DOI: 10.1002/chem.201404035
Full Paper
& Supramolecular Chemistry
A Peptide Dendron-Based Shrinkable Metallo-hydrogel for Charged Species Separation and Stepwise Release of Drugs Long Qin, Fan Xie, Pengfei Duan, and Minghua Liu*[a]
Abstract: A shrinkable supramolecular metallo-hydrogel based on the l-glutamic acid dendron and magnesium showed reversible volume-phase transition depending on pH changes. The hydrogel further showed selective shrinkage upon addition of positively charged species, while it remained in the gel state when negatively charged species were incorporated. Based on this property, the gel could be used as the matrix to efficiently separate ionic dye mixtures,
in which the cationic dye was incorporated predominantly in the shrunken gel, while the negatively charged dye was released into the aqueous solution. More interestingly, the shrinkable gel can be used as a model drug-delivery vehicle for the stepwise release of a two-component drug system, in which the negatively charged drug is released first and then the second component is released with a pH trigger.
Introduction
cussed.[12] Although these systems have showed the shrinkable properties, its potential application has not been well developed. We have recently found that a series of divalent metal ions can trigger the shrinkage of supramolecular hydrogel formed by an l-glutamic acid peptide dendron, N-octadecanoyl-1,5bis(l-glutamic acid)-l-glutamic diamide (OGAc).[13] With different kinds of divalent metal ions, the supramolecular hydrogel displays various shrinkage properties. It was found that the hydrogel containing magnesium exhibited the most obvious shrinkage performance; meanwhile, the shrinkage process is a continuous, reversible and shape-dependent macroscopic volume phase transition. Based on this work, herein, we further fabricated a shrinkable metallo-hydrogel by combining the dendron gelator OGAc with magnesium ions, which exhibited some new responsive properties. First, the shrinkage process is pH-dependent. The phase behaviour can be reversibly regulated by pH change. The shrunken gel can convert to the stable gel at higher or lower pH values. Second, the metallo-hydrogel showed selective shrinkage based on the additive charged species. It was found that the addition of positively charged species can accelerate the shrinkage of the supramolecular hydrogel, while negatively charged additives inhibited hydrogel shrinkage. Based on this property, the shrinkable metallo-hydrogel could be used as a matrix to efficiently separate ionic dye mixtures. That is, when an ionic mixture of dyes was mixed with the OGAc/Mg2 + metallo-hydrogel, the anionic dye was released into the aqueous solution phase through shrinkage while the cationic dye was maintained in the gel skeleton. Besides the separation of dye mixtures, the shrinkable metallohydrogel was proposed for use as a vehicle for the step-wise release of two-component drugs. In this gel, the anionic drug was first released through gel shrinkage and then by pH trigger, the second cationic drug was released through gel collapse. Although the system is still not very sophisticated, to
Supramolecular hydrogels, as an important class of soft materials formed through the self-assembly of low molecular weight hydrogelators in water via various non-covalent interactions, have attracted great interest in recent years.[1] In comparison with the conventional polymer hydrogels, supramolecular hydrogels are more apt to be regulated for their thermo-reversibility, stimuli-responsiveness,[2] and biocompatibility since the hydrogelators can be derived from biological components including amino acids,[3] sugars[4] and steroids.[5] Many functional supramolecular hydrogels have been thus developed and found applications in tissue engineering,[6] sensing,[7] biomaterials[8] and drug delivery.[9] However, due to the inherent weak character of non-covalent interactions, supramolecular hydrogels still suffer the reality that the brittle 3D structures have difficulty in maintaining physical change such as folding and curling of the supramolecular chains as in polymer hydrogels to accordingly exhibit phase transition in volume (shrinkage– swelling)[10] and shape (deformation–recovery),[11] which limits their many potential uses as biomaterials. For example, while many polymer hydrogels can show volume phase transition, it is still a great challenge and highly significant to develop supramolecular hydrogels with volume phase transition. A thermally and pH-responsive supramolecular hydrogel based on glycosylated amino acetate was first reported by Hamachi et al. and the unique properties of hydrogel shrinkage as well as the mechanism of phase volume transition were dis[a] L. Qin, F. Xie, Dr. P. Duan, Prof. M. Liu Beijing National Laboratory for Molecular Science (BNLMS) CAS Key Laboratory of Colloid, Interface and Chemical Thermodynamics Institute of Chemistry, Chinese Academy of Sciences (P. R. China) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404035. Chem. Eur. J. 2014, 20, 1 – 8
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Full Paper the best of our knowledge, this is the first report of a shrinkable supramolecular hydrogel that can be used as a smart vehicle for stepwise release of multicomponent drugs, and might show great significance in the development of complex drugdelivery systems.
Results and Discussion pH responsiveness and gel shrinkage Since the amphiphilic dendron OGAc contains four carboxylic acids, the phase behaviour of OGAc/Mg2 + metallo-hydrogel was highly dependent on pH. Therefore, potentiometric pH titration was carried out at 25 8C to determine the protonation constant of OGAc, as shown in Figure S1 in the Supporting Information. The pH titration curve shows OGAc has two protonation constants pKa1 and pKa2 with the values of 3.2 0.1 and 5.7 0.1, respectively. The compositions of OGAc at [OGAc]total = 1.0 mm were calculated from the Henderson–Hasselbalch equation and the negative charge distribution from 0 to 4 under different pH values was obtained.[14] Based on the above results, the phase behaviour and micromorphology of OGAc/Mg2 + hydrogel was investigated under selected pH values (as shown in Figure 1 and Figure 2 A–D). It was found that when the gel shrinks, the system pH value was located at 3.2. The randomly distributed and nicely separated nanofibres with the thickness range 16–20 nm were investigated in the shrunken hydrogel. However, when the system pH was changed to 1.8, a stable semi-transparent hydrogel was formed, at which point the carboxylic acid can hardly be ionised. Morphologic measurements showed that entangled nanofibres with diameter from 20 to 30 nm were formed. Moreover, increasing the pH to 4.3 induced the formation of a transparent hydrogel, in which ultrafine nanofibres with an average diameter of about 4 nm were obtained. This is close to the thickness of an OGAc-formed interdigitated bilayer structure. Finally, when the pH reached 6.2 or above, the ionised head group increased the hydrophilicity of the gelator, which destroyed the hydrogelation of OGAc leading to the formation of a sol. TEM investigation showed that here irregular nanosheets were formed. Meanwhile, it was observed that hydrogel shrinkage is located at a pH range from 2.8 to 3.5. The gel cannot shrink at a system pH value below or above the critical points. Moreover, the pH responsiveness in OGAc/Mg2 + hydrogel is a reversible phase transition. The phase behaviour can be reversibly transformed by changing the system pH value several times (Figure S2 in the Supporting Information). In order to further understand such phase transition at different pH values, the X-ray diffraction (XRD) and FT-IR spectra were analysed, as shown in Figure 2 E and F. FT-IR spectra showed that the driving forces for the formed nanofibres are hydrogen bonds between the amide groups, the hydrophobic interaction between alkyl chains and the electrostatic interaction between metal ions and carboxyl groups. The bands that appeared at 3298, 1637 and 1549 cm1 are attributed to stretching N=H vibration, amide I and II, respectively. With an increase in pH, the N=H vibration gradually decreased and the &
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Figure 1. Molecular structure of OGAc and the pH responsiveness of OGAc/ Mg2 + shrinkable metallo-hydrogel. The gel shrunk at pH 3.2 and converted to a semitransparent gel at pH 1.8, transparent gel at pH 4.3 and sol at pH 6.2. The blue and yellow balls refer to the unprotonated and protonated head groups of l-glutamic acid dendron, respectively.
