Facile synthesis of a reduction-responsive amphiphilic triblock polymer via a selective thiol–disulfide exchange reaction

Abstract

A reduction-responsive amphiphilic triblock copolymer mPEG-b-PDS-b-mPEG was synthesized via polycondensation between a dithiol and dipyridyl disulfide, followed by a selective thiol–disulfide exchange reaction. The reduction-triggered release of Nile Red by DTT was further demonstrated using fluorescence spectroscopy.

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The disulfide bond (–S–S–) is extremely valuable in a variety of chemical and biological agents due to the advantages of the disulfide bond in terms of biocompatibility, stability in the bloodstream, and responsiveness to redox stimuli.^1–4 In addition, it can be reduced by the abundance of free thiols, including glutathione, in cells, especially cancer cells, which have enhanced glutathione levels.^5–7 Hence, researchers have developed various disulfide-containing polymers as nanocarriers.^8–22 Sun reported the preparation of redox responsive micelles via linking poly(ethylene glycol) and poly(ε-caprolactone) by disulfide bonds. The DOX-loaded micelles released DOX rapidly in the presence of 10 mM DTT in pH 7.4 PB buffer at 37 °C and achieved a virtually quantitative release after 12 h.²³ Cheng reported the synthesis of disulfide-based cross-linked micelles via the ring opening polymerization of 5-(4-(prop-2-yn-1-yloxy)-benzyl)-1,3-dioxolane-2,4-dione and click chemistry.²⁴ The cross-linked micelles could retain their structural stability under extreme dilution conditions but released the payload quickly in the presence of dithiothreitol (DTT). However, they all used a small disulfide compound as the linker. Recently, a mild and versatile synthetic route for preparing poly(disulfide)s was developed by Ghosh.²⁵ Telechelic poly(disulfide)s with a predictable molecular weight and reactive disulfide group at both the terminals of the chain were obtained via polycondensation between a dithiol and dipyridyl disulfide. Furthermore, the terminal pyridyl disulfide group can be quantitatively replaced by a functional thiol using selective thiol–disulfide exchange to produce functional telechelic poly(disulfide)s.

Using a similar strategy, we developed a facile route for the synthesis of ABA type amphiphilic triblock copolymer via the thiol–disulfide exchange reaction of telechelic pyridyl disulfide terminated poly(disulfide)s with a hydrophilic thiol-terminated polymer (Scheme 1). Molecular structures, self-assembly, and reduced-triggered drug release were thoroughly investigated by NMR, gel permeation chromatography (GPC), dynamic light scattering (DLS), transmission electron microscope (TEM), and fluorescence spectrophotometer.

Scheme 1 Synthesis of ABA triblock copolymer.

Telechelic pyridyl disulfide terminated poly(disulfide)s (PDS) were synthesized according to the same strategy reported by Ghosh.²⁵ The condensation reaction between 2,2′-dithiodipyridine (M₁) and 1,6-hexanethiol (M₂) with three different mole ratios gave solid powder PDS in about 70% yield. ¹H NMR end-group analysis showed that the obtained PDS(1) with a monomer mole ratio of 1.045 : 1 had a number average molecular weight (M_n) of 5.4 kDa (Table 1 and Fig. 1). GPC measurements demonstrated a unimodal distribution with a M_n of 6.4 kDa (polystyrene standards) and a moderate polydispersity index (PDI) of 1.66 (Table 1 and Fig. 2). The thiol-terminated hydrophilic polymer mPEG-SH was obtained by the esterification of monomethoxy poly(ethylene glycol) (M_n = 1.9 kDa) with mercaptoacetic acid using p-toluenesulfonic acid as the catalyst. Then, triblock copolymer mPEG-b-PDS-b-mPEG was readily prepared via a thiol–disulfide exchange reaction between PDS and PEG-SH. The successful exchange reactions of the thiol–disulfide bonds were demonstrated with ¹H NMR and GPC. In the ¹H NMR spectrum of mPEG-b-PDS(1)-b-mPEG (Fig. 1), the disappearance of the peak of the proton in the region of 7.0–8.5 ppm (corresponding to the terminal pyridyl disulfide group) and the appearance of the peaks in the region of 3.3–4.3 ppm (corresponding to the mPEG protons) confirmed the successful thiol–disulfide exchange reaction. Moreover, the GPC analysis revealed that the obtained triblock copolymer also possessed a unimodal distribution with a narrow PDI value (Table 1 and Fig. 2). The M_n of the corresponding triblock copolymer mPEG-b-PDS(1)-b-mPEG from PDS(1) increased from 6.4 kDa to 12.1 kDa, and the PDI changed from 1.66 to 1.29.

