of CSPNIPAM28 with CSPNIPAM28-Ab (Figure 7), it was clear that the Ab was successfully conjugated. The amount of Ab was quantitatively analyzed using a calibration curve of pure Ab (Figure S9A), and revealed that the conjugation efficiency of Ab onto CSPNIPAM28 was about 45%.

Figure 7. (A) Concentration of Ab as evaluated by ELISA technique and appearances of the samples (a) CSPNIPAM28, and (b) Ab conjugated on CSPNIPAM28, CSPNIPAM28-Ab 19 ACS Paragon Plus Environment


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(rLipL32: recombinant Leptospirosis antibody), and (B) Concentration of Ag as evaluated by ELISA technique and appearances of the samples (c) CSPNIPAM28-Ab (no Ag (control)), (d) CSPNIPAM28+Ag (no Ab conjugated on CSPNIPAM28 (control)) , and (e) CSPNIPAM28-Ab after treating with Ag, CSPNIPAM28-Ab-Ag. (C) Concentration of Ag as evaluated by ELISA technique and appearances of CS conjugated with MAG via covalent bond (represented as commercial product) (f) MAG-CS-Ab (no Ag (control)), and (g) MAGCS-Ab after treating with Ag, MAG-CS-Ab-Ag. Concentrations of Ab and Ag are 30 ppm, and 300 ng/mL respectively. Data were obtained from three independent experiments.

The CSPNIPAM28-Ab was added to the Ag (recombinant Leptospirosis Ag) solution followed by MAG to the system to obtain the MAG CSPNIPAM28-Ab-Ag. As shown in Scheme 1, the temperature was then shifted to above the LCST so that Ag entrapment was accomplished. After collecting the brownish colloidal particles of MAG CSPNIPAM28-AbAg, the solution was decanted, deionized water was added and the obtained suspension was cooled to below the LCST. The MAG were then harvested by the strong magnet. Figure 7B shows the specific binding of CSPNIPAM28-Ab, in comparison with the two controls of CSPNIPAM28-Ab (no Ag in the system) and CSPNIPAM28+Ag (No conjugated Ab). After treating with Ag, the specific binding of CSPNIPAM28-Ab with Ag can be observed, as seen from the concentration of Ag in the solution. Moreover, it was clear that after shifting the temperature to below the LCST, the MAG were separated. The solution showed an Ag content (detection level) of 46%. Comparison between CSPNIPAM28-Ab-Ag and MAG-CSAb-Ag revealed that although the concentration of Ag (amount of entrapped Ag) was the same in both cases, the background was much higher for the MAG-CS-Ab (Figure 7C (f)) than with CSPNIPAM28-Ab (Figure 7B (c)) and CSPNIPAM28+Ag (Figure 7B (d)). This implies that for Ag detection, the CSPNIPAM28-Ab is more convenient to apply as it does


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not need the background correction and so the data obtained is directly related to the amount of extracted Ag and the developed color is a primary result that allows confirmation of the existence of Ag by naked eyes.

3. CONCLUSIONS The present work demonstrated the design and synthesis of magneto-thermo-responsive CS nanoparticles. The radical polymerization of NIPAM in the presence of mercaptoacetic acid followed by conjugation onto CS-NHS in a water-based system led to CSPNIPAM. The CSPNIPAM had a LCST of 33 °C and the LCST could be fine-tuned by alteration of the %DS of PNIPAM and the PNIPAM CL (HLB value). By simply mixing the CSPNIPAM with MAG, it entrapped the MAG efficiently by H-bonds, as confirmed by the temperature dependent FTIR and XPS analysis. The evaluation of the remaining CSPNIPAM after removal of the MAG CSPNIPAM by a magnet allowed the determination of the optimal CSPNIPAM: MAG (w/w) ratio as 1:10. The MAG CSPNIPAM showed entrapablereleasable MAG by shifting the temperature below and above the LCST, respectively. Instead of mixing with MAG, the mixing with 3-NiSA enabled us to confirm the formation of Hbonds between CSPNIPAM and 3-NiSA using DOSY NMR. The work was extended to a demonstration of a biosensor to show the simple, effective and efficient Ag collection by MAG CSPNIPAM-Ab. The conjugating of the rLipL32 Ab (anti-recombinant Leptospirosis Ab) on CS-PNIPAM followed by mixing with MAG yielded the MAG CSPNIPAM-Ab probe for specific interaction with the Ag. Shifting the temperature led to the release of the MAG and the isolation of the CSPNIPAM-Ab-Ag, allowing the analysis of Ag without any MAG contamination. As demonstrated in this work, the dual functions of thermo- and magneto-responsive MAG CSPNIPAM not only showed its advantages in MAG entrapment-