band of amide I shifted to the high wavenumber and finally to 1647 cm1 when the pH reached 6.2. This indicated that the hydrogen bonding between amide groups decreased with pH change. The C=O vibration that mainly appeared at 1730 cm1 gradually moved to 1735 cm1 and disappeared at pH 6.2. Meanwhile, the typical vibration of COO at around 1600 cm1 became more obvious and finally changed to a broad band at 1585 cm1. All these changes indicate that with increasing pH, the hydrogen bonding between head groups slowly decreased and the carboxyl groups were gradually protonated and interacted with the metal ions.[15] The XRD patterns of the xerogels obtained at different pH values showed that the lamellar structure was formed in all the gels. The layer distance of 4.0, 3.9 nm for the shrunken and transparent hydrogel at pH 3.2, 4.3 and d-spacing value of 5.4 nm for the semitransparent-hydrogel obtained at pH 1.8 are more than one molecular length (estimated for 2.7 nm) but less than or equal to twice that of the molecular thickness, which indicated the formation of different bilayer structures. Based on the above results, a possible mechanism for pH responsiveness is proposed. Under the strongly acidic condition (pH 1.8), the ionisation of the carboxyl groups cannot occur, which makes the gelator more hydrophobic and thereby contributes to the formation of the wormlike micelle. The micelles further entangle through hydrogen bonds to form the threedimensional superstructures. A similar assembly process to form the nanofibres with interdigitated bilayer structures can be found in the hydrogel at pH 3.2. However, between the formed molecular-thick nanofibres, magnesium ions can slowly bridge the adjacent nanofibres through electrostatic interactions with the protonated carboxyl groups to expel the bound water, as shown in Figure 6 B, which finally causes the volume transition at the macro-level. But the further protonation of carboxyl groups will enhance the surface electronegativity of the nanofibres and prevent additional entanglement between the nanofibres. Therefore, a transparent hydrogel containing molecular-thick nanofibres are observed at pH 4.3. 2
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Full Paper Figure 4 A. Upon mixing with the dyes, a series of transparent gels were formed and the UV/Vis spectra show that the absorption is very close to their solution absorption. For the asprepared hydrogel, there is no CD signal either in the OGAc/ Mg2 + hydrogel mixed with cationic or anionic dyes. However, accompanying the volume transition, a series of negative Cotton effects were observed at the corresponding absorption peak for cationic-dye-doped shrunken metallo-hydrogels. Morphological investigation showed that the formed nanofibres in these gels were several nanometers thicker than the nanostructure obtained in the shrunken gel without dyes. On the other hand, when anionic dyes were used, no CD peak was observed even after resting for several weeks. TEM analysis showed that there were some irregular nanodots randomly distributed in gel nanofibrillar networks. EDX investigation confirmed that no relevant metal atoms were present in the nanodots (Figure S3 in the Supporting Information), suggesting that the nanodots most probably correspond to the aggregated anionic dyes. These results indicate that when the dyes, either cationic or anionic, were mixed in the gels, the gelator molecules self-assembled into nanofibre structures, while the dyes distributed in the aqueous phase at the beginning. Upon resting, the cationic dye interacted with the gelator fibres and formed a chiral complex on the surface of nanofibre, as illustrated in Figure 4 B, which accompanied with the shrinkage of the gel. For the anionic dyes, due to the electrostatic repulsion between sulfonate and carboxylate, the dyes remained in the aqueous phase and prevented the shrinkage of hydrogel.
Figure 2. A)–D) TEM images, and E), F) FT-IR and XRD spectra of OGAc/Mg2 + hydrogel (mole ratio 5:1) at different pH values (scale bar = 500 nm).
Selective shrinkage of the gel with charged species The metallo-hydrogel can incorporate water-soluble dyes with good capacity. During the shrinkage, the gel showed different responsiveness to the charged dyes. A series of water-soluble ionic dyes with OGAc/Mg2 + hydrogel matrix (mole ratio dye/ OGAc = 1:20) at pH 3.2, among which cationic dyes such as methyl violet (MV), methylene blue (MB), acridine yellow (AY), neutral red (NR) and anionic dyes including methyl orange (MO), acid red 26 (AR), were chosen as the model compounds (molecular structures and basic parameters are shown in Table S1 in the Supporting Information). The phase behaviour of the OGAc/Mg2 + hydrogel with doped amount of ionic dye was observed after the gel was allowed to rest for 20 h. It was found that shrinkage took place in the hydrogel mixed with cationic dyes, so that more than half the volume of water was expelled from the original hydrogel. In addition, the UV/Vis spectra of the expelled water showed that there was nearly no dye in this phase, which means that the cationic dyes were almost entirely incorporated in the shrunken gel matrix. In the case of anionic dyes, the transparent hydrogel was formed and maintained its original phase state even after resting for several weeks. A control experiment with the OGAc/Mg2 + hydrogel with an equal amount of NaCl(aq.) revealed that the gel could shrink under these conditions, which suggests that it was the anionic dyes that prevented the hydrogel shrinkage. In order to understand these changes, CD spectroscopy of the dye-incorporated gel and TEM observation of the corresponding xerogels were performed, as shown in Figure 3 and Chem. Eur. J. 2014, 20, 1 – 8
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Figure 3. TEM images and photographs of ionic-dye-doped OGAc/Mg2 + (0.13 wt %) hydrogel after being allowed to rest for 20 h (cationic dyes: neutral red, methyl violet, acridine yellow, methylene blue, anionic dyes: methyl orange, acid red 26); scale bar = 200 nm.
Separation of dye mixture using the shrunken gel as matrix We further investigated the shrinkage property of the gel incorporated by a series of cationic and anionic dye mixtures. The OGAc/Mg2 + hydrogel mixed with the same molar amounts of two different dyes was firstly fabricated and then allowed to rest for 20 h. It was interesting to find that the ionic dye mix3
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Figure 4. A) UV and CD spectra of ionic-dye-doped shrunken OGAc/Mg2 + (0.13 wt %) hydrogel after being allowed to rest for 20 h. B) Schematic illustration of the interaction between ionic dyes and gel networks. The cationic dyes well incorporated and aligned with the gel fibre structures through strong electrostatic interactions (a). However, the anionic dyes were distributed between the solvent phase and immobilised by the 3D gel frameworks (b).
