Poly(N-isopropylacrylamide)-coated gold nanorods mediated by thiolated chitosan layer: thermo-pH responsiveness and optical properties

2.1 Materials

Tetrachloroauric III acid (HAuCl₄), silver nitrate (AgNO₃), ascorbic acid, CTAB, NaBH₄, 3-mercaptopropionic acid (3-MPA), 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), N-hydroxysuccinimide (NHS), NaOH, low molecular weight chitosan (LMWC) 100,000 g/mol with degree of deacetylation (70–80%) and viscosity of 20–300 cp, N,N′-Methylenebisacrylamide (BIS) 154.17 g/mol (99.5%), isopropylacrylamide (NIPAM) 113.16 g/mol (98%), tetramethylethylenediamine 116.9 g/mol (99%) and ammonium persulfate (APS) 228.20 g/mol (98%) were purchased from Sigma-Aldrich. Nitrogen gas was obtained from a local supplier.

2.2 GNR preparation

Synthesis of GNR was performed following the seed-mediated method previously reported by Nikoobakht El-Sayed (24). The seed solution was prepared mixing 10 ml of 200 mm CTAB solution with 10 ml of 0.5 mm HAuCl₄. Next, a solution 10 mm of NaBH₄ was added to the gold seed solution. A growth solution was prepared by pouring 10 ml of 200 mm CTAB solution into 0.30 ml of 4 mm AgNO₃ solution at room temperature. Then 10 ml of 1 mm of HAuCl₄ was poured into this solution, and after gentle mixing, 0.14 ml of 80 mm ascorbic acid was added. Finally, 0.024 ml of the seed solution was added to the growth solution at room temperature and left for 1 h to react.

2.3 Synthesis of thiomer chitosan

Chemical modification of native chitosan was carried out following a two-step coupling reaction. An aqueous solution of 3-MPA reacted with an equimolar amount of EDAC and an equimolar quantity NHS. EDAC bound carboxyl groups of 3-MPA to NHS, resulting in a new intermediate compound with terminations of thiol and NHS. O-acylisourea was also obtained from reaction. Then the conjugation of primary amines from chitosan was carried out by adding, drop by drop, a solution of the new intermediate compound to obtain the thiomer chitosan. The thiomer chitosan was purified by precipitation in 1 m NaOH solution and subsequently characterized by FT-IR spectroscopy in order to confirm the structural modification of native chitosan. Also, for the same reaction to obtain chitosan thiomer, a functionalization degree of 11% is reported (25).

2.4 Stabilization of GNR with thiomer chitosan

Stabilizer exchange reaction for gold nanoparticles was previously demonstrated by Wang et al. (9). In this work, we used this modification step to prepare the metallic surface of GNR to carry out in situ polymerization of the NIPAM by the free radical method. Briefly, an aliquot of 10 ml of 1.5×10⁻¹¹ M of GNR colloidal suspension (data obtained from a viscosimetry analysis technique) was poured into dialysis tubing 15 kDa MWCO and stirred for 2 h to reach equilibrium. Once achieved, 3.33 μmol thiomer chitosan was dissolved in 10 ml of 1 mm acetic acid solution. Polymer solution was slowly added in the same dialysis tube, and the membrane was suspended and stirred for 48 h in ultrapure water to promote the diffusion process. The colloidal suspension was analyzed in order to confirm structural and physical properties. The product obtained was chitosan stabilized GNR (GNR-Ch).

2.5 In situ NIPAM polimerization on GNR-Ch (GNR-Ch-PNIPAM)

Typical free radical polymerization of NIPAM was performed with some adjustments (26). One hundred and fifty millimoles of NIPAM was poured into a three-necked round-bottom flask and dissolved in 10 ml of ultrapure water. In the second step, the reactor flask was charged by adding 20 mmol of the cross-linking agent BIS and 20 mmol of surfactant SDS. The role of the SDS surfactant in the nucleation stage was to increase the colloidal stability of the precursor nanoparticles. Then the GNR-Ch colloidal suspension was added in volume ratios of GNR-Ch (μl):PNIPAM (ml) of 10:1 (P10), 20:1 (P20), 30:1 (P30) and 40:1 (P40) drop by drop and stirred for 1 h. NIPAM solution was heated to 70°C using a water bath. The physical removal of dissolved oxygen was a critical process in the synthesis procedure and achieved by the flow of N₂ bubbling directly into solution. Finally, 1.33 ml of 5 mm of APS was slowly added in the reaction flask. The nitrogen flow and magnetic stirring conditions were maintained for 2 h at 70°C. We observed an opalescent suspension free of polymer aggregates.

