Article pubs.acs.org/Biomac
Delivery of Dermatan Sulfate from Polyelectrolyte ComplexContaining Alginate Composite Microspheres for Tissue Regeneration Yanhong Wen,† Lisbeth Grøndahl,‡ Monica R. Gallego,§ Lene Jorgensen,† Eva H. Møller,† and Hanne M. Nielsen*,† †
Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100, Copenhagen, Denmark ‡ School of Chemistry and Molecular Bioscience, University of Queensland, Australia § Global R&D, Coloplast A/S, Denmark
ABSTRACT: Dermatan sulfate (DS) is a glycosaminoglycan (GAG) with a great potential as a new therapeutic agent in tissue engineering. The aim of the present study was to investigate the formation of polyelectrolyte complexes (PECs) between chitosan and dermatan sulfate (CS/DS) and delivery of DS from PEC-containing alginate/chitosan/dermatan sulfate (Alg/CS/ DS) microspheres for application in tissue regeneration. The CS/DS complexes were initially formed at different conditions including varying CS/DS ratio (positive/negative charge ratio), buffer, and pH. The obtained CS/DS complexes exhibited stronger electrostatic interaction, smaller complex size, and more stable colloidal structure when chitosan was in large excess (CS/DS 3:1) and prepared at pH 3.5 as compared to pH 5 using acetate buffer. The CS/DS complexes were subsequently incorporated into an alginate matrix by spray drying to form Alg/CS/DS composite microspheres with a DS encapsulation efficiency of 90−95%. The excessive CS induced a higher level of sustained DS release into Tris buffer (pH 7.4) from the microspheres formulated at pH 3.5; however, the amount of CS did not have a significant effect on the release from the microspheres formulated at pH 5. Significant cell proliferation was stimulated by the DS released from the microspheres in vitro. The present results provide a promising drug delivery strategy using PECs for sustained release of DS from microspheres intended for site-specific drug delivery and ultimately for use in tissue engineering.
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INTRODUCTION Glycosaminoglycans (GAGs) are a group of large complex molecules participating in a wide range of physiological and pathological processes.1 The recent progress in understanding the GAG structure−function relationship provides great potential for promoting GAGs as novel pharmaceuticals. GAGs interact with a variety of proteins including growth factors, enzymes, adhesion molecules, which mediate cell−cell and cell−matrix signaling for the function and development of complex multicellular organisms.2 In addition to the great attention to heparin, heparan sulfate, and chondroitin sulfate in many previous studies,3,4 dermatan sulfate (DS) (Figure 1A) is a particularly attractive GAG molecule, which functions as a cofactor in a variety of therapeutics, especially in cell-mediated tissue repair and wound healing.5 DS, predominantly present in the skin, plays an important role in binding and activating © 2012 American Chemical Society
extracellular molecules and growth factors for many important biological processes, including cell adhesion, migration, proliferation and differentiation.5 The superior function of DS compared to other GAGs is attributed to the strong binding to heparin cofactor-II in antithrombotic activity.1 However, so far, the use of DS in clinical trials is limited as drug delivery strategies have not yet been applied to promote the therapeutic functions of DS. Polyelectrolyte complexes (PECs) formed between polycations and polyanions have been extensively investigated for drug delivery.6 PECs formed between relatively large and highly charged molecules often give rise to occurrence of colloidal Received: December 21, 2011 Revised: January 20, 2012 Published: February 1, 2012 905
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stability and degradability of the heparin/CS PECs were also shown to considerably depend on the formulation pH.16 The investigation of the stoichiometry of dextran sulfate/CS PEC formation indicated different mechanisms of the complexation depending on which of the two polyions was in excess.6 In spite of vast amounts of studies on various GAG/CS PEC systems, dermatan sulfate/CS (DS/CS) PECs have not yet been explored for the purpose of controlled drug delivery. Alginate is a linear block copolymer composed of α-Lguluronic and β-D-mannuronic acid residues (Figure 1C), which can be cross-liked by calcium ions via carboxyl groups to form physical gels.17 The alginate/chitosan (Alg/CS) complexes formed between the carboxyl groups on alginate and the amine groups on chitosan often lead to colloidal structures, for example, nano/microparticles,18 which showed profound properties of sustained release of encapsulated bioactive substances and stronger colloidal strength than either alginate or chitosan alone.19,20 For the delivery of anionic biomacromolecules, for example, DNA, strong DNA/CS PECs were produced at different CS/DNA ratios and subsequently complexed with alginate resulting in high drug loading and controlled DNA release from the colloidal Alg/CS drug carriers.21 Similarly, delivery of a single strand nucleotide intended for antisense therapy from Alg/CS nanoparticles was found to be dependent on the CS/Alg ratio and the addition of CaCl2.20 Hence, it is highly relevant to investigate the factors controlling the complex formation between CS and anionic macromolecules such as DS, and the drug delivery properties of such Alg/CS/DS microparticles. Potentially, drug delivering particles could be combined with tissue-engineered products, for instance, a composite or hybrid system, by immobilizing drug carriers in a hydrogel or scaffold for tissue regenerative applications.22−24 The objective of the present work is to achieve sustained delivery of dermatan sulfate (DS) from Alg/CS/DS microspheres intended for site-specific DS delivery. The hypothesis is that this can be achieved by precomplex formation of CS and DS. The CS/DS PECs were formed initially and investigated with respect to the CS/DS ratio (the negative:positive charge ratio), buffer and pH. The chemical compositions, complex size, zeta potential, and thermal stability of the CS/DS PECs prepared at different conditions were systematically studied. The CS/DS PECs were further complexed with alginate to produce microspheres by spray drying. The Alg/CS/DS microspheres were further examined in terms of physical characteristics, stability, drug release, and ability to induce cell proliferation.