Figure 5. Photographs of mixed-dyes-doped OGAc/Mg2 + (0.13 wt %) hydrogel (top) and efficient separation of these oppositely charged dyes through hydrogel shrinkage process (middle) and corresponding separation efficiency (bottom).
tures could be easily and spontaneously separated through the hydrogel shrinkage. The cationic dyes remained in the gel phase. However, the anionic dyes were released into the aqueous phase. Experimental results indicated that the shrinkable hydrogel can efficiently separate the mixed quaternary ammonium (+) and sulfonic acid () dyes, as shown in Figure 5. The corresponding separation efficiency was indirectly evaluated by the distribution ratio of dye molecules in the aqueous phase when gel shrinkage reached equilibrium. Through contrasting UV spectra of the extruded water phase after shrinkage with the original mixed dye aqueous solution, the distribution ratio of the mixed dyes were calculated (calculation details are shown in Experimental Section). It is very obvious that the distribution ratio of anionic dyes in expelled water is generally more than 70 % and less than 10 % for all the cationic dyes. Although many hydrogels have been used to entrap dyes,[16] our results show that through the shrinkable supramolecular hydrogel, the dyes can be separated from the charged mixtures.
techniques, well-controlled stepwise release of the multicomponent drugs is becoming necessary and showing potential prospects. The above-separation results inspired us to perform the stepwise release of two-component water-soluble ionic drugs through supramolecular hydrogel shrinkage in vitro tests. We designed the two-component drug-loaded shrinkable hydrogel to firstly release one part of the drugs in the tissue with the acidic pH range and then to continuously release the remaining parts in the tissues with a neutral or alkaline pH value. Herein pralidoxime iodide (PI) and phenol red (PR) were used as the model drugs of hydrophilic small molecules. The phase behaviour of OGAc/Mg2 + hydrogel loaded with 5 mol % of the two hydrophilic drugs was firstly observed (Figure S5 in the Supporting Information). As with the dye-doped experiment, the volume of hydrogel loaded with PI gradually shrunk over time. However, the hydrogel containing PR remained in the original state. CD spectra and TEM analysis indicated that PR was immobilised in the water phase by the hydrogel skeleton structure, while PI adhered to the 3D network of hydrogel. Finally, the selected drug PI and PR were mixed with equimolar ratio and entrapped in OGAc/Mg2 + hydrogel matrix to conduct the controlled-release experiments. The drug loading content (DLC) and drug loading efficiency (DLE) calculation shows that the hydrogel was able to encapsulate the two drugs (PI DLC = 1.97 % and DLE = 99.2 %; PR DLC = 2.61 % and DLE = 98.7 %) with a high loading efficiency.
Stepwise release of two-component ionic drugs Stimuli-responsive hydrogel is frequently used as a matrix for controlled drug release, especially some intelligent polymer hydrogels with the volume phase transition property.[17] With the development of intelligent drug carriers and controlled release &
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Full Paper Conclusion
The release profile was obtained by detecting time-dependent UV/Vis spectra of release media taken at desired time intervals (Figure S6 in the Supporting Information). As shown in Figure 3, in vitro release studies of OGAc hydrogel revealed that the release rate of PI and PR exhibited a similar trend. The slight difference in the cumulative release ratio of PI and PR is most likely due to the electrostatic interactions between the gelator and PI. However, to our delight, the release behaviour in OGAc/Mg2 + shrinkable hydrogel shows obvious selectivity. With the shrinkage of the hydrogel, the release of encapsulated PR is significantly faster than PI. The results show that compared with the OGAc hydrogel, the release of PR is accelerated in the shrunken gel. Nearly 90 % of the PR was released at 22 h. However, the release of PI was evidently decreased and less than 30 % of PI escaped from the gel matrix when the release process reached equilibrium. The system pH value was then adjusted to 6.2, at which point the shrunken hydrogel collapsed to the sol to further release the remaining drug PI at 8 h. Based on the above results, the stepwise release property renders this shrinkable supramolecular hydrogel a new promising carrier for targeted drug delivery (Figure 6).
Unlike most reported supramolecular hydrogels, here we discovered a shrinkable metallo-hydrogel that shows reversible volume-phase transition with pH-stimulus and the phase behaviour from stable, shrunken hydrogel to homogenous solution can be well regulated by pH change. The shrinkable hydrogel also exhibits charged-species responsive property and can selectively incorporate cationic molecules, which inspired us to utilise the shrinkable metallo-hydrogel as an intelligent carrier to efficiently separate ionic dye mixtures and sophisticated controlled stepwise release of loaded two-component drugs. By introducing this novel stimuli-responsive shrinkable metallo-hydrogel, we anticipate further progress in the design of sophisticated multifunctional materials based on supramolecular self-assembly with broad application prospects.
Experimental Section Materials The gelator OGAc was synthesised according to our previous work.[3a] The ionic dyes: methyl violet (MV), methylene blue (MB), acridine yellow (AY), neutral red (NR), methyl orange (MO), acid red 26 (AR) and model drugs pralidoxime iodide (PI) and phenol red (PR) used in all the cases were purchased from Acros Organics or Sigma Aldrich and were used without any additional purification. Milli-Q water (18.2 MW cm) was used in all experiments. All the organic solvents were purified and dried according to standard methods.
Instruments and methods The UV/Vis and CD spectra were detected using JASCO UV-550 and JASCO J-810 spectrophotometers, respectively. Fourier transform infrared (FT-IR) spectra were recorded on a JASCO FT/IR-660 plus spectrophotometer with a wavenumber resolution of 4 cm1 at room temperature. X-ray diffraction (XRD) was achieved on a Rigaku D/Max-2500 X-ray diffractometer (Japan) with CuKa radiation (l = 1.5406 ), which was operated at 45 kV, 100 mA. Transmission electron microscopy (TEM) images were obtained on a JEM1011 electron microscope operating at accelerating voltages of 10 and 200 kV, respectively. All the photographs of the gel were taken by using a Canon EOS60D.
Procedures for preparation and characterisation of gel The typical hydrogel was prepared by adding OGAc (1.34 mg; 0.002 mmol) and Mg(NO3)2 (8 mL; 50 mm) aqueous solution to pure water (1 mL), which was slightly heated to a transparent solution and then placed at room temperature for 20 h. The pH value of the hydrogel was adjusted with HCl and NaOH aqueous solution. As for the reversible phase-transition experiment, 1.0 or 0.1 m HCl and NaOH aqueous solution were used to regulate the system pH between 1.8, 4.3, 6.2 and 3.2. For the TEM measurements, a small amount of diluted hydrogel was placed onto carbon coated copper grid (unstained) and the sample was cryodesiccated in a freeze dryer at 40 8C for 24 h. In the case of preparing samples for XRD measurements, hydrogels were cast onto glass plates and dried in vacuum. Platelets made from the mixture of vacuum-dried xerogels with KBr powder and the sample nipped by CaF2 platelets were used for FT-IR spectral measurements.
Figure 6. A) Two-component drug release in OGAc semi-hydrogel and OGAc/Mg2 + shrinkable hydrogel. At the initial process of drug release in the OGAc/Mg2 + hydrogel, the system pH was firstly kept at 3.2. After release for 22 h, the system pH was adjusted to 6.2 to continuously release for 8 h. B) Schematic illustration for the stepwise release of two-component drugs. At the beginning of the shrinkage, the charged species were immobilised in the 3D gel networks. In the remaining time, the magnesium ions can slowly bridge the adjacent carboxyl groups in the proposed manner and expel the bound water to show the volume transition at the macro-level, during which the positively charged drugs are incorporated with nanofibres; the negatively charged drug was released first by hydrogel shrinkage. After the system pH was changed to 6.2, the 3D network of the hydrogel collapsed and the positively charged drug was gradually released. Chem. Eur. J. 2014, 20, 1 – 8
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Full Paper Potentiometric pH titration
Stepwise release for two-component ionic drugs
OGAc was dissolved in water at a concentration of 1.0 mm and the solution pH was adjusted to 10 with a small volume of 0.1 m NaOH. Then, calibrated 0.1 m HCl was gradually added into this solution in small portions. The pH values were recorded with a pHS2C acidity meter and the temperature was kept at 25.0(0.1) 8C throughout the measurements. The electrode was calibrated with a standard buffer solution before being used. Three titrations were performed and the mean pKa values were calculated.
