Temperature-switched controlled release nanosystems based on molecular recognition and polymer phase transition

Abstract

Environmental stimuli-responsive nanosystems that can release their encapsulated or loaded substances in spatial-, temporal- and dosage-controlled fashions have attracted tremendous attention, especially from the field of nanomedicine. In this work, a novel temperature-switched controlled release nanosystem based on the molecular recognition of beta-cyclodextrin (β-CD) and thermosensitivity based on the phase transition of poly(N-isopropylacrylamide) (PNIPAM) is developed. This smart nanosystem consists of magnetic Fe₃O₄ colloidal nanocrystal clusters (MCNCs) core grafted with linear PNIPAM chains attached to numerous β-CD units, (denoted as Fe₃O₄@PNG–CD). β-CD units act as containers for the loading of hydrophobic model drugs, PNIPAM chains serve as intelligent “microvalves” for controlling the release of loaded model drugs, and MCNCs core can achieve a “site-specific targeting” function. The gating mechanism involves that temperature can significantly affect the association constants of β-CD toward model drug molecules in β-CD modified PNIPAM chains. The resultant Fe₃O₄@PNG–CD nanoparticles have excellent thermo-reversible adsorption/desorption properties for the loaded model drug 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS), and demonstrate a positive temperature “on–off” release fashion. By simply changing the temperature, the delivery rate can be effectively controlled. At temperature above the lower critical solution temperature (LCST) of the grafted PNG–CD chains (open gate), the delivery rate of ANS is fast, but very slow at temperature below the LCST (closed gate). These multifunctional nanoparticles provide a new mode for designing and engineering intelligent controlled release nanosystems.

---

1. Introduction

Environmental stimuli-responsive nanomaterials that can release their encapsulated or loaded drugs or substances in spatial-, temporal- and dosage-controlled fashions via a physical stimulus (e.g. variations in temperature, magnetic field, ultrasound intensity, light or electric pulse),^1–11 or a chemical stimulus (e.g. changes in pH, enzyme concentrations or redox gradients),^1–7,9–15 have attracted tremendous attention in recent years. Magnetic colloidal nanocrystal clusters (MCNCs) formed by self-assembly of numerous iron oxide (typical Fe₃O₄) nanocrystals into submicrometer scale particles are especially attractive, due to their high magnetic susceptibility as well as low coercive and superparamagnetism.^16–26 MCNCs, when used as drug carriers, have many prominent advantages compared with other drug carriers (liposome and small molecules and so on),^27–30 such as high drug loading efficiency, low cytotoxicity, excellent biocompatibility and reproducibility, easy control for particle size, and simple and facile preparation, especially unique magnetic responsive properties, enable them to be easily guided to targeted organs or tissues inside the body by an external magnetic field.^1–3,16 This is greatly beneficial to protect the body tissues or organs from potential toxicity before drugs reach specifically desirable sites, thus improving the therapeutic effect.^1–3,23,24 However, MCNCs without any surface modification are usually colloidal unstable in biological fluids and might be cleared from the bloodstream rapidly before arriving at the targeting sites.^13,16,31 Moreover, MCNCs are generally suitable for the loading of water-soluble or hydrophilic drugs or chemicals,^23,24 and releasing their cargos in a simple Fickian diffusion fashion.^1,2 Most importantly, it is very difficult to achieve a controlled-release function using MCNCs as drug vehicles due to their limited functions.^16,23,24,32 All above the disadvantages of MCNCs certainly will compromise their practical applications, especially in the biomedical field.

By functionalizing MCNCs with stimuli-responsive grafted polymer chains or crosslinked networks as functional switches to achieve stimuli-responsive smart delivery systems is a promising strategy to address these problems.^5,7,14,32,33 Delivery rate of the loaded substances from the stimuli-responsive delivery systems can be flexibly controlled and regulated by the functional switches.³³ In addition, the stability of drug carriers can also be greatly improved.³⁴ Most importantly, by further incorporating of some host molecules, such as beta-cyclodextrin (β-CD) that can recognize many hydrophobic guest molecules, into the functional switches to obtain MCNCs-based stimuli-responsive nanocarriers seem to be ideal candidates for hydrophobic drug controlled release.³⁵ Among the stimuli, temperature is an important stimulus for stimuli-responsive delivery systems due to its ease of manipulation in practical applications.³⁶ Moreover, temperature are also capable of affecting the structural changes of thermosensitive materials, thus resulting in release of their loaded cargos.^1–3,9 Poly(N-isopropylacrylamide) (PNIPAM) is a well-known thermosensitive polymer, which can undergo dramatic swelling/shrinking change as environmental temperature is changed across its lower critical solution temperature (LCST) of around 32 °C³⁷ and therefore, has been widely utilized to engineer smart controlled release systems.^1,2,38–40 β-CD, a torus-shaped cyclic oligosaccharide consists of seven α-1,4-linked D-glucopyranose units, features a hydrophobic internal cavity and hydrophilic external surface. Through a series of weak intermolecular forces, such as hydrophobic, van der Waals, and/or hydrogen-bonding interactions, β-CDs can accommodate specific hydrophobic guest molecules into their interior cavities and form host–guest inclusion complexes. Due to its unique molecular-recognition property,^41–44 nontoxicity and chemical stability, β-CDs have been extensively used as hydrophobic or poorly water-soluble drug carriers, not only enhancing the drug stability, solubility and bioavailability, but also minimizing the undesired side effects associating with the corresponding drugs.^45–47 It has been reported that the phase transition of PNIPAM in response to temperature change can dramatically affect the association constants of β-CDs toward guest molecules in the PNIPAM–β-CD systems (Fig. S1†). At temperature above the LCST of PNIPAM, the association constants of β-CDs toward the guest molecules are much smaller than those at temperature below the LCST, due to the steric hindrance caused by the shrunken and hydrophobic PNIPAM chains.^41–43,48 Inspired by those interesting results, we speculate that by adjusting the association constants of β-CDs toward guest molecules with the phase transition of PNIPAM and the high magnetic responsiveness of MCNCs, it is possible to achieve a smart nanosystem that simultaneously possesses temperature-switched controlled delivery property for loaded hydrophobic drugs and site-specific targeting function.

In this study, we develop a novel temperature-switched controlled release nanosystem based on the molecular recognition of β-CD and the thermosensitivity based on the phase transition of PNIPAM. This smart controlled release nanosystem composed of MCNCs core grafted with linear poly(N-isopropylacrylamide-co-glycidyl methacrylate) chains appended numerous β-CD units (abbreviated as Fe₃O₄@PNG–CD) is schematically shown in Fig. 1. In this smart controlled release nanosystem, β-CD units act as containers for the loading of hydrophobic model drugs, PNIPAM chains serve as intelligent “microvalves” for controlling and regulating the release of the loaded model drugs, and MCNCs core can obtain a site-specific targeting function. The gating mechanism involves that the thermo-triggered polymer phase transition of PNIPAM affects the association constants of β-CDs toward the hydrophobic model drugs, 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS). As ambient temperature is below the LCST of the grafted PNG–CD chains on the MCNCs surface, such as at room temperature, the PNG–CD grafting chains is in swollen state and hydrophilic, and thus have little effect on the association constants of β-CDs toward the hydrophobic model drugs. Under this condition, β-CDs can capture drug molecules and form stable β-CDs/drugs inclusion complexes. As environmental temperature is increased above the LCST of PNG–CD grafting chains, these chains will change from a swollen state to a shrunken one responding to temperature change. Due to the PNIPAM phase transition triggered reduction in association constants of β-CDs toward drug molecules, the loaded drugs can be released from the Fe₃O₄@PNG–CD NPs. Therefore, the delivery rate of drugs from these NPs can be effectively controlled and regulated by simply changing the temperature of delivery media above/or below the LCST of the grafted PNG–CD chains on the MCNCs surface.

