Calcium-Ion-Triggered Co-assembly of Peptide and Polysaccharide into a Hybrid Hydrogel for Drug Delivery
© Xie et al. 2016
Received: 16 January 2016
Accepted: 4 April 2016
Published: 12 April 2016
We report a new approach to constructing a peptide–polysaccharide hybrid hydrogel via the calcium-ion-triggered co-assembly of fluorenylmethyloxycarbonyl-diphenylalanine (Fmoc-FF) peptide and alginate. Calcium ions triggered the self-assembly of Fmoc-FF peptide into nanofibers with diameter of about 30 nm. Meanwhile, alginate was rapidly crosslinked by the calcium ions, leading to the formation of stable hybrid hydrogel beads. Compared to alginate or Fmoc-FF hydrogel alone, the hybrid Fmoc-FF/alginate hydrogel had much better stability in both water and a phosphate-buffered solution (PBS), probably because of the synergistic effect of noncovalent and ionic interactions. Furthermore, docetaxel was chosen as a drug model, and it was encapsulated by hydrogel beads to study the in vitro release behavior. The sustained and controlled docetaxel release was obtained by varying the concentration ratio between Fmoc-FF peptide and alginate.
Hydrogels are a class of soft materials with attractive applications in the field of biomedicine because of their high water content, biocompatibility, and tissue-like elastic properties [1–4]. Particularly, peptide-based hydrogels fabricated by supramolecular self-assembly have received much attention because they are highly biocompatible, biodegradable in vivo, injectable, and amenable to molecular design [5–7]. To date, a wide variety of peptide-based hydrogels have been constructed from fluorenylmethyloxycarbonyl (Fmoc) peptides [8–11], peptide amphiphiles [12, 13], NAP peptides [14, 15], Fc peptides [16, 17], EAK16/RAD , multidomain peptides , and peptide polymers [20, 21]. These materials exhibit attractive prospects for application in the fields of drug delivery , tissue regeneration , three-dimensional (3D) cell cultures , and biosensors , which require certain properties such as good biocompatibility, suitable mechanical strength, high stability, environmental stimuli responsiveness, cell compatibility, and porous structures . However, it is still difficult to meet all the above-mentioned demands for most of the peptide hydrogels. Therefore, the construction of a new peptide-based hydrogel with a set of desired properties is a significant topic of research.
One potential solution is to introduce other functional components into the self-assembled peptide systems to form complex hydrogels. Nanoparticles , graphene oxide , synthetic polymers , proteins [7, 28], and polysaccharides [22, 29] have been widely used as the functional components. For example, Stupp et al. reported the heparin-induced self-assembly of a peptide amphiphile into a polysaccharide–peptide hybrid hydrogel, which exhibited good performance in promoting angiogenesis . Our group also created a self-assembling hybrid hydrogel composed of fluorenylmethyloxycarbonyl-diphenylalanine (Fmoc-FF) peptide and konjac glucomannan (KGM), Fmoc-FF–KGM, which showed much higher stability than the Fmoc-FF hydrogel, and the controlled docetaxel release from this hybrid hydrogel was also achieved .
The polysaccharide–protein complex widely exists in biology and contributes to many unique functions of biological organisms, such as chondroitin sulfate proteoglycan. This has greatly inspired us to develop a new peptide–polysaccharide hybrid hydrogel with a specific set of the desired properties. Herein, alginate, a kind of natural polysaccharide, is proposed as a hybrid component in view of its good hydrophilicity, high water-absorption ability, and ease of gelation through ionic crosslinking [30, 31]. However, the ionic crosslinks of this alginate hydrogel are easily broken because of ion exchange, which greatly limits its biological application. Thus, we also expect the self-assembled peptide–alginate hybrid hydrogel to have enhanced stability due to the coexistence of noncovalent (e.g., π–π stacking, hydrogen bonding) and ionic interactions when compared with peptide or alginate hydrogel alone.