2.6 Nanoparticle characterization

FT-IR spectra were collected from spectrometer Spectrum Two (Perkin-Elmer, MA, USA). The IR spectra were collected 16 times (spectral resolution 4 cm⁻¹) and analyzed using Spectrum software. Spectra were recorded and analyzed for signal assignation. Size distribution and ζ potential measurements were obtained by DLS in the equipment Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK). The DLS measurements were performed in intensity mode, and ζ potential measurements were performed at pH5, except for the experiments varying pH. AFM images were obtained by an instrument (JSPM-4210, JEOL, MA, USA) in no contact mode. A cantilever (OMCL-AC160TS, Olympus) with resonant frequency of 300 kHz and spring constant of 42 N/m was used. Nanoparticles were diluted in ultrapure water 1:100 and poured in a new cleaved mica surface. Images were treated and analyzed in order to elucidate morphology and size of particles. UV-Vis measurements were performed in a Lambda 850 UV-Vis spectrophotometer (Perkin Elmer, Whaltham, MA, USA). The morphology of nanocomposites was analyzed by scanning electron microscopy (SEM) through a field emission scanning electron microscope (JSM-7800F, JEOL, Pleasanton, CA, USA). Samples were placed on a silicon wafer and dried before analysis.

3.1 Stabilization of GNR-thiomer chitosan

Functionalization of metallic surfaces by exchange reactions results in the improvement of physicochemical, morphological and biological properties as a function of the stabilizer used. In our case, we focused our efforts to achieve the modification of the GNR surface with thiomer chitosan by a interchange reaction (thiomer chitosan replaces CTAB). DLS was used to determine the average value of GNR size after being treated with thiomer chitosan solution (GNR-Ch). Also, ζ-potential measurements were carried out by electrophoretic mobility. Figure 1 presents average size values, in which an increase in size was evident. GNR shift in size was from 70 to 110 nm for the GNR-Ch, and the polydispersity index (PDI) value was modified proportionately (from 0.18 to 0.20). In previous research, Polystyrene-stabilized gold nanoparticles were synthesized by Muriel-Neil. They described a dispersion-matrix method with a tunable position of surface plasmon band resonance (27). Similarly, GNR could also be stabilized with thiolated low molecular weight chitosan and a subsequent functionalization with folic acid. Results indicated a significant increase in size distribution from 66 to 84.9 nm (9). In recent work, Yang et al. (28) synthesized a GNR platform following a layer-by-layer methodology. Nanoplatforms were obtained in the first step of the procedure (GNR-Ch) with an average size of 103.2 nm. Notably, this stabilizer exchange strategy can be followed in order to successfully modify a GNR colloidal system. Hence, according to our results, the increase in particle size distribution suggests that ligand modification was carried out correctly.

Figure 1:

DLS and ζ potential measurements for samples GNR and GNR-Ch. Bars symbolize the hydrodynamic diameters, and ζ potentials are represented by points.

Given the critical role of nanoparticle surface charge in the stability of colloidal systems, we proposed a modification of this parameter to increase stability by an exchange stabilizer reaction with thiolated chitosan. Figure 1 shows the ζ potential data for GNR and GNR-Ch. These values presented in noticeable increments from 38.93 to 48.53 mV for GNR and GNR-Ch, respectively. The data support the surface incorporation of the polycationic chains. Benefiting from chitosan stabilization, the platform can be widely used as biocompatible drug carrier or a precursor for multicomponent nanomaterials due to the chitosan reactivity and pH sensitivity. The results coincided with Rayavarapu et al. (29), who evaluated the properties of PEGylated and PSS nanorods using the standard wet chemistry method with CTAB as a stabilizer. In both cases, the measurements of ζ potential indicated significant modification by the effect of surface modification. These potentials were 37.7 mV (GNR), −60.5 mV (PSS-GNR) and −5.2 mV (PEGylated GNR), respectively. Charan et al. (30) synthesized a multifunctional gold nanorod polymer-based platform by EDAC coupling reaction. The resulting oligosaccharide-modified GNR presented changes in size distribution, and above all in surface charge, they reported an important increase of ζ potential to 40 mV.