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release but the CS retains a certain amount of functional groups (NH2 and OH) to further modify with specific molecules, such as a specific Ab, leading to various targeting MAG.

4. EXPERIMENTAL SECTION Materials. The CS (95% DD, Mw of 7.0 x 105) was supplied by Seafresh Chitosan (Lab) Co., Thailand. Hydrochloric acid (HCl; 37%), toluene, acetone, ethanol, hexane, tetrahydrofuran, and sodium hydroxide (NaOH; 99%) were bought from RCI Labscan, Thailand. The EDC was purchased from TCI, Japan. Ammonium hydroxide (NH4OH), dimethylformamide (DMF),

ferric

chloride

hexahydrate

(FeCl3·6H2O),

ferrous

chloride

tetrahydrate

(FeCl2·4H2O) and NHS were purchased from Merck, Germany. Acetic acid and sodium acetate were purchased from Univar, Australia. Oleic acid, NIPAM and thioglycolic acid were purchased from Sigma-Aldrich, Inc., USA and recrystallized in hexane before use. The free radical initiator AIBN was bought from Fluka, purum and recrystallized in methanol before use. Dialysis was performed using a cellulose membrane with a molecular weight cutoff of 12,000 g/mol. All chemicals were analytical grade and used without any purification unless specified. The anti-recombinant Leptospirosis Ab (rLipL32) and Leptospirosis Ag were from Faculty of Medicine Chulalongkorn, University.

Preparation of MAG. The MAG were prepared as previously published.32 Briefly, FeCl3·6H2O (6.77 mg) and FeCl2·4H2O (2.50 mg) were dissolved in deionized water at a 2:1 (w/w) ratio under a nitrogen (N2) atmosphere. After 30 minutes, 25% NH4OH solution (14 mL) was added dropwise within 15 minutes to the mixture and then heated at 85 ˚C for 1 h. Ammonium ions were removed from the reaction mixture by dialyzing against deionized water before freeze-drying to obtain black particles of MAG. The morphology and size of the MAG are shown in Figure S10.


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Synthesis of CSPNIPAM. Radical polymerization of PNIPAM-COOH was performed as previously reported.22 The recrystallized NIPAM monomer (0.505 g, 4.463 mmol) was added in a round-bottom flask followed by adding AIBN (0.040 g, 0.246 mmol) in toluene (10 mL). The mixture was degassed by purging with N2 for 30 min. The polymerization was performed at 80 °C for 12 h followed by adding mercaptoacetic acid (35 µL, 0.492 mmol) and the reaction proceeded for 12 h. The PNIPAM-COOH was obtained by precipitating in diethyl ether before drying under vacuum (yield 75%). Next, CS was conjugated with PNIPAMCOOH as follows: CS (1 wt%, 0.5 g, 3.049 mmol) was mixed with NHS at three molar ratios to CS (1.388 g, 9.146 mmol) in deionized water before adding dropwise to the PNIPAMCOOH (6.098 g, 3.049 mmol) with EDC (1.753 g, 9.147 mmol) in deionized water at 4 °C. The conjugating reaction proceeded for 24 h at 4 °C and the resulting product was dialyzed against DW to remove impurities and unreacted reactants.

Characterization. The FTIR spectra were collected using a Bruker ALPHA Fourier transform infrared spectrophotometer, and

1

H-NMR spectra were obtained from an

Ultrashield 500 Plus Bruker spectrometer (500 MHz). The anhydrous size and morphology of the samples were observed by TEM using an H-7650 Hitachi transmission electron microscope. The DLS was obtained using a Malvern Zetasizer Nano ZS at various temperatures. The H-bond between CSPNIPAM and MAG was analyzed by XPS using a Kratos Axis Ultra DLD X-ray photoelectron spectroscope (Shimadzu, Japan) using monochromatic Mg KR radiation (1253.6 eV) The binding energy was referenced at 284.6 eV to the C 1s peak, and the high-resolution scans of core level spectra were set to 15 eV pass energy.



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Immobilization of Ab onto CSPNIPAM. The Ab solution (30 ppm) was mixed with CSPNIPAM28 (20 mg) in PBS buffer (pH 7) at 4 °C. Then EDC (17 mg) and NHS (17 mg) were added to the mixture and stirred for 24 h at 4 °C to avoid denaturing the Ab, and then dialyzed well before use. Sandwich ELISA, as shown in Figure S8 (a), was used to determine the conjugation efficiency of the Ab.

Detection of Ag by MAG CSPNIPAM-Ab. The efficiency of Ag (recombinant Leptospirosis) detection was evaluated using sandwich ELISA as shown in Figure S8 (b). For assays, Ag (300 ng/mL) was spiked into the solution that contained a mixture of CSPNIPAM28-Ab (5 mg/mL) and MAG (50 mg). The total volume after adding all the reagents was 1 mL. For Ag-Ab separation, the sample mixture was incubated at 40 °C for 5 min and then the MAG were pulled out using the high power magnet. Next, the supernatant, which consists of impurities and unreacted reactants, was cautiously removed and the MAG CSPNIPAM28-Ab-Ag was re-dispersed in 1 mL PBS buffer (pH 7). The temperature was shifted to 6 °C for 20 min before using a strong magnet to entrap the MAG. The supernatant was removed and sandwich ELISA used to determine the efficiency of Ag detection.

Entrapable and releasable MAG-CSPNIPAM. The CSPNIPAM (2.8 mg) was dissolved in deionized water (1 mL) at 6 °C and mixed with MAG at various weight ratios. The mixture was heated to 40 °C and incubated for 5 min. The aggregated MAG CSPNIPAM was entrapped with the magnet and separated from the supernatant. The supernatant was collected and heated to 40 °C before measuring the A650. For the release cycle, deionized water was added to the entrapped MAG CSPNIPAM and the temperature was decreased to 6 °C and incubated for 20 min. The MAG were entrapped with a strong magnet and the supernatant


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was removed and heated to 40 °C prior to measuring the A650. The amount of CSPNIPAM entrapped and released was then calculated from the A650 calibration curve.

ASSOCIATED CONTENT Supporting Information The Supporting Information Available: Additional information on structural characterization; the calculation of HLB; Calibration curve of the turbidity; method applied to qualify/quantify of antibody and antigen. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail address: [email protected]. Conflict of Interest: The authors declare no competing financial interest. ACKNOWLEDGEMENTS The authors would like to thank Assistant Professor Dr. Kanitha Patarakul, and Mr. Prayoon Lae-ngee, Faculty of Medicine, Chulalongkorn University for the Ab and Ag. The authors acknowledge The Royal Golden Jubilee,

Thailand Research Fund (Grant No.

PHD/0124/2557), and the National Research University Project, Office of Higher Education Commission (WCU-58-015-FW) for the research funds. The authors extend their special thanks to Professor Ica Manas-Zloczower, Macromolecular Science & Engineering, Case Western Reserve University for valuable comments and English corrections. REFERENCES 1.

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Table of Contents (TOC)


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Thermo-Magnetoresponsive Dual Function Nanoparticles: An

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