Frank
van de Manakker
a,
Loes M. J.
Kroon-Batenburg
b,
Tina
Vermonden
a,
Cornelus F.
van Nostrum
a and
Wim E.
Hennink
*a
aDepartment of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Sorbonnelaan 16, P.O. Box 80082, 3508 TB, Utrecht, The Netherlands. E-mail: w.e.hennink@uu.nl; Fax: +31 30 251 7839; Tel: +31 30 253 6964
bDepartment of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
First published on 3rd November 2009
Supramolecular hydrogels are highly interesting for drug delivery and tissue engineering applications, especially those systems that display a combination of tunable properties, high mechanical strength and easy preparation from well-available and biocompatible building blocks. In the present paper, we show that the combination of free β-cyclodextrin (βCD) and 8-arm or linear cholesterol-derivatized poly(ethylene glycol) (PEG–chol) in aqueous solution resulted in the formation of almost fully elastic gels with storage moduli in the range of 10–500 kPa. X-Ray diffraction measurements demonstrated the presence of crystalline βCD domains in the hydrogel networks. Rheological experiments further proved that hydrogel formation is based on inclusion complex formation between these βCD clusters and cholesterol coupled to the terminal end of PEG. The observation that the gels were weakened by addition of the competitive βCD–guest molecule adamantanecarboxylic acid (ACA) supported the proposed gelation mechanism. The gel mechanical properties were dependent on temperature, concentration of cholesterol-derivatized PEG and/or βCD, PEG's molecular weight and its architecture. This hydrogel system can be considered as an excellent candidate for future applications in the biomedical and pharmaceutical fields.
A physical driving force that has recently been exploited for the design of reversible, self-assembled hydrogels, is the inclusion complex formation between β-cyclodextrin (βCD) and lipophilic guest molecules.34–41 βCD molecules are cyclic ‘doughnut’-shaped oligosaccharides composed of 7 dextrose units that are coupled via α-1,4-glucosidic linkages.42 All hydroxyl groups are located at the outer surface of the molecule, which renders the inner cavity of βCD relatively hydrophobic. Driven by hydrophobic and van der Waals interactions,43,44 this cavity can act as a binding site for a wide range of lipophilic compounds (e.g. adamantane, cholesterol and aromatic compounds34–41). Kretschmann et al. designed a thermoreversible hydrogel system based on adamantane-grafted N-isopropylacrylamide copolymers and a βCD dimer.34 Other researchers prepared hydrogels by combining adamantane- and βCD-derivatized chitosan35 or hyaluronic acid.36,37 Molecules other than adamantane were utilized as guest compounds as well. For example, Hashidzume et al. described stimuli-responsive gels formed by poly(acrylamide) derivatized with either βCD or aromatic guest molecules.38 Recently, our group reported on self-assembling hydrogels obtained by combining cholesterol- and βCD-derivatized 8-arm star-shaped poly(ethylene glycol) (PEG8) in aqueous solution.39–41 This gel system is highly versatile as its mechanical properties could easily be controlled by the polymer concentration, βCD–cholesterol stoichiometry, the PEG molecular weight and other parameters.39,40 Degradation of these hydrogels is mainly mediated by a surface erosion process, which led to quantitative and nearly zero-order release of entrapped model proteins.41
Although the hosting properties of βCD have been investigated for more than four decades,42,45 only recent studies have demonstrated that native, unmodified βCD molecules self-aggregate.46–50 Light scattering experiments on aqueous solutions of βCD, in the concentration range of 4–12 mM, showed the formation of βCD nano-aggregates with average diameters of about 200 nm.46,47 Bonini et al. visualized these nanostructures with transmission electron microscopy at cryogenic temperature (Cryo-TEM).48 It was found that polydisperse nearly spherical structures with diameters in the range of 100–200 nm were predominant at low concentrations, while at higher concentrations (≥6 mM) micrometer planar aggregates became the main structures.
The ability of cyclodextrins to crystallize has previously been used to design so-called polypseudorotaxane-based hydrogel systems.51–55 Polypseudorotaxanes are inclusion complexes in which cyclic molecules, e.g. αCD, a cyclodextrin subtype composed of 6 dextrose units, are threaded onto a polymer chain.56 The dense packing of multiple αCD units onto the PEG chains leads to crystalline domains that act as physical cross-links in hydrogels.51–55 Because of its larger inner cavity, βCD is not able to stably complex with PEG, but instead the bulkier polymer poly(propylene glycol) (PPG) was used as guest polymer to develop hydrogels.57 Besides these polypseudorotaxane-based systems, no other hydrogels have been designed yet in which crystalline cyclodextrin domains are exploited.
In the present paper, we describe a novel self-assembled hydrogel system, which consists of cholesterol-derivatized 8-arm PEG and free βCD molecules in an aqueous solution. In these hydrogels, cholesterol–βCD inclusion complexes as well as βCD self-association are the driving forces in network formation. The presence of crystalline βCD nanoclusters was studied by X-ray diffraction measurements and the tailorability of the gel mechanical properties was investigated by rheological analyses.
β-Cyclodextrin (βCD), 6-monodeoxy-6-monoamino-β-cyclodextrin (βCD–NH2) and 1-adamantanecarboxylic acid (ACA) were provided by Sigma-Aldrich (Zwijndrecht, The Netherlands). Ammonium acetate (NH4OAc) was purchased from Merck (Darmstadt, Germany) and 37% HCl solution in water was ordered from Acros Chimica (Geel, Belgium).
Rheological characterization of the hydrogels was done with an AR-G2 rheometer (TA instruments, Etten-Leur, The Netherlands) equipped with a 1° steel cone geometry of 20 mm diameter and solvent trap. Using a spatula or pipette (for liquids), approximately 55 µL sample were placed between the pre-heated (40 °C) plates of the rheometer. Rheological gel characteristics were monitored by oscillatory time sweep experiments at 4, 20 and 37 °C and temperature sweep experiments. During time sweep experiments the G′ (shear storage modulus) and G″ (loss modulus) were measured for a period of 5 min. Temperature sweep experiments from 4 to 80 °C were done at a heating rate of 2 °C min−1 (30 s equilibration per point). The point at which G″/G′ (= tan δ) = 1 is considered as the gel transition temperature (Tgel).59 All experiments were performed at a frequency of 1 Hz and 1% strain.
Fig. 1 shows a photograph of hydrated mixtures containing 10% (w/w) cholesterol-derivatized and non-derivatized 8-arm PEG with or without 2 eq. free unmodified βCD relative to the number of cholesterol or hydroxyl groups of the corresponding PEG component. A solution of non-derivatized 8-arm PEG (PEG820K–OH) behaved as a viscous liquid (D). 10% (w/w) PEG820K–OH in the presence of 2 eq. βCD led to a viscous suspension (C) likely due to βCD's low maximum solubility of ∼16 mM (1.8% (w/w)).48 However, when using cholesterol-derivatized PEG (PEG820K–chol5.6) with or without βCD, hydrogels were formed. Gels composed of PEG820K–chol5.6 were almost transparent (A). Their formation is likely caused by the amphiphilicity of the polymer, where hydrophobic interactions occur between the PEG-bound cholesterol groups. Sample B in Fig. 1 shows that a white opalescent gel was formed by mixing PEG820K–chol5.6 and 2 eq. βCD. This gel formation was somewhat surprising, since it was found in previous studies that disruption of self-assembling systems based on hydrophobic interactions occurred by adding small amounts of βCD (<16 mM).37,60,61 For example, Akiyoshi et al. added βCD molecules to hydrogel nanoparticles based on cholesterol-grafted pullulan60 or poly(L-lysine),61 causing the hydrophobic cholesterol groups to be captured by βCD complexation and consequently disrupting the nanogels. It should be mentioned that we used higher amounts of βCD (5.5% (w/w)), i.e. well above the solubility limit of βCD. This interesting phenomenon prompted us to investigate the gelation mechanism in more detail.