In this study, we created a new peptide–polysaccharide hybrid hydrogel via calcium-ion-triggered co-assembly of Fmoc-FF peptide and alginate. The physicochemical properties including morphology, stability, and supramolecular structures were investigated. For comparison, hydrochloric acid (HCl) was introduced into a CaCl2 solution to trigger co-assembly of Fmoc-FF peptide and alginate, and the morphological structures of the resulting hybrid hydrogel beads were also characterized. Furthermore, the release kinetics of Fmoc-FF–alginate hydrogel beads was evaluated, using docetaxel as a drug model, for its potential application in drug delivery. We also investigated the effect of concentration ratio of Fmoc-FF and alginate on the release of docetaxel.
The lyophilized form of Fmoc-FF peptide was purchased from Bachem (Bubendorf, Switzerland). Alginate (M w: 32–250 kDa) was obtained from Aladdin Industrial Corp. (Shanghai, China). Docetaxel (purity >98 %) was purchased from Cenway Pharmaceutical Co. (Tianjin, China). All other chemicals, including calcium chloride, sodium hydroxide, Tween 80 (polysorbate 80), and acetonitrile, were of analytical grade and were obtained from commercial sources.
Calcium-Ion-Triggered Self-Assembly of Fmoc-FF
In a typical experiment, 3 mg of Fmoc-FF was added to 1 mL of deionized water and homogeneously dispersed by ultrasound for 15 min, after which, it was solubilized by dropwise addition of 0.5 M NaOH. Next, 2 mL of a CaCl2 (2 wt.%) solution was slowly added to 1 mL of the fresh Fmoc-FF stock solution, and the mixture was aged at room temperature for 2 h without disturbance, leading to formation of transparent hydrogels.
Calcium-Ion-Triggered Co-Assembly of Fmoc-FF and Alginate
First, fresh Fmoc-FF stock solution (3 mg/mL) was prepared by the above method. Next, 3 mg of alginate powder was slowly added to 1 mL of the fresh Fmoc-FF stock solution and stirred (1000 rpm) at room temperature for 30 min, resulting in a uniform and translucent mixed solution. The mixture was then centrifuged at 2000 rpm for 2 min to remove bubbles. Finally, the Fmoc-FF–alginate solution was added drop by drop to an abundant volume of an aqueous solution of CaCl2 (2.0 wt.%), using a syringe needle with an inner diameter of 0.7 mm. Hydrogel beads could be formed in a few seconds when the Fmoc-FF–alginate liquid droplets were brought into contact with the CaCl2 solution. In addition, we used an aqueous solution of CaCl2-HCl (pH = 6) as a control to study the effect of HCl on the structure of the hydrogel beads.
where R represents the fraction of unbroken hydrogel at 24 h, W represents the mass of freeze-dried unbroken hydrogels at 24 h, W 0 represents the mass of freeze-dried hydrogels before adding water or buffer solutions.
Zeta Potential Measurement
A Zetasizer Nano ZS (Malvern Instruments Ltd., UK) was used to measure zeta potential of the Fmoc-FF peptide solutions and alginate solutions. To prepare fresh solutions, 1.5 mg of Fmoc-FF peptide was dissolved in 15 mL of double-distilled water (ddH2O), and 1.5 mg of alginate was dissolved in 15 mL of ddH2O. The pH values of the resulting solutions were adjusted with 0.5 M NaOH or 0.1 M HCl. The pH values of all the solutions were measured using a pH meter.