Fig. 1 Schematical illustration of temperature-switched controlled release Fe₃O₄@PNG–CD nanosystem.

2. Experimental

2.1. Materials

N-Isopropylacrylamide (NIPAM) was obtained from Tokyo Chemical Industry (Tokyo, Japan) and purified by recrystallization with a mixture of hexane/acetone (v/v, 50/50). Ferric chloride hexahydrate (FeCl₃·6H₂O), poly (4-styrenesulfonic acid-co-maleic acid, SS : MA = 3 : 1) sodium salt (PSSMA, M_W = 20 000) and sodium acetate trihydrate (CH₃COONa·3H₂O, NaAc) were purchased from Aladdin (Beijing, China). 3-Aminopropyltriethoxysilane (APS), 2-bromoisobutyryl bromide (BiBB) and glycidyl methacrylate (GMA) were obtained from Acros (USA). 2,2′-Bipyridine (Bpy) and 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS) (97%) were bought from J&K Chemicals (Shanghai, China). β-CD, cuprous bromide (CuBr), N,N-dimethylmethanamide (DMF) was purchased from Kelong Chemicals (Chengdu, China) and purified before use. Ethylenediamine-modified β-CD (EDA–β-CD) was synthesized according to the previous reports^49,50 (please see the ESI†). All other chemicals were of analytical grade and used as received. Deionized water was used throughout the experiments.

2.2. Preparation of the Fe₃O₄@PNG–CD smart NPs

The Fe₃O₄@PNG–CD smart NPs are fabricated by a multi-step process as shown in Fig. 2. Firstly, PSSMA-stabilized MCNCs are synthesized through a modified solvothermal method.²¹ Then, the β-CD-containing PNIPAM chains, acting as the functional switches of drug-controlled delivery, are grafted on the MCNCs surface by a combination of surface-initiated atom transfer free radical polymerization (SI-ATRP)⁵¹ and ring-opening reaction.^52–54 To obtain the macroinitiators of SI-ATRP (Fe₃O₄–Br), MCNCs cores are first coated with APS to achieve amino groups-functionalized MCNCs (Fe₃O₄–APS).⁵⁵ Then, the resulted Fe₃O₄–APS are reacted with BiBB to prepare the macroinitiators of SI-ATRP (Fe₃O₄–Br). Due to the lack of active groups in PNIPAM and parent β-CD molecules, it is difficult to initiate reaction between PNIPAM and β-CDs.⁵⁴ Therefore, GMA is copolymerized with NIPAM to achieve the epoxy-functionalized thermosensitive PNG copolymer chains on the MCNCs surface by SI-ATRP.⁵¹ After that, β-CDs are easily attached on the PNG copolymer chains by a ring-opening reaction between the epoxy groups and the amino groups of EDA–β-CDs,^52–54 and finally resulting in the formation of PNG–CD functional switches on the MCNCs surface.

Fig. 2 Schematic illustration of fabrication process of the proposed smart Fe₃O₄@PNG–CD NPs.

To enhance the loading capacity of the prepared smart NPs for model drug ANS effectively via the formation of β-CD/ANS complexes, sufficient amount of β-CD units should be introduced on the PNG copolymer chains. The incorporation of β-CD into the PNG copolymer chains is through the ring-opening reaction between epoxy groups of the PNG and the amino groups of EDA–β-CD. Therefore, the dosage of GMA monomer in the SI-ARTP reaction will affect the grafting amounts of β-CD on the PNG chains, and correspondingly affect the loading capacity of ANS. On the other hand, the content of PNIPAM, serving as smart “microvalves” for controlling and regulating the release of loaded ANS from the Fe₃O₄@PNG–CD NPs, affects the gating performance of PNG–CD chains on the MCNCs surface. In other words, the higher the PNIPAM content in PNG–CD grafting chains, the better the controlled-release performance of PNG–CD. However, on the other hand, the larger the β-CD content in PNG–CD grafting chains, the higher the loading capacity of ANS. Therefore, two kinds of β-CDs-functionalized thermosensitive MCNCs with different content of PNIPAM and β-CD on the MCNCs surface (Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs) are prepared and used to investigate the temperature-responsive controlled release properties and switched performance in this study.

2.2.1. Synthesis of MCNCs. PSSMA-stabilized MCNCs were prepared by a modified solvothermal reaction.²¹ Briefly, PSSMA (1.0 g), FeCl₃·6H₂O (1.08 g) and NaAc (3.0 g) were firstly added in ethylene glycol (40 mL). After vigorous stirring for 30 min, the obtained homogeneous red-brown solution was transferred in a Teflon-lined stainless-steel autoclave (100 mL), and heated at 200 °C for 10 h, after natural cooling to room temperature. The black product was magnetically isolated, washed five times with deionized water, and dried in vacuum at 30 °C overnight. The obtained product was denoted as MCNCs.

2.2.2. Synthesis of amino-functionalized MCNCs (Fe₃O₄–APS). Amino-functionalized MCNCs were prepared according to a previous report.⁵⁵ Briefly, MCNCs power (0.30 g) was first dispersed in a mixture of ethanol and deionized water (200 mL/1.5 mL) by ultrasonication. Next, APS (1.5 mL) was added in the above solution, and continuously stirred for 12 h under nitrogen atmosphere at room temperature. The suspended substance was then collected with magnet and rinsed five times with ethanol. The obtained product was dried in vacuum at 30 °C overnight and denoted as Fe₃O₄–APS.

2.2.3. Synthesis of macroinitiators of SI-ATRP (Fe₃O₄–Br). Macroinitiators of SI-ATRP were synthesized by the following procedure: Fe₃O₄–APS (0.30 g) was ultrasonically dispersed in a mixture of dichloromethane (CH₂Cl₂) and triethylamine (150 mL/9 mL). Next, a mixture of CH₂Cl₂ (20 mL) and BiBB (2.0 mL) was added dropwise in the above solution in an ice-water bath under stirring. After half an hour, the ice-water bath was retreated, and further reacted for another 4 h under stirring at room temperature. The prepared products were collected with magnet, and rinsed with CH₂Cl₂ and ethanol several times, respectively, and finally dried in vacuum at 30 °C overnight. The obtained product was denoted as Fe₃O₄–Br.

2.2.4. Synthesis of epoxy groups-functionalized thermosensitively MCNCs (Fe₃O₄@PNG). Epoxy groups-functionalized thermosensitively MCNCs (Fe₃O₄@PNG) were prepared via SI-ATRP method using the above-prepared Fe₃O₄–Br as the macroinitiators,⁵¹ NIPAM and GMA as the comonomers. To ascertain effect of the comonomer dosage on the loading capacity of model drug and the gating performance of PNG–CD grafting chains on the MCNCs surface, two kinds of Fe₃O₄@PNG with different PNIPAM and epoxy group content were prepared via varying the molar ratio of NIPAM to GMA during the SI-ATRP reaction in this study, while keeping other conditions unchanged. Fe₃O₄@PNG-1 refers to the NPs prepared with a molar ratio of NIPAM to GMA of 1 : 1, and Fe₃O₄@PNG-2 stands for the NPs synthesized with the molar ratio of NIPAM to GMA of 2 : 1.