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Fig. 1 Photographs of a 10% (w/w) PEG820K–chol5.6 hydrogel (A), a hydrogel composed of 10% (w/w) PEG820K–chol5.6 + 2 eq. βCD (B), an aqueous mixture of 10% (w/w) PEG820K–OH + 2 eq. βCD (C), and of a 10% (w/w) PEG820K–OH solution (D). |
The cluster formation of βCD was investigated by X-ray diffraction analysis. Fig. 2 shows that for the PEG820K–chol5.6 powder (A) two major diffraction peaks were observed at 2θ (λ = 0.7107 Å) = 8.8° and 10.8° together with less intense peaks at 2θ = 12.3, 16.4, 18.7 and 20.6°. By using Bragg's law of diffraction62 (2d sin θ = λ, where d, θ and λ represent the crystal lattice spacing, the diffraction angle and the wavelength, respectively), this pattern can be attributed to the monoclinic unit cell of crystalline PEG.63–65 The βCD powder (Fig. 2, trace B) also showed a high degree of crystallinity with many diffraction peaks, which were found to correspond to the reported X-ray structure of the βCD hydrate.66 To estimate the size of the crystallite spheres in the βCD powder, the peak at 2θ = 5.7° was selected and the Scherrer equation67 was applied:
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Fig. 2 X-Ray diffraction patterns of PEG820K–chol5.6 (A) and βCD (B) powders and 15% (w/w) gels composed of PEG820K–chol5.6 with 1 (C) or 2 (D) eq. βCD or without βCD (E). Patterns for (C), (D) and (E) were corrected with a background profile collected from deuterated water. |
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Fig. 3 Rheological properties at 4 and 37 °C of mixtures containing 10% (w/w) PEG820K–chol5.6 and 10% (w/w) PEG820K–chol5.6 + 2 eq. βCD (measurement frequency = 1 Hz). Left axis: G′ and G″; right axis: tan δ. Data are shown as mean ± standard deviation, n = 3. |
To further investigate the thermosensitivity of the viscoelastic gels, their G′ and G″ were measured while gradually increasing the temperature from 4 to 80 °C. With increasing temperature, G′ (and to a lower extent G″) gradually decreased, and at a certain temperature (Tgel) G′ equaled G″, which indicates a gel-to-sol transition. Above Tgel, G″ exceeded G′ and the mixtures behaved as viscous liquids. Besides higher gel strengths, the presence of βCD also gave the 10% (w/w) PEG820K–chol5.6–βCD gel a higher Tgel of 53 ± 3 °C as compared to 42 ± 4 °C for the 10% (w/w) PEG820K–chol5.6 gel (see ESI†, Table S3). We previously described a hydrogel system with a similar thermosensitive behavior.39,40 In that system, composed of cholesterol- and βCD-derivatized 8-arm PEG (i.e. PEG820K–chol6.1 and PEG820K–βCD7.4; see Experimental part for polymer nomenclature), gel formation is driven by inclusion complex formation of cholesterol and βCD moieties coupled to the terminal ends of 8-arm PEG. An elevation of the temperature in this system resulted in a reduced number of βCD–cholesterol complexes, which ultimately led to a gel-to-sol transition. At a total PEG content of 10% (w/w), the Tgel of this particular gel system was 20 ± 1 °C, so that at 37 °C it behaved as a viscous liquid with low G′ and G″ between 2 and 35 Pa.39 Compared to this previously reported PEG820K–chol6.1–PEG820K–βCD7.4 system, gels composed of PEG820K–chol5.6 and free βCD showed higher Tgel and improved mechanical strength at elevated temperatures (G′ ≈ 500 kPa), which is beneficial for future applications.
The observation that an aqueous mixture of βCD molecules and cholesterol-derivatized 8-arm PEG forms gels that are stronger than the gels containing only PEG820K–chol suggests that the βCD molecules dramatically affect the hydrogel network structure. As discussed in the Introduction section, several groups have previously reported on the formation of βCD aggregates in water.46–50 The increased G′ and lower tan δ of the PEG820K–chol5.6/βCD gels as compared to PEG820K–chol5.6 gels indicate that βCD increases the cross-link density of PEG8–chol based hydrogels. This strongly suggests that the βCD aggregates form host–guest inclusion complexes with PEG-bound cholesterol moieties. In this way, the βCD nano-aggregates serve as cross-linkers in the polymer network, as depicted in Fig. 4.