Table 1 Molecular characteristics of PDS and PEG-b-PDS-b-PEG

Entry	M1 : M2 ratio	Yield (%)	Mw/Mn^a	Mn,GPC^a (kDa)	Mn,NMR^b (kDa)
a Both molecular weight (Mn,GPC) and the polydispersity (Mw/Mn) of the polymers were determined by GPC.b Mn,NMR was determined by ^1H NMR.
PDS(1)	1.045 : 1	73	1.66	6.4	5.4
PDS(2)	1.025 : 1	71	1.42	11.6	8.6
PDS(3)	1.01 : 1	75	1.30	17.9	13.7
mPEG-b-PDS(1)-b-mPEG	—	88	1.29	12.1	9.1
mPEG-b-PDS(2)-b-mPEG	—	84	1.25	15.6	12.3
mPEG-b-PDS(3)-b-mPEG	—	86	1.23	20.8	17.5

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Fig. 1 ¹H NMR spectra of PDS(1) and PEG-b-PDS(1)-b-PEG.

Fig. 2 GPC curves of PDS and the corresponding mPEG-b-PDS-b-mPEG.

The amphiphilic nature of the triblock copolymer mPEG-b-PDS-b-mPEG provided an opportunity to form micelles in water. Micelles of mPEG-b-PDS(1)-b-mPEG were prepared by a dialysis method. The hydrophobic dye Nile Red (NR) was chosen as the fluorescence probe because it fluoresces when solubilized by the hydrophobic micelle core; however, its emission intensity decreases when the micelles is disrupted bringing the dye molecule in contact with water. The critical micelle concentration (CMC) could be calculated by tracking the fluorescence intensity of NR as a function of the sample concentration.^26,27 As shown in Fig. S2,† the fluorescence intensity of the NR remained nearly constant below a certain concentration and then increased rapidly, which reflected that the dye was encapsulated into the hydrophobic region of micelles. The corresponding concentration of the inflection point in Fig. 3(a) was identified as the CMC of mPEG-b-PDS(1)-b-mPEG, which was about 0.024 mg mL⁻¹.

Fig. 3 Fluorescence emission intensity vs. log of micelle concentrations (a), mean size distribution (b), and TEM photograph (c) of mPEG-b-PDS(1)-b-mPEG micelles.

Both the morphology and the average size of the self-assembled PEG-b-PDS-b-PEG micelles were investigated by TEM and DLS. DLS measurements (Fig. 3(b)) showed that mPEG-b-PDS(1)-b-mPEG formed micelles with a number-average hydrodynamic diameter of 29.9 nm and a polydispersity of 0.021, which indicated that the micelles had a narrow size distribution. As shown in Fig. 3(c), the TEM micrograph confirms that the micelles of mPEG-b-PDS(1)-b-mPEG had a spherical morphology with an average size of ∼25 nm. The average size determined from TEM analysis is smaller than that determined by DLS, which was most likely due to the shrinkage of the PEG shell upon drying.

The reducible moiety PDS on the micelles can be degraded in the presence of reducing reagents, which was demonstrated by Ghosh et al.²⁵ They used ¹H NMR to monitor the degradation of triblock copolymer poly(lactide)-b-PDS-b-poly(lactide) in the presence of the reducing reagent DTT and confirmed the cleavage of the polydisulfide. The demicellization process of the mPEG-b-PDS(1)-b-mPEG micelles induced by DTT was investigated by fluorescence spectroscopy using NR as the probe.^28–31 As shown in Fig. 4, in the presence of DTT, the fluorescence emission intensity of the NR-loaded micelles (0.2 mg mL⁻¹) decreased rapidly, and about 48% NR was released from the micelle core into the aqueous solution within the first 8 h. After 48 h, the fluorescence intensity remained at about 30%, which was probably because the precipitation of the short hydrophobic PDS chain from the aqueous solution could enclose some NR molecules and keep them in the hydrophobic environment during the chain scission process. In contrast, the fluorescence emission intensity did not change clearly in the absence of DTT and only about 6% NR was released from the micelles. The results indicate that the encapsulated guest release from mPEG-b-PDS-b-mPEG micelles can be triggered by reduction.

Fig. 4 Fluorescence emission spectra of Nile Red at varying times in mPEG-b-PDS(1)-b-mPEG micelles in the presence of 10 mM DTT (a), and the reduction-triggered NR release profiles in aqueous solution with a micelle concentration of 0.2 mg mL⁻¹ with and without DTT (b).

Conclusions

In summary, we report a facile synthetic method for the preparation of reduction-responsive amphiphilic triblock copolymers composed of two hydrophilic blocks of mPEG and a hydrophobic block of PDS. The molecular structures, self-assembly, and reduction-triggered release of mPEG-b-PDS-b-mPEG were thoroughly investigated. mPEG-b-PDS(1)-b-mPEG could self-assemble into spherical micelles in water with an average size of ∼30 nm and a critical micelle concentration of 0.024 mg mL⁻¹. Fluorescence spectroscopy showed that the encapsulated NR can be released as reductive DTT was introduced. The reduction-responsive micelles are promising for applications in the efficient delivery and release of potential hydrophobic anticancer drugs.

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Footnote

† Electronic supplementary information (ESI) available: Experimental details, including polymer synthesis and NMR spectra. See DOI: 10.1039/c4ra07792j

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