Typically, to prepare Fe₃O₄@PNG-1, Fe₃O₄–Br (0.10 g) was first added in a mixture of methanol/deionized water (23 mL/2 mL) in a three-neck round bottom flask with a capacity of 50 mL. Then the NIPAM (1.058 g) and GMA (1.23 mL) (n_NIPAM/n_GMA = 1 : 1) were added in the above solution and bubbled for 10 min with N₂. After adding CuBr (0.12 g) and Bpy (0.36 g), and further bubbling with N₂ for another 30 min, reaction was allowed to last for another 10 h at 50 °C under N₂ atmosphere at a stirring speed of 200 rpm. Then the reaction mixture was cooled down to room temperature and exposed to air. The synthesized product was magnetically collected and washed several times with ethanol and water respectively, and finally dried in vacuum at 30 °C for 24 h. As for the preparation of Fe₃O₄@PNG-2, all the other conditions were kept unchanged, except that the dosage of NIPAM and GMA were replaced with 1.448 g and 0.67 mL (n_NIPAM/n_GMA = 2 : 1), respectively.

2.2.5. Synthesis of β-CDs-functionalized thermosensitively MCNCs (Fe₃O₄@PNG–CD). β-CDs-functionalized thermosensitively MCNCs (Fe₃O₄@PNG–CD) were prepared via a ring-opening reaction between the epoxy groups of PNG copolymer chains on the MCNCs surface and the amino groups of EDA–β-CD.^52–54 Typically, to prepare the Fe₃O₄@PNG-1–CD NPs, the Fe₃O₄@PNG-1 (0.8 g) was first dispersed in DMF (15 mL) containing EDA–β-CD (0.75 g) by ultrasonication, and then allowed them to react at 60 °C for 24 h. Finally, the reaction mixture was cooled down to room temperature and rinsed four times with DMF, water and ethanol in turn to remove the unreacted substances, and dried in vacuum at 30 °C for 24 h. For preparing the Fe₃O₄@PNG-2–CD NPs, all the other conditions were kept unchanged expect that the Fe₃O₄@PNG-1 was substituted with equivalent Fe₃O₄@PNG-2.

2.3. Investigation on the temperature-responsive molecular recognition properties of Fe₃O₄@PNG–CD NPs

The temperature-responsive molecular recognition properties of the prepared Fe₃O₄@PNG–CD NPs were performed by adsorption experiments using ANS as the guest molecules. The prepared Fe₃O₄@PNG-1–CD or Fe₃O₄@PNG-2–CD NPs (50 mg) were first dispersed in ANS aqueous solution of 20 mL (0.125 mM, 39.55 mg L⁻¹) with slight stirring. At preset time intervals, the ANS-adsorbed Fe₃O₄@PNG-1–CD or Fe₃O₄@PNG-2–CD NPs were collected by a magnet. Supernatant solution (2.5 mL) was taken out for absorbance measurement by UV-vis spectrophotometer (TU-1950, Persee, Beijing, China) at a wavelength of 350 nm. After reaching the adsorption equilibrium at 25 °C, the temperature was promptly switched to 45 °C. The absorbance of ANS solution was similarly analyzed at preset time intervals until new adsorption equilibrium was reached. The low/high-temperature courses (namely “25 °C → 45 °C → 25 °C → 45 °C”) were performed in duplicate to study the dynamic temperature-dependent adsorption/desorption behaviours. Moreover, to verify the adsorption behaviours of Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs toward the ANS is via the formation of β-CD/ANS inclusion complexes, the controlled trials were also carried out using the β-CD-free Fe₃O₄@PNG-1 and Fe₃O₄@PNG-2 NPs with the same procedure. The adsorption and desorption experiments were performed at 25 °C and 45 °C respectively is due to the fact that the temperature is lower or higher than the LCST of PNG–CD grafting chains on the MCNCs surface. The amounts of ANS adsorbed on the particles were calculated from the following equation:where V (L) is the volume, C₀ and C_e (mg L⁻¹) are the initial and equilibrium concentrations of ANS, respectively, and m (mg) is the mass of Fe₃O₄@PNG-1, Fe₃O₄@PNG-2, Fe₃O₄@PNG-1–CD or Fe₃O₄@PNG-2–CD NPs.

2.4. Investigation on the temperature-responsive controlled release and switched performances of Fe₃O₄@PNG–CD NPs

To study the temperature-responsive controlled release and switched performances of the prepared Fe₃O₄@PNG–CD NPs, hydrophobic model drug ANS was first loaded on the Fe₃O₄@PNG-1–CD or the Fe₃O₄@PNG-2–CD NPs by adding 50 mg Fe₃O₄@PNG-1–CD or Fe₃O₄@PNG-2–CD NPs in 20 mL ANS aqueous solution (0.5 mM, 158 mg L⁻¹). After stirring for 36 h at 25 °C to reach the adsorption equilibrium in dark, the ANS-loaded Fe₃O₄@PNG-1–CD or Fe₃O₄@PNG-2–CD NPs were collected by magnet. Then the weakly-adsorbed ANS molecules on the particle surface were removed via a rinsing with deionized water twice (20 mL for each time), the ANS-loaded Fe₃O₄@PNG-1–CD or Fe₃O₄@PNG-2–CD NPs were redispersed in pH 7.4 phosphate buffer solution (PBS) (30 mL) and stirred at 200 rpm in a thermostatic water-bath heater maintained at 25 °C or 45 °C. At preset time intervals, an incubation solution (2.5 mL) was taken out and replaced with a fresh PBS with the same volume and temperature. The accumulative release amounts of ANS were obtained by determining the supernatant solution using UV-vis spectrophotometer at 350 nm. In order to verify that the release of ANS from the Fe₃O₄@PNG–CD NPs is mainly triggered by the reduction of association constants between the β-CD of the PNG–CD grafting chains and ANS molecules, causing by the phase transition of PNIPAM segments on the PNG–CD grafting chains, the controlled trials were also performed using the PNIPAM-free Fe₃O₄@PG–CD NPs with the same procedure.

2.5. Characterization

¹H NMR and Fourier transform infrared (FT-IR) spectra were recorded on a 400-MR DD2 NMR (Agilent, America) spectrometer with DMSO-d₆ and D₂O as the solvent and on an IR200 (Thermo Nicolet, USA) spectrometer. Transmission electron microscope (TEM) images were taken on a JEM-2010 (JEOL, Japan) transmission electron microscope. The samples were prepared by mounting a drop of the particle dispersion on a carbon-coated Cu grid and allowing the samples to dry in air. The hydrodynamic diameters and size distributions of particles were determined by a DLS instrument (Zetasizer Nano-ZS, Malvern Instruments, UK). Powder X-ray diffraction (XRD) patterns were obtained on a DX-1000 (Dandong Fangyuan Instrument Co., Ltd, China) diffractometer with Cu Kα radiation operating at 40 kV and 36 mA. Thermogravimetric analyses (TGA) were conducted on a Mettler TGA/SDTA851e° (Switzerland) under following N₂ atmosphere at a heating rate of 10 °C min⁻¹. Magnetic property measurements were carried out on a vibrating sample magnetometer (VSM) on a Model 6000 physical property measurement system (Quantum Design, USA) at 300 K.