The typical procedure for stepwise release of two-component drugs is as follows. OGAc (2.68 mg; 0.004 mmol), 400 mL cationic and anionic drug (0.5 mm) aqueous solution with or without (control experiment) Mg(NO3)2 (16 mL; 50 mm) aqueous solution were mixed in a sealed tube with pure water (1.2 mL). The mixtures were slightly heated to obtain a transparent solution and were then allowed to cool to room temperature undisturbed, to obtain the hydrogel. The drug-loaded hydrogel was then transferred to a dialysis bag with a molecular weight cut-off (MWCO) 3500 and then the dialysis bag was carefully dropped into 5 mL aqueous solution (pH 3.2). Sampling was carried out over a specified time interval and sample volume (200 mL). The total volume of the system was kept at 5 mL by returning the solution after analysis. After the release reached equilibrium at 22 h, the system pH was adjusted to 6.2 to continuously monitor the release process. The released amount of both drugs was calculated based on UV/Vis spectra of the samples at 294 nm for PI and 432 nm for PR. For determination of drug-loading content and loading efficiency, the drug-loaded hydrogels were dissolved in methanol. Then the concentration of the two drugs was obtained based on the UV/Vis spectra. Drugloading content (DLC) and drug-loading efficiency (DLE) were calculated according to the following formulas: DLC = (weight of loaded drug/weight of gelator) 100 %, DLE = (weight of loaded drug/weight of drug in feed) 100 %.
Selective separation for mixed ionic dye mixture The typical procedure for selective separation of mixed ionic dyes was as follows. OGAc (2.68 mg; 0.004 mmol), Mg(NO3)2 (16 mL; 50 mm) aqueous solution and 400 mL cationic and anionic dye (0.5 mm) aqueous solution were mixed in a sealed tube with pure water (1.2 mL). The mixtures were slightly heated to obtain a transparent solution and then allowed to cool to room temperature unaffectedly to obtain the hydrogel. After the gel was allowed to rest for 20 h to shrink, the gelation state was recorded and the separation ratio was obtained by detecting the UV spectra of expelled water phase. Due to the interference between UV/Vis spectra of the two-component ionic dye mixtures, the separation ratio was calculated based on simultaneous equation method which includes the following steps. 1) A series of cationic (X) and anionic (Y) dye standard solutions with linear concentration gradient were prepared (including total 5 2 samples with the five concentrations: 1 104, 8 105, 6 105, 4 105 and 2 105 mol L1). The absorbance of the above five samples at the maximum absorption wavelength of the two dyes (l1 and l2) was measured to obtain the four absorbance–concentration curve:
al1 ¼ e1X bC X
ð1Þ
al2 ¼ e2X bC X
ð2Þ
al1 ¼ e1Y bC Y
ð3Þ
al2 ¼ e2Y bC Y
ð4Þ
Acknowledgements This work was supported by the Basic Research Development Program (2010CB833305, 2013CB834504), the National Natural Science Foundation of China (Nos. 91027042, 21021003, 21227802, 21321063), the Strategic Priority Research Program of the Chinese Academy of Sciences, Grant No. XDB12020200. Keywords: dendrons · drug delivery · selective separation · supramolecular chemistry · stimuli-responsiveness [1] a) L. A. Estroff, A. D. Hamilton, Chem. Rev. 2004, 104, 1201 – 1218; b) M. de Loos, B. L. Feringa, J. H. van Esch, Eur. J. Org. Chem. 2005, 3615 – 3631; c) A. R. Hirst, B. Escuder, J. F. Miravet, D. K. Smith, Angew. Chem. 2008, 120, 8122 – 8139; Angew. Chem. Int. Ed. 2008, 47, 8002 – 8018; d) R. G. Weiss, P. Terech, in Molecular Gels: Materials with Self-Assembled Fibrillar Networks, Springer, Dordrecht, 2006, pp. 613 – 648. [2] a) M. D. Segarra-Maset, V. J. Nebot, J. F. Miravet, B. Escuder, Chem. Soc. Rev. 2013, 42, 7086 – 7098; b) S. Roy, N. Javid, P. W. J. M. Frederix, D. A. Lamprou, A. J. Urquhart, N. T. Hunt, P. J. Halling, R. V. Ulijn, Chem. Eur. J. 2012, 18, 11723 – 11731; c) M. Ikeda, T. Tanida, T. Yoshii, I. Hamachi, Adv. Mater. 2011, 23, 2819 – 2822; d) C. J. Bowerman, B. L. Nilsson, J. Am. Chem. Soc. 2010, 132, 9526 – 9527; e) G. O. Lloyd, J. W. Steed, Nat. Chem. 2009, 1, 437 – 442; f) W. Deng, H. Yamaguchi, Y. Takashima, A. Harada, Angew. Chem. 2007, 119, 5236 – 5239; Angew. Chem. Int. Ed. 2007, 46, 5144 – 5147; g) H. J. Kim, J. H. Lee, M. Lee, Angew. Chem. 2005, 117, 5960 – 5964; Angew. Chem. Int. Ed. 2005, 44, 5810 – 5814; h) R. V. Ulijn, J. Mater. Chem. 2006, 16, 2217 – 2225. [3] a) P. Duan, L. Qin, X. Zhu, M. Liu, Chem. Eur. J. 2011, 17, 6389 – 6395; b) Y. Liu, T. Wang, Z. Li, M. Liu, Chem. Commun. 2013, 49, 4767 – 4769; c) Y. Liu, T. Wang, M. Liu, Chem. Eur. J. 2012, 18, 14650 – 14659; d) A. M. Smith, R. J. Williams, C. Tang, P. Coppo, R. F. Collins, M. L. Turner, A. Saiani, R. V. Ulijin, Adv. Mater. 2008, 20, 37 – 41; e) S. Fleming, S. Debnath, P. W. J. M. Frederix, T. Tuttle, R. V. Ulijin, Chem. Commun. 2013, 49, 10587 – 10589; f) J. B. Matson, S. I. Stupp, Chem. Commun. 2012, 48, 26 – 33.
where e1X, e2X, e1Y and e2Y are the molar absorption coefficients of X and Y at l1 and l2 ; b refers to the length of optical path. Equations (1) + (3) and (2) + (4) were used to obtain the absorbance– concentration curve of the two-component dye mixtures at l1, l2, respectively:
Al1 ¼ e1X bC X þ e1Y bC Y
ð5Þ
Al2 ¼ e2X bC X þ e2Y bC Y
ð6Þ
2) After the hydrogel was allowed to rest for 20 h to shrink, the absorbance of the expelled water phase at l1 and l2 was detected and the concentration of the two dyes was calculated based on above equation. 3) The separation ratio was finally obtained based on the following equation:
ð7Þ
R ¼ C X V m =C 0 V 0
where Cx and C0 are the concentration of the dye X in the expelled-water and initial sample; and Vm and V0 are the volume of expelled-water and initial sample.
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Received: June 19, 2014 Published online on && &&, 0000
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FULL PAPER & Supramolecular Chemistry
The incredible shrinking gel: A shrinkable supramolecular metallo-hydrogel was developed (see figure), which could serve as a matrix to efficiently separate ionic dye mixtures or as an intelligent drug vehicle for stepwise release of two-component ionic drugs.
L. Qin, F. Xie, P. Duan, M. Liu* && – && A Peptide Dendron-Based Shrinkable Metallo-hydrogel for Charged Species Separation and Stepwise Release of Drugs
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& Supramolecular Chemistry
A Peptide Dendron-Based Shrinkable Metallo-hydrogel for Charged Species Separation and Stepwise Release of Drugs Long Qin, Fan Xie, Pengfei Duan, and Minghua Liu*[a]
Abstract: A shrinkable supramolecular metallo-hydrogel based on the l-glutamic acid dendron and magnesium showed reversible volume-phase transition depending on pH changes. The hydrogel further showed selective shrinkage upon addition of positively charged species, while it remained in the gel state when negatively charged species were incorporated. Based on this property, the gel could be used as the matrix to efficiently separate ionic dye mixtures,
in which the cationic dye was incorporated predominantly in the shrunken gel, while the negatively charged dye was released into the aqueous solution. More interestingly, the shrinkable gel can be used as a model drug-delivery vehicle for the stepwise release of a two-component drug system, in which the negatively charged drug is released first and then the second component is released with a pH trigger.
Introduction
cussed.[12] Although these systems have showed the shrinkable properties, its potential application has not been well developed. We have recently found that a series of divalent metal ions can trigger the shrinkage of supramolecular hydrogel formed by an l-glutamic acid peptide dendron, N-octadecanoyl-1,5bis(l-glutamic acid)-l-glutamic diamide (OGAc).[13] With different kinds of divalent metal ions, the supramolecular hydrogel displays various shrinkage properties. It was found that the hydrogel containing magnesium exhibited the most obvious shrinkage performance; meanwhile, the shrinkage process is a continuous, reversible and shape-dependent macroscopic volume phase transition. Based on this work, herein, we further fabricated a shrinkable metallo-hydrogel by combining the dendron gelator OGAc with magnesium ions, which exhibited some new responsive properties. First, the shrinkage process is pH-dependent. The phase behaviour can be reversibly regulated by pH change. The shrunken gel can convert to the stable gel at higher or lower pH values. Second, the metallo-hydrogel showed selective shrinkage based on the additive charged species. It was found that the addition of positively charged species can accelerate the shrinkage of the supramolecular hydrogel, while negatively charged additives inhibited hydrogel shrinkage. Based on this property, the shrinkable metallo-hydrogel could be used as a matrix to efficiently separate ionic dye mixtures. That is, when an ionic mixture of dyes was mixed with the OGAc/Mg2 + metallo-hydrogel, the anionic dye was released into the aqueous solution phase through shrinkage while the cationic dye was maintained in the gel skeleton. Besides the separation of dye mixtures, the shrinkable metallohydrogel was proposed for use as a vehicle for the step-wise release of two-component drugs. In this gel, the anionic drug was first released through gel shrinkage and then by pH trigger, the second cationic drug was released through gel collapse. Although the system is still not very sophisticated, to
Supramolecular hydrogels, as an important class of soft materials formed through the self-assembly of low molecular weight hydrogelators in water via various non-covalent interactions, have attracted great interest in recent years.[1] In comparison with the conventional polymer hydrogels, supramolecular hydrogels are more apt to be regulated for their thermo-reversibility, stimuli-responsiveness,[2] and biocompatibility since the hydrogelators can be derived from biological components including amino acids,[3] sugars[4] and steroids.[5] Many functional supramolecular hydrogels have been thus developed and found applications in tissue engineering,[6] sensing,[7] biomaterials[8] and drug delivery.[9] However, due to the inherent weak character of non-covalent interactions, supramolecular hydrogels still suffer the reality that the brittle 3D structures have difficulty in maintaining physical change such as folding and curling of the supramolecular chains as in polymer hydrogels to accordingly exhibit phase transition in volume (shrinkage– swelling)[10] and shape (deformation–recovery),[11] which limits their many potential uses as biomaterials. For example, while many polymer hydrogels can show volume phase transition, it is still a great challenge and highly significant to develop supramolecular hydrogels with volume phase transition. A thermally and pH-responsive supramolecular hydrogel based on glycosylated amino acetate was first reported by Hamachi et al. and the unique properties of hydrogel shrinkage as well as the mechanism of phase volume transition were dis[a] L. Qin, F. Xie, Dr. P. Duan, Prof. M. Liu Beijing National Laboratory for Molecular Science (BNLMS) CAS Key Laboratory of Colloid, Interface and Chemical Thermodynamics Institute of Chemistry, Chinese Academy of Sciences (P. R. China) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404035. Chem. Eur. J. 2014, 20, 1 – 8
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Full Paper the best of our knowledge, this is the first report of a shrinkable supramolecular hydrogel that can be used as a smart vehicle for stepwise release of multicomponent drugs, and might show great significance in the development of complex drugdelivery systems.
Results and Discussion pH responsiveness and gel shrinkage Since the amphiphilic dendron OGAc contains four carboxylic acids, the phase behaviour of OGAc/Mg2 + metallo-hydrogel was highly dependent on pH. Therefore, potentiometric pH titration was carried out at 25 8C to determine the protonation constant of OGAc, as shown in Figure S1 in the Supporting Information. The pH titration curve shows OGAc has two protonation constants pKa1 and pKa2 with the values of 3.2 0.1 and 5.7 0.1, respectively. The compositions of OGAc at [OGAc]total = 1.0 mm were calculated from the Henderson–Hasselbalch equation and the negative charge distribution from 0 to 4 under different pH values was obtained.[14] Based on the above results, the phase behaviour and micromorphology of OGAc/Mg2 + hydrogel was investigated under selected pH values (as shown in Figure 1 and Figure 2 A–D). It was found that when the gel shrinks, the system pH value was located at 3.2. The randomly distributed and nicely separated nanofibres with the thickness range 16–20 nm were investigated in the shrunken hydrogel. However, when the system pH was changed to 1.8, a stable semi-transparent hydrogel was formed, at which point the carboxylic acid can hardly be ionised. Morphologic measurements showed that entangled nanofibres with diameter from 20 to 30 nm were formed. Moreover, increasing the pH to 4.3 induced the formation of a transparent hydrogel, in which ultrafine nanofibres with an average diameter of about 4 nm were obtained. This is close to the thickness of an OGAc-formed interdigitated bilayer structure. Finally, when the pH reached 6.2 or above, the ionised head group increased the hydrophilicity of the gelator, which destroyed the hydrogelation of OGAc leading to the formation of a sol. TEM investigation showed that here irregular nanosheets were formed. Meanwhile, it was observed that hydrogel shrinkage is located at a pH range from 2.8 to 3.5. The gel cannot shrink at a system pH value below or above the critical points. Moreover, the pH responsiveness in OGAc/Mg2 + hydrogel is a reversible phase transition. The phase behaviour can be reversibly transformed by changing the system pH value several times (Figure S2 in the Supporting Information). In order to further understand such phase transition at different pH values, the X-ray diffraction (XRD) and FT-IR spectra were analysed, as shown in Figure 2 E and F. FT-IR spectra showed that the driving forces for the formed nanofibres are hydrogen bonds between the amide groups, the hydrophobic interaction between alkyl chains and the electrostatic interaction between metal ions and carboxyl groups. The bands that appeared at 3298, 1637 and 1549 cm1 are attributed to stretching N=H vibration, amide I and II, respectively. With an increase in pH, the N=H vibration gradually decreased and the &
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Figure 1. Molecular structure of OGAc and the pH responsiveness of OGAc/ Mg2 + shrinkable metallo-hydrogel. The gel shrunk at pH 3.2 and converted to a semitransparent gel at pH 1.8, transparent gel at pH 4.3 and sol at pH 6.2. The blue and yellow balls refer to the unprotonated and protonated head groups of l-glutamic acid dendron, respectively.