3.2 In situ NIPAM polimerization on GNR-Ch (GNR-Ch-PNIPAM)

The chemical modification of native chitosan with 3-MPA was performed successfully. FT-IR analysis was carried out in order to confirm the presence of most representative bands of thiol-derivative compounds. Representative bands of free Chitosan thiomer appear at 1670 cm⁻¹ for amide group and 1560 cm⁻¹ for Amine (CONH-) (Figure 2). In addition, we performed four different experiments varying the GNR-Ch concentration to obtain GNR-Ch-PNIPAM systems (P10, P20, P30 and P40), whereas the other parameters, such as temperature and monomer concentration, remained constant. An important shift of the free Chitosan thiomer bands (1670 and 1560 cm⁻¹) was observed due to interactions of amide carbonyl coordinated with metallic gold surface. We observed the typical glucosamide -O-stretching, located around 980 cm⁻¹. A distinctive band for thiol-derivative compounds was observed (Figure 2) near to 1250 cm⁻¹ for samples (P10, P20, P30 P40 and free chitosan thiomer). This signal is associated with C-SH stretching. After the polymerization of NIPAM by the free radical method (GNR-Ch-PNIPAM) for samples P10–P40, a significant band located at 1450 cm⁻¹ associated this signal to the methyl group (CH₃) stretching (31), is observed (Figure 2); this band confirmed the presence of PNIPAM after the polymerization process. Stabilization of colloidal metal surfaces are commonly associated with electrostatic interactions between functional groups, such as amine, carbonyl or thiol and the orbital of metallic surfaces. In some cases, these interactions are triggered in significant shifts due to the vibration frequencies of specific functional groups. For example, Regiel-Futyra et al. (32) described the synthesis and characterization of colloidal composites. Spherical gold nanoparticles were synthesized using polymer as a reducing agent. The results showed an important change in the wave number for samples treated with colloidal gold, 1590 cm⁻¹ for the free amine NH₂ and 1560 cm⁻¹ for amine groups in the presence of gold nanoparticles. The collection of spectra showed an important difference for samples treated with GNR. In this analysis, it was possible to appreciate the specific peak due to carbonyl stretching in free chitosan (C=O, 1670 cm⁻¹) and amine stretching (NH₂, 1560 cm⁻¹). On the other hand, samples P10–P40 showed important shifts at lower wave number due to the double electrostatic interaction of the amine and carbonyl groups. For example, sample P10 presented displacement up to 1630 cm⁻¹ for the band associated with carbonyl groups treated with GNR-Ch, but the same sample showed a wave number shift from 1560 to 1532 cm⁻¹ in the case of amine groups. This suggests that some functional groups could interact by an electrostatic mechanism with gold nanoparticles.

Figure 2:

FT-IR spectra of free chitosan thiomer and samples of GNR-Ch-PNIPAM (P10–P40).