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Fig. 4 Schematic representation of 8-arm PEG–chol/βCD gels. Cholesterol groups at the termini of the 8-arm PEG's form inclusion complexes with crystalline nanoclusters of βCD's (A). βCD clusters with arbitrary size and crystal packing are shown. Dependent on the relative number of cholesterol and βCD moieties, hydrophobic cholesterol–cholesterol interactions might also occur (B). |
Fig. 5 shows G′ and G″ at 37 °C of aqueous mixtures containing 20% (w/w) PEG820K–chol5.6, linear bifunctional PEG6K–chol1.8 or monofunctional mPEG5K–chol0.9, combined with 2 eq. βCD (relative to PEG-bound cholesterol). This figure shows that mixtures based on monofunctional linear PEG–cholesterol and βCD did not result in the formation of viscoelastic networks (tan δ ≫ 1). This is not unexpected, because with only 1 interacting cholesterol group per PEG chain it is not possible to form a network structure. Fig. 5 also shows that the mixtures based on 8-arm PEG–cholesterol + 2 eq. βCD or linear bifunctional PEG–cholesterol + 2 eq. βCD both formed almost fully elastic and strong hydrogels with G′ values of 360 ± 13 kPa and 180 ± 20 kPa and tan δ values of 0.15 ± 0.01 and 0.21 ± 0.01, respectively. The approximately two-fold difference in G′ between these gels can be explained by the lower cross-link density in gels containing the linear PEG–cholesterol. To explain, it should be noted that the concentration of cholesterol (0.05 mmol g−1 gel) and βCD (0.1 mmol g−1 gel) was equal in both hydrogels and therefore, the difference in G′ and thus cross-link density between the 8-arm PEG820K–chol5.6 and linear PEG6K–chol1.8 containing gels are due to the architecture of the cholesterol-derivatized PEG's. The 8 arms of PEG820K–chol are covalently connected to a core unit, and consequently networks based on this polymer have a higher cross-link density, which leads to networks with higher G′ values. Fig. 5 also displays the G′ and G″ of mixtures in which the PEG–chol component was replaced by non-derivatized linear or 8-arm PEG (PEG8–OH) and of an 11% (w/w) suspension of βCD. As represented by their very low G′ and G″, these mixtures behaved as Newtonian liquids. This demonstrates that the use of cholesterol-derivatized PEG with ≥2 functionalities per PEG molecule is crucial for network formation. A combination of inclusion complexes between cholesterol and βCD insoluble nano-aggregates (see also the above X-ray diffraction analysis) and hydrophobic cholesterol–cholesterol interactions is likely responsible for the network formation.
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Fig. 5 Storage modulus (G′) and loss modulus (G″) measured at 37 °C (measurement frequency = 1 Hz) of mixtures of PEG820K–chol5.6 + 2 eq. βCD, PEG6K–chol1.8 + 2 eq. βCD, mPEG5K–chol0.9 + 2 eq. βCD, non-functionalized PEG8–OH or PEG6K–OH + 2 eq. βCD and an aqueous 11% (w/w) βCD suspension. For all mixtures, the concentration of derivatized (or non-derivatized) PEG was 20% (w/w). Data are shown as mean ± standard deviation, n = 3. |
Fig. 6 shows the gel mechanical properties of an aqueous mixture of PEG820K–chol5.6 with 1 eq. of a 6-monodeoxy-6-monoamino-functionalized βCD derivative (βCD–NH2), in comparison with gels containing no or unmodified βCD. The figure demonstrates again the strengthening of a PEG820K–chol5.6 containing gel in the presence of βCD. This figure also shows that the presence of βCD–NH2 led to a collapse of the gel structure (tan δ > 1). The monoamino-derivatized βCD is able to form host–guest inclusion complexes,68 however, its aqueous solubility is 40 times higher than that of βCD.69 Furthermore, it likely has a lower tendency to aggregate, because the aminomethyl group is positively charged in the aqueous solutions (pKa = 8.72),69 which results in repulsion between the βCD–NH2 molecules. The dissolution of the gel can therefore be explained by complexation of the PEG-bound cholesterol groups in the cavities of soluble βCD–NH2, which does not result in network formation.
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Fig. 6 Rheological characteristics (4 °C) of mixtures containing 10% (w/w) PEG820K–chol5.6, 10% (w/w) PEG820K–chol5.6 + 1 eq. βCD or 10% (w/w) PEG820K–chol5.6 + 1 eq. βCD–NH2 (measurement frequency = 1 Hz). Left axis: G′ and G″; right axis: tan δ. Data are shown as mean ± standard deviation, n = 3. |
To get further insight into the gelation mechanism, an excess (3 eq. relative to the present cholesterol groups) of a competitive inclusion complex forming compound, 1-adamantanecarboxylic acid (ACA), was added to the 20% PEG820K–chol5.6/2 eq. βCD gel system. The binding affinity of ACA towards βCD is 2× higher than that of cholesterol (Ka (in water): 3.2 × 104 M−1vs. 1.6 × 104 M−1).60,70,71Fig. 7 shows that addition of the competitor substantially weakened the PEG820K–chol5.6/βCD gel, as reflected by a 2-fold decrease in G′ and a higher G″ (resulting in a 3 times higher tan δ). The addition of ACA did not completely break the gel structure, but led to gel properties that approached those of a hydrogel composed of PEG820K–chol5.6. Likely, when the cholesterol moieties are displaced from the βCD cavities by ACA, the hydrophobic interactions between these groups return. The gel strength of the PEG820K–chol5.6/βCD/ACA mixture was slightly higher than that of the PEG820K–chol5.6 gel (Student's t-test, P < 0.05), which is probably due to a remaining fraction of βCD-complexed cholesterol.
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Fig. 7 Storage modulus (G′) and loss modulus (G″) at 4 °C of hydrogels containing 20% (w/w) PEG820K–chol5.6, 20% (w/w) PEG820K–chol5.6 + 2 eq. βCD or 20% (w/w) PEG820K–chol5.6 + 2 eq. βCD + 3 eq. ACA (measurement frequency = 1 Hz). Left axis: G′; right axis: G″. Data are shown as mean ± standard deviation, n = 3. |
In summary, the results in this section give evidence that the network formation of the hydrogel systems based on cholesterol-derivatized PEG combined with βCD is due to small crystalline βCD clusters (proven by X-ray diffraction measurements) combined with cholesterol–βCD inclusion complexes.
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Fig. 8 Storage modulus (G′) at 20 °C of hydrogels containing 22.5% (w/w) PEG820K–chol5.6 with increasing amounts of βCD (measurement frequency = 1 Hz). For all gels, tan δ < 0.1. Data are shown as mean ± standard deviation, n = 3. |
Fig. 9 shows the rheological properties at 4 and 37 °C of hydrogels composed of 8-arm or linear PEG–chol and 1 eq. βCD at 4 and 37 °C with increasing solid content of the gels. At solid contents <12.8% (w/w) (10% (w/w) PEG–chol; 2.8% (w/w) βCD), all mixtures showed liquid-like behavior with G′ < 0.3 kPa and tan δ > 1 (not shown in figure). Here, the concentration of interacting cholesterol and βCD groups is likely too low to obtain a strong physical network. However, at a solid content of 12.8% (w/w) or higher, G′ started to dominate G″. Furthermore, with increasing PEG–chol/βCD content, G′ (and also G″ to a lower extent) gradually increased. In agreement with these observations, also the observed Tgel's increased with higher solid contents, which reached values between 38 and more than 100 °C (Tgel's > 80 °C by extrapolation; see ESI†, Table S3). These observations are in line with expectations, as higher concentrations of PEG–cholesterol and βCD result in more interacting cholesterol moieties and βCD clusters and subsequently a higher cross-link density. At solid contents ≥28.7% (w/w) (22.5% (w/w) PEG–chol; 6.2% (w/w) βCD), both G′ and G″ leveled off. This can be ascribed to the solubility of both linear and 8-arm PEG–cholesterol, which approached its limit at concentrations ≥22.5% (w/w). Because at higher concentrations PEG–cholesterol is not fully dissolved, only the dissolved molecules contribute to network formation.
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Fig. 9 Storage modulus (G′) and loss modulus (G″) at 4 and 37 °C of PEG820K–chol5.6 + 1 eq. βCD (A) or PEG6K–chol1.8 + 1 eq. βCD (B) hydrogels as a function of the percentage solid content (measurement frequency = 1 Hz). Data are shown as mean ± standard deviation, n = 3. |
Fig. 10 shows the G′ of PEG8–chol/βCD gels as a function of the 8-arm PEG's molecular weight. It shows that the highest gel strength was reached for mixtures based on 20 kDa star shaped PEG. At equal concentrations, the use of the higher molecular weight PEG8–chol (PEG840K–chol5.6) implies that the concentration of cholesterol moieties decreases, which in turn will lead to a lower number of cross-links. Based on the higher concentration of cholesterol groups present in the PEG810K–chol5.6–βCD gels, stronger networks were expected. However, the use of the lower molecular weight PEG810K–chol5.6 led to a 7-fold decrease of G′. As suggested previously,39 this can be ascribed to the poor solubility of PEG810K–chol5.6, and as a consequence not all cholesterol units are available to form inclusion complexes with βCD, which leads to weaker networks.
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Fig. 10 Storage modulus (G′) at 37 °C of hydrogels containing 8.3% (w/w) βCD and 15% (w/w) PEG8–chol5.6±0.1 with different molecular weights (measurement frequency = 1 Hz). Data are shown as mean ± standard deviation, n = 3. |
Footnote |
† Electronic supplementary information (ESI) available: Studied hydrogel compositions with corresponding gel transition temperatures. See DOI: 10.1039/b916378f |
This journal is © The Royal Society of Chemistry 2010 |