Figure 1. Chemical structures of dermatan sulfate (A), chitosan (B), and alginate (C).
structures, such as nanoparticles or submicrometer particles, which represent great potential for drug delivery purposes.7,8 Polysaccharides such as chitosan and alginate are well-known for their excellent properties of biodegradability and biocompatibility for biomedical applications.9 These polysaccharides are highly positively or negatively charged under certain conditions (i.e., below or above their pKa) and are capable of PEC formation.10 Chitosan (Figure 1B), derived from chitin, is the second most abundant polysaccharide in nature and possesses cationic charges at acidic condition. It has been widely investigated as an absorption promoter due to its tremendous mucoadhesive and permeability enhancing abilities.11,12 Structurally, GAG is a linear and negatively charged polysaccharide decorated with sulfate and carboxyl groups, which can readily interact with polycations, such as chitosan.13 The colloidal nano/submicrometer structures of GAG/CS PECs represent useful and promising means for delivery of GAGs or other incorporated bioactive macromolecules.14 Many studies have reported on the formation, properties, and applications of GAG/CS PECs.6,14 The properties of GAG/ CS PECs significantly depend on the preparation conditions, such as the charge ratios (polycation/polyanion ratio), ionic strength, and pH.15 For instance, the colloidal stability of the heparin/CS PECs was found to depend on the charge ratios, which led to different particle sizes and zeta potentials. The decrease in particle size was correlated with an increase in the surface charge (>+20 mV), which correspondingly increased the colloidal stability of the PEC nanoparticles.14 The thermal
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MATERIALS AND METHODS
Materials. Chitosan (75−85% deacetylation degree, Mw 50−190 kDa) and dermatan sulfate (Mw ∼ 46 kDa) were purchased from Sigma Aldrich (St. Louis, MO, U.S.A.). Sodium alginate (Protanal LF 10/60 FT, viscosity 45−60 mPa, G/M: 65−75/25−35) was kindly provided by FMC BioPolymer (Cork, Ireland). Fluorescein isothiocyanate isomer I (FITC) was purchased from Invitrogen (Carlsbad, CA, U.S.A.). Ethylene-diamine tetra-acetic acid (EDTA, potassium salt), Hanks Balanced Salt Solution (HBSS), and PMS reagent were purchased from Sigma Aldrich (St. Louis, MO, U.S.A.) and MTS reagent from Promega (Madison, WI, U.S.A.). All the other chemicals were obtained commercially at analytical grade. Milli-Q water was used throughout the studies. Preparation of FITC-DS. FITC-labeled DS was obtained as reported previously.25 Briefly, 20 μL of 50 mg/mL FITC in absolute ethanol solution was mixed with 1 mL of a 10 mg/mL dermatan sulfate
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Microsphere Preparation. Alginate (Alg) was dissolved in acetate buffer (at either pH 3.5 or 5) to the final concentration of 5 mg/mL. A total of 10% (v/v) of the FITC-labeled DS was mixed with the nonlabeled DS to obtain the FITC-DS solution. A 10 mL aliquot of either CS/FITC-DS or CS/DS complexes (formed at different conditions as described above) was added dropwise to 40 mL of a 5 mg/mL alginate solution with the same pH and stirred for at least 30 min. The formed Alg/CS/DS complex solution were spray dried using a Büchi 190 mini-Spray Dryer (Büchi Laboratorium, Flawil, Switzerland) at the following operating conditions: inlet temperature 145 °C, outlet temperature 65−70 °C, pump rate 3 mL/min, nozzle gas flow rate 14 L/min, aspirator 85−95%. For Alg/DS microspheres, 10 mL of 1 mg/mL FITC-DS or DS (at either pH 3.5 or 5) was directly added to 40 mL of 5 mg/mL alginate solution and spray dried in the same way as described above. The spray-dried microspheres were collected at room temperature in 20 mg/mL CaCl2 in 90% (v/v) ethanol aqueous solution and magnetically stirred for 24 h at room temperature to cross-link the alginate. Finally, the microspheres were vacuum-dried at 40 °C for 24 h. Microsphere Characterization. Particle Size. The mean particle size of the microspheres was