Synthesis and characterization of thermosensitive and polarity-sensitive fluorescent PNIPAM-coated gold nanoparticles

2.1 Materials

Tetraoctylammonium bromide (TOAB), HAuCl₄, α-lipoic acid, 2-hydroxyethyl methacrylate (HEMA), 4-dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide (DCC) were purchased from Sinopharm (Shanghai, China) and used without further purification. N-isopropylacrylamide (NIPAM) was purchased from TCI (Shanghai, China) and recrystallized from toluene and hexane. β-Cyclodextrin (β-CD) (J&K Scientific, Shanghai, China) was recrystallized twice from water and dried at 90°C. Azobisisobutyronitrile (AIBN) (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) was purified by recrystallization from ethanol. 4-Methacryloyloxy-4′-dimethylaminochalcone (MADMAC) was prepared by the condensation of 4-hydroxyl-4′-dimethylaminochalcone with methacryloyl chloride in solution of tetrahydrofuran and triethylamine according to our previous procedure (15). 2-(Methacryloyloxy)ethyl α-lipoate (MAEL) was synthesized by the condensation of α-lipoic acid with 2-hydroxyethyl methacrylate in tetrahydrofuran in the presence of dicyclohexylcarbondiimide (DCC) and 4-dimethylaminopyridine (DMAP) at room temperature for 12 h (16). All other reagents were supplied analytically pure and were not further purified.

2.2 Preparation of MAEL-capped AuNPs

MAEL-capped AuNPs [2-(methacryloyloxy)ethyl α-lipoate-capped AuNPs] were prepared by use of a two-step method (16). First, TOAB-stabilized AuNPs were prepared in a two-phase water/TOAB/toluene system without thiol ligand. Then, MAEL-capped AuNPs were prepared through the ligand exchange reaction between TOAB and MAEL on the surface of Au nanoparticles.

2.3 Preparation of AuNPs@P(NIPAM-co- MADMAC)

AuNPs@P(NIPAM-co-MADMAC) were synthesized by the radical “grafting through” copolymerization method. The MAEL-capped AuNPs (20 mg), NIPAM (452 mg, 4 mmol), MADMAC (variable), and AIBN (16.4 mg, 0.1 mmol) were dissolved in THF solution of 70 ml. The mixtures were stirred in a nitrogen atmosphere at 65°C for 8 h. The resulting mixture was precipitated three times from THF into diethyl ether to obtain a dark solid. Then, the dark solid was dialysed in water for 2 weeks. The pure AuNPs@P(NIPAM-co-MADMAC) were obtained by lyophilization.

2.4 Measurement methods

FT-IR spectra were measured on a Nicolet 560 FT-IR spectrometer using KBr pellets. UV-Vis spectra were obtained from a Purkinje General 1901 spectrophotometer. TEM was performed on a JEM-100CX with an 80 kV accelerating voltage. A Bruker AMX300 NMR spectrometer was used to measure the samples’ ¹H NMR spectra. A TA Q600 with a scan rate of 10°C·min⁻¹ under N₂ was used to perform thermogravimetric analysis (TGA). XPS was used to characterize the chemical composition of the AuNPs. LCST is definited as the temperature at which transmittance fell by 50%. This was determined by measuring the transmittance of 1 gl⁻¹ AuNPs aqueous dispersion at 750 nm at a 0.5°C·min⁻¹ warming rate. A Hitachi 850 fluorescence spectrometer was used to measure fluorescence spectra.

3.1 Preparation and characterization of AuNPs@P(NIPAM-co-MADMAC)

First, AuNPs@P(NIPAM-co-MADMAC) were prepared by the radical “grafting through” polymerization of MADMAC, polymerizable double bond-containing AuNPs (MAEL-capped AuNPs), and NIPAM in a THF solution using AIBN as an initiator. To prevent crosslinking among AuNPs, the “grafting through” polymerization was carried out in a dilute solution, which we proved in a previous work to be a feasible approach (16). AuNPs@P(NIPAM-co-MADMAC) were purified through precipitation and dialysis. Finally, the pure AuNPs@P(NIPAM-co-MADMAC) solid was obtained by lyophilization. Due to the polymer attached to their surfaces, these AuNPs can be effectively dispersed in water, ethanol, DMF, THF, chloroform, and so on. Furthermore, we investigated the effect of molar feed ratio of MADMAC versus NIPAM on the composition of AuNPs. As shown in Table 1, organic polymer content among the AuNPs is almost unchanged when the molar feed ratio of MADMAC varies between 1.5 and 3%, which was determined by TGA. Based on the methine proton (PNIPAM) to aromatic protons of the chalcone unit, the molar ratio of MADMAC versus NIPAM in the AuNPs was estimated by ¹H NMR spectroscopy. When there is more feed of MADMAC monomer, it has higher content of MADMAC unit in the AuNPs. And the composition of MADMAC unit in the AuNPs is less than that in feed probably owing to monomer reactivity ratios.