band of amide I shifted to the high wavenumber and finally to 1647 cm1 when the pH reached 6.2. This indicated that the hydrogen bonding between amide groups decreased with pH change. The C=O vibration that mainly appeared at 1730 cm1 gradually moved to 1735 cm1 and disappeared at pH 6.2. Meanwhile, the typical vibration of COO at around 1600 cm1 became more obvious and finally changed to a broad band at 1585 cm1. All these changes indicate that with increasing pH, the hydrogen bonding between head groups slowly decreased and the carboxyl groups were gradually protonated and interacted with the metal ions.[15] The XRD patterns of the xerogels obtained at different pH values showed that the lamellar structure was formed in all the gels. The layer distance of 4.0, 3.9 nm for the shrunken and transparent hydrogel at pH 3.2, 4.3 and d-spacing value of 5.4 nm for the semitransparent-hydrogel obtained at pH 1.8 are more than one molecular length (estimated for 2.7 nm) but less than or equal to twice that of the molecular thickness, which indicated the formation of different bilayer structures. Based on the above results, a possible mechanism for pH responsiveness is proposed. Under the strongly acidic condition (pH 1.8), the ionisation of the carboxyl groups cannot occur, which makes the gelator more hydrophobic and thereby contributes to the formation of the wormlike micelle. The micelles further entangle through hydrogen bonds to form the threedimensional superstructures. A similar assembly process to form the nanofibres with interdigitated bilayer structures can be found in the hydrogel at pH 3.2. However, between the formed molecular-thick nanofibres, magnesium ions can slowly bridge the adjacent nanofibres through electrostatic interactions with the protonated carboxyl groups to expel the bound water, as shown in Figure 6 B, which finally causes the volume transition at the macro-level. But the further protonation of carboxyl groups will enhance the surface electronegativity of the nanofibres and prevent additional entanglement between the nanofibres. Therefore, a transparent hydrogel containing molecular-thick nanofibres are observed at pH 4.3. 2
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Full Paper Figure 4 A. Upon mixing with the dyes, a series of transparent gels were formed and the UV/Vis spectra show that the absorption is very close to their solution absorption. For the asprepared hydrogel, there is no CD signal either in the OGAc/ Mg2 + hydrogel mixed with cationic or anionic dyes. However, accompanying the volume transition, a series of negative Cotton effects were observed at the corresponding absorption peak for cationic-dye-doped shrunken metallo-hydrogels. Morphological investigation showed that the formed nanofibres in these gels were several nanometers thicker than the nanostructure obtained in the shrunken gel without dyes. On the other hand, when anionic dyes were used, no CD peak was observed even after resting for several weeks. TEM analysis showed that there were some irregular nanodots randomly distributed in gel nanofibrillar networks. EDX investigation confirmed that no relevant metal atoms were present in the nanodots (Figure S3 in the Supporting Information), suggesting that the nanodots most probably correspond to the aggregated anionic dyes. These results indicate that when the dyes, either cationic or anionic, were mixed in the gels, the gelator molecules self-assembled into nanofibre structures, while the dyes distributed in the aqueous phase at the beginning. Upon resting, the cationic dye interacted with the gelator fibres and formed a chiral complex on the surface of nanofibre, as illustrated in Figure 4 B, which accompanied with the shrinkage of the gel. For the anionic dyes, due to the electrostatic repulsion between sulfonate and carboxylate, the dyes remained in the aqueous phase and prevented the shrinkage of hydrogel.
Figure 2. A)–D) TEM images, and E), F) FT-IR and XRD spectra of OGAc/Mg2 + hydrogel (mole ratio 5:1) at different pH values (scale bar = 500 nm).
Selective shrinkage of the gel with charged species The metallo-hydrogel can incorporate water-soluble dyes with good capacity. During the shrinkage, the gel showed different responsiveness to the charged dyes. A series of water-soluble ionic dyes with OGAc/Mg2 + hydrogel matrix (mole ratio dye/ OGAc = 1:20) at pH 3.2, among which cationic dyes such as methyl violet (MV), methylene blue (MB), acridine yellow (AY), neutral red (NR) and anionic dyes including methyl orange (MO), acid red 26 (AR), were chosen as the model compounds (molecular structures and basic parameters are shown in Table S1 in the Supporting Information). The phase behaviour of the OGAc/Mg2 + hydrogel with doped amount of ionic dye was observed after the gel was allowed to rest for 20 h. It was found that shrinkage took place in the hydrogel mixed with cationic dyes, so that more than half the volume of water was expelled from the original hydrogel. In addition, the UV/Vis spectra of the expelled water showed that there was nearly no dye in this phase, which means that the cationic dyes were almost entirely incorporated in the shrunken gel matrix. In the case of anionic dyes, the transparent hydrogel was formed and maintained its original phase state even after resting for several weeks. A control experiment with the OGAc/Mg2 + hydrogel with an equal amount of NaCl(aq.) revealed that the gel could shrink under these conditions, which suggests that it was the anionic dyes that prevented the hydrogel shrinkage. In order to understand these changes, CD spectroscopy of the dye-incorporated gel and TEM observation of the corresponding xerogels were performed, as shown in Figure 3 and Chem. Eur. J. 2014, 20, 1 – 8
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Figure 3. TEM images and photographs of ionic-dye-doped OGAc/Mg2 + (0.13 wt %) hydrogel after being allowed to rest for 20 h (cationic dyes: neutral red, methyl violet, acridine yellow, methylene blue, anionic dyes: methyl orange, acid red 26); scale bar = 200 nm.
Separation of dye mixture using the shrunken gel as matrix We further investigated the shrinkage property of the gel incorporated by a series of cationic and anionic dye mixtures. The OGAc/Mg2 + hydrogel mixed with the same molar amounts of two different dyes was firstly fabricated and then allowed to rest for 20 h. It was interesting to find that the ionic dye mix3
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Figure 4. A) UV and CD spectra of ionic-dye-doped shrunken OGAc/Mg2 + (0.13 wt %) hydrogel after being allowed to rest for 20 h. B) Schematic illustration of the interaction between ionic dyes and gel networks. The cationic dyes well incorporated and aligned with the gel fibre structures through strong electrostatic interactions (a). However, the anionic dyes were distributed between the solvent phase and immobilised by the 3D gel frameworks (b).
