Synthesis, characterization and applications of selenocysteine-responsive nanoprobe based on dinitrobenzene sulfonyl-modified poly(carbonate) micelles

Abstract

In trace amounts, selenium (Se) participates in different physiological functions of the human body. Its biological significance is manifested in its presence as selenocysteine (Sec) in genetically encoded selenoproteins that are important in redox regulation, anti-inflammation, and cancer treatment. Recently, stimuli-responsive micelles were developed as biosensors, but there are no nanomicelle probes for the selective imaging of Sec under biological conditions (pH = 7.4). Herein, we report the design and preparation of a 2,4-dinitrobenzenesulfonyl-decorated block poly(carbonate) copolymer, viz. PMPC-Dns, for Sec imaging. We found that PMPC-Dns trapped with the fluorescent drug doxorubicin (DOX) selectively responds to Sec, while getting little interference from biological thiols, amines or alcohols. We applied the PMPC-Dns probe successfully to image endogenous Sec in cervical cancer tissues as well as in HeLa cells. In the course of these studies, we observed simultaneous release of the trapped DOX. Hence, besides Sec imaging, the probe can be used for controlled delivery of hydrophobic molecules for biomedical applications.

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Introduction

Selenium (Se) is considered as an essential micronutrient and its participation in physiological functions is well recognized.¹ Various kinds of Se metabolites, such as hydrogen selenide, selenocysteine (Sec), selenite, selenophosphate, selenodiglutathione, and charged Sec-tRNA,^2,3 are biosynthesized in animals. The anticancer activity of Se was discovered in 1969.² Nonetheless, it is noted that a number of diseases are related to Se intake at abnormal levels.^2,4 Selenium compounds of low molecular weight, such as Sec and methylselenol,^5,6 are key metabolites in cancer prevention.^7,8 Sec is a Se-containing amino acid encoded by a UGA stop codon⁹ that is located in the active sites of selenoproteins (SePs). It functions in redox signalling and anti-inflammation as well as in the production of active thyroid hormones. It is therefore essential to determine the pathological roles of Sec because the majority of the in vivo functions of Se-containing entities are performed by it. For this kind of study, a biocompatible tool is needed to monitor the metabolites in living systems.

Stimuli-responsive micelles have advantages in terms of stability, simplicity, sensitivity, portability, cost-effectiveness, and storage convenience, and have been studied for the development of biosensors. The commonly used stimuli are pH, temperature, light, redox potential, ultrasound, charge, gases, biomolecules, and enzymes.^10–18 To the best of our knowledge, the use of nanomicelle probes for selective recognition of Sec under biological conditions (pH = 7.4) has never been reported. Up to now, only a few fluorescent probes of small molecules have been studied for the detection and imaging of Sec^19,20 or SePs.²¹ In 2006, Maeda et al.²² reported the use of a fluorescent probe to discriminate between Sec and cysteine (Cys) based on the fact that the selenol group of Sec is inclined to ionize, unlike the thiol group of Cys at pH 5.8. In 2014, Zhang et al.¹⁹ reported the design, synthesis, and biological evaluation of a series of potential Sec probes based on the same mechanism under physiological conditions. Recently, Kong et al.²⁰ developed a fluorescence method to investigate the inhibitory mechanism of Se in tumor cells. However, due to the enhanced permeability and retention (EPR) effect,²³ the reported methods cannot be widely applied in biomedical research as a result of poor biocompatibility and low photobleaching resistance.²⁴

In this paper, we report the use of amphiphilic polymer micelles for the detection of Sec. A 2,4-dinitrobenzenesulfonyl-decorated block poly(carbonate) copolymer, viz. PMPC-Dns, was synthesized. Then, by means of the ring-opening polymerization (ROP) of cyclic carbonates, we generated amphiphilic block copolymers with poly(carbonate) hydrophobic chains. Finally, by means of the copper-catalysed azide–alkyne Huisgen 1,3-dipolar cycloaddition (a coupling reaction), we produced Sec-responsive micelles in which the fluorescent drug doxorubicin (DOX) was trapped. The copolymer that self-assembles into micelles consists of a hydrophobic core surrounded by a hydrophilic shell. It should be noted that the hydrophobic ends are the sites for Sec recognition. In the presence of Sec, 2,4-dinitrobenzenesulfonyl is activated and the generation of hydrophilic ends in the destabilized micelles occurs. The consequence is the release of DOX for fluorescence imaging. In this study, we investigated the synthesis and cytotoxicity of the micelle probe, as well as the release of DOX as induced by Sec.

Results and discussion

Synthesis and self-assembly of amphiphilic copolymers

The monomers of azide-functionalized dinitrobenzenesulfonate (Dns-N₃) and 5-methyl-5-propargyloxycarbonyl-1,3-dioxan-2-one (MPC) were synthesized according to the procedure shown in Scheme 1. The Sec-responsive copolymer PMPC-Dns was prepared through a two-step method. Firstly, the PEG-b-poly(MPC) copolymer was synthesized via the ROP of MPC with poly(ethylene glycol) (PEG) being the macroinitiator (Scheme 1b). The copolymer was characterized using ¹H NMR and gel permeation chromatography (GPC) techniques. The ¹H NMR spectrum of PEG-b-poly(MPC) in CDCl₃ is shown in Fig. S1c.† The degree of polymerization (DP) of the polycarbonate backbone was determined to be 10 by comparing the integral of the peak at δ = 3.36 (CH₃O–, methyl protons of PEG end group) with that at δ = 4.28 (–C(O)OCH₂CCH₂O–, the methylene protons of the carbonate units), which is close to the theoretical value of the protons of the carbonate units, as shown in Table S1.† The results of GPC investigation reveal that the Sec-responsive copolymer has a narrow polydispersity index (PDI) of 1.04 and number-average molecular weight (M_n) of 7263 g moL⁻¹, which are in good agreement with the results of ¹H NMR end group analysis. Secondly, we modified PEG-b-poly(MPC) to prepare the PMPC-Dns copolymer via the click reaction (Scheme 1c), and purified the product by dialysis. We compared the ¹H NMR spectra of Dns-N₃, PMPC-Dns, and PEG-b-poly(MPC) (Fig. S1†). The signal at δ = 2.54 in Fig. S1,† assigned to the protons of the alkynyl groups of PEG-b-poly(MPC), disappears after click chemistry modification (Fig. S1b†), indicating that there is complete reaction of the propargyl groups of PEG-b-poly(MPC) with Dns-N₃. Examination of Fig. S1a–c† reveals that for the PMPC-Dns copolymer, the Dns-N₃ signals are at δ 2.21, 3.47, 4.24–4.43, 8.49, 8.74 and 9.07, while the signals owing to PEG-b-poly(MPC) are at δ 1.22, 3.37, 3.65 and 4.26. Moreover, a weak signal at δ 7.72 that is attributable to the proton of the triazole ring provides evidence for the attachment of Dns-N₃ to the polyester backbone. The GPC results reveal that the PMPC-Dns copolymer has a narrow PDI of 1.07 and M_n of 11 353 g mol⁻¹, close to that calculated by ¹H NMR end group analysis (Table S1†). The GPC trace of the graft copolymer shows a slight shift to lower retention time while maintaining a narrow distribution with a dispersity similar to that of the unmodified copolymer PEG-b-poly(MPC) (Fig. S2†). It is apparent that the PMPC-Dns graft copolymer can be readily prepared from propargyl-functionalized polycarbonate via the click reaction.

Scheme 1 Synthesis of Dns-N₃, MPC, PEG-b-poly(MPC) and amphiphilic copolymer PMPC-Dns.

The amphiphilic copolymers PEG-b-poly(MPC) and PMPC-Dns self-assemble into micelles with hydrophobic cores that are stabilized with hydrophilic PEG coronae (Fig. 1). Their self-assembly behaviours were investigated in detail using fluorescence spectroscopy, as well as dynamic light scattering (DLS) and scanning electron microscopy (SEM) techniques. Using hydrophobic Nile Red (NR) as the fluorescent dye, the use of fluorescence spectroscopy can conveniently monitor the self-assembly of micelles and determine the critical micelle concentration (CMC) of amphiphiles.³⁰ As shown in Fig. S3,† the fluorescence emission intensity gradually increases with increasing amphiphile concentration, suggesting the spontaneous self-assembly of micelles. PMPC-Dns shows a CMC value similar to that of PEG-b-poly(MPC) (0.01245 mg mL⁻¹ vs. 0.01159 mg mL⁻¹), suggesting that the self-assembled micelles are thermodynamically stable. These values are consistent with literature data reported for graft copolymers.^30–32 Then, SEM (Fig. 2a and b) and DLS (Fig. S4a and b†) were applied to measure the size and morphology of the self-assembled micelles. In the SEM images, a spherical morphology was observed with average sizes of 60 and 90 nm for the PEG-b-poly(MPC) and PMPC-Dns micelles, respectively. Despite being common in morphology, the PMPC-Dns micelles are larger than the PEG-b-poly(MPC) micelles in the DLS-determined diameter, which can be attributed to the expanded hydrophobic nucleus of the former. In the presence of Sec, the 2,4-dinitrobenzenesulfonyl recognition site of the PMPC-Dns micelles is activated, generating intertwining hydrophilic ends in the destabilized micelles. One can see that there are micelles that are not activated in Fig. 2d, and upon destabilization of the micelles there is an obvious change in the DLS average size from 118 (Fig. S4b†) to 13 nm (Fig. S4d†). After Sec activation, the product was dialyzed in aqueous solution and lyophilized. In ¹H NMR analysis, one can see that the signals at δ 8.49, 8.74, and 9.07 disappear and a weak signal at δ 7.72 appears (Fig. S5†), meaning that the reaction proceeds with the release of the 2,4-dinitrobenzenesulfonate. However, the PEG-b-poly(MPC) micelles with no recognition sites show no change in morphology (Fig. 2c) and DLS average size (Fig. S4c†) compared to Fig. 2a and S4a,† respectively.

Fig. 1 Working principle and response mechanism of the PMPC-Dns probe for Sec detection.

Fig. 2 SEM photographs of the micelles: (a) PEG-b-poly(MPC), (b) PMPC-Dns, (c) PEG-b-poly(MPC) treated with Sec, and (d) PMPC-Dns treated with Sec.

Working principle and feasibility of PMPC-Dns micelles for Sec detection

The working principle of the Sec-responsive PMPC-Dns copolymer is schematically shown in Fig. 1. The Sec recognition site, 2,4-dinitrobenzenesulfonate, is copolymerized with the polycarbonate backbone at the hydrophobic end. When the PMPC-Dns copolymer and the DOX drug are put into aqueous solution, the micelles assemble and the hydrophobic DOX is wrapped inside. In the absence of Sec, the Sec recognition sites are stable under physiological conditions. In the presence of Sec, however, the sites react with Sec and there is cleavage of 2,4-dinitrobenzenesulfonate. In the process, the hydrophobic ends change into hydroxyl ends that are hydrophilic, leading to the breaking up of the micelles and unwrapping of DOX. Since the intensity of fluorescence reflects the amount of released DOX, the concentration of Sec can be measured as a degree of fluorescence intensity enhancement.

To validate the feasibility of the copolymer micelles for Sec detection, the Sec-responsive PMPC-Dns micelles were prepared with DOX trapped inside. The drug loading efficiency (DLE) and drug loading content (DLC) of the DOX-loaded PMPC-Dns micelles are found to be 6.5% and 7.63%, respectively. The fluorescence response of the PMPC-Dns micelles towards Sec was examined (Fig. S6a†). Compared to that in the absence of Sec, there is a 2.78-fold enhancement in fluorescence intensity at 590 nm when Sec (1 mM) is introduced to the solution of micelles (0.5 mg mL⁻¹). To further evaluate the feasibility, time-dependent fluorescence changes of the micelles at 590 nm were monitored in the absence and presence of Sec (Fig. S6b†). Without Sec, no fluorescence changes at 590 nm are observed within 10 min, which indicates that there is no release of DOX and the micelles are stable. On the contrary, in the presence of Sec, there is an obvious enhancement in fluorescence intensity that reaches a plateau after 1.6 h, indicating the release of DOX. The results clearly prove that the PMPC-Dns micelles respond to Sec.

Sensitive and specific detection of Sec

To demonstrate the applicability of the approach for qualitative identification of Sec, we measured the fluorescence spectra of PMPC-Dns micelles in phosphate-buffered saline (PBS) solution treated with different concentrations of Sec. As shown in Fig. 3, with increasing concentration of Sec, there is an increase in fluorescence intensity, displaying a concentration-dependent trend. A linear range is obtained between 0 and 25 μM. With a further increase in Sec concentration, the fluorescence intensity reaches a plateau. Based on the 3σ rule, the detection limit is calculated to be 0.05 μM. The results reflect the cleavage of 2,4-dinitrobenzenesulfonate and destabilization of the micelles in the presence of Sec. With a higher concentration of Sec, more DOX is released and hence a higher fluorescence intensity is observed.

Fig. 3 Fluorescence spectra of PMPC-Dns micelles (0.5 μg mL⁻¹) in PBS (pH 7.4, 20 mM) solution treated with increasing amounts of Sec. The arrow indicates the signal changes with increase in Sec concentration (0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 10.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0 and 24.0 μM). Inset: the F/F₀ ratio of micelles as a function of Sec concentration, where F₀ and F are fluorescence intensity of PMPC-Dns micelles at 595 nm in the absence and presence of Sec, respectively.

Due to the high selectivity of the interaction of 2,4-dinitrobenzenesulfonate with Sec, as described by Zhang et al.,¹⁹ the detection of Sec using the PMPC-Dns micelles is highly selective. In the presence of a high concentration of various reducing reagents, including thiols, beta-mercaptoethanol (b-ME), thioredoxinreductase (TrxR), dithiothreitol (DTT), N-acetyl-L-cysteine (NAC), vitamin C, hydrogen sulfide (H₂S), Na₂SeO₃, and a mixture of Na₂SeO₃, Cys, and GSH, there is no detectable fluorescence change, as shown in Fig. 4. The selectivity was further verified by ¹H NMR analysis, as shown in Fig. S7.† There is no obvious change in the ¹H NMR features of PMPC-Dns after the PMPC-Dns micelles were treated with the thiols and sulfur compounds. The results confirm that the PMPC-Dns probe shows no response toward the reducing reagents. It is apparent that H₂S and the reducing reagents with a sulfhydryl group do not respond to the probe. It is noted that vitamin C also shows a negative response, while DTT (50 μM) only exhibits a slight response. One probable reason for TrxR not responding is that due to steric effects, it is hard for the Sec in SePs to get close to the recognition sites.

Fig. 4 The fluorescence of PMPC-Dns micelles (0.5 μg mL⁻¹) in PBS (pH 7.4, 20 mM) solution treated with thiols and other sulfur compounds for 90 min (1 μM).

Detection of Sec in cells and tissues by PMPC-Dns micelles

For suitability in biological applications, the probe has to be nontoxic. The long-term cellular toxicity of the PEG-b-poly(MPC) and PMPC-Dns micelles towards the HeLa cell line was determined by means of a standard MTT (methyl thiazolyl tetrazolium) assay. As shown in Fig. S8a,† when the concentration of micelles is 1 mg mL⁻¹, the cell viability still remains at ca. 93%, demonstrating their low cytotoxicity. The result can be attributed to the excellent biocompatibility of PEG and poly(carbonate)s. Moreover, the stability of the PMPC-Dns micelles under the condition of a high concentration of Fetal Bovine Serum (FBS) (Fig. S8b†) is also of importance, especially in the detection of Se in tissues. We recorded the fluorescence spectra of the probe after different periods of storage time. The results show that the PMPC-Dns micelles are stable under biological conditions for a long time. Hence, the PMPC-Dns micelles can serve as a selective and sensitive probe for qualitative analysis of Sec. In addition, the Sec can be used as a target agent for drug delivery in cancer therapy.

The imaging of Sec in live cells was performed using HeLa cells as a model. Since the physiological concentration of Sec is low in cells, we first determined whether the PMPC-Dns micelles could respond to exogenous Sec. After culturing the HeLa cells with Sec for 2 to 12 h, we detect a bright red fluorescence signal when the probe is applied (Fig. 5b and c), while there is no obvious enhancement in fluorescence intensity in the case of the control cells (Fig. 5a). We further determined the endogenous Sec in the cells. Sodium selenite (Na₂SeO₃) is a precursor of Sec biosynthesis, and the supplementation of cells with sodium selenite could significantly increase the Sec level in the cells.³³ After the cells were stimulated with sodium selenite for 12 h, there is a notable appearance of fluorescence (Fig. 5e). A short treatment time of 2 h gives a weak enhancement in the fluorescence signal (Fig. 5d). The results demonstrate that the probe is suitable for imaging Sec in live cells.

Fig. 5 HeLa cells were treated with Sec (5 μM) for 2 to 12 h, followed by incubation with PMPC-Dns micelles (0.5 μg mL⁻¹), resulting in the appearance of a bright red fluorescence signal (b and c), whereas in the case of the control cells there was no obvious fluorescence enhancement (a). For the cells stimulated with sodium selenite for 12 h, there was a notable appearance of fluorescence (e). A short treatment time (2 h) resulted in a weak but obviously enhanced signal (d). The images in the last row were acquired using Image Pro Plus software. The colour strip shows the pseudo colour change with Sec.

To further investigate the response of the PMPC-Dns micelles under biological conditions, we selected cervical neoplasm for imaging. Fig. 6 shows the micelle staining of frozen slices of cervical tumor tissue. A strong fluorescence signal (Fig. 6c) was observed in the cervical neoplasm slices that were treated with Sec for 12 h. The tissue slice (Fig. 6b) treated with Na₂SeO₃ for the same time period also gives a fluorescence signal, but with slightly weaker intensity. As for the neoplasm slice not treated with Sec or Na₂SeO₃, there is no obvious fluorescence enhancement (Fig. 6a). The results are in good agreement with those of HeLa cell imaging. The confocal images clearly demonstrate that the PMPC-Dns micelles can be used for qualitative analysis of the Sec level in cervical cancer cells under biological conditions.

Fig. 6 Cervical tumor cells were treated with Sec (5 μM) for 12 h, followed by incubation with PMPC-Dns micelles (0.5 mg mL⁻¹), resulting in the appearance of a bright red fluorescence signal (c), whereas in the case of the control cells there was no obvious fluorescence enhancement (a). When the cells were stimulated with sodium selenite for 12 h, there was a notable appearance of fluorescence (b). The images in the last column were acquired using Image Pro Plus software. The colour strip shows the pseudo colour change with Sec.

Conclusions

We designed and prepared a novel 2,4-dinitrobenzenesulfonyl-decorated block poly(carbonate) copolymer, viz. PMPC-Dns, via a convenient click conjugation of Sec-responsive 2,4-dinitrobenzenesulfonyl molecules to propargyl-functionalized poly(carbonate). DLS and SEM measurements revealed that the polymer self-assembles in aqueous solution into spherical micelles with an average diameter of 100 nm. The CMC of the micelles was determined to be 0.01245 mg mL⁻¹ by fluorescence spectroscopy using NR as a fluorescent dye. The follow-up studies demonstrated that PMPC-Dns with the fluorescent drug DOX trapped inside could selectively respond to Sec and other selenols under biological conditions (pH = 7.4). It was found that there is little interference from biological thiols, amines, or alcohols. The PMPC-Dns probe was successfully applied to image the endogenous Sec in HeLa cells as well as that in live cervical neoplasm. It is noted that during Sec imaging, there is simultaneous release of the entrapped hydrophobic DOX molecules under physiological conditions. To the best of our knowledge, PMPC-Dns is the first Sec-induced probe selective for Sec imaging. The work opens up a way to study the role of Sec in biological, pathological and also tumor xenograft model systems. Furthermore, the approach provides a methodology for the controlled delivery of hydrophobic molecules in biomedical applications.

Experimental section

Materials

Monomethoxypoly(ethyleneglycol) (PEG_5k, M_n = 5000), ethyl chloroformate, 2,4-dinitrobenzenesulfonyl chloride, 3-bromo-1-propanol and 2,2-bis(hydroxyl methyl)propionic acid were purchased from Alfa Aesar and used as received. We dried 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), dichloromethane (DCM), triethylamine, dimethylformamide (DMF) over calcium hydride for 24 h at room temperature (RT) and distilled them under reduced pressure. The thiourea catalyst (TU) was synthesized as reported previously and recrystallized from dry methylene chloride.²⁵ We synthesized (Sec)₂ according to the procedure reported by Braga et al.²⁶ The Sec was generated from (Sec)₂ in the presence of DTT (100 μM, from 50 μM (Sec)₂ and 50 μM DTT). Tetrahydrofuran (THF) was dried by means of refluxing over a benzophenone–sodium mixture until deep blue in appearance, and then subjected to distillation.

Characterization

UV-Vis absorption spectra were recorded with a Hitachi U-4100 UV/Vis spectrophotometer using quartz cuvettes of 1 cm path length. The steady-state fluorescence emission spectra were recorded using equipment from Photon Technology International. Fluorescence emission spectra were collected using a bandwidth of 5 nm and 0.2 × 1 cm² quartz cuvettes containing 500 μL of solution. ¹H and ¹³C NMR spectra were recorded on an Inova-400 (Varian) spectrometer and referenced to solvent signals. The number-average molecular weight (M_n) and molecular weight distribution (PDI = M_w/M_n) of the polymers were determined at RT using a Waters GPC liquid chromatograph equipped with four TSK HXL series of polystyrene divinylbenzene gel columns (300 × 7.8 mm). Calibration was established with polystyrene standards from Polymer Laboratories. THF with a flow rate of 1 mL min⁻¹ was used as the solvent. SEM images were obtained on an S-4800 scanning electron microscope (Hitachi) with a working voltage of 5 kV. A drop of the aqueous micelle solution (0.05 mg mL⁻¹) was deposited onto a silicon slice and allowed to dry at RT before measurement. The mean size of the micelles was determined by DLS using a Malvern Nano S instrument (Malvern, UK).

Synthesis of 3-bromopropyl 3-azido-propanol (1)

To a solution of 3-bromo-propan-1-ol (1.02 g, 7.3 mmol) in water (10 mL) was added sodium azide (0.95 g, 14.6 mmol) and the solution was heated at 80 °C for 18 h. The aqueous solution was extracted with EtOAc (4 × 20 mL). The organic layers were washed with brine (30 mL), dried over MgSO₄, subjected to filtration, and concentrated under reduced pressure to give pure 3-azido-propanol as a colourless oil (0.57 g, yield = 76.5%).

¹H NMR (400 MHz, CDCl₃): δ = 3.73 (t, 2H, OHCH₂), 3.44 (t, 2H, CH₂CH₂N₃), 2.51 (s, 1H, OH), 1.80–1.86 (m, 2H, CH₂CH₂CH₂).

¹³C NMR (100 MHz, CDCl₃): δ = 59.65, 48.41, 31.43.

Synthesis of 3-azidopropyl 2,4-dinitrobenzenesulfonate (Dns-N₃)

A solution of 3-azido-propanol (0.57 g, 5.6 mmol) and Et₃N (1.56 mL, 11.2 mmol) in dry CH₂Cl₂ (10 mL) was cooled to 0 °C. With the addition of 2,4-dinitrobenzene-1-sulfonyl chloride (1.7 g, 6.7 mmol) in dry dichloromethane (10 mL), the solution was stirred at RT for 18 h. After the quenching of the reaction with water (40 mL), the organic layer was separated, dried over MgSO₄, subjected to filtration, and concentrated under reduced pressure. The as-obtained crude oil was purified by column chromatography (eluent: ethyl acetate/pet. ether = 1/5, v/v) to yield 3-bromopropyl 2,4-dinitrobenzenesulfonate as a colourless oil (1.33 g, yield = 71.1%).

¹H NMR (400 MHz, CDCl₃): δ = 9.00 (s, 1H, ArH), 8.67 (d, 1H, ArH), 8.45 (d, 1H, ArH), 4.03–4.25 (m, 2H, CH₂CH₂CH₂), 3.32 (t, 2H, OCH₂CH₂), 1.86 (t, 2H, CH₂CH₂N₃).

¹³C NMR (100 MHz, CDCl₃): δ = 152.19, 145.22, 142.19, 123.88, 123.47, 115.71, 62.00, 42.49, 24.34.

Synthesis of 2,2-bis (hydroxyl methyl) propionate (2)

To a 250 mL round-bottom flask, 2,2-bis(hydroxyl methyl)-propionic acid (2.24 g, 16.72 mmol), KOH (1.01 g, 18.04 mmol), and DMF (100 mL) were added. The mixture was stirred at 100 °C for 2 h, and then propargyl bromide (2.13 g, 18.04 mmol) was added dropwise over a 30 min period. After 72 h of reaction, the reaction mixture was subjected to filtration, and the solvent was evaporated under reduced pressure. The residue was dissolved in 50 mL of DCM and washed three times with saturated salt water (20 mL × 3). The organic phase was concentrated to yield the crude product, which was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 1/5, v/v). Yield: 1.3 g (45.1%).

¹H NMR (400 MHz, CDCl₃): δ = 4.76 (d, 2H, CHCCH₂CO), 3.93 (d, 2H, CH₂OH), 3.73 (d, 2H, CH₂OH), 2.87 (s, 2OH), 2.51 (t, 1H, CHCCH₂CO), 1.11 (s, 3H, CH₃CC).

¹³C NMR (100 MHz, CDCl₃): δ = 175.01, 75.20, 67.82, 60.37, 52.43, 49.29, 16.95.

Synthesis of 5-methyl-5-propargylxycarbonyl-1,3-dioxane-2-one (MPC)

Compound 2 (1.18 g, 6.88 mmol) was mixed with ethyl chloroformate (1.48 g, 13.76 mmol) and THF (20 mL) in a sealed vessel that was purged with nitrogen and cooled in an ice bath. After stirring for an hour, triethylamine (1.39 g, 13.76 mmol) was added dropwise over a 30 min period under nitrogen atmosphere. The reaction was conducted at 0 °C with stirring for 3 h, then at 25 °C with stirring overnight. The solution was then subjected to filtration, evaporated to dryness, and the as-obtained product was precipitated in a mixture of ethyl acetate and diethyl ether (1 : 1) as white crystals. Yield: 1.24 g (91.1%).

¹H NMR (400 MHz, CDCl₃): δ = 4.82 (d, 2H, CHCCH₂CO), 4.75 (d, 2H, CH₂OCO), 4.27 (d, 2H, CH₂OCO), 2.57 (t, 1H, CHCCH₂CO), 1.39 (s, 3H, CH₃CC).

¹³C NMR (100 MHz, CDCl₃): δ = 170.31, 147.21, 76.35, 75.94, 72.70, 53.47, 40.17, 17.39.

Synthesis of PEG-b-poly(MPC)

The ROP of MPC was carried out under an inert atmosphere of nitrogen using the standard Schlenk-line technique. In a typical experiment, MPC (0.594 g, 3 mmol), PEG_5k(0.601 g, 0.12 mmol), TU (0.055 g, 0.15 mmol), DBU (0.005 g, 0.03 mmol) and dried DCM (10 mL) were placed in a dried Schlenk tube fitted with a rubber septum. The solution was further degassed through three freeze–pump–thaw cycles. The resulting mixture was stirred at RT for 7 h, followed by precipitation in ice-cold diethyl ether and centrifugation. The resulting product was collected by filtration and dried under vacuum to yield a white powder. Yield: 1.1 g (92.0%).

¹H NMR (400 MHz, CDCl₃): δ = 4.73 (d, OCH₂CCH), 4.28–4.32 (m, OC(O)OCH₂), 3.65 (s, OCH₂CH₂O), 3.38 (s, CH₃O), 2.55 (s, CH₂CCH), 2.19 (s, OH), 1.29 (s, CH₃).

GPC (THF, RI): M_n (PDI) = 7263 g mol⁻¹ (1.04).

Synthesis of PMPC-Dns via ‘‘click’’ chemistry

Into a Schlenk tube, PEG-b-poly(MPC) (200 mg, propargyl group, 0.023 mmol), Dns-N₃ (76 mg, 0.23 mmol), sodium ascorbate (4.5 mg, 0.023 mmol), and DMF (4 mL) were introduced. The tube was fitted with a rubber septum. The solution was further degassed through three freeze–pump–thaw cycles. A DMF solution of copper sulfate (2.8 mg, 0.012 mmol) was then added to the Schlenk tube. The solution was stirred at RT for 24 h. The crude material was purified by dialysis (dialysis tubing 3500 MWCO) against deionised (DI) water that was renewed regularly. After 3 days, the final product, PMPC-Dns, was obtained by lyophilization. Yield: 190.9 mg (81.3%).

¹H NMR (400 MHz, CDCl₃): δ = 9.07 (s, ArH), 8.75 (d, ArH), 8.49 (s, ArH), 7.72 (s, N₃CHC), 4.73 (s, C(O)OCH₂), 4.31 (s, C(O)OCH₂), 4.28–4.24 (m, OCH₂CH₂CH₂N), 3.64 (s, OCH₂CH₂O), 3.47 (t, CH₂CH₂O), 3.38 (s, CH₃O), 2.17 (s, NCH₂CH₂), 1.29 (s, CH₃).

GPC (THF, RI): M_n (PDI) = 11353 g mol⁻¹ (1.07).

Preparation of micelles

Micelles of PEG-b-poly(MPC) and PMPC-Dns were prepared using a dialysis method. First, 25.0 mg copolymer was dissolved in DMF (1 mL), and then DI water (10 mL) was slowly added with vigorous stirring. After vigorous stirring for another 2 h at RT, the micelles were obtained and subjected to further dialysis against DI water for 24 h to remove DMF (MWCO 1000 Da). The final polymer concentration was adjusted by adding DI water to 0.5 mg mL⁻¹.

Measurement of the critical micelle concentration (CMC)

The CMC values of the PEG-b-poly(MPC) and PMPC-Dns amphiphiles were determined using a dye solubilization method with NR as the probe molecule. NR in THF (0.1 mg mL⁻¹, 30 μL) was added to a glass vial using a microsyringe. After the evaporation of THF, a portion of the micelle solution (2 mL) was added. The concentration of the micelle solution was varied from 0.1 to 5 × 10⁻⁴ mg mL⁻¹. Then, the solution was stirred for 24 h. Fluorescence measurements were taken at an excitation wavelength of 550 nm and the emission was monitored from 570 to 750 nm.

Preparation of DOX-loaded PMPC-Dns micelles and calculation of drug loading content (DLC)

In brief, PMPC-Dns (15.0 mg) and DOX (1.0 mg) were simultaneously added to dimethyl sulfoxide (DMSO, 5 mL) under vigorous stirring until complete dissolution. Then, the mixture was transferred into a dialysis bag (MWCO 3500 Da) and subjected to dialysis in aqueous solution for 3 days for the formation of the DOX-loaded PMPC-Dns micelles. During the 3 day period, the DI water was renewed regularly. To determine the DLC and DLE, the DOX-loaded PMPC-Dns micelles were incubated in Sec solution for 24 h, then lyophilized, and dissolved in DMSO again. The drug concentration was determined by measuring the fluorescence intensity of DOX (excited at 490 nm). The DLC and DLE were calculated according to the following equations:

DLC (%) = W_loaded/(W_polymer + W_loaded) × 100%

DLE (%) = W_loaded/W_total × 100%

where W_total, W_loaded and W_polymer are the weights of the total DOX used, loaded DOX, and PMPC-Dns micelles, respectively.

Cytotoxicity assay

Using HeLa cells as a model, the cellular cytotoxicity of the probe was evaluated using a standard MTT cell viability assay.²⁷ HeLa cells were seeded into a 96-well plate at a concentration of 5 × 10³ cells per well in 100 μL of MEM with 10% FBS. The plates were maintained at 37 °C in a 5% CO₂ 95% air incubator for 24 h. Then, the medium was removed and replaced with 200 μL polymer micelles. The aggregate concentrations of each formulation were prepared by serial dilution with DMEM. The cells incubated with the culture medium only were used as controls. The cells were washed with PBS solution three times, and then 100 μL MTT solution (0.5 mg mL⁻¹ in PBS solution) was added to each well. After the addition of DMSO (150 μL per well), the assay plate was shaken at RT for 10 min. Experiments were done in triplicate. The cell viability was calculated based on the measurement of UV-Vis absorption at 570 nm using the following equation, where OD₅₇₀ represents the optical density.^28,29

Cell viability = [OD_570(sample) − OD_570(blank)]/[OD_570(control) − OD_570(blank)]

Cell incubation and imaging

The HeLa cells were from the Biomedical Engineering Centre of Hunan University (Changsha, China). The cells were cultured using high-glucose DMEM (GIBCO) with 1% penicillin–streptomycin (10 000 U mL⁻¹, 10 000 μg mL⁻¹, Invitrogen) and 10% FBS (GIBCO) in an atmosphere of 5% CO₂ and 95% air at 37 °C. For the detection of exogenously produced Sec, the cells were co-incubated with Sec (5 μM) for 2 to 12 h. After washing three times with PBS solution (pH = 7.4) to remove the remaining Sec, the cells were further incubated with PMPC-Dns micelles (0.05 μg mL⁻¹) for 60 min at 37 °C. Then the incubated cells were washed with 3 × 1 mL of PBS solution (pH = 7.4), and fresh medium was added before imaging. To induce the endogenous Sec, the HeLa cells were exposed to sodium selenite (5 μM) for 2 to 12 h, and then the cells were further incubated with the PMPC-Dns micelles (0.05 μg mL⁻¹) for 30 min at 37 °C. After washing the cells three times with PBS solution (pH = 7.4), fresh medium was added. All the fluorescence images were acquired using an Olympus FV1000 laser confocal microscope and analyzed using Image Pro Plus software.

Preparation and staining of HeLa cancer tissue slices

All experiments were performed with the relevant laws and institutional guidelines of the biomedical engineering center of Hunan University (Changsha, China) and the committee had approved the experiments. Tissue slices were prepared from HeLa cancer neoplasm. A total of 2 × 10⁶ HeLa cancer cells diluted in 100 mL of serum-free DMEM were injected subcutaneously into the right flank of 6 to 8 week-old BALB/c nude mice for tumor inoculation. After 15–20 days, the mice were sacrificed, and the tumors were transferred and embedded with O.C.T (Sakura Finetek, USA, Torrance, CA) for frozen sections. The tissues were cut into 50 μm thick slices using a vibrating-blade microtome. For detection of exogenously produced Sec, the slices were co-incubated with Sec (20 μM) for 12 h. After washing three times with PBS solution (pH = 7.4) to remove the remaining Sec, the slices were further incubated with the PMPC-Dns micelles (0.2 μg mL⁻¹) for another 12 h at 4 °C. Afterward, the incubation slices were washed with 3 × 3 mL of PBS solution (pH = 7.4) before imaging. To induce the endogenous Sec, the tissues were exposed to sodium selenite (20 μM) for 12 h, and then the slices were further incubated with the PMPC-Dns micelles (0.2 μg mL⁻¹) for 12 h at 4 °C. After washing three times with PBS solution (pH = 7.4), the slices were mounted with 10% glycerol and sealed with nail varnish on a glass substrate. The fluorescence images of the HeLa cancer tissue were acquired on an Olympus FV1000 laser confocal microscope and analysed using Image Pro Plus software.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 21273068 and 21373003). C.T. Au thanks the HNU for an adjunct professorship.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra12314c

‡ The authors contributed equally to this work.

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