Secondly, the structure of AuNPs@P(NIPAM-co-MADMAC) was analyzed by XPS, UV-Vis, TEM, ¹H NMR, and FT-IR spectroscopy. Due to changes in the matter coated on the surface of AuNPs, the SPR (surface plasmon resonance) absorption of AuNPs@P(NIPAM-co-MADMAC)-3 blueshifted to 527 nm, while MAEL-capped AuNPs was 532 nm in UV-Vis spectra (Figure 1). There was an absorption band around 415 nm, which was attributed to the chalcone group in AuNPs. From the TEM images (Figure 2), it was found that the AuNP diameters were between 3 and 12 nm after “grafting through” copolymerization, and the AuNPs were moderately dispersed. Some aggregation resulted from the TEM sample preparation, and not from inter-nanoparticles crosslinking. XPS spectra (Figure 3) of AuNPs@P(NIPAM-co-MADMAC)-3 showed the characteristic signals of nitrogen, carbon, oxygen, and gold. Gold signals were very weak and sulfur signals did not appear due to large amounts (65%, as determined by TGA) of organic polymers on AuNP surfaces. Figure 4 shows the decomposition cure of the AuNPs and the poly(NIPAM-co-MADMAC) copolymer in which MADMAC unit content is 1.9% and 1.8% separately. Except gold element, the remaining substances should be poly(NIPAM-co-MADMAC) copolymer coated on the surface of the AuNPs among AuNPs@P(NIPAM-co-MADMAC)-3. At high temperature, the remaining material is very small in the case of the copolymer (curve b). Thus, in the case of curve a, the remaining material is probably Au. This was used to determine the polymer shell content and the results were shown in Table 1. The chemical compositions of the organic compound on the AuNP surface was analyzed by ¹H NMR and FT-IR spectroscopy (Figures 5 and 6); they were confirmed to be consistent with the poly(NIPAM-co-MADMAC) copolymer. The above results showed that AuNPs@P(NIPAM-co-MADMAC) were successfully synthesized by the radical “grafting through” copolymerization.

Figure 1:

UV-vis absorption spectra of MAEL-capped AuNPs in toluene (A), MADMAC in ethanol (B) and AuNPs@P(NIPAM-co-MADMAC)-3 in chloroform (C).

Figure 2:

TEM image of AuNPs@P(NIPAM-co-MADMAC)-3.

Figure 3:

XPS spectrum of AuNPs@P(NIPAM-co-MADMAC)-3.

Figure 4:

TGA curve of AuNPs@P(NIPAM-co-MADMAC)-3 (A) and poly(NIPAM-co-MADMAC) (B).

Figure 5:

¹H NMR spectra of AuNPs@P(NIPAM-co-MADMAC)-3 in CDCl₃.

Figure 6:

FT-IR spectra of AuNPs@P(NIPAM-co-MADMAC)-3.

3.2 Thermosensitivity of AuNPs@P(NIPAM-co-MADMAC)

As shown in Figure 7A, the transmittance at 750 nm for the 1 gl⁻¹ AuNPs@P(NIPAM-co-MADMAC) aqueous dispersion decreased rapidly when the temperature was increased to reach the phase transition point. The LCST is listed in Table 1. The LCST decreases as the MADMAC unit content in the AuNPs increases, resulted from the increasing of hydrophobic group. Furthermore, the temperature-responsive phase transition exhibited reversibility (Figure 7B), and a macroscopically transparent to opaque phenomenon could be obviously observed when the temperature fluctuated between 25 and 45°C. Here, the thermosensitivity of the prepared polymer-coated AuNPs originated from the polymer on the AuNP surfaces.

Figure 7:

Transmittance related to temperature for the AuNPs aqueous solution (A) and the reversible change of transmittance with respect to the temperature fluctuation for AuNPs@P(NIPAM-co-MADMAC)-3 (B).

3.3 Fluorescence of AuNPs@P(NIPAM-co-MADMAC)

The sensitive fluorescent properties of AuNPs@P(NIPAM-co-MADMAC) were investigated, shown in Figure 8. First, there was no fluorescence at 25°C in water for AuNPs@P(NIPAM-co-MADMAC)-3. AuNPs@P(NIPAM-co-MADMAC)-3 aqueous dispersion exhibited weak fluorescence when the temperature was increased to 45°C, or when β-CD was added. Furthermore, the fluorescent intensity of AuNPs@P(NIPAM-co-MADMAC)-3 was rapidly enhanced, and the fluorescent wavelength underwent a blue shift when the solvent was changed from water to ethanol or chloroform. 4′-Dimethylaminochalcone (DMAC) is a fluorophore with a charge donor and acceptor parts in its molecular structure, which causes solvent polarity sensitivity (17), (18). With the weakening in the solvent polarity, nonradiative effects may be decreased by the n-π* state far away from the π-π* state, which results in increased fluorescent intensity. In our work, the DMAC unit was covalently immobilized into AuNPs by the “grafting through” technique, which endowed the AuNPs with solvent-sensitive fluorescent properties. Temperature increases induced the aggregation of AuNPs@P(NIPAM-co-MADMAC) because thermosensitive poly(NIPAM-co-MADMAC) chains on AuNP surfaces arose from the phase separation. The polarity near the DMAC unit was weakened due to the weak polar polymers instead of strong polar water, which sharply enhanced fluorescent intensity. Similarly, the addition of β-CD reduced the polarity surrounding DMAC fluorophore for the formation of the inclusion complex between DMAC and β-CD, and improved the fluorescent intensity of AuNPs@P(NIPAM-co-MADMAC).

Figure 8:

Fluorescent spectra of AuNPs@P(NIPAM-co-MADMAC)-3 in different solvents (A) and water at different temperature (B).