Figure 5. Photographs of mixed-dyes-doped OGAc/Mg2 + (0.13 wt %) hydrogel (top) and efficient separation of these oppositely charged dyes through hydrogel shrinkage process (middle) and corresponding separation efficiency (bottom).
tures could be easily and spontaneously separated through the hydrogel shrinkage. The cationic dyes remained in the gel phase. However, the anionic dyes were released into the aqueous phase. Experimental results indicated that the shrinkable hydrogel can efficiently separate the mixed quaternary ammonium (+) and sulfonic acid () dyes, as shown in Figure 5. The corresponding separation efficiency was indirectly evaluated by the distribution ratio of dye molecules in the aqueous phase when gel shrinkage reached equilibrium. Through contrasting UV spectra of the extruded water phase after shrinkage with the original mixed dye aqueous solution, the distribution ratio of the mixed dyes were calculated (calculation details are shown in Experimental Section). It is very obvious that the distribution ratio of anionic dyes in expelled water is generally more than 70 % and less than 10 % for all the cationic dyes. Although many hydrogels have been used to entrap dyes,[16] our results show that through the shrinkable supramolecular hydrogel, the dyes can be separated from the charged mixtures.
techniques, well-controlled stepwise release of the multicomponent drugs is becoming necessary and showing potential prospects. The above-separation results inspired us to perform the stepwise release of two-component water-soluble ionic drugs through supramolecular hydrogel shrinkage in vitro tests. We designed the two-component drug-loaded shrinkable hydrogel to firstly release one part of the drugs in the tissue with the acidic pH range and then to continuously release the remaining parts in the tissues with a neutral or alkaline pH value. Herein pralidoxime iodide (PI) and phenol red (PR) were used as the model drugs of hydrophilic small molecules. The phase behaviour of OGAc/Mg2 + hydrogel loaded with 5 mol % of the two hydrophilic drugs was firstly observed (Figure S5 in the Supporting Information). As with the dye-doped experiment, the volume of hydrogel loaded with PI gradually shrunk over time. However, the hydrogel containing PR remained in the original state. CD spectra and TEM analysis indicated that PR was immobilised in the water phase by the hydrogel skeleton structure, while PI adhered to the 3D network of hydrogel. Finally, the selected drug PI and PR were mixed with equimolar ratio and entrapped in OGAc/Mg2 + hydrogel matrix to conduct the controlled-release experiments. The drug loading content (DLC) and drug loading efficiency (DLE) calculation shows that the hydrogel was able to encapsulate the two drugs (PI DLC = 1.97 % and DLE = 99.2 %; PR DLC = 2.61 % and DLE = 98.7 %) with a high loading efficiency.
Stepwise release of two-component ionic drugs Stimuli-responsive hydrogel is frequently used as a matrix for controlled drug release, especially some intelligent polymer hydrogels with the volume phase transition property.[17] With the development of intelligent drug carriers and controlled release &
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Full Paper Conclusion
The release profile was obtained by detecting time-dependent UV/Vis spectra of release media taken at desired time intervals (Figure S6 in the Supporting Information). As shown in Figure 3, in vitro release studies of OGAc hydrogel revealed that the release rate of PI and PR exhibited a similar trend. The slight difference in the cumulative release ratio of PI and PR is most likely due to the electrostatic interactions between the gelator and PI. However, to our delight, the release behaviour in OGAc/Mg2 + shrinkable hydrogel shows obvious selectivity. With the shrinkage of the hydrogel, the release of encapsulated PR is significantly faster than PI. The results show that compared with the OGAc hydrogel, the release of PR is accelerated in the shrunken gel. Nearly 90 % of the PR was released at 22 h. However, the release of PI was evidently decreased and less than 30 % of PI escaped from the gel matrix when the release process reached equilibrium. The system pH value was then adjusted to 6.2, at which point the shrunken hydrogel collapsed to the sol to further release the remaining drug PI at 8 h. Based on the above results, the stepwise release property renders this shrinkable supramolecular hydrogel a new promising carrier for targeted drug delivery (Figure 6).
Unlike most reported supramolecular hydrogels, here we discovered a shrinkable metallo-hydrogel that shows reversible volume-phase transition with pH-stimulus and the phase behaviour from stable, shrunken hydrogel to homogenous solution can be well regulated by pH change. The shrinkable hydrogel also exhibits charged-species responsive property and can selectively incorporate cationic molecules, which inspired us to utilise the shrinkable metallo-hydrogel as an intelligent carrier to efficiently separate ionic dye mixtures and sophisticated controlled stepwise release of loaded two-component drugs. By introducing this novel stimuli-responsive shrinkable metallo-hydrogel, we anticipate further progress in the design of sophisticated multifunctional materials based on supramolecular self-assembly with broad application prospects.
Experimental Section Materials The gelator OGAc was synthesised according to our previous work.[3a] The ionic dyes: methyl violet (MV), methylene blue (MB), acridine yellow (AY), neutral red (NR), methyl orange (MO), acid red 26 (AR) and model drugs pralidoxime iodide (PI) and phenol red (PR) used in all the cases were purchased from Acros Organics or Sigma Aldrich and were used without any additional purification. Milli-Q water (18.2 MW cm) was used in all experiments. All the organic solvents were purified and dried according to standard methods.
Instruments and methods The UV/Vis and CD spectra were detected using JASCO UV-550 and JASCO J-810 spectrophotometers, respectively. Fourier transform infrared (FT-IR) spectra were recorded on a JASCO FT/IR-660 plus spectrophotometer with a wavenumber resolution of 4 cm1 at room temperature. X-ray diffraction (XRD) was achieved on a Rigaku D/Max-2500 X-ray diffractometer (Japan) with CuKa radiation (l = 1.5406 ), which was operated at 45 kV, 100 mA. Transmission electron microscopy (TEM) images were obtained on a JEM1011 electron microscope operating at accelerating voltages of 10 and 200 kV, respectively. All the photographs of the gel were taken by using a Canon EOS60D.
Procedures for preparation and characterisation of gel The typical hydrogel was prepared by adding OGAc (1.34 mg; 0.002 mmol) and Mg(NO3)2 (8 mL; 50 mm) aqueous solution to pure water (1 mL), which was slightly heated to a transparent solution and then placed at room temperature for 20 h. The pH value of the hydrogel was adjusted with HCl and NaOH aqueous solution. As for the reversible phase-transition experiment, 1.0 or 0.1 m HCl and NaOH aqueous solution were used to regulate the system pH between 1.8, 4.3, 6.2 and 3.2. For the TEM measurements, a small amount of diluted hydrogel was placed onto carbon coated copper grid (unstained) and the sample was cryodesiccated in a freeze dryer at 40 8C for 24 h. In the case of preparing samples for XRD measurements, hydrogels were cast onto glass plates and dried in vacuum. Platelets made from the mixture of vacuum-dried xerogels with KBr powder and the sample nipped by CaF2 platelets were used for FT-IR spectral measurements.
Figure 6. A) Two-component drug release in OGAc semi-hydrogel and OGAc/Mg2 + shrinkable hydrogel. At the initial process of drug release in the OGAc/Mg2 + hydrogel, the system pH was firstly kept at 3.2. After release for 22 h, the system pH was adjusted to 6.2 to continuously release for 8 h. B) Schematic illustration for the stepwise release of two-component drugs. At the beginning of the shrinkage, the charged species were immobilised in the 3D gel networks. In the remaining time, the magnesium ions can slowly bridge the adjacent carboxyl groups in the proposed manner and expel the bound water to show the volume transition at the macro-level, during which the positively charged drugs are incorporated with nanofibres; the negatively charged drug was released first by hydrogel shrinkage. After the system pH was changed to 6.2, the 3D network of the hydrogel collapsed and the positively charged drug was gradually released. Chem. Eur. J. 2014, 20, 1 – 8
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Stepwise release for two-component ionic drugs
OGAc was dissolved in water at a concentration of 1.0 mm and the solution pH was adjusted to 10 with a small volume of 0.1 m NaOH. Then, calibrated 0.1 m HCl was gradually added into this solution in small portions. The pH values were recorded with a pHS2C acidity meter and the temperature was kept at 25.0(0.1) 8C throughout the measurements. The electrode was calibrated with a standard buffer solution before being used. Three titrations were performed and the mean pKa values were calculated.
