Spectroscopic and electron microscopic analysis of bi-ligand functionalized glycopolymer/FITC–gold nanoparticles

Abstract

Herein, we report a strategy for quantifying the relative proportion of di-block copolymers of poly(ethylene glycol) (PEG) on fluorescein isothiocyanate (FITC) functionalized gold nanoparticles (AuNP) by releasing the FITC via the self-assembly process. The self-assembly processes of terminally functionalized trithiocarbonate PEG and homoglycopolymers were also assessed to demonstrate the usefulness of the terminal functional groups of the glycopolymers on the surface of well-dispersed colloidal suspensions of AuNP. The synthesized AuNP colloidal suspension showed a surface plasmon resonance (SPR) peak at 517 nm and a red shift of about 19 nm, observed after the addition of FITC to the AuNP suspension. The transmission electron microscopy (TEM) images and histogram of the AuNP nanoparticles size distribution showed an average diameter of 8 nm, where the FITC functionalization of AuNP led to agglomeration. The fluorescence spectra reveal that the PEG-I binds to the FITC–AuNP and subsequently, FITC is proportionately released from the FITC–AuNP surface. This indicates that PEG-I possesses a stronger binding affinity for AuNP. Released concentrations of FITC fluorescence were calculated based on the amount of PEG-I added to the colloid of FITC–AuNPs and a linear response was observed. The homoglycopolymer, poly[6′-(acryloxy)hexyl-2,3,4,6-tetra-O-acetyl-D-glucopyranoside] [P(AHTAGP)], showed a weak binding affinity towards the AuNP surface, which was significantly improved in the PEG based glycopolymer, PEG-b-P(AHTAGP), with similar functional moiety. The di-block copolymer with glucose moiety of greater pendant spacer length showed better binding affinity for AuNP, whereas the bi-ligand functionalization of AuNP with the glycopolymer and FITC was more effective with glucose moieties of shorter pendant spacer length. The variation of the pendant spacer length of the functional moiety in the di-block glycopolymers has the potential advantage of serving multiple purposes in targeted delivery and detection.

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1. Introduction

Inorganic nanomaterials have been extensively used for biomedical applications in diagnostics,^1,2 drug delivery,³ tissue engineering,^4,5 cancer therapy⁶ and bio-imaging.⁷ For instance, gold nanoparticles (AuNPs) have gained significant research interest due to their potential biological applications resulting from the tunability of their size, shape and surface dependent properties.⁸ Citrate capped AuNPs with sizes between 10 and 20 nm are commonly used, where the sizes of the particles depend upon the ratio of citrate and gold salt.⁹ Reduction of gold salt by sodium borohydride (NaBH₄) can provide highly dispersed gold nanoparticles with sizes in the range of 5–8 nm, without using any capping agents.¹⁰ There are reports that show even smaller sized gold nanoparticles (1–2 nm) in a controlled reduction with thiolic capping ligands.¹¹ It has also been reported that stable gold nanoparticles can be achieved with appropriate capping, but the capping introduces toxicity, which is not desirable for biological applications.^12,13 Agarwal et al.¹⁴ described an effective, rapid and facile hydrosol approach for the synthesis of monodisperse ultra-small gold nanoparticles (∼2 nm) by the reduction of gold salt by lithium borohydride.

Wangoo et al.¹⁵ reported a method for protein conjugation on gold salt reduced by glutamic acid, whereas, high dispersity was achieved in tryptophan capped gold nanoparticles.¹⁶ Functionalized gold nanoparticles were prepared and fluorescence resonance energy transfer (FRET) estimated in bio-conjugate AuNPs.^17,18 However, in the development of FRET based immune sensors, the dye molecules were conjugated with antibody molecules, and were quenched by the AuNPs.¹⁹ The release of fluorescence occurred quite specifically, while these antibody immobilized gold nanoparticles were bound to cancer cells. On the other hand, there were challenges of stability when the metal nanoparticles were functionalized with multiple molecules of different types to develop FRET based sensors.^20–22 Previously, stable bi-ligand conjugates on ultra-small gold nanoparticles, reduced by lithium borohydride, were prepared and used for the FRET based estimation of collagen in mice.¹⁴ These particles showed higher stability towards bi-ligand co-functionalization with FITC and fluorescent lysine molecules. The replacement of bi-ligand fluorophores by collagen and the reappearance of fluorescence were used for the sensitive fluorometric detection of rat tail collagen.

In recent years, the use of glycopolymers in biology has increased, due to their multivalent nature, biocompatibility and functional response, similar to the natural systems.^23–26 The multivalent nature of glycopolymers can have multiple advantages, as these can simultaneously be used as drug carriers and attach to target specific molecules. Several studies have shown the usefulness of metal surface functionalization with multifunctional glycan in biomedical applications.^27–29 Previously, living radical polymerization with reversible addition–fragmentation chain transfer (RAFT) reagent was used to synthesize glycopolymer substituted gold nanoparticles.³⁰ An extensive review of the direct polymerization of glycomonomers or post-glycosylation of pre-formed polymers was carried out to show the progress in the synthesis of functional glycopolymers.³¹ A detailed and systematic investigation on the stability and efficacy of the cationic polyethylenimine conjugated AuNPs for biological systems reported that chemical functionality most likely influences colloidal stability.³² Papp et al.³³ reported the synthesis and selectivity towards the binding response of multivalent glyco-architectures of hyperbranched polyglycerols. The use of gold complexes with sugar ligands for studying the anti-proliferative effect against human ovarian carcinoma cells showed the high stability and low toxicity issues faced by discrete gold based chemotherapeutics.³⁴