determined by laser diffraction using a Malvern Mastersizer 2000 (Malvern Instruments, Worcestershire, U.K.) equipped with a Scirocco 2000 device from Malvern for dry powder measurement. All the measurements were performed at vacuum in triplicates. Morphology. The surface morphology of the microspheres was observed using an environmental scanning electron microscope (ESEM, tabletop TM-3000, Hitachi, Tokyo, Japan). The microspheres were spread on a metal specimen stub and coated with gold prior to the imaging. Representative images were selected from images of three different batches. Drug Loading. The drug loading was determined by extracting the FITC-DS from all batches of spray-dried microspheres. A 2 mg aliquot of the microspheres was dispersed in 1 mL of 4 mM EDTA aqueous solution and mixed for 2 h to dissolve the alginate microspheres. A total of 100 μL of the resulting solution was added to a 96-well plate (Nunc black, Thermo Scientific, Lake Barrington, IL, U.S.A.) and measured using a platereader (FLUOstar OPTIMA, BMG Labtech, Offenburg, Germany) at an excitation wavelength of 485 nm and emission wavelength of 520 nm. The drug loading (% (w/w)) was calculated as the ratio of loaded DS to the total microsphere, and the encapsulation efficiency was calculated as described previously.24 The measurements were performed on three different batches and each batch was in duplicate. Microsphere Physical Stability. A total of 10 mg of Alg/DS and Alg/CS/DS microspheres (prepared at either pH 3.5 or pH 5) with and without Ca2+ cross-linking were dispersed in 1 mL Tris buffer (pH 7.4, 10 mM) and kept at 37 °C in a water bath with gentle shaking. The microspheres in dispersion were collected after 1 and 3 days and placed on a metal specimen stub. The morphological changes of microspheres were monitored using the ESEM, as described above (n = 3). In Vitro DS Release. For the spray-dried microspheres, the release of DS was evaluated over 10 days. For each type of the FITC-DSlabeled microspheres, 10 mg of the microspheres was suspended in 1 mL of Tris-buffer (pH 7.4, 10 mM). All suspensions were incubated at 37 °C in a linear shaking water bath at a shaking rate of 0.18 min−1 (Grant Instruments, Cambridgeshire, U.K.) at preset time points. At every time point, the samples were ultracentrifuged at 10 000 g for 5 min at room temperature, aliquots of the supernatants (200 μL) were collected for analysis, and an equal volume of the fresh buffer was used for replenishment. The amount of FITC-DS in all collected supernatants was determined by fluorescence intensity using the plate reader, as described above. All measurements were performed in triplicate. In Vitro Cell Proliferation. Mouse fibroblasts (NIH 3T3 cell line, ATCC, VA, U.S.A.) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% (v/v) fetal calf serum (FCS) at 37 °C with 5% CO2. The fibroblasts were seeded in clear 96-well plates (Nunc Transparent, Thermo Scientific, Lake Barrington, IL, U.S.A.) at
aqueous solution. The mixture was wrapped in aluminum foil to prevent photobleaching and reacted for 2 h at room temperature before transfering to a dialysis cassette (Slide-A-Lyzer Dialysis Cassette, Mw 7000 cutoff, Thermo Scientific, Lake Barrington, IL, U.S.A.). The dialysis was run in 1 L Milli-Q water for at least 5 days and the outer media was replaced with fresh Milli-Q water every 24 h until no FITC (limit of detection 0.5 μg/mL) was detected by a fluorometer (FLUOstar OPTIMA, BMG Labtech, Offenburg, Germany). Preparation of Polyelectrolyte Complexes. CS/DS PECs were prepared at different conditions: CS/DS ratios (w/w) 1:1, 2:1, or 3:1; pH 3.5 or 5; citrate or acetate buffer. The following procedure was used: CS was dissolved in either citrate or acetate buffer adjusted to pH 3.5. An aliquot of the CS solution was adjusted to pH 5 using 1 M NaOH. The CS stock solutions at pH 3.5 and 5 had a final concentration of 3 mg/mL and were diluted with the same buffer to a concentration of 1 and 2 mg/mL, respectively. Dermatan sulfate (DS) was dissolved in either citrate or acetate buffer (at pH 3.5 or 5) to a