Polymerization of NIPAM by free radicals has been regarded as an interesting methodology for encapsulation of several molecules because of the great specificity and outstanding stimuli-responsive material properties. The incorporation of PNIPAM coating offers a viable improvement by introducing temperature-sensitive conformations and an ability to incorporate biological molecules like peptides, protein and antibodies. The average sizes of samples P10–P40 are presented in the Figure 3. As the GNR-Ch concentration increased, a proportional increment of hydrodynamic diameter was observed. We reported increments in the range from 359 nm (P10) to 372 nm (P40). We believe that increasing the number of GNR-Ch available at the NIPAM polymerization will increase the number of rods encapsulated on the composite. No statistical significance was observed between samples (Table 1). However, the PDI value presented increments at higher concentrations of nanorods (P10 PDI=10%, P20 PDI=11%, P30 PDI=13% and P40 PDI=14%), as presented in Table 1. By increasing the amount of NIPAM, a thicker shell was generated by a higher polymerization degree. In addition, more GNR-Ch could have adhered to the nanoparticle surface. In recent years, different approaches have been proposed in order to prepare a hybrid nanoshell with reference to external stimuli, like temperature. Kang et al. (33) developed a PNIPAM@gold nanorods core-shell prepared in a two-step methodology. The methodology began with a single-layer PEG-SH deposition in order to prepare a more compatible surface. TEM characterization confirmed the core-shell-like structure, and detailed AFM image showed a bump in the center for all the particles. UV-Vis characterization revealed the optical activity of the SPR, which is located at 860 nm. Thermoresponsive properties showed the volume transition at 30°C and that a reversibility process was possible by cooling (33). In a more recent work, Keerati et al. stabilized gold nanoparticles with a thermoresponsive polymer prepared by the graf method of PNIPAM-thiolated terminated microgel with gold nanoparticles stabilized with citrate. The prepared nanoparticles exhibited a semihemispherical shape and homegeneous size distribution under 10%. UV-Vis characterization detailed the surface plasmon band, which presented a shift from 520 nm for citrate-stabilized nanoparticles and 530 nm for PNIPAM-SH@goldNps (34). In comparison, Liz-marza et al. (35) prepared a near-infrared-responsive nanogel. They used surface-initiated atom transfer polymerization and further analyzed morphology and particle size distribution. The incorporation of a polymeric coating aims to improve the structural-responsive properties without affecting the colloidal stability and plasmon resonance localization. We have shown, so far, that the addition of specific concentration of GNR-Ch increased the size distribution. This behavior could be explained in terms of the polymerization mechanism, which is driven solely by factors such as the arrangement of polymer chains.

Figure 3:

DLS measurements of samples GNR-Ch-PNIPAM (P10–P40), evolution of size average as a function of GNR-Ch concentration.

In order to determine if surface charge is influenced by PNIPAM attachment, a systematic study of ζ potential evolution for different concentration of GNR-Ch was carried out. We found that the addition of PNIPAM coating on GNR-Ch induced a dramatic change of ζ potential. Figure 4 presents ζ potential measurements. Despite the fact that samples P10 to P40 had values of ζ potential close to the isoelectric point, no precipitation or aggregation was observed by DLS, even 8 weeks after preparation (results not shown). This result could be due to steric effects from chitosan polymer chains. In comparison with previous research, Bradley and Garcia-risueño (36) synthesized hybrid temperature and pH-responsive nanogel PNIPAM-co-DMAPMA@AuNps, reporting a similar ζ potential near to zero for a pH of 6 (36). This effect was attributed to the interaction of carboxylic groups because at pH 7, the carboxyl groups are protonated and the ζ potential was equal to zero. The ζ potential for each sample is shown in Figure 4. In the case of pure GNR, this ζ potential corresponded to that reported when stabilized with CTAB. GNR-Ch increased the ζ potential to 48.53 mV compared to GNR due to exposure of amine groups (NH₂). This functional group could be protonated in these conditions.

Figure 4:

ζ potential measurements of samples of GNR-Ch-PNIPAM (P10–P40), GNR-Ch and GNR obtained at pH 5.

After NIPAM polymerization, it was evident that polymer concentration could produce a screen effect over the positive charge of chitosan layer. Notably, an important change in surface charge occurred in all samples of GNR-Ch-PNIPAM from P10 to P40 with no significant difference. We associated this to the correct attachment of PNIPAM to the GNR-Ch surface. These results and others showed that the incorporation of PNIPAM coating produces composite nanoparticles with unique properties of electrophoretic mobility. This polymeric shell provides stability and protection conserving their relative ratio-volume and structural transition of PNPAM.