In this work we report the synthesis of a bi-ligand functionalized multivalent glycopolymer–gold complex. At first, stable colloidal suspensions of gold nanoparticles were synthesized by the reduction of gold salt by sodium borohydride, followed by the preparation of FITC functionalized gold nanoparticles, with complete quenching of FITC fluorescence to load controlled concentrations of the RAFT generated glycopolymer onto gold nanoparticles. This enabled us to monitor the glycopolymer loading on the gold nanoparticles by the FITC ligand replacement process, in terms of restored fluorescence energy, by releasing FITC from the gold surface. Further, we investigated the effect of the pendant alkyl chain length of the glucose moiety of the glycopolymer segment of the di-block copolymer on the binding of AuNPs.

2. Experimental section

2.1 Materials

Gold(III) chloride (AuCl₃), sodium borohydride (NaBH₄), fluorescein isothiocyanate (FITC) and methanol (all obtained from Sigma-Aldrich), anhydrous D-glucose (from Fisher Scientific Pvt. Ltd.), acetic anhydride and sodium acetate (from Sisco Research Laboratories Pvt. Ltd.), methylethylketone (MEK), acryloylchloride, triethylamine (Et₃N), molecular sieves, 4 Å (from S.D. Fine-Chem Limited), azobisisobutyronitrile (AIBN, from Avra Synthesis Pvt. Ltd.), boron trifluoride diethyl etherate (BF₃·Et₂O), 1,6-hexanediol, 2-cyano-2-propyldodecyltrithiocarbonate (CPDTC), poly(ethylene glycol) methyl ether (4-cyano-4-pentanoate dodecyl trithiocarbonate) (PEG-DT, number average molecular weight (M_n): 2400 (751634-5G), from Sigma-Aldrich), chloroform and 1,4-dioxane (from Spectro Chem), were used as received. Tetrahydrofuran (THF) (Finar Chemicals Ltd.) was distilled over sodium wire with benzophenone under nitrogen atmosphere. Dichloromethane (DCM) (Finar Chemicals Ltd) was dried over calcium hydride with stirring overnight and distilled.

2.2 Methods

2.2.1 Synthesis of gold nanoparticles. A 2 ml solution of 1% (w/v) AuCl₃ was added to the solution of NaBH₄ (0.33, 0.66 and 1.32 mM in 198 ml Milli Q water) and the mixture was left at room temperature overnight. Colloidal gold nanoparticles were formed and described as AuNP(a), AuNP(b) and AuNP(c), respectively, based on the use of the various NaBH₄ concentrations (0.33, 0.66 and 1.32 mM). In the past, Storhoff et al.¹ prepared stabilizer-free colloidal AuNPs with the use of NaBH₄ as reducing agent, with the possibility of anion capping of the boron based ions of the reducing agent. The color of the aqueous colloidal suspension of AuNP was stable without using stabilizing agents for the capping. To test the stability of the AuNP colloidal suspension at various pH scales, 1 M HCl and 1 M NaOH were used to change the pH scale. The pH of the solution was measured using MERCK pH indicator paper to avoid any effects of electronic signals generated while using electronic automated pH meters.

2.2.2 Preparation of FITC–gold nanoparticle conjugates. A solution containing 3 μl of 500 μM of FITC in 5 ml of alcohol (99.9% ethyl alcohol) was mixed with 3 ml of AuNP(b) colloidal gold nanoparticles. Finally, 0.5 μM of FITC were incubated in 3 ml of AuNP at room temperature for 30 min to obtain FITC–gold nanoparticle conjugates.

2.2.3 Monomer synthesis. Glycomonomers, acryl-2,3,4,6-tetra-O-acetyl-D-glucopyranoside (ATAGP), (acryloxy)butyl-2,3,4,6-tetra-O-acetyl-D-glucopyranoside (ABTAGP) and 6′-(acryloxy)hexyl-2,3,4,6-tetra-O-acetyl-D-glucopyranoside (AHTAGP) were prepared (Scheme S1, ESI†) according to the reported methods.^35–37

2.2.4 Synthesis of poly[6′-(acryloxy)hexyl-2,3,4,6-tetra-O-acetyl-D-glucopyranoside] [P(AHTAGP)]. The AHTAGP was polymerized by the RAFT process using CPDTC as the RAFT agent with AIBN as a radical initiator in THF solvent to produce poly[6′-(acryloxy)hexyl-2,3,4,6-tetra-O-acetyl-D-glucopyranoside] [P(AHTAGP)].³⁸ Yield: 68%. FT-IR (KBr, cm⁻¹, ν): 2963 (–CH–), 1756 (–O–C=O–), 1634 (–C=S–), 1437, 1372 (–O–C–, ester) and 1226 (–C–O–C–). ¹H NMR (CDCl₃, 300 MHz, δ): 6.40–6.24, 5.84–5.59, 5.53–5.37, 5.37–5.21, 5.21–4.95, 4.48–4.20, 4.20–4.03, 3.99–3.79, 3.76–3.54, 2.74–2.25, 2.19–1.94, 1.60–1.50, 1.40–1.23 and 0.93–0.81 ppm. ¹³C NMR (CDCl₃, 75 MHz, δ): 20.45, 68.26–67.27, 70.46–69.60, 72.61, 91.75, 169.11, 169.32, 169.30, 170.51 and 170.61 ppm.