final concentration of 1 mg/mL. Subsequently, 10 mL of the DS solution was added dropwise to 10 mL of 1, 2, or 3 mg/mL CS solution dissolved in the same buffer (either at pH 3.5 or 5). The CS/ DS complexes were formed and stirred magnetically at room temperature for at least 30 min. The resulting suspension was analyzed by light scattering. For further analysis, the resulting CS/DS complexes were centrifuged at 4500 rpm for 20 min at room temperature, and the precipitates of the complexes were dried in the oven at 50 °C for 24 h and weighed prior to analysis. Characterization of Polyelectrolyte Complexes. X-ray Photoelectron Spectroscopy (XPS). XPS survey and highresolution spectra of the CS, DS, and CS/DS complexes were obtained using an Axis Ultra XPS spectrometer (Kratos Analytical, Manchester, U.K.) with a monochromatic Al Kα Xray source operating at 15 kV, 10 mA for all the data acquisitions. Survey spectra were collected at analyzer pass energy of 160 eV for a binding energy range of 0−1200 eV. High-resolution spectra of C 1s and N 1s were collected at analyzer pass energy of 20 eV. Binding energies were chargecorrected to 285.0 eV for C 1s peak of adventitious carbons.26 High-resolution spectra were resolved into individual Gaussian−Lorentzian peaks using least-squares fitting program (CasaXPS, Casa Software, Estepona, Spain). Complex Size and Zeta Potential. The mean complex size of the CS/DS complexes (in diameter) was determined by dynamic light scattering using a Malvern NanoZS (Malvern Instruments, Worcestershire, U.K.) equipped with 633 nm laser and 173° detection optics. The zeta potential of the complexes was measured by the laser Doppler electrophoresis technique using the Malvern NanoZS. A nanosphere size standard (60 ± 2.7 nm, Duke Scientific, Palo Alto, CA, U.S.A.) and a zeta potential transfer standard (−68 ± 6.8 mV) from Malvern were used to verify the performance of the instrument. All the samples were measured in triplicate. Malvern DTS v.510 software (Malvern Instruments, Worcestershire, U.K.) was used for data acquisition and analysis. For viscosity and refractive index, the value of pure water was used. Element Microanalysis. The element composition of the CS, DS, and CS/DS complexes was analyzed using a Carlo Erba NA 1500 Elemental Analyzer (Thermo Scientific, Lake Barrington, IL, U.S.A.) for C, H, N, S. A 1 mg aliquot of sample was placed in a lightweight tin capsule and dropped at preset intervals into a vertical quartz combustion tube maintained at 1020 °C with helium running as drift gas. All the samples were measured in triplicate. Differential Scanning Calorimetry (DSC). Calorimetric analysis of the CS, DS polymers and the precipitates of the CS/DS PECs was performed using a differential scanning calorimeter (DSC; TAC7/DX, Perkin-Elmer, Waltham, MA, U.S.A.). DSC thermograms of the samples (2−5 mg) were recorded in sealed aluminum pans at a scanning rate of 10 °C/min ranging from 40 to 400 °C in a nitrogen atmosphere (n = 3). Pyris-Instrument managing software (PerkinElmer, Waltham, MA, U.S.A.) was used for data acquisition and analysis. 907
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a density of 10 000 cells/well and cultured for 24 h prior to use. The culture medium was exchanged with 200 μL of sample solution consisting of either Alg/DS or Alg/CS/DS microspheres dispersion in the cell culture medium at concentrations of 1, 2, 5, or 10 mg/mL. The cells were incubated with the sample solutions for 48 h. As a measure of cell proliferation, the metabolic activity of cells was measured by the MTS/PMS assay. Briefly, after 48 h of incubation, the microsphere suspensions were removed from the plate and the cells were rinsed twice with HBSS supplemented with 10 mM HEPES (hHBSS buffer, pH 7.4). A total of 100 μL of the MTS/PMS reagents containing 240 μg/mL MTS and 2.4 μg/mL PMS in the hHBSS buffer was added to the cells and incubated for 1.5 h at 37 °C, and the absorbance was measured at 492 nm using the plate reader. Cell culture medium and pure DS solution was used as negative and positive control, respectively.