In Vitro Release of Docetaxel
The release of docetaxel from different hydrogel beads was carried out in the PBS buffer at 37 °C. The docetaxel-loaded hydrogel beads were prepared according to the following protocol: Docetaxel was first dissolved in the solvent dimethyl sulfoxide (DMSO) with a concentration of 45 mg/mL. Next, 20 μL of the docetaxel-loaded DMSO solution was added to 3 mL of the fresh Fmoc-FF or Fmoc-FF–alginate stock solution, and the resulting mixture was added to 10 mL of an aqueous solution of CaCl2 (2.0 wt.%) using a syringe needle with an inner diameter of 0.7 mm to form drug-loaded hydrogel beads. The docetaxel-loaded hydrogel beads were then placed inside small vials containing 8 mL of PBS solution (10 mM PBS, pH 7.4), 0.1 % (w/v) Tween 80, and 0.02 % (w/v) NaN3. The vials were incubated in a rotary shaker at 100 rpm and 37 °C to perform the drug release experiments. An aliquot of 0.5 mL of the supernatant solution was removed from each vial at the designated sampling intervals and replaced with the same amount of fresh buffer. The docetaxel concentration in the supernatant solution was analyzed by high-performance liquid chromatography (HPLC). All the samples were centrifuged at 10 000 rpm for 5 min before they were tested by the chromatograph. The HPLC measurements were performed on an Agilent 1200 HPLC system (Agilent Technologies, USA) operating with an Aglient Eclipse XDB-C18 column. The parameters used in the HPLC analysis were as follows: the mobile phase consisted of acetonitrile water (60:40, v/v); the wavelength of UV detector was set at 229 nm; flow rate was set at of 1.0 mL min–1; the injection volume was 20 μL.
The morphologies of the hydrogels were characterized using a S-4800 field-emission scanning electron microscope (SEM; Hitachi High-Technologies Co., Japan) at an acceleration voltage of 3 kV. All samples were freeze-dried and sputter-coated with platinum using an E1045 Pt-coater (Hitachi High-technologies Co., Japan) before characterization. Fourier-transform infrared (FTIR) spectra of Fmoc-FF, alginate, and Fmoc-FF–alginate hydrogels were recorded on a Nicolet-560 FTIR spectrometer (Nicolet Co., USA). The hydrogels were freeze-dried and deposited on the surface of the KBr plate, and the FTIR spectra were recorded across the range of 400–4000 cm–1 with 20 scans and a resolution of 4 cm–1.
Results and Discussion
where M t/M ∞ represents the fraction of drug released from hydrogel beads at time t, k represents the kinetic constant characteristic of the drug/hydrogel matrix, and n is the diffusional exponent characteristic of the release mechanism. When n = 0.5, the drug release is purely controlled by Fickian diffusion; when 0.5 < n < 1, more than one mechanism is at work; when n = 1, the drug release is controlled by hydrogel swelling (case II transport).
Release exponent (n), rate constant (k), and correlation coefficient (R 2) obtained from the power law equation
Fmoc-FF/alginate = 2
Fmoc-FF/alginate = 1
A new peptide–polysaccharide hybrid hydrogel bead was prepared by calcium ion-triggered co-assembly of Fmoc-FF peptide and alginate. The calcium ions triggered self-assembly of Fmoc-FF peptide into nanofibers with a diameter of about 30 nm by decreasing their charge; at the same time, they allowed alginate to be crosslinked, leading to the formation of hybrid hydrogel beads. SEM results verified that the hybrid hydrogel beads had a dense surface structure on the outside and a structure of stacked sheets with a porous nanofibrous network in its interior. Compared to the alginate or Fmoc-FF hydrogel beads, the hybrid hydrogel beads had higher stability in both water and PBS, probably because of the synergistic effect of noncovalent and ionic interactions. Furthermore, we embedded docetaxel in the hydrogel beads during the hydrogel formation process and studied the drug release behavior. By varying the concentration ratio of Fmoc-FF to alginate, controlled release of docetaxel could be obtained. The release exponent n suggests that docetaxel was mainly released from the hybrid beads in a Fickian diffusion-mediated mode. We thus conclude that our method provides a way to fabricate new supramolecular materials with fascinating properties.
This work was supported by the Natural Science Foundation of China (Nos. 21306134 and 21476165), 863 Program of China (No. 2013AA102204), and Ministry of Education (No. 20130032120029).
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