2.2.5 Synthesis of PEG based di-block glycopolymers. The PEG based di-block glycopolymers were prepared by using the corresponding acrylate monomers in the RAFT process with PEG-DT as the RAFT agent and AIBN as the initiator in 1,4-dioxane solvent for a polymerization period of 30 h. Di-block glycopolymer spectral data are provided in ESI.†
PEG-b-poly(acryl-2,3,4,6-tetra-O-acetyl-D-glucopyranoside) [PEG-b-P(ATAGP)]. Yield: 53%. FT-IR (KBr, cm⁻¹, ν): 2963 (–CH–), 1755 (–O–C=O–), 1631 (–C=S–), 1438, 1372 (–O–C–, ester) and 1224 (–C–O–C–). ¹H NMR (CDCl₃, 300 MHz, δ): 6.52–6.19, 5.89–5.60, 5.54–5.36, 5.34–5.24, 5.21–4.99, 4.52–4.22, 4.21–4.01, 3.99–3.80, 3.70–3.58, 3.54–3.41 (glucose –CH and –OCH₂– PEG protons), 2.80–2.28 (acrylate protons adjacent to the terminal end group), 2.26–1.89 (acetylated –CH₃ protons), 1.87–1.36 (–CH₂ protons and acrylate back bone protons) and 1.32–0.97 ppm (terminal end –CH₃ protons). ¹³C NMR (CDCl₃, 75 MHz, δ): 19.16–21.33, 30.65, 32.95–36.73, 38.49–42.68, 60.63, 61.65, 67.51, 69.02, 69.42, 70.36, 72.51, 89.11, 91.65, 169.29, 169.92 and 170.47 ppm.
PEG-b-poly[4′-(acryloxy)butyl-2,3,4,6-tetra-O-acetyl-D-glucopyranoside] [PEG-b-(ABTAGP)]. Yield: 30%. FT-IR (KBr, cm⁻¹, ν): 2962 (–CH–), 1755 (–O–C=O–), 1634 (–C=S–), 1439, 1372 (–O–C–, ester) and 1226 (–C–O–C–). ¹H NMR (CDCl₃, 300 MHz, δ): 5.80–5.56, 5.51–4.63, 4.42–4.16, 4.15–3.92, 3.91–3.78, 3.76–3.68, 3.67–3.39, 3.38–3.18 (glucose –CH and –OCH₂ PEG protons), 2.61–2.25 (acrylate protons adjacent to terminal end), 2.21–1.77 (acetylated –CH₃ protons), 1.75–1.46, 1.43–1.08 (–CH₂ protons and acrylate backbone protons) and 0.97–0.76 ppm (terminal end –CH₃ protons). ¹³C NMR (CDCl₃, 75 MHz, δ): 14.09, 20.49, 22.59, 25.72, 25.46, 28.47, 29.12, 29.51, 31.74, 40.11, 42.46, 59.06, 61.42, 67.17, 67.95, 68.35, 69.40, 69.92, 70.44, 71.74, 72.53, 90.18, 169.41, 169.94 and 170.46 ppm.
PEG-b-poly[6′-(acryloxy)hexyl-2,3,4,6-tetra-O-acetyl-D-glucopyranoside] [PEG-b-(AHTAGP)]. Yield: 52%. FT-IR (KBr, cm⁻¹, ν): 2945 (–CH–), 1756 (–O–C=O–), 1633 (–C=S–), 1439, 1372 (–O–C–, ester) and 1226 (–C–O–C–). ¹H NMR (CDCl₃, 300 MHz, δ): 6.45–6.20, 5.93–5.57, 5.54–5.37, 5.37–5.22, 5.22–4.91, 4.57–4.20, 4.19–4.01, 4.00–3.81, 3.80–3.37, 3.56–3.52, 3.52–3.46, 3.18–3.04, 2.81–2.3025, 2.26–1.88, 1.87–1.49 and 1.48–1.08 ppm. ¹³C NMR (CDCl₃, 75 MHz, δ): 8.42, 20.37, 25.39, 28.32, 29.24, 30.66, 31.70–35.66, 37.82, 39.0–41.90, 45.61, 58.81, 61.47, 64.18, 67.45, 69.57, 70.31, 72.4, 73.2, 91.97, 169.20, 169.84 and 170.37 ppm.

2.2.6 Deacetylation of acetylated glycopolymers. Acetylated homoglycopolymer or PEG based di-block copolymer (200 mg) was dissolved in an anhydrous methanol and chloroform solution (2 : 1, total volume: 10 ml) and left stirring under a nitrogen atmosphere. A solution of NaOMe (101 mg) in anhydrous methanol (2 ml) was added through a syringe and the reaction mixture was allowed to stir for 90 min. Afterward, methanol and chloroform were removed by vacuum evaporation and neutralized with 2 M HCl solution by stirring over night. Subsequently, the solution was passed through the basic alumina to neutralize the solution. Excess water was removed by lyophilisation and the polymer was recovered as a white powder by precipitation in excess acetone.