in the citrate buffer was near the theoretical value, while the precipitates formed in the acetate buffer at this pH amounted to around 1/3 of the theoretical mass at each CS/DS ratio. XPS survey scans displayed the major elemental components of the pure biopolymers and their precipitates. Table 2 lists the S% determined from XPS survey scans and reveals that sulfur (S) was only present in DS (2.19%) and not in CS, in agreement with the chemical composition of the biopolymers (Figure 1). Therefore, the S-content could be used to evaluate the compositions of the precipitates formed in the CS/DS mixture. At pH 5, the precipitates formed in citrate and acetate buffers gave rise to a decrease in S-content with increasing CS/ DS ratio correlating with mixing ratios. However, at pH 3.5 there was a significant difference in S%, depending on the buffer used. The S% was constant and close to the theoretical value for a 1:1 PEC in the precipitates formed in the acetate buffer, whereas S was not detected in the precipitates formed in the citrate buffer. The elemental composition of the precipitates formed in the citrate buffer was further investigated by element microanalysis and the data is tabulated in Table 3. At pH 5, the S% of the precipitates showed a similar trend to that observed by XPS, and it was confirmed that no S could be detected in the precipitates formed in citrate buffer at pH 3.5. The elemental CHN composition of the precipitates formed at pH 3.5 was similar to that of stoichiometric CS/citrate (1:1). The precipitates formed at pH 5 displayed N% in-between that of the two biopolymers. In combination with the S% of these precipitates, it indicates the appropriate formation of CS/DS PEC at pH 5. From this data it is clear that, although CS/DS PECs do appear to form at pH 5 in citrate buffer, near theoretical amounts of precipitates indicate that they are in the form of aggregates with low colloidal stability. In addition, CS/citrate complexes appear to form at pH 3.5 instead of CS/DS complexes. In contrast, based on the XPS survey scans the precipitates formed in acetate buffer are DS/CS PECs. Therefore, the following studies were limited to CS/DS complexes prepared in acetate buffer. Figure 2 shows the XPS N 1s narrow scans of the six types of CS/DS complexes. These spectra displayed a shape, which requires three peaks for curve fitting: amine (NH2) at 399.5 eV, amide (NHCO) at 400.3 eV, and protonated amine (NH3+) at
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RESULTS Characterization of CS/DS Complexes. Composition of Polyelectrolyte Complexes. The CS/DS PECs were formed between the cationic CS and the anionic DS at various CS/DS ratios, different pH values, and different buffers. The precipitates that formed in these mixtures were collected and analyzed for their chemical composition using XPS and elemental microanalysis. Table 1 shows the mass of the
Table 1. Dry Mass of the Precipitates Formed in the CS/DS Mixtures under Different Conditionsa mass of the precipitates (mg) theoretical mass (mg) CS/ DSb 1:1 2:1 3:1
20 30 40
citrate buffer
acetate buffer
pH 5
pH 3.5
pH 5
pH 3.5
16.7 ± 1.0 31.0 ± 1.2 39.7 ± 0.8
5.7 ± 0.7 4.4 ± 0.5 5.5 ± 0.3
7.8 ± 0.5 11.6 ± 1.0 15.0 ± 1.2
6.5 ± 0.3 5.1 ± 0.4 6.0 ± 0.5
a Sample values represent the mean of three determinations ± standard deviation. bMixtures of CS/DS at w/w ratios.
obtained precipitates in the mixture of CS/DS. The amount of precipitates formed at pH 3.5 was constant at all CS/DS ratios for both the citrate and the acetate buffer and amounted to approximately 1/4 of the theoretical PEC mass at the CS/DS ratio 1:1. In contrast, the amount of precipitate formed at pH 5
Table 2. Atomic S% and N% of the Biopolymer CS and DS and the Precipitates of CS/DS Obtained from XPS Survey Scans sample
pH
CS DS CS/DS 1:1b CS/DS 2:1c CS/DS 3:1d CS/DS 1:1b CS/DS 2:1c CS/DS 3:1d
5
citrate buffer 1.18
S%
N%
N/Aa 2.19
5.78 3.14 acetate buffer 1.02
citrate buