Reduction-controlled substrate release from a polymer nanosphere based on a viologen-cavitand

Department of Supramolecular Chemistry Physical Chemistry, Kazan Scientic Cente str. 8, Kazan 420088, Russia. E-mail: az@io A.M. Butlerov Institute of Chemistry, Kazan Kazan 420018, Russia Interdisciplinary Center of Analytical M Kremlevskaya str. 18, Kazan 420018, Russia Kazan Institute of Biochemistry and Bio Lobachevskii str. 2/31, Kazan 420008, Russi † Electronic supplementary informa 10.1039/c6ra15165e Cite this: RSC Adv., 2016, 6, 70072


Introduction
The application eld of polymer nanoparticles (PNP) is rapidly expanding. 1-3 PNP have found their place in different areas: in electronics and photonics, in medicine and biotechnology. In recent decades, PNP due to their favorable properties such as good biocompatibility, easy design and preparation, a variety of structures and interesting biomimetic character are widely used as biomaterials. [4][5][6] In the eld of smart drug delivery, PNP play a signicant role because they can store and stabilize drugs and effectively deliver them to their target sites. [7][8][9][10] Among the chemically cross-linked polymer particles, widely used ones are disulde cross-linkers. [11][12][13] The disulde bond is a dynamic covalent bond, which can be easily detached and reformed again. 14 Disuldes play an important role in pharmaceutical and biological applications because of their stability under normal conditions and degradation in a reductive environment. 15 Reducing agents such as dithiothreitol (DTT), glutathione (GSH, biologically available) easily cleave the disulde bonds. [16][17][18][19] Chemical reduction of disulde bonds has been operated for the cellular drug release or cellular imaging. 20,21 Resorcinarenes are well known class of macrocyclic compounds obtained by the condensation of resorcinol with aldehydes. 22 Resorcinarenes exhibit conformational mobility; however, their exible macrocyclic structure can be toughened by the intramolecular crosslinking of hydroxyl groups to produce cavitands. 23 The upper rim of cavitands can be easily modied by various groups, so this class of macrocycles is widely used in supramolecular chemistry for the selective binding of various guests. Cavitands have found application in different areas of science mainly in the separation technique 24 and catalysis. 25 Methylviologen cavitands (MVCA, Scheme 1) are redox-active macrocycle consisting four methylviologen groups on the upper rim. 26 MVCA effectively bind multiply charged anions and donors due to the cooperative action of the viologen groups to form host-guest complexes. 27,28 Viologens are 4,4 0bipyridinium salts which can be easily reduced to cation-radical and neutral state. 29 Viologens are used as a electroswitchable component in the molecular devices and molecular machines. 30 Viologens show antiviral and antimicrobial activity. [31][32][33] Multiviologen can be used in the gene and nucleic acids delivery. 34-36 MVCA reveal amphiphilic properties and selfassembly in aqueous media. 37,38 Earlier, it was demonstrated that MVCA, in a manner similar to cyclodextrins, [39][40][41] stabilizes o/w emulsions. 42 This behavior was used to creation of the thermosensitive polymeric nanocapsules. The nanocapsules were synthesized by the microemulsion polymerization followed by removing an organic part leaded to the porous and hollow nanocapsules formation. The MVCA-based nanocapsules were applied for the temperature-controlled delivery of hydrophilic species. 42 Work was continued by designing a new nanocarrier, which acts for hydrophobic substrates. For this purpose, a crosslinker with disulde bonds sensitive to the reducing agents was used in the MVCA polymerization. The crosslinker forms a monolithic core for the hydrophobic species while MVCA cover the core through covalent bonds making the nanocarrier well soluble in water. Herein, we report a synthesis of a core-shell polymer nanoparticle obtained by the polymerization of MVCA (shell component) with diallyl disulde (core component) and study the possibility of their use in the delivery of hydrophobic substrates.

Results and discussion
The microemulsion polymerization has been applied to obtain monodisperse polymer particles. Diallyl disulde (SS) represented both as a copolymer of MVCA and an organic part of the microemulsion. SS exhibits two double bonds and its polymerization results in the formation of the branched rigid polymers. A mixing SS (75 mM) with the aqueous solution of MVCA (5 mM) and the ensuing ultrasonic treatment under an argon atmosphere generate an 'oil-in-water' emulsion where SS drops are stabilized with MVCA. Its polymerization results in the formation of the polymer nanoparticles (p(MVCA-co-SS), Scheme 1). The polymerization was carried out in 12 h at 70 C in the presence of the initiator (NH 4 ) 2 S 2 O 8 . Aer completion of the reaction, p(MVCA-co-SS) was isolated, dialyzed for three days to elimination of the initial reagents and being washed with acetone. The size of p(MVCA-co-SS) is about 120 nm according to SEM image (Fig. 1). The molecular weight of p(MVCA-co-SS) is in the range of 1270 AE 65 kDa, as determined by static light scattering (Fig. S1 in the ESI †). The IR spectrum of p(MVCA-co-SS) exhibits the vibration bands of C]O at 1714 cm À1 and C]N at 1637 cm À1 and the shi and overlay bands of the alkyl and alkene groups at 2800-3100 cm À1 (Fig. S2 in the ESI †).
The multi-charged MVCA on the surface of p(MVCA-co-SS) make the nanoparticle well soluble and stable in water. 13 C { 1 H} NMR spectrum of p(MVCA-co-SS) displays the signals of the viologen groups while the resonance signals from the hydrophobic part are dramatically broadened and could not be detected (Fig. 2). In the 1 H-NMR spectrum of p(MVCA-co-SS), the signals at 5.5-6.0 ppm of the acrylic protons of MVCA completely disappeared, and new broad signals related to the polymerized diallyl disulde appeared at 1-2.5 ppm, approving the polymerization of MVCA with SS ( Fig. S3 in ESI †).
The core-shell structure of p(MVCA-co-SS) is conrmed by TEM where the 110-120 nm nanosphere has an amorphous monolithic structure (Fig. 1B). The hydrodynamic diameter of p(MVCA-co-SS) is not much larger than its size because of the rigidity of the p(MVCA-co-SS) (Fig. 1C). The inexible structure of p(MVCA-co-SS) is approved by the dynamic light scattering (DLS) through a temperature ramping experiment (Fig. 1D). A temperature increase does not affect the size of p(MVCA-co-SS) and its hydrodynamic diameter almost does not change.
The hydrophobic core of p(MVCA-co-SS) represents an area for the storage of the poorly water-soluble species. It is well illustrated by the examples of the environment-sensitive dyes: pyrene (Py), rhodamine B (RhB) and uorescein (Fl). The synthesis of p(MVCA-co-SS) was carried out in the dye solutions followed by the dialysis for three days at room temperature, results in the nanoparticles with the encapsulated dye (D@p(MVCA-co-SS), where D is Py, RhB, Fl). The encapsulated Py exhibits an excimer emission at 470 nm and the decrease of the rst emission band at 374 nm (Fig. 3A) indicating the  This journal is © The Royal Society of Chemistry 2016 location of the dye in the hydrophobic limited space. 43,44 A signicant bathochromic shi, from 580 to 605 nm is observed for RhB inside p(MVCA-co-SS) also conrming the organic nature of the SS core (Fig. 3B). 45 Fl@p(MVCA-co-SS) displays a greater shi in the emission and absorption peaks in comparison with free Fl (Fig. 3C). It is necessary to note that only the encapsulation into the SS core causes to such signicant spectral changes. The cavitand MVCA does not affect qualitatively the dye spectral characteristics. Adding up to ten equivalents of MVCA with pentyl tails to the dyes does not cause to any shi of the spectral bands in the UV and uorescence spectra (Fig. S4 in ESI †). In the case of Fl, a decrease of the emission intensity without the bands shi appears due to an electron transfer from Fl to viologen moieties ( Fig. S4 in ESI †). 46 The nanospheres D@p(MVCA-co-SS) are quite stable. There is no release of the dye molecules from the core of p(MVCA-co-SS) during their storage at room temperature for more than ten days (Fig. S5 in ESI †).
p(MVCA-co-SS) is sensitive to the thiol-reducing agents affecting disulde bonds. We investigated the changes in p(MVCA-co-SS) aer the addition of the natural antioxidant glutathione (GSH) and redox reagent with two SH groupsdithiothreitol (DTT). GSH is an important tripeptide performing many biological functions such as neutralization of free radicals and reactive oxygen compounds, regulation of the nitric oxide cycle, involvement in metabolism and in progression of the cell cycle. 47,48 Adding p(MVCA-co-SS) (8 mg ml À1 ) into the water containing 2.5 mg ml À1 (8 mM) of GSH causes to the degradation of the polymeric nanospheres. DLS data shows that a distribution peak at 130 nm decreases and multiple peaks in the range 2.5-500 nm appear. Polydispersity index (PDI) increases from 0.29 to 1 (Fig. S6 in ESI †).
The sensitivity of p(MVCA-co-SS) to GSH can be applied in the GSH-controlled species release. The addition of GSH to D@p(MVCA-co-SS) causes to the yield the dye molecules to the bulk aer the breakdown of the p(MVCA-co-SS) core. In uorescence of Py the rst emission peak increases while the excimer band decreases (Fig. S7 in ESI †). In the cases of RhB and Fl the hypsochromic shi of the uorescence peaks are observed.
The dye yield is the most clearly seen on the Fl, monitoring this action by uorescence. As shown in Fig. 4A, the emission intensity increases almost 50 times upon at once and the emission peak shis from 550 to 520, thus conrming the dye release and complete destruction of the SS core. The color of the solution changes from yellow to colorless, indicating the change in the uorescein environment.
Opposite to GSH, DTT does not completely destroy the hydrophobic core of p(MVCA-co-SS). It only changes the size of the cross-linked nanoparticles. According to the DLS data, the addition of 9.3 mg ml À1 (60 mM) of DTT leads to an increase of the hydrodynamic size of p(MVCA-co-SS) from 130 to 210 nm and a decrease of PDI till 0.01 (Fig. S6 in ESI †). The molecular weight increases slightly to 1320 AE 35 kDa (Fig. S1 in ESI †). Evidently, the DTT inuence is more complex. DTT does not only reduces the disulde bonds, it can embed in the SS core increasing the SS core size and its friability. Therefore, the impact of DTT does not cause the dye out of the cavity (Scheme 2). The addition of DTT to Fl@p(MVCA-co-SS) increases the intensity of the uorescence at three times without the bands shi. This demonstrates that the dye molecules do not yield from the hydrophobic SS core into water. Their motion in the enlarged core is facilitated, leading to enhancement of uorescence emission of Fl (Fig. 4B).

Experimental
NMR spectroscopic experiments were carried out with an Avance 600 spectrometer (Bruker, Germany) equipped with a pulsed gradient unit capable of producing magnetic-eld pulse gradients in the z direction of about 56 G cm À1 . D 2 O   was used as a solvent in all experiments. Chemical shis were reported relative to HDO (d ¼ 4.7 ppm) as an internal standard. UV-vis spectra were recorded with a Perkin-Elmer Lambda 25 UV/vis spectrometer. Fluorescence emission spectra were recorded with a Cary Eclipse uorescence spectrophotometer (USA). A quartz cell of 1 cm path length was used for all uorescence measurements. Imaging of the polymer nanoparticles was carried out by intermittent contact mode. Transmission electron microscope (TEM) images were obtained by using JEM-1200EX (JEOL, Japan) instrument at an accelerating voltage of 120 kV. The samples (2-5 ml) were applied on formvar coated copper grids, and then dried at room temperature. The morphology of sample surfaces were characterized in plan-view by scanning electron microscopy (SEM) using high-resolution microscope Merlin Carl Zeiss combined with ASB (Angle Selective Backscattering) and SE InLens (Secondary Electrons Energy selective Backscattering) detectors. A Zetasizer Nano instrument (Malvern, UK) equipped with a 4 mW He:Ne solidstate laser operating at 633 nm was used for SLS and DLS experiments. Malvern dispersion technology soware 5.0 was used for the analysis of particle size and molecular weight.

Synthesis of p(MVCA-co-SS)
Diallyl disulde (0.070 ml, 0.5 mmole) was added to a solution of MVCA 42 in water (C ¼ 5 mM, V ¼ 9.7 ml). The mixture was bubbled with argon for 30 min and then sonicated in the argon atmosphere until the complete homogenization (approximately 80 min). The suspension was heated at 70 C for 30 min under argon and then ammonium persulfate (20 mg in 0.3 ml water) was added. The suspension heating at 70 C was continued for 12 h. The nal colloidal solution was dialyzed for 3 hour (10 ml versus 3 Â 800 ml water). Water was removed under reduced pressure. The solid formed was washed with acetone and dried in atmosphere. p(MVCA-co-SS) (0.136 g, 80%). Mp > 300 C. Found: C, 48

Synthesis of D@p(MVCA-co-SS), where D -Py, RhB or Fl
D@p(MVCA-co-SS) were synthesized similarly to p(MVCA-co-SS) using an aqueous solution of D (5 mM) instead of water. The nal colloidal solutions were dialyzed for 3 days (2 ml versus 3 Â 800 ml water).

Reduction of p(MVCA-co-SS) by GSH
To an aqueous solution of p(MVCA-co-SS) (C ¼ 8 mg ml À1 , V ¼ 0.5 ml), a solution of GSH (C ¼ 8 mM, V ¼ 0.5 ml) was added at room temperature. Aer 10 min, the particle size was determined by DLS. Reduction of p(MVCA-co-SS) by DTT to an aqueous solution of p(MVCA-co-SS) (C ¼ 8 mg ml À1 , V ¼ 0.5 ml), a solution of DTT (C ¼ 60 mM, V ¼ 0.5 ml) was added at room temperature. Aer 10 min, the particle size was determined by DLS.

Conclusions
In conclusion, we have prepared a new redox-responsive watersoluble nanosphere with rigid monolithic core by the microemulsion polymerization of viologen-resorcinarene cavitand with diallyl disulde. The nanosphere can be applied for the hydrophobic species storage. The inuence of the reducing agents GSH and DTT on the structure, stability and the species release is investigated. It is shown that GSH disrupts the nanospheres resulting in the species release while DTT only reorganizes the nanospheres core (Scheme 2).