PAHGP: FT-IR (KBr, cm⁻¹, ν): 3425 (–OH), 2927 (–CH–), 1755 (–O–C̲=O̲–), 1635 (–C=S–), 1430, 1383 (–O–C–, ester) and 1239 (–C–O–C–). PEG-b-PAGP: FT-IR (KBr, cm⁻¹, ν): 3425 (–OH), 2929 (–CH–), 1730 (–O–C̲=O̲–), 2200 (–CN), 1592 (–C=S–) and 1059 (C–O–C). PEG-b-PABGP: FT-IR (KBr, cm⁻¹, ν): 3420 (–OH), 2927 (–CH–), 1736 (–O–C̲=O̲–), 2200 (–CN), 1629 (–C=S–) and 1053 (C–O–C). PEG-b-PAHGP: FT-IR (KBr, cm⁻¹, ν): 3424 (–OH), 2926 (–CH–), 1738 (–O–C̲=O̲–), 2200 (–CN), 1594 (–C=S–) and 1079 (C–O–C).

2.2.7 Bio-conjugate formation with glycopolymers on FITC functionalized AuNPs. Stock solutions of 0.002 g ml⁻¹ of all the glycopolymers used were prepared in water and vertex for 1 h to obtain complete dispersion. Then, each polymer solution was added to the 3 ml of FITC–AuNPs colloid to get the final five concentrations of 60 to 300 μg ml⁻¹, with 60 μg ml⁻¹ increments, and incubated for 30 min at every step.

2.2.8 Equipment. The FT-IR (Thermo Nicolet Nexus 670 spectrometer) spectra were recorded at a resolution of 4 cm⁻¹ using KBr optics at room temperature and a minimum of 32 scans were signal-averaged. The proton nuclear magnetic resonance (¹H NMR) and carbon nuclear magnetic resonance (¹³C NMR) spectra were recorded on an AVANCE-300 or INOVA-500 spectrometer in CDCl₃ with tetramethylsilane as an internal standard. Gel permeation chromatography (GPC) was performed by using a Shimadzu system built-in with one (300 × 7.5 mm) PLgel 5 μm 10 × 10³ Å column (VARIAN) and evaporative light-scattering (PL-ELS) detector (Polymer Laboratories). The eluent was DMF with a flow rate of 0.5 ml min⁻¹ at 30 °C. Calibration for detector response was gained using a single narrow PS standard (molecular weights: 550; 1480; 3950; 10 680 and 31 420 from Polymer Labs). A 1 mg sample of polymer was dissolved in 1 ml of DMF. Values of M_n and M_w/M_n were determined using LC Solution for Windows software. Thermogravimetric analyses were carried out on a TGA Q-500 thermal analyzer at a heating rate of 10 °C min⁻¹ under nitrogen atmosphere. Glycopolymers thermal transitions were obtained by differential scanning calorimeter, DSC Q100 at a heating rate of 10 °C min⁻¹ under nitrogen atmosphere. The data from second heating and cooling curves are reported. Absorption spectra of AuNPs colloids were measured by a UV-2600 SHIMADZU spectrophotometer. The F-7000 HITACHI Fluorescence spectrophotometer was used with 2.5 nm of slit width for both emission and excitation. The fluorescence spectra of FITC and FITC–gold nanoparticle conjugates were obtained at 488 nm, excitation and emission spectra were acquired between 495 and 650 nm. The TEM micrographs of the AuNPs were obtained by a 120 kV Transmission Electron Microscope from JEOL, Japan.

3. Result and discussion

3.1 FITC functionalised AuNPs

The UV-Vis spectra and electron microscope images of synthesized AuNP colloids with different concentrations are shown in Fig. 1. The sizes of the nanoparticles obtained from various NaBH₄ concentrations were calculated from the TEM images and UV-Vis spectra, and the data are presented in Table 1. The particles synthesized with lower concentrations of NaBH₄ showed a very good monodispersity, and with the use of 1.32 mM NaBH₄, an increase in AuNP population density was observed in the TEM images. Using the mathematical model developed by Haiss et al.,³⁹ the AuNP size and density were also estimated from spectroscopic observations. The values of λ_SPR (surface plasmon resonance) at <525 nm are indicative of small size of particles; hence, the estimation of particle size was carried out with the ratio of absorbance at λ_SPR/λ₄₅₀, as described in literature.³⁹ The average particle size for AuNP was ∼7 nm which is consistent with the TEM observations. Fig. 2 shows the UV-Vis spectra of AuNP colloidal suspension at various pH values and the λ_SPR peak position of AuNP was varied by about 8 nm, with the variation of the pH scale. The resulting absorbance confirmed that the AuNPs reduced by 0.66 mM NaBH₄ were stable in the pH range of 2 to 10.

Fig. 1 UV-Vis spectra (A–C) and TEM images (D–F) of synthesized AuNPs at NaBH₄ concentrations of (A and D) 0.33 mM, (B and E) 0.66 mM and (C and F) 1.32 mM.

Table 1 Experimental conditions for synthesis of AuNPs, and the resulting AuNPs characteristics

Code	AuCl3/mM	NaBH4/mM	λSPR^a/nm	A450^b	ASPR^c	C^d/nM	Particle diameter/nm
a SPR peak position from UV-Vis spectra.b Absorbance peak at 450 nm.c Absorbance at SPR peak.d Concentration estimated from UV-Vis spectroscopy data.
AuNP(a)	0.2	0.33	516.5	0.58	0.84	9.04	8.5
AuNP(b)	0.2	0.66	516.5	0.61	0.9	9.53	9.2
AuNP(c)	0.2	1.32	519	0.65	0.94	10.14	8.4

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Fig. 2 (A) UV-Vis spectra of AuNP at pH of (a) 2, (b) 3, (c) 4, (d) 5.4, (e) 7, (f) 8 and (g) 10. (B) SPR peak position of AuNP dependence at various pH values. (C) Optical image of AuNP colloidal suspension at different pH values.

Fluorescence spectra of the FITC solution before and after the addition of various concentrations of AuNP colloids are shown in Fig. 3A. The characteristic peak emission between 495 and 650 nm was observed for FITC solution excited at 488 nm. There was a blue shift in the fluorescence with the addition of AuNP colloids to the FITC solution, which indicates the possible functionalization of the AuNP surfaces by FITC (–NCS group).³⁹ A gradual decrease in fluorescence intensity of the FITC solution was observed with the increasing concentration of AuNPs, which was quenched completely with the use of AuNPs synthesized using 0.66 mM of NaBH₄ [AuNP(b)]. We optimized the FITC adsorption at the surface of AuNP by measuring the fluorescence reduction with an increase in AuNP concentration, for a constant amount of FITC (0.5 μM) (ESI, Fig. S1†). Therefore, an optimum concentration was estimated by adjusting the concentrations FITC (0.5 μM) and AuNPs (3 ml) until the disappearance of the FITC peak in the fluorescence spectra, which we concluded to be an indicator of complete coverage of AuNP nanoparticles with FITC.

Fig. 3 (A) Fluorescence spectra of FITC (0.5 μM) and FITC–AuNP at various concentrations of AuNP and the arrow indicates the ratio of AuNP and FITC solution at 1 : 6, 1 : 5, 1 : 4, 1 : 3, 1 : 2 and 1 : 1, (B) UV-vis spectra of (a) FITC and (b)–(f) for FITC–AuNP at various concentrations of AuNP : FITC as (a) 1 : 6, (b) 1 : 4, (c) 1 : 3, (d) 5 : 12, (e) 1 : 2 and (f) 2 : 3, and (g) only AuNP, (C) TEM image of AuNP synthesized at 0.66 mM of NaBH₄, (D) histogram of particle size distribution the colloids of (C) and (E) TEM image of (0.5 μM) FITC functionalized AuNP.

Fig. 3B shows the UV-Vis spectra of FITC, AuNP and FITC functionalized AuNP (FITC–AuNP). The absorption bands at 275, 255 and 478 nm correspond to the FITC dye molecules in the water,⁴⁰ whereas synthesized AuNP(b) showed a SPR peak at 517 nm. The red colour of the gold nanoparticles colloid changed to blue with the addition of the FITC–AuNP suspension. The UV-Vis spectra of the FITC–AuNP at various concentrations showed a red shift in the SPR peak of about 19 nm, after the addition of the FITC solution. The increase in the AuNP concentration in FITC solution resulted in an increase in the broadening of the SPR peak, and a shift towards higher wavelength.

The TEM images and histogram of the synthesized AuNP revealed that they had an average particle size of 8 nm (Fig. 3C and D). After functionalization with FITC, nanoparticles were agglomerated as shown in Fig. 3E.

3.2 Characteristics of synthesized glycopolymers

Homopolymers of AHTAGP and PEG based di-block copolymers with the glycoacrylates ATAGP, ABTAGP and AHTAGP were prepared by the RAFT process using CPDTC and PEG-methyl ether (4-cyano-4-pentanoate dodecyl trithiocarbonate) as RAFT agents, respectively, with AIBN initiator. The resulting P(AHTAGP) and PEG-b-P(ATAGP), PEG-b-P(ABTAGP) and PEG-b-P(AHTAGP) polymers pendant 2,3,4,6-tetra-O-acetyl-D-glucopyranoside units were deacetylated⁴¹ in the presence of a sodium methoxide/chloroform/methanol mixture to obtain the P(AHGP) and PEG-b-P(AGP), PEG-b-P(ABGP) and PEG-b-P(AHGP), respectively. The confirmation of the structures of the polymers are given in ESI,† and PEG-I and di-block glycopolymer structures are presented in Fig. 4.

Fig. 4 Structures of the PEG initiator and PEG based di-block copolymers.

The P(AHTAGP) and PEG-b-P(ATAGP), PEG-b-P(ABTAGP) and PEG-b-P(AHTAGP) polymers and their deacetylated polymers molar masses with polydispersities (M_w/M_n) are summarized in Table 2. Covalently bonded pendant glucose moieties in the di-block copolymers were in the range of 31–46 wt%. The deacetylated glycopolymers are soluble in water; with acetylated moieties, they were insoluble. The 2,3,4,6-tetra-O-acetyl signal integration of the acetylated polymers glucose pendants occurred at δ 1.8–2.3 ppm, whereas, after deacetylation, signals at δ 3.8–4.0 ppm corresponding to the –OH groups of the glucose moieties⁴² were observed in the ¹H NMR spectra. The FT-IR spectra of the acetylated polymers showed characteristic strong absorptions for C=O stretching centred at about 1756 cm⁻¹, whereas the deacetylated polymers exhibited the C=O stretching peaks in the range of 1730–36 cm⁻¹. The –OH signals of the deacetylated polymers showed characteristic strong absorptions centred at 3425 cm⁻¹. The temperatures of 10% (T_d10%) weight loss of the acetylated polymers were higher than T_d10% of the deacetylated polymers, as shown in Table 2. The glass transition temperature (T_g) of the acetylated copolymers occurred from 50 to 92 °C, depending on the pendant spacer length of the glucose moiety in the glycoacrylate segment. With the increase in the pendant spacer length of the glucose moiety in the di-block copolymers, a decrease in the T_g values was observed. After deacetylation, the T_g values of the copolymers decreased in the range of −21 to 12 °C.

Table 2 Characteristics of the glycopolymers

Polymer code	GPC data^a		Acetylated/deacetylated glucose weight^b/%	Td10%^c/°C	Tg^d/°C
	Mw	Mw/Mn
a DMF used as a eluent at room temperature.b Weight = 100 × Dp[GMFW/polymer molecular weight (GPC)], where, Dp: degree of polymerization,^38 GMFW: acetylated or deacetylated glucose moiety formula weight.c Temperature of 10% weight of the sample decomposition calculated from TGA curve.d From DSC curve on second heating.
Acetylated
P(AHTAGP)	11 726	1.07	67	264	71
PEG-b-P(ATAGP)	8845	1.17	63	271	92
PEG-b-P(ABTAGP)	13 944	1.24	61	283	69
PEG-b-P(AHTAGP)	13 294	1.25	57	255	50
Deacetylated
P(AHGP)	4394	1.03	49	239	7
PEG-b-P(AGP)	4022	1.01	31	173	12
PEG-b-P(ABGP)	9290	1.30	46	163	−21
PEG-b-P(AHGP)	11 228	1.16	42	186	−17

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3.3 Bi-ligand functionalization of AuNPs with FITC and various glycopolymer chains

Initially, the release of FITC from the FITC–AuNP colloidal suspension was optimised with the thiolated-PEG [poly(ethylene glycol) methyl ether thiol, average M_n: 800]. Time and concentration dependent binding of thiolated-PEG on fluorescence quenched FITC–AuNP were optimised and results are shown in the ESI (Fig. S2 and S3†). Bi-ligand functionalized AuNP were prepared by the addition of PEG-I solutions of various concentrations to FITC–AuNP colloids. Fig. 5A shows the UV-Vis spectra of FITC–AuNP with the addition of PEG-I of various concentrations. The increase in the PEG-I concentration in the FITC–AuNPs colloid exhibited a relative increase in the peak at 478 nm, which is indicative of the release of FITC from the FITC–AuNP conjugates, and the formation of the PEG-I–FITC–AuNP complex. The functionalization takes place at the surface of the AuNP by the attachment of the –NCS group of FITC, and is followed by terminal trithiocarbonate PEG-I attachment. The fluorescence spectra (Fig. 5B) reveal that when the PEG-I binds to the FITC–AuNP proportionately, FITC is released from the FITC–AuNP surface. This indicates that PEG-I possesses stronger binding affinity for AuNP than FITC. Released concentrations of FITC, based on fluorescence, were calculated based on the amount of PEG-I added to the colloid of FITC–AuNPs, as shown in Fig. 5C.

Fig. 5 (A) UV-Vis and (B) fluorescence spectra of FITC functionalized AuNP colloidal suspensions at (a) 0, (b) 60, (c) 120, (d) 180, (e) 240 and (f) 300 μg ml⁻¹ of PEG-I. (C) Quantitative response of FITC fluorescence at different concentrations of PEG-I.

Fig. 6A shows the UV-Vis spectra after the addition of P(AHGP) solution of various concentrations to FITC–AuNP colloids. The FITC characteristic peak at 478 nm did not gradually increase as it did with the presence of PEG-I in the FITC–AuNP colloids. This was expected because the hydroxyl groups of the glucose moiety of the glycopolymer did not have as strong a binding affinity as the FITC, in addition to the non-approach of the terminal trithiocarbonate to the FITC–AuNP. The fluorescence spectra of FITC–AuNP colloids after the addition of P(AHGP) are presented in Fig. 6C. We did not observe a significant change in the amount of FITC released from the FITC–AuNP surface with the addition of P(AHGP) solution, as compared to the presence of various concentrations of PEG-I solutions.

Fig. 6 UV-Vis spectra of FITC functionalized AuNP colloid suspensions with various concentrations of (A) P(AHGP) and (B) PEG-b-P(AHGP). Fluorescence spectra of FITC functionalized AuNP colloid suspensions with various concentrations of (C) P(AHGP) and (D) PEG-b-P(AHGP). Concentrations were (a) 0, (b) 60, (c) 120, (d) 180, (e) 240 and (f) 300 μg ml⁻¹.

To further validate the selective binding of glycopolymers with FITC–AuNP, we used the synthesized PEG containing di-block glycopolymer, PEG-b-P(AHGP), and measured UV-Vis (Fig. 6B) and fluorescence (Fig. 6D) spectra of FITC–AuNP at various concentrations of PEG-b-(AHGP) solution. The PEG-b-P(AHGP) has the dodecyltrithiocarbonate group at one end of the macromolecular chain and it was expected that the PEG would increase the hydrophilic nature of the P(AHGP) segment and the relatively hydrophobic terminal trithiocarbonate group could access the hydrophobic FITC–AuNPs particles. It was previously demonstrated that the trithiocarbonate terminated glycopolymer has binding affinity for AuNP due to the formation of Au–(S)₂ bonds;^43–45 therefore, it has been hypothesized that the addition of these glycopolymers to the FITC–AuNP initiated the formation of Au–(S)₂ bonds on the Au particles and the release of FITC occurred. The increase in the concentrations of the PEG-b-P(AHGP) showed a relative increase in the FITC characteristic peak fluorescence, which is indicative of the release of FITC from the FITC–AuNPs surface. Further, the glycopolymer in the di-block copolymer was modified by reducing the pendant alkyl chain length of [PEG-b-P(AGP) and PEG-b-P(ABGP)] the glucose moiety, and employed to obtain bi-ligand gold nanoparticles. Fluorescence and UV-Vis spectra of FITC–AuNP in the presence of PEG-b-P(AGP) and PEG-b-P(ABGP) at various concentrations were measured.

The released concentrations of FITC fluorescence were calculated based on the amount of P(AHGP), PEG-b-P(AGP), PEG-b-P(ABGP) and PEG-b-P(AHGP) added to the FITC–AuNP colloid, and the data are presented in Fig. 7. The presence of di-block copolymers in the FITC–AuNP colloids resulted in a higher release of FITC than for P(AHGP). The increase in the pendant spacer length between the glucose moiety and the backbone of the polymer chain showed relatively higher proportions of release of the FITC for the di-block glycopolymers. Thus, di-block copolymers having higher glucose moiety pendant spacer lengths showed better binding affinity with AuNP. The slower release of FITC from FITC–AuNP with the addition of di-block polymers with shorter glucose moiety pendant spacer length has an advantage for better bi-ligand functionalization of glycopolymer and FITC on AuNP.

Fig. 7 Quantitative response of FITC fluorescence release with different concentrations of P(AHGP), PEG-b-P(AGP), PEG-b-P(ABGP) and PEG-b-P(AHGP) glycopolymers.

Fig. 8 shows representative TEM images of the primary conjugation of the gold nanoparticles with FITC dye molecules, and the bi-functional attachment of different glycopolymers with FITC–AuNP in the form of the bi-ligand. Fig. 8A shows the aggregation of AuNP when the dye molecules are present at the surface of AuNP. After the bi-functional attachment of PEG-I, P(AHGP), PEG-b-P(ABGP) and PEG-b-P(AGP) to the gold nanoparticles, the TEM images (Fig. 8B–E) show disaggregation, with better dispersion of the particles, whereas the functionalization with PEG-b-P(AHGP) exhibited agglomerations of the particles (Fig. 8F). The TEM images of the PEG-I, P(AHGP) and PEG-b-P(AHGP) were obtained and results indicate that the polymers do not show micelle formation (ESI Fig. S4†). Hence, the change in surface morphology and dispersion obtained in the TEM images are specific to glycopolymer interactions on the AuNP surface. The gold nanoparticles with quantified FITC and PEG-based glycopolymers provide a new and promising strategy in the development of selective drug loading and targeting for site-specific applications.

Fig. 8 TEM images of (A) FITC (0.5 μM)–AuNP, and FITC–AuNP treated with 300 μg ml⁻¹ of (B) PEG-I, (C) P(AHGP), (D) PEG-b-P(AGP), (E) PEG-b-P(ABGP) and (F) PEG-b-P(AHGP) solution.

4. Conclusions

We have synthesized stable AuNPs with diameter of ∼8 nm in aqueous media, and prepared AuNPs bioconjugates with fluorescein isothiocyanate (FITC). Di-block copolymers of poly(ethylene glycol) (PEG) based glycopolymers with glucopyranoside moieties having three different pendant spacer lengths were produced by a reversible addition–fragmentation chain transfer process, and the pendant acetyl glucose moieties of the resulting polymers were deacetylated. The effect of the PEG segmented di-block copolymers with glucose moieties of varying spacer lengths on gold nanoparticles loaded by the FITC ligand replacement process was investigated by fluorescence resonance energy transfer (FRET) in terms of restored fluorescence energy of the FITC released from the gold surface. It was demonstrated that the glycopolymers having shorter glucose moiety pendant spacer lengths showed very good bi-ligand formation capability, whereas an increase in the pendant spacer length of the glucose moiety allowed higher loading capacity of glycopolymers on AuNP. This study could provide the possibility for the synthesis of bio-conjugates without biomolecules having isoelectric points for functionalization.

Acknowledgements

AVSS and MD thank the Council for Scientific and Industrial Research, India for financial support in network projects CSC 0134 and BSC-0112, respectively. KD is a Project Student at CCMB from Centre for Converging Technologies, University of Rajasthan, Jaipur 302004, India. NNMR and MT thank UGC and CSIR for providing fellowships, respectively.

References

1. J. J. Storhoff, R. Elghanian, C. R. Mucic, C. A. Mirkin and R. L. Letsinger, J. Am. Chem. Soc., 1998, 120, 1959–1964.
2. A. J. Mieszawska, W. J. M. Mulder, Z. A. Fayad and D. P. Cormode, Mol. Pharmaceutics, 2013, 10(3), 831–847.
3. O. V. Salata, J. Nanobiotechnol., 2004, 2, 3.
4. M. Shevach, S. Fleischer, A. Shapira and T. Dvir, Nano Lett., 2014, 14, 5792–5796.
5. X. Huang, P. K. Jain, I. H. E. Sayed and M. A. E. Sayed, Nanomedicine, 2007, 2, 681–693.
6. R. Popovtzer, A. Agrawal, N. A. Kotov, A. Popovtzer, J. Balter, T. E. Carey and R. Kopelman, Nano Lett., 2008, 8, 4593–4596.
7. D. Kim, Y. Y. Jeong and S. Jon, ACS Nano, 2010, 4, 3689–3696.
8. C. Ziegler and A. Eychmüller, J. Phys. Chem. C, 2011, 115, 4502–4506.
9. G. Frens, Nat. Phys. Sci., 1973, 241, 20–22.
10. A. K. Samal, T. S. Sreeprasad and T. Pradeep, J. Nanopart. Res., 2010, 12, 1777–1786.
11. Y. Yu, Z. Luo, Y. Yu, J. Y. Lee and J. Xie, ACS Nano, 2012, 6, 7920–7927.
12. H. Qian, Y. Zhu and R. Jin, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 696–700.
13. I. Hussain, S. Graham, Z. Wang, B. Tan, D. C. Sherrington, S. P. Rannard, A. I. Cooper and M. Brust, J. Am. Chem. Soc., 2005, 127, 16398–16399.
14. S. V. Agarwal, S. S. Reddy and M. Dhayal, RSC Adv., 2014, 4, 18250–18256.
15. N. Wangoo, K. K. Bhasin, S. K. Mehtab and C. R. Suri, J. Colloid Interface Sci., 2008, 323, 247–254.
16. P. R. Selvakannan, S. Mandal, S. Phadtare, A. Gole, R. Pasricha, S. D. Adyanthaya and M. Sastry, J. Colloid Interface Sci., 2004, 269, 97–102.
17. P. C. Ray, A. Fortner and G. K. Darbha, J. Phys. Chem. B, 2006, 110(42), 20745–20748.
18. M. Iosin, F. Toderas, P. L. Baldeck and S. Astilean, J. Mol. Struct., 2009, 924–926, 196–200.
19. M. Arruebo, M. Valladares and Á. González-Fernández, J. Nanomater., 2009, 2009, 1–24.
20. D. Gao, Z. Wang, B. Liu, L. Ni, M. Wu and Z. Zhang, Anal. Chem., 2008, 80, 8545–8553.
21. L. Fan, Y. Hua, X. Wangb, L. Zhang, F. Li, D. Han, Z. Li, Q. Zhang, Z. Wanga and N. Liu, Talanta, 2012, 101, 192–197.
22. A. Ambrosi, F. Airò and A. Merkoçi, Anal. Chem., 2010, 82, 1151–1156.
23. H. Otsuka, Y. Akiyama, Y. Nagasaki and K. Kataoka, J. Am. Chem. Soc., 2001, 123, 8226–8230.
24. K. El-Boubbou and X. F. Huang, Curr. Med. Chem., 2011, 18, 2060–2078.
25. S. G. Spain, L. Albertin and N. R. Cameron, Chem. Commun., 2006, 40, 4198–4200.
26. Y. Miura, Polym. J., 2012, 44, 679–689.
27. A. L. Parry, N. A. Clemson, J. Ellis, S. S. R. Bernhard, B. G. Davis and N. R. Cameron, J. Am. Chem. Soc., 2013, 135, 9362–9365.
28. C. Boyer, A. Bousquet, J. Rondolo, M. R. Whittaker, M. H. Stenzel and T. P. Davis, Macromolecules, 2010, 43, 3775–3784.
29. B. Ebeling and P. Vana, Macromolecules, 2013, 46, 4862–4871.
30. M. Toyoshima and Y. Miura, J. Polym. Sci., Part A: Polym. Chem., 2009, 47, 1412–1421.
31. Q. Zhang and D. M. Haddleton, Adv. Polym. Sci., 2013, 269, 39–60.
32. T. J. Cho, J. M. Pettibone, J. M. Gorham, T. M. Nguyen, R. I. MacCuspie, J. Gigault and V. A. Hackley, Langmuir, 2015, 31, 7673–7683.
33. I. Papp, J. Dernedde, S. Enders and R. Haag, Chem. Commun., 2008, 44, 5851–5853.
34. S. Pearson, W. Scarano and M. H. Stenzel, Chem. Commun., 2012, 48, 4695–4697.
35. M. Sim, H. Kondo and C. H. Wong, J. Am. Chem. Soc., 1993, 115, 2260–2267.
36. J. W. Woodcock, X. Jiang, R. A. E. Wright and B. Zhao, Macromolecules, 2011, 44, 5764–5775.
37. Z. Petrovica, S. Konstantinovic and A. Spasojevic, Indian J. Chem., Sect. B: Org. Chem. Incl. Med. Chem., 2004, 43, 132–134.
38. M. Trinadh, G. Kannan, T. Rajasekhar, A. V. Sesha Sainath and M. Dhayal, RSC Adv., 2014, 4, 37400–37410.
39. W. Haiss, N. T. K. Thanh, J. Aveyard and D. G. Fernig, Anal. Chem., 2007, 79, 4215.
40. Y.-M. Chen, T.-L. Cheng and W.-L. Tseng, Analyst, 2009, 134, 2106–2112.
41. C. R. Bertozzi and L. L. Kiessling, Science, 2001, 291, 2357–2364.
42. G. Pasparakis and C. Alexander, Angew. Chem., Int. Ed., 2008, 47, 4847–4850.
43. M. Álvarez-Paino, V. Bordegé, R. Cuervo-Rodríguez, A. Muñoz-Bonilla and M. Fernández-García, Macromol. Chem. Phys., 2014, 215, 1915–1924.
44. A. Aqil, H. Qiu, J.-F. Greisch, R. Jérôme, E. D. Pauw and C. Jérôme, Polymer, 2008, 49, 1145–1153.
45. B. Ebeling and P. Vana, Macromolecules, 2013, 46, 4862–4871.

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Footnote

† Electronic supplementary information (ESI) available: Detailed characterisation of glycopolymer and UV-Vis and fluorescence spectra of bi-ligand AuNPs. See DOI: 10.1039/c6ra04273b

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