The typical procedure for stepwise release of two-component drugs is as follows. OGAc (2.68 mg; 0.004 mmol), 400 mL cationic and anionic drug (0.5 mm) aqueous solution with or without (control experiment) Mg(NO3)2 (16 mL; 50 mm) aqueous solution were mixed in a sealed tube with pure water (1.2 mL). The mixtures were slightly heated to obtain a transparent solution and were then allowed to cool to room temperature undisturbed, to obtain the hydrogel. The drug-loaded hydrogel was then transferred to a dialysis bag with a molecular weight cut-off (MWCO) 3500 and then the dialysis bag was carefully dropped into 5 mL aqueous solution (pH 3.2). Sampling was carried out over a specified time interval and sample volume (200 mL). The total volume of the system was kept at 5 mL by returning the solution after analysis. After the release reached equilibrium at 22 h, the system pH was adjusted to 6.2 to continuously monitor the release process. The released amount of both drugs was calculated based on UV/Vis spectra of the samples at 294 nm for PI and 432 nm for PR. For determination of drug-loading content and loading efficiency, the drug-loaded hydrogels were dissolved in methanol. Then the concentration of the two drugs was obtained based on the UV/Vis spectra. Drugloading content (DLC) and drug-loading efficiency (DLE) were calculated according to the following formulas: DLC = (weight of loaded drug/weight of gelator) 100 %, DLE = (weight of loaded drug/weight of drug in feed) 100 %.
Selective separation for mixed ionic dye mixture The typical procedure for selective separation of mixed ionic dyes was as follows. OGAc (2.68 mg; 0.004 mmol), Mg(NO3)2 (16 mL; 50 mm) aqueous solution and 400 mL cationic and anionic dye (0.5 mm) aqueous solution were mixed in a sealed tube with pure water (1.2 mL). The mixtures were slightly heated to obtain a transparent solution and then allowed to cool to room temperature unaffectedly to obtain the hydrogel. After the gel was allowed to rest for 20 h to shrink, the gelation state was recorded and the separation ratio was obtained by detecting the UV spectra of expelled water phase. Due to the interference between UV/Vis spectra of the two-component ionic dye mixtures, the separation ratio was calculated based on simultaneous equation method which includes the following steps. 1) A series of cationic (X) and anionic (Y) dye standard solutions with linear concentration gradient were prepared (including total 5 2 samples with the five concentrations: 1 104, 8 105, 6 105, 4 105 and 2 105 mol L1). The absorbance of the above five samples at the maximum absorption wavelength of the two dyes (l1 and l2) was measured to obtain the four absorbance–concentration curve:
al1 ¼ e1X bC X
ð1Þ
al2 ¼ e2X bC X
ð2Þ
al1 ¼ e1Y bC Y
ð3Þ
al2 ¼ e2Y bC Y
ð4Þ
Acknowledgements This work was supported by the Basic Research Development Program (2010CB833305, 2013CB834504), the National Natural Science Foundation of China (Nos. 91027042, 21021003, 21227802, 21321063), the Strategic Priority Research Program of the Chinese Academy of Sciences, Grant No. XDB12020200. Keywords: dendrons · drug delivery · selective separation · supramolecular chemistry · stimuli-responsiveness [1] a) L. A. Estroff, A. D. Hamilton, Chem. Rev. 2004, 104, 1201 – 1218; b) M. de Loos, B. L. Feringa, J. H. van Esch, Eur. J. Org. Chem. 2005, 3615 – 3631; c) A. R. Hirst, B. Escuder, J. F. Miravet, D. K. Smith, Angew. Chem. 2008, 120, 8122 – 8139; Angew. Chem. Int. Ed. 2008, 47, 8002 – 8018; d) R. G. Weiss, P. Terech, in Molecular Gels: Materials with Self-Assembled Fibrillar Networks, Springer, Dordrecht, 2006, pp. 613 – 648. [2] a) M. D. Segarra-Maset, V. J. Nebot, J. F. Miravet, B. Escuder, Chem. Soc. Rev. 2013, 42, 7086 – 7098; b) S. Roy, N. Javid, P. W. J. M. Frederix, D. A. Lamprou, A. J. Urquhart, N. T. Hunt, P. J. Halling, R. V. Ulijn, Chem. Eur. J. 2012, 18, 11723 – 11731; c) M. Ikeda, T. Tanida, T. Yoshii, I. Hamachi, Adv. Mater. 2011, 23, 2819 – 2822; d) C. J. Bowerman, B. L. Nilsson, J. Am. Chem. Soc. 2010, 132, 9526 – 9527; e) G. O. Lloyd, J. W. Steed, Nat. Chem. 2009, 1, 437 – 442; f) W. Deng, H. Yamaguchi, Y. Takashima, A. Harada, Angew. Chem. 2007, 119, 5236 – 5239; Angew. Chem. Int. Ed. 2007, 46, 5144 – 5147; g) H. J. Kim, J. H. Lee, M. Lee, Angew. Chem. 2005, 117, 5960 – 5964; Angew. Chem. Int. Ed. 2005, 44, 5810 – 5814; h) R. V. Ulijn, J. Mater. Chem. 2006, 16, 2217 – 2225. [3] a) P. Duan, L. Qin, X. Zhu, M. Liu, Chem. Eur. J. 2011, 17, 6389 – 6395; b) Y. Liu, T. Wang, Z. Li, M. Liu, Chem. Commun. 2013, 49, 4767 – 4769; c) Y. Liu, T. Wang, M. Liu, Chem. Eur. J. 2012, 18, 14650 – 14659; d) A. M. Smith, R. J. Williams, C. Tang, P. Coppo, R. F. Collins, M. L. Turner, A. Saiani, R. V. Ulijin, Adv. Mater. 2008, 20, 37 – 41; e) S. Fleming, S. Debnath, P. W. J. M. Frederix, T. Tuttle, R. V. Ulijin, Chem. Commun. 2013, 49, 10587 – 10589; f) J. B. Matson, S. I. Stupp, Chem. Commun. 2012, 48, 26 – 33.
where e1X, e2X, e1Y and e2Y are the molar absorption coefficients of X and Y at l1 and l2 ; b refers to the length of optical path. Equations (1) + (3) and (2) + (4) were used to obtain the absorbance– concentration curve of the two-component dye mixtures at l1, l2, respectively:
Al1 ¼ e1X bC X þ e1Y bC Y
ð5Þ
Al2 ¼ e2X bC X þ e2Y bC Y
ð6Þ
2) After the hydrogel was allowed to rest for 20 h to shrink, the absorbance of the expelled water phase at l1 and l2 was detected and the concentration of the two dyes was calculated based on above equation. 3) The separation ratio was finally obtained based on the following equation:
ð7Þ
R ¼ C X V m =C 0 V 0
where Cx and C0 are the concentration of the dye X in the expelled-water and initial sample; and Vm and V0 are the volume of expelled-water and initial sample.
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Received: June 19, 2014 Published online on && &&, 0000
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FULL PAPER & Supramolecular Chemistry
The incredible shrinking gel: A shrinkable supramolecular metallo-hydrogel was developed (see figure), which could serve as a matrix to efficiently separate ionic dye mixtures or as an intelligent drug vehicle for stepwise release of two-component ionic drugs.
L. Qin, F. Xie, P. Duan, M. Liu* && – && A Peptide Dendron-Based Shrinkable Metallo-hydrogel for Charged Species Separation and Stepwise Release of Drugs
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ÝÝ These are not the final page numbers!