3. Results and discussion

3.1. Materials characterization

The morphologies and sizes of NPs are characterized by TEM. As shown in Fig. 3a, the Fe₃O₄–Br NPs are nearly spherical in shape with an average diameter of about 190 nm. After grafting PNG copolymer chains from the Fe₃O₄–Br surface, the Fe₃O₄@PNG-1 NPs exhibit core/shell structure with the dark MCNCs core covered with about 20 nm light-gray PNG-1 grafting layers (Fig. 3b). After further incorporating β-CD units into the PNG-1 chains, the morphology and size of particles does not change evidently (Fig. 3c and d). That is to say, the introduction of β-CD units on the PNG-1 chains has little influence on the particle morphology and size. Similar phenomena are also observed in β-CD polymers-functionalized MCNCs without PNIPAM moieties in our recent work.⁵⁶ Moreover, slight aggregation among the Fe₃O₄@PNG-1 or Fe₃O₄@PNG-1–CD NPs are observed due to the incorporation of hydrophilic PNIPAM and β-CD moieties into the grafting chains on the MCNCs surface.^56–60 Hydrodynamic diameters and size distributions of the Fe₃O₄–Br, Fe₃O₄@PNG-1 and Fe₃O₄@PNG-1–CD samples were studied by DLS at 25 °C and shown in Fig. 3e. The mean hydrodynamic diameters of the Fe₃O₄–Br, Fe₃O₄@PNG-1 and Fe₃O₄@PNG-1–CD are 258, 892 and 1067 nm and their corresponding polydispersity index (PDI) values are 0.117, 0.343 and 0.196, respectively. All the mean hydrodynamic diameters are larger than that of the TEM results due to the formation of hydrate layer between the water molecules and the grafting substances on the Fe₃O₄–Br, Fe₃O₄@PNG-1 or Fe₃O₄@PNG-1–CD surface.⁶⁰

Fig. 3 Typical TEM images of Fe₃O₄–Br (a), Fe₃O₄@PNG-1 (b), Fe₃O₄@PNG-1–CD NPs ((c) and (d)). Scale bar ((a) and (b)) 200 nm, (c) 300 nm and (d) 100 nm. Hydrodynamic size distributions of Fe₃O₄–Br (○),Fe₃O₄@PNG-1 (□), Fe₃O₄@PNG-1–CD NPs (◇).

The chemical compositions of MCNCs, Fe₃O₄–APS, Fe₃O₄–Br, Fe₃O₄@PNG-1 and Fe₃O₄@PNG-1–CD NPs are characterized by FT-IR spectrometer as shown in Fig. 4. For the MCNCs, the characteristic peaks at 1181 and 1124 cm⁻¹ are ascribed to the asymmetric stretching vibrations of sulfonate group in PSSMA, and 580 cm⁻¹ is assigned to the Fe–O stretching vibrations²¹ (Fig. 4a). After modification with APS, the adsorption bands at 1632 and 1550 cm⁻¹ corresponding to the bending vibrations of N–H of –NH₂ can be observed⁶¹ (Fig. 4b). The adsorption bands for Fe₃O₄–Br at the peak of 1706 cm⁻¹ is attributed to the C=O stretching vibrations of O=C–N group in BiBB,^56,62 indicating the successful immobilization of Br atoms on the MCNCs surface (Fig. 4c). Furthermore, compared with the Fe₃O₄–Br, the characteristic peaks at 1720 and 909 cm⁻¹ attributed to the C=O stretching vibrations and antisymmetric deformation vibrations of epoxy group of PGMA in the spectrum of Fe₃O₄@PNG-1 NPs are also observed (Fig. 4d). The bands at 1650 and 1550 cm⁻¹ are attributable to the C=O stretching vibrations and N–H deformation vibrations of PNIPAM.⁵³ Those results indicate that PNG copolymer chains have been successfully grafted on the MCNCs surface. Besides, after further functionalization with β-CD, the antisymmetric glycosidic ν_a(C–O–C) vibrations and the coupled ν(C–C/C–O) stretching vibrations at 1030 and 1157 cm⁻¹ are also found in the spectrum of Fe₃O₄@PNG-1–CD NPs (Fig. 4e).^61,63 Meanwhile, the characteristic peak of epoxy ring group at 909 cm⁻¹ disappears, indicating that β-CD units have been introduced on the PNG copolymer chains. The Fe₃O₄@PNG–CD NPs have been successfully synthesized.^44,53,56

Fig. 4 FT-IR spectra of MCNCs (a), Fe₃O₄–APS (b), Fe₃O₄–Br (c), Fe₃O₄@PNG-1 (d) and Fe₃O₄@PNG-1–CD NPs (e).

To investigate whether the conjugation of PNG–CD grafting chains on the MCNCs surface changes the crystal phase of magnetic Fe₃O₄ CNCs or not, the XRD patterns of MCNCs, Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs are measured and shown in Fig. 5. The broad bands located at 2θ = (30.32°, 36.56°, 43.36°, 53.76°, 57.20°, 62.96°) marked by their indices (220, 311, 400, 422, 511, 440), can be evidently observed for the MCNCs, Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs, which is consistent with the previous reports.^61,63 This result indicates that the grafting of PNG–CD chains on the MCNCs surface does not change the crystal phase of MCNCs.

Fig. 5 XRD patterns of MCNCs (a), Fe₃O₄@PNG-1–CD (b) and Fe₃O₄@PNG-2–CD NPs (c).

TGA is used to quantitatively characterize the amount of organics grafted on the MCNCs surface in this study, and the results are displayed in Fig. 6 and Fig. S2.† The weight loss of each sample increases gradually due to the organics decomposition and similar results are also observed in our recent work.⁵⁶ In comparison with the Fe₃O₄@PNG-1 and Fe₃O₄@PNG-2, around 3.64% and 2.48% of β-CD are introduced on the PNG-1 and PNG-2 copolymer chains, respectively. The grafting amounts of β-CD are calculated as 36.4 and 24.8 mg g⁻¹ (the results are listed in Table 1). These results confirm again the successful grafting of PNG–CD polymer chains on the MCNCs surface. Besides, to verify that the loading of ANS on the Fe₃O₄@PNG–CD NPs is via the formation of β-CD/ANS inclusion complexes, TGA is also used to analyze the weight loss of the in-vacuum dried Fe₃O₄@PNG-2–CD/ANS, and around 1.55% of weight loss is observed compared with that of the Fe₃O₄@PNG-2–CD without ANS adsorption (Fig. S2†), indicating that the loading of ANS on the Fe₃O₄@PNG-2–CD NPs is mainly via the formation of β-CD/ANS inclusion complexes.

Fig. 6 TGA curves of MCNCs (a), Fe₃O₄–APS (b), Fe₃O₄–Br (c), Fe₃O₄@PNG-1 (d) and Fe₃O₄@PNG-1–CD NPs (e).

Table 1 The grafting results calculated by TGA

Samples	TGA^a/%	Grafting percentage^b/%
a The remaining weight percent after TGA.b The grafting percentage is defined as the weight percentage of newly grafted component.
MCNCs	84.40	—
Fe3O4–APS	83.41	1.17
Fe3O4–Br	82.35	1.27
Fe3O4@PNG-1	78.74	4.38
Fe3O4@PNG-2	77.68	5.67
Fe3O4@PNG-1–CD	75.87	3.64
Fe3O4@PNG-2–CD	75.75	2.48

---

3.2. Magnetic responsive properties of Fe₃O₄@PNG–CD NPs

Drug release at specific pathological sites can effectively reduce the side effects. The MCNCs cores enable the Fe₃O₄@PNG–CD NPs with magnetically guided to targeted organs or tissues inside the body by an external magnetic field. The prepared Fe₃O₄@PNG-1–CD NPs exhibit excellent dispersion properties in pH 7.4 PBS (Fig. 7a, the left) and magnetic-responsive aggregation at a specific site within two minutes under an external magnetic field (Fig. 7a, the right). The magnetic properties of NPs are investigated at room temperature using VSM. Fig. 7b and S3† show the variations of magnetization values at room temperature with applied magnetic field for the NPs. As can be seen from the magnetic curves, all the NPs show superparamagnetic behaviours. Decreased magnetization values of 33.4 and 45.2 emu g⁻¹ respectively for the Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs comparing with that of MCNCs (62.1 emu g⁻¹) are due to the existence of nonmagnetic organics on the MCNCs surface.^58–63 Besides, the hysteresis and coercivity for all the NPs are almost undetectable, suggesting all the prepared NPs have superparamagnetism. The superparamagnetic properties of the Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs are critical for their practical applications, which prevent them from aggregation and enable them to redisperse rapidly while removing the external magnetic field. The magnetism studies indicate that the prepared Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs possess excellent magnetic responsive properties and have potentials in magnetically guided drug delivery application.