3.3 LCST evaluation for GNR-Ch-PNIPAM

LCST phase activity after the polymerization and influence of GNR-Ch concentration in response to thermal stimuli for GNR-Ch-PNIPAM were analyzed. To monitor the particle transition induced by temperature, we set up a temperature profile from 23°C to 41°C. Change in particle size was measured using DLS. All nanogels (P10–P40) showed temperature-sensitive discontinuous phase transition. In Figure 5, we observed initial average sizes close to 370 without a significant statistical difference for all samples. This first state was associated with an expanded or swollen structure due to hydrogen bonds with water molecules. The increase in temperature led to an important decrease in particle diameter when the temperature was near to LCST. A collapsed state was achieved before the hydrogen bonds dissipated and hydrophobic interactions predominated, which led to a more compact arrangement.

Figure 5:

DLS measurements as a function of temperature for samples P10–P40 of GNR-Ch-PNIPAM.

Kawano et al. (37) reported the preparation of hybrid nanorods with a typical core-shell structure core made up of single PNIPAM@nanorod. Core-shell structure demonstrated photothermal phase transition at 34°C for all Nrds concentrations. In addition, core-shell structure showed a blue shift of the longitudinal plasmon band at 809 nm due to the local refractive index cause by the PNIPAM shell. Thermal analysis by DLS measurements showed a similar phase-transition when compared with our results. Average size at the swollen state was close to 340 nm, whereas in the collapsed state, the nanoparticle size suddenly decayed to 150 nm. LCST temperature was 34°C in all the samples. This confirmed the temperature-structure dependence due to the PNIPAM shell in average size from 340 to 130 nm.

3.4 pH-responsive evaluation of GNR-Ch-PNIPAM

The pH-responsive properties of samples P10–P40 of GNR-Ch-PNIPAM were evaluated. Particle size of nanostructures after exposure to different pH environments was investigated. GNR-Ch-PNIPAM size distribution increased to values around 360 nm in acidic conditions for all samples, whereas a notable decrease around 80 nm diameter size for all samples was achieved when the pH was alkalized (Figure 6). We suggest that amine groups of thiolated chitosan interacted inside the nanoshell with the hydronium ions, favoring the protonated form of amine (NH3+). Protonated polycation chains could lead to repulsive interactions between chains with the same charge, leading to more expanded structure. In addition, protonated state of amine groups promoted the hydrophilic interactions, so a swollen structure was favored.

Figure 6:

Average size evolution of samples P10–P40 of GNR-Ch-PNIPAM as a function of pH for different concentrations of GNR-Ch (increasing from P10 to P40).

Chuang et al. (38) reported thermo- and pH-responsive copolymer nanoparticles of acrylic acid, N-isopropylacrylamide and chitosan by free radical polymerization with an average diameter of 200 nm. As we described, polycation chitosan plays a very important role in stabilization and formation of nanoparticles. According to their results, diameters reached a minimum value around pH 4–5 and were larger under acidic conditions. At low pH value, ionization states of amine groups are complete, while carboxyl groups of PNIPAM remain neutral. When the pH was acid, repulsive interactions predominated, causing increments in the particle size. ζ potential decreased as result of total ionization of PNIPAM carbonyl group leading to more compact structures.

3.5 Optical evaluation of GNR-Ch-PNIPAM

Synthesis of metallic nanoparticles with a thermo- or pH-responsive polymer provides an interesting model for multicomponent smart material. In case of plasmonic particles, their optical properties depend highly on factors as such as refractive index, shell morphology and thickness of coating polymeric material. Optical characterization for the set of experiments was carried out by UV-Vis spectroscopy, varying nanorods concentration in order to elucidate optimum conditions for preparing nanoparticles with PRS activity. The set of spectra presented in Figure 7 corresponded to samples P10–P40. These results revealed no aggregation process after the polimerization of PNIPAM, whereas transversal and longitudinal plasmon bands were well defined.

Figure 7:

Surface plasmon resonance for GNR and GNR-Ch-PNIPAM (P10–P40).

It is known that the surface plasmon band of GNR is sensitive to the gold particles size and the environment parameters. Therefore, the UV-Vis spectra of GNR present a longitudinal surface plasmon band (LSPB) was located at 773 nm and transversal surface plasmon band (TSPB) at 521 nm. However, a blue shift was observed in the case of PNIPAM nanocomposites. TSPB was recorded as follows: P10 (759 nm–519 nm), P20 (757 nm–515 nm), P30 (759 nm–517 nm) and P40 (519 nm–755 nm). Displacement of the surface plasmon band was correlated to the polymeric coverage thickness because this property was affected by the refractive index, causing a deviation of the incident radiation. Rayavarapu et al. (29) evaluated the optical properties of GNR modified with different polymers on the surface. They measured an LSPB at 773 nm, and a blue shift was recorded with PEGylated (760 nm) GNR and PSS (765). In a similar study, Kawano et al. (37) prepared a hybrid GNR-PNIPAM with a core-shell-like structure nanogel. PNIPAM polymerization by free radicals reaction was carried out in situ on silica-GNR using KPS as an initiator. UV-Vis spectra exhibited the same blue-shift in comparison with pure GNR. They attributed this to the variation of thickness in the PNIPAM shell. On the other hand, a red shift has been observed when GNR-PNIPAM hybrids were evaluated against higher temperature. As discussed, optical properties as SPB can be valuable for a potential application in photo-thermal therapy. Also, the advantageous response to pH and temperature could lead to the design of nanoplatforms for specific therapies.

3.6 Morphology analysis of GNR-Ch-PNIPAM

The nanocomposite GNR-Ch-PNIPAM shape was studied using AFM in a no-contact mode. Figure 8 illustrates that all nanocomposites exhibited in general an elongated shape are very similar to the characteristic structure of free nanorods. Also, Figure 8 shows that the AFM image for sample P10 with the lowest concentration of GNR-Ch and rod-like morphology can be observed with a significant increase in size, close to 200 nm. In pure GNR-Ch, the average size reported is around 70 nm. Subsequently, the addition of a higher concentration of GNR-Ch (sample P20) resulted in the increase of particle size close to 220 nm. The shape of sample P30 was semihemispherical, with a rod in the center; it was possible to observe a PNIPAM coating. Images of PNIPAM for P30 nanoparticles were analyzed, and measurements revealed a size near to 240 nm in diameter. Meanwhile, particles P40 with the highest GNR-Ch concentration clearly show a core of GNR-Ch. Measurements suggested that its size was around 200 nm in the center, and the diameter was close to 250 nm. Similarly, in Figure 9, we observed a scanning electron micrograph of sample P40, where the composite is clearly presented. In order to observe the core, the composites were exposed for around 20 s to an electron beam. After that time, some melting of the polymer was observed, and the rods were exposed (Figure 9). Singh and Lyon (39) explored a new synthetic route of PNIPAM nanogels by growing a PNIPAM shell on gold nanoparticles. The thermo-responsive nanocomposite was created by the physical absorption of amine groups (NH₂) on Au nanoparticles. PNIPAM coated onto Au nanoparticles collapsed near the LCST. This property could be interesting for drug delivery application. Jaber et al. (40) prepared Au-PNIPAM core-shell nanocrystals using a convective deposition and spin-coating method. The interspacing particle was controlled by the shell polymeric thickness and could be modulated from 5 to 100 nm. Au-PNIPAM coreshell optical properties were studied by Murphy et al. (41). In their work, they reported the synthesis of hybrid coreshell and the LSPR shift as a function of the hydrophilic-hydrophobic state. Photothermal heating by local excitation of plasmon resonance resulted in the rapid collapse of the core-shell. They also reported on architecture and morphology, demonstrated by AFM and SEM microscopy, which resulted in a single gold core coating with homogenous PNIPAM layer. According to previous work, gold nanoparticles (spheres and rods) could be coated with a PNIPAM layer following direct or indirect methods in which prior treatments are given in order to potentiate the interactions between the metallic surface and the polymeric layer. In the present work, thiolated chitosan was deposited on GNR surface via interaction between metallic surface-thiol groups. The presence of the chitosan layer increased the compatibility with the polymeric layer of PNIPAM.

Figure 8:

AFM images of GNR-Ch-PNIPAM samples P10–P40. Evolution of morphology as a function of GNR-Ch concentration.

Figure 9:

SEM image of GNR-Ch-PNIPAM sample P40.