Fig. 7 Fe₃O₄@PNG-1–CD NPs dispersed in pH 7.4 PBS at room temperature without and with an external magnetic field (a), and magnetic hysteresis loops of Fe₃O₄ CNCs, Fe₃O₄ CNCs–Br, Fe₃O₄@PNG-1 and Fe₃O₄@PNG-1–CD NPs at room temperature (b).

3.3. Temperature-responsive properties of Fe₃O₄@PNG–CD NPs

The temperature-responsive properties of the prepared Fe₃O₄@PNG–CD NPs are investigated by DLS. Fig. 8 shows the temperature-dependent hydrodynamic diameter change of the Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs. As expected, both two samples exhibit excellent temperature-responsive deswelling properties upon raising environmental temperature. DLS results show a larger particle diameter than that of TEM measurements. This is because the DLS measurements reflect the hydrodynamic diameters, while the TEM tests show the dry-sample sizes. Hydration and swelling of the PNG–CD grafting chains on the MCNCs surface lead to the difference in particle size between two measurements.^57,58,60 Moreover, both PNG-1–CD and PNG-2–CD grafting chains exhibit slightly higher LCST than that of pure PNIPAM (about 32 °C). And the LCST of PNG-1–CD grafting chains is higher compared with that of PNG-2–CD grafting chains. The LCST is defined as the temperature around which the polymer chains undergo a sharp conformational change in aqueous solution.

Fig. 8 Temperature-responsive property of Fe₃O₄@PNG-1–CD (circle) and Fe₃O₄@PNG-2–CD (square) NPs in deionized water.

Such a positive shift in LCST is caused by the incorporation of hydrophilic β-CD moieties on the grafted copolymer chains.^49,52 The larger the content of hydrophilic moieties in copolymer chains, the higher the LCST of grafting chains. Due to the higher content of β-CD moieties in PNG-1–CD grafting chains, the PNG-1–CD shows a higher LCST compared with the PNG-2–CD.⁵⁵ As mentioned above, PNG–CD functional switches are grafted on the MCNCs surface by SI-ATRP using NIPAM and GMA as comonomers and subsequent a ring-opening reaction. For preparing the PNG copolymer chains, the total dosage of NIPAM and GMA is fixed. And the content of PNIPAM and epoxy groups in PNG copolymer chains can be easily adjusted by varying the molar ratio of NIPAM to GMA, thus regulating the temperature-responsive gating performance of PNG–CD grafting chains and the content of β-CD moieties. For fabricating PNG-1–CD grafting chains on the MCNCs surface, the dosage of GMA in the SI-ATRP is twice as much as that of the PNG-2–CD. Therefore, PNG-1 copolymer chains have relatively larger content of epoxy groups. As a result, more β-CD units are introduced on the PNG-1 copolymer chains by subsequent ring-opening reaction. Furthermore, due to relatively higher content of PNIPAM in PNG-2–CD, so PNG-2–CD have larger equilibrium-deswelling ratio (EDR) (D_{50 °C}/D_{20 °C}) than that of PNG-1–CD upon increasing temperature. Suggesting that Fe₃O₄@PNG-2–CD NPs have better temperature-responsive gating performance compared with that of Fe₃O₄@PNG-1–CD NPs. However, on the other hand, the higher the compositions of PNIPAM, the lower the content of PGMA in PNG–CD grafting chains, thus resulting in reduction of β-CD content in PNG-2–CD chains upon increasing the PNIPAM components. This certainly affects the loading capacity of ANS. On the contrary, Fe₃O₄@PNG-1–CD NPs have larger loading capacity for ANS due to higher content of β-CD units in the PNG-1–CD chains, though smaller EDR of PNG-1–CD. Therefore, both Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs were used to investigate the temperature-responsive molecular-recognition and controlled delivery properties for hydrophobic model drug ANS in the following studies.

3.4. Temperature-responsive molecular-recognition properties of Fe₃O₄@PNG–CD NPs

The temperature-responsive molecular recognition properties of Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs for ANS in two low/high-temperature courses are shown in Fig. 9. The controlled trials were also carried out with the β-CD-free Fe₃O₄@PNG-1 and Fe₃O₄@PNG-2 NPs using the same protocols. At 25 °C, below the LCST of PNG-1–CD and PNG-2–CD grafting chains, for the Fe₃O₄@PNG-1–CD NPs, the amount of ANS absorbed on the Fe₃O₄@PNG-1–CD NPs increases sharply during the first 140 min, and then gradually tends to the maximal adsorption capacity (q_max) of 7.69 mg g⁻¹ in the following 60 min. When suddenly changed the temperature from 25 °C to 45 °C, above the LCST of PNG-1–CD and PNG-2–CD grafting chains, the adsorption capacity q reduces rapidly from the q_max 7.69 mg g⁻¹ to 3.87 mg g⁻¹ within the following 50 min, and then keeps nearly unchanged. The similar phenomena are observed for the subsequent absorption/desorption course in the next low/high-temperature cycles. While the temperature is switched to 25 °C again, the q value increases until the second adsorption equilibrium is reached, and decreases again until the following another desorption equilibrium is achieved at 45 °C. For the Fe₃O₄@PNG-2–CD NPs, similar phenomena of adsorption at a low temperature (25 °C) and desorption at a high temperature (45 °C) are observed. In contrast with the Fe₃O₄@PNG-1–CD NPs, Fe₃O₄@PNG-2–CD NPs have smaller q_max value toward the ANS (5.80 mg g⁻¹) (Fig. 9c and d).

Fig. 9 Thermo-triggered adsorption and desorption properties of ANS from Fe₃O₄@PNG-1 and Fe₃O₄@PNG-1–CD ((a) and (c)), Fe₃O₄@PNG-2 and Fe₃O₄@PNG-2–CD ((b) and (d)) NPs in pH 7.4 PBS at different temperatures (25 °C and 45 °C). The ANS concentration in PBS is 0.125 mM (39.55 mg L⁻¹).

Such thermo-induced decrease in ANS-adsorption capacity of the Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs is mainly caused by the reduction in the association constants of β-CD/ANS complexes upon increasing temperature.^41–43,48 At 25 °C, the PNG-1–CD grafting chains on the MCNCs surface are in swollen state and hydrophilic. As a result, the association constants of β-CD/ANS complexes are high, β-CD cavities can capture ANS molecules efficiently and tightly.^41–43,48 Therefore, ANS can be effectively loaded on the Fe₃O₄@PNG-1–CD NPs. As the temperature increases, the PNG-1–CD grafting chains change from a swollen state to a shrunken state in response to temperature change, the association constants of β-CD/ANS complex decreases. Therefore, part of captured ANS release from β-CD cavities of the Fe₃O₄@PNG-1–CD NPs at high temperature.^41–43,48 The difference in q_max value for ANS is due to the difference of β-CD content in PNG–CD grafting chains. Comparing with the Fe₃O₄@PNG-2–CD NPs, the Fe₃O₄@PNG-1–CD NPs have higher β-CD content in PNG-1–CD grafting chains, thus β-CD units can accommodate more ANS molecules and form stable β-CD/ANS inclusion complexes at temperature below the LCST of PNG–CD grafting chains. Moreover, both Fe₃O₄@PNG-1 and Fe₃O₄@PNG-2 NPs also show a certain capability of adsorption at low temperature and desorption at high temperature for ANS (Fig. 9c and d). Hydrophobic interactions between PGMA segments and ANS molecules maybe responsible for the small adsorption of ANS at a low temperature course.^53,56 Upon increasing temperature above the LCST of PNG copolymer chains, hydrophobic interactions between the PGMA segments and ANS molecules become weak. As a result, ANS adsorbed on the PNG copolymer chain detach from particle surface.⁵⁸ Additionally, Fe₃O₄@PNG-1 NPs exhibit larger adsorption capacity toward ANS than that of Fe₃O₄@PNG-2 NPs due to relatively higher content of hydrophobic PGMA segments. All above the results verify that the resulted Fe₃O₄@PNG–CD NPs have excellent temperature-responsive molecular-recognition properties, which can be potentially applied in temperature-responsive controlled release for hydrophobic drugs. While environmental temperature below the LCST of on grafted PNG–CD chains, hydrophobic drugs can be loaded on the Fe₃O₄@PNG–CD NPs via the formation of β-CD/drug molecules complexes. When the drug-loaded Fe₃O₄@PNG–CD NPs are magnetically guided in specific lesion sites inside the body, upon heating the local lesion site by near infrared (NIR) irradiation above the LCST of PNG–CD grafting chains, the loaded drugs can be effectively delivered.

3.5. Temperature-responsive controlled release properties of Fe₃O₄@PNG–CD NPs

Temperature-responsive controlled release properties of Fe₃O₄@PNG–CD NPs for a hydrophobic model drug ANS are carried out at two representative temperatures (25 °C and 45 °C), below or above the LCST of PNG–CD grafting chains. The accumulative release profiles of ANS from ANS-loaded Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs are shown in Fig. 10. The loading amounts of ANS on the Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs are respectively (1.073 ± 0.055) mg and (0.831 ± 0.053) mg by the UV measurement. The difference in loading amounts is due to the different content of β-CD on the PNG–CD chains. The higher the content of β-CD units, the larger the loading amounts of ANS. Moreover, the delivery profiles show that the release of ANS is positively temperature-dependent for both the Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs. The released amounts of ANS are high at 45 °C, and while low at 25 °C. For the ANS-loaded Fe₃O₄@PNG-1–CD system, an initial small burst release of physically-adsorbed ANS on the PNG-1–CD grafting chains and followed fast release are observed. This is mainly due to the reduction in association constants of the β-CD/ANS complexes, causing by the shrinking of PNG-1–CD grafting chains at high temperature.^41–43,48 This tested system shows rapid release of ANS and a cumulative released amount of ANS of 0.87 mg within 11 h, corresponding to 81% of the total ANS amounts loaded on the Fe₃O₄@PNG-1–CD NPs. By contrast, a relatively low and smooth release of ANS is observed at 25 °C. The release of ANS reaches a plateau of 0.38 mg within 5 h and then keeps little changed over hours due to relatively larger association constants of the β-CD/ANS complexes at the temperature below the LCST.^41–43,48 Similar release trends are also observed when the ANS-loaded Fe₃O₄@PNG-2–CD system is studied at the same conditions, but the cumulative released amount of ANS is relatively smaller due to lower content of β-CD units on the PNG-2 copolymer chains.

Fig. 10 Accumulative release profiles of ANS from ANS-loaded Fe₃O₄@PNG-1–CD (circle), Fe₃O₄@PNG-2–CD (diamond) and Fe₃O₄@PG–CD (triangle) NPs in pH 7.4 PBS at different temperatures (25 °C and 45 °C). The ANS concentration in PBS is 0.5 mM (158 mg L⁻¹). The loading amounts of ANS onto the Fe₃O₄@PNG-1–CD, Fe₃O₄@PNG-2–CD and Fe₃O₄@PG–CD NPs are (1.073 ± 0.055) mg, (0.831 ± 0.053) mg and (0.547 ± 0.015) mg, respectively.

To further prove that the release of ANS from the ANS-loaded Fe₃O₄@PNG–CD system is triggered by the reduction in association constants of β-CD/ANS complexes, causing by the phase transition of PNIPAM segment on the PNG–CD grafting chains, the release of ANS from the PNIPAM-free Fe₃O₄@PG–CD NPs was also studied and the result was also shown in Fig. 10. Compared with the PNIPAM-contained Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs, a smaller loading amount of ANS can be observed ((0.547 ± 0.015) mg). The loading amounts of ANS on the Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs are (1.073 ± 0.055) mg and (0.831 ± 0.053) mg, respectively. This is due to a smaller association constant between β-CDs of PG–CD grafting chains toward the ANS molecules. The association constant of β-CDs toward ANS is about 80 M⁻¹ at 20 °C, much smaller than that of PNIPAM-modified β-CD (around 1000 M⁻¹) at the same temperature. Besides, the ANS amount released from the ANS-loaded Fe₃O₄@PG–CD system at 45 °C is a little higher than that of 25 °C, which can be explained that the association constants of β-CDs on the PG–CD grafting chains toward ANS molecules is affected by the environmental temperature.^41,48,64,65 Association constants of β-CDs toward drug molecules decrease with increasing the environmental temperature. The association constant at 25 °C is approximately twice as much as that at 45 °C. Moreover, the ANS amounts released from the Fe₃O₄@PG–CD NPs at 45 °C and 25 °C do not exhibit significant difference as much as that from the Fe₃O₄@PNG-1–CD or Fe₃O₄@PNG-2–CD NPs at the same temperature. This is because the association constants of β-CDs on the PNG-1–CD or PNG-2–CD grafting chains toward ANS are significantly affected by the PNIPAM segments.^41,48 At temperature below the LCST of PNG–CD grafting chains, the association constant of β-CD on the PNG–CD grafting chains toward ANS is nearly ten times as much as that at temperature above the LCST of PNG–CD grafting chains.^41,48 The above results indicate that the PNIPAM segments on the PNG–CD grafting chains serve as the intelligent “microvalves” for controlling the release of loaded model drug ANS from the Fe₃O₄@PNG–CD NPs. By simply changing the environmental temperature, the delivery rate can be effectively controlled. At temperature above the LCST of the PNG–CD grafting chains (open gate), the delivery rate of ANS is fast, while very slow at temperature below the LCST (closed gate).

3.6. Temperature-switched controlled release properties of Fe₃O₄@PNG–CD NPs

To further verify the temperature-switched controlled release performance of the PNG–CD grafting chains, we also investigate the release of ANS from the ANS-loaded Fe₃O₄@PNG–CD NPs via periodically changing the temperature of delivery media. The temperature-responsive “on and off” release of ANS from the Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs are demonstrated in Fig. 11. Both two investigated systems exhibit rapid release at a high temperature (45 °C), and slow and even shut-off release at a low temperature (25 °C). As mentioned above, at 45 °C, above the LCST of PNG–CD grafting chains, the association constant of β-CD/ANS complexes is small due to the shrinking of PNG–CD responding to temperature. Therefore, part of captured ANS molecules release from β-CD cavities of the Fe₃O₄@PNG-1–CD NPs. However, as the temperature of delivery media is switched from 45 °C to 25 °C (below the LCST of PNG-1–CD grafting chains), the PNG-1–CD grafting chains change from a shrunken state to a swollen one responding to a temperature change. Under this condition, the association constant of β-CD/ANS complexes is large. Therefore, few ANS molecules inside the β-CD cavities can be released.^41–43,48 Moreover, during the initial four-hour period, the release rate of ANS from the Fe₃O₄@PNG-2–CD NPs is slightly faster than that from the Fe₃O₄@PNG-1–CD NPs due to higher content of PNIPAM segments in the PNG-2–CD grafting chains, as a result, having larger swelling/shrinking capability in response to temperature change. Thereby, release rate of ANS is slightly quicker than that from the Fe₃O₄@PNG-1–CD NPs. However, during the following two-hour period, the release rate and released ANS amount from the Fe₃O₄@PNG-1–CD NPs exceed that from the Fe₃O₄@PNG-2–CD NPs, which can be attributed to a higher loading amount of ANS on the Fe₃O₄@PNG-1–CD NPs. The cumulative released amount of ANS from the Fe₃O₄@PNG-1–CD NPs is 0.046 mg larger than that from the Fe₃O₄@PNG-2–CD NPs. The aforesaid results indicate that the “on” and “off” switches of ANS delivery can be repeated several times and a reversible controlled-delivery property of the “on” and “off” behaviors of the PNG–CD grafting chains which allow the delivery of ANS depending on environmental temperature.

Fig. 11 Temperature “on and off” properties for ANS release from ANS-loaded Fe₃O₄@PNG-1–CD (circle) and the Fe₃O₄@PNG-2–CD (diamond) NPs in pH 7.4 PBS. The ANS concentration is 0.5 mM (158 mg L⁻¹). The loading amounts of ANS on the Fe₃O₄@PNG-1–CD and Fe₃O₄@PNG-2–CD NPs are (1.073 ± 0.055) mg and (0.831 ± 0.053) mg, respectively.

From the aforementioned results, we can conclude that a temperature-switched controlled release nanosystem based on the molecular recognition of β-CD and the polymer phase transition of PNIPAM has been successfully developed. The PNG–CD grafting chains on the MCNCs surface play an important role in controlling the release of hydrophobic model drug. β-CD units on the PNG–CD grafting chains act as the containers of hydrophobic model drug, PNIPAM chains serve as the intelligent “microvalves” to control and regulate the release rate of model drug, and the MCNCs cores can behave the “site-specific targeting” function.

4. Conclusions

In summary, we have successfully developed a novel temperature-switched controlled release nanosystem based on the molecular recognition of β-CD and the thermosensitivity based on the phase transition of PNIPAM. These smart NPs are fabricated via grafting linear PNIPAM chains attached numerous β-CD units on the MCNCs surface by SI-ATRP and subsequent a ring-opening reaction. The prepared smart Fe₃O₄@PNG–CD NPs demonstrate excellent thermo-reversible adsorption/desorption properties for a hydrophobic model drug ANS and remarkable positive temperature “on–off” controlled-release fashion. By simply changing the temperature of delivery media, the release rate of ANS from those smart NPs can be effectively controlled and tuned. At temperature above the LCST of the PNG–CD grafting chains, the Fe₃O₄@PNG–CD NPs show fast release for ANS, while slow and even shut-off release at temperature below the LCST. Such smart NPs with temperature-switched controlled drug delivery and site-specific targeting function have a great potential application in nanomedicine.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21106116), the project of academic innovation team, Southwest University for Nationalities (XN2014-4).

Notes and references

1. S. Mura, J. Nicolas and P. Couvereur, Nat. Mater., 2013, 12, 991–1003.
2. E. Fleige, M. A. Quadir and R. Haag, Adv. Drug Delivery Rev., 2012, 64, 866–884.
3. Q. Yin, J. Shen, Z. Zhang, H. Yu and Y. Li, Adv. Drug Delivery Rev., 2013, 65, 1699–1715.
4. R. Cheng, F. Meng, C. Deng, H. A. Klok and Z. Zhong, Biomaterials, 2013, 34, 3647–3657.
5. B. Sahoo, K. S. P. Devi, R. Banerjee, T. K. Maiti, P. Pramanik and D. Dhara, ACS Appl. Mater. Interfaces, 2013, 5, 3884–3893.
6. L. A. Tziveleka, P. Bilalis, A. Chatzipavlidis, N. Boukos and G. Kordas, Macromol. Biosci., 2014, 14, 131–141.
7. S. F. Lee, X. M. Zhu, Y. X. J. Wang, S. H. Xuan, Q. You, W. H. Chan, C. H. Wong, F. Wang, J. C. Yu, C. H. K. Cheng and K. C. F. Leung, ACS Appl. Mater. Interfaces, 2013, 5, 1566–1574.
8. Y. Wang, B. Li, L. Zhang, H. Song and L. Zhang, ACS Appl. Mater. Interfaces, 2013, 5, 11–15.
9. J. Liu, C. Detrembleur, A. Debuigne, M. C. D. Pauw Gillet, S. Mornet, L. V. Elst, S. Laurent, E. Duguet and C. Jérôme, J. Mater. Chem. B, 2014, 2, 1009–1023.
10. E. Aznar, M. D. Marcos, R. Martínez-Mánez, F. Sancenón, J. Soto, P. Amorós and C. Guillem, J. Am. Chem. Soc., 2009, 131, 6833–6843.
11. N. Bertrand, J. Wu, X. Y. Xu, N. Kamaly and O. C. Farokhzad, Adv. Drug Delivery Rev., 2014, 66, 2–25.
12. A. Bernardos, E. Aznar, M. D. Marcos, R. Martínez-Mánez, F. Sancenón, J. Soto, J. M. Barat and P. Amorós, Angew. Chem., Int. Ed., 2009, 121, 5998–6001.
13. C. Park, H. Kim, S. Kim and C. Kim, J. Am. Chem. Soc., 2009, 131, 16614–16615.
14. J. Liu, C. Detrembleur, A. Debuigne, M. C. D. Pauw-Gillet, S. Mornet, L. V. Elst, S. Laurent, C. Labrugère, E. Duguet and C. Jérôme, Nanoscale, 2013, 5, 11464–11477.
15. Y. Liu, J. Rohrs and P. Wang, Nano LIFE, 2014, 4, 1441007–1441016.
16. Z. Lu and Y. Yin, Chem. Soc. Rev., 2012, 41, 6874–6887.
17. H. Deng, X. Li, Q. Peng, X. Wang, J. Chen and Y. Li, Angew. Chem., Int. Ed., 2005, 44, 2782–2785.
18. J. Ge, Y. Hu, M. Biasini, W. P. Beyermann and Y. Yin, Angew. Chem., Int. Ed., 2007, 46, 4342–4345.
19. J. Liu, Z. Sun, Y. Deng, Y. Zou, C. Li, X. Guo, L. Xiong, Y. Gao, F. Li and D. Zhao, Angew. Chem., Int. Ed., 2009, 48, 5875–5879.
20. J. Guo, W. Yang and C. Wang, Adv. Mater., 2013, 25, 5196–5214.
21. J. Gao, X. Ran, C. Shi, H. Cheng, T. Cheng and Y. Su, Nanoscale, 2013, 5, 7026–7033.
22. M. Lin, H. Huang, Z. Liu, Y. Liu, J. Ge and Y. Fang, Langmuir, 2013, 29, 15433–15441.
23. S. Xu, C. Sun, J. Guo, K. Xu and C. Wang, J. Mater. Chem., 2012, 22, 19067–19075.
24. D. Li, J. Tang, J. Guo, S. Wang, D. Chaudhary and C. Wang, Chem.–Eur. J., 2012, 18, 16517–16524.
25. Y. Pan, X. W. Du, F. Zhao and B. Xu, Chem. Soc. Rev., 2012, 41, 2912–2942.
26. Y. Pan, M. J. C. Long, H.-C. Lin, L. Hedstrom and B. Xu, Chem. Sci., 2012, 3, 3495–3499.
27. H. L. Gao, Q. Y. Zhang, Z. Q. Yu and Q. He, Curr. Pharm. Biotechnol., 2014, 15, 210–219.
28. Z. Q. Yu, R. M. Schmaltz, T. C. Bozeman, R. Paul, M. J. Rishel, K. S. Tsosie and S. M. Hecht, J. Am. Chem. Soc., 2013, 135, 2883–2886.
29. B. R. Schroeder, M. I. Ghare, C. Bhattacharya, R. Paul, Z. Q. Yu, P. A. Zaleski, T. C. Bozeman, M. J. Rishel and S. M. Hecht, J. Am. Chem. Soc., 2014, 136, 13641–13656.
30. C. Bhattacharya, Z. Q. Yu, M. J. Rishel and S. M. Hecht, Biochemistry, 2014, 53, 3264–3266.
31. B. Haley and E. Frenkel, Urol. Oncol., 2008, 26, 57–64.
32. A.-H. Lu, E. L. Salabas and F. Schüth, Angew. Chem., Int. Ed., 2007, 46, 1222–1244.
33. S. F. Medeiros, A. M. Santos, H. Fessi and A. Elaissari, Int. J. Pharm., 2011, 403, 139–161.
34. S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. V. Elst and R. N. Muller, Chem. Rev., 2008, 108, 2064–2110.
35. L. Wang, L. L. Li, Y. S. Fan and H. Wang, Adv. Mater., 2013, 25, 3888–3898.
36. Y. R. Liu, L. Xiao, K.-I. Joo, B. Hu, J. Fang and P. Wang, Biomacromolecules, 2014, 15, 3836–3845.
37. H. G. Schild, Prog. Polym. Sci., 1992, 17, 163–249.
38. Y. Qiu and K. Park, Adv. Drug Delivery Rev., 2001, 53, 321–339.
39. A. Chilkoti, M. R. Dreher, D. E. Meyer and D. Raucher, Adv. Drug Delivery Rev., 2002, 54, 613–630.
40. J. Zhang, H. Chen, L. Xu and Y. Gu, J. Controlled Release, 2008, 131, 34–40.
41. T. Nozaki, Y. Maeda, K. Ito and H. Kitano, Macromolecules, 1995, 28, 522–524.
42. H. Ohashi, Y. Hiraoka and T. Yamaguchi, Macromolecules, 2006, 39, 2614–2620.
43. M. Yanagioka, H. Kurita, T. Yamaguchi and S. Nakao, Ind. Eng. Chem. Res., 2003, 42, 380–385.
44. R. Xie, S. B. Zhang, H. D. Wang, M. Yang, P. F. Li, X. L. Zhu and L. Y. Chu, J. Membr. Sci., 2009, 326, 618–626.
45. K. Uekama, F. Hirayama and T. Irie, Chem. Rev., 1998, 98, 2045–2076.
46. F. Hirayama and K. Uekama, Adv. Drug Delivery Rev., 1999, 36, 125–141.
47. F. van de Manakker, T. Vermonden, C. F. van Nostrum and W. E. Hennink, Biomacromolecules, 2009, 10, 3157–3175.
48. T. Nozaki, Y. Maeda and H. Kitano, J. Polym. Sci., Part A: Polym. Chem., 1997, 35, 1535–1541.
49. Y. Y. Liu, X. D. Fan and L. Gao, Macromol. Biosci., 2003, 3, 715–719.
50. R. C. Petter, J. S. Salek, C. T. Sikorski, G. Kumaravel and F. T. Lin, J. Am. Chem. Soc., 1990, 112, 3860–3868.
51. R. Barbey, L. Lavanant, D. Paripovic, N. Schüwer, C. Sugnaux, S. Tugulu and H. A. Klok, Chem. Rev., 2009, 109, 5437–5527.
52. M. Yang, L. Y. Chu, H. D. Wang, R. Xie, H. Song and C. H. Niu, Adv. Funct. Mater., 2008, 18, 652–663.
53. M. Yang, L. Y. Chu, R. Xie and C. Wang, Macromol. Chem. Phys., 2008, 209, 204–211.
54. Y. Y. Liu, X. D. Fan and Y. B. Zhao, J. Polym. Sci., Part A: Polym. Chem., 2005, 33, 3516–3524.
55. H. Cao, J. He, L. Deng and X. Gao, Appl. Surf. Sci., 2009, 255, 7974–7980.
56. S. N. Lv, M. Q. Zhao, C. J. Cheng and Z. G. Zhao, J. Nanopart. Res., 2014, 16, 2393–2404.
57. S. N. Lv, Y. B. Song, Y. Y. Song, Z. G. Zhao and C. J. Cheng, Appl. Surf. Sci., 2014, 305, 747–752.
58. B. Luo, X. Song, F. Zhang, A. Xia, W. Yang, J. Hu and C. Wang, Langmuir, 2010, 26, 1674–1679.
59. S. Xu, W. Ma, L. You, J. Li, J. Guo, J. Hu and C. Wang, Langmuir, 2012, 28, 3271–3278.
60. J. Zheng, C. Ma, Y. Sun, M. Pan, L. Li, X. Hu and W. Yang, ACS Appl. Mater. Interfaces, 2014, 6, 3568–3574.
61. K. Cai, J. Li, Z. Luo, Y. Hu, Y. Hou and X. Ding, Chem. Commun., 2011, 47, 7719–7721.
62. J. L. Liu, W. He, L. Zhang, Z. Zhang, J. Zhu, L. Yuan, H. Chen, Z. Cheng and X. Zhu, Langmuir, 2011, 27, 12684–12692.
63. Z. Luo, K. Cai, Y. Hu, J. Li, X. Ding, B. Zhang, D. Xu, W. Yang and P. Liu, Adv. Mater., 2012, 23, 431–435.
64. P. K. Zarzychi and H. Lamparczyk, J. Pharm. Biomed., 1998, 18, 165–170.
65. M. I. Sancho, E. Gasull, S. E. Blanco and E. A. Castro, Carbohydr. Res., 2011, 346, 1978–1984.

---

Footnote

† Electronic supplementary information (ESI) available: Schematical illustration for the effect of the phase transition of PNIPAM responding to temperature change on the association constants of β-CD/ANS complexes and temperature-dependent association constants of β-CD/ANS complexes in PNIPAM–β-CD system (Fig. S1); synthesis of EDA–β-CD, Fe₃O₄@PG, Fe₃O₄@PG–CD, TGA curves of MCNCs, Fe₃O₄–APS, Fe₃O₄–Br, Fe₃O₄@PNG-2, Fe₃O₄@PNG-2–CD and Fe₃O₄@PNG-2–CD/ANS (Fig. S2); magnetic hysteresis loop of Fe₃O₄@PNG-2–CD NPs (Fig. S3). See DOI: 10.1039/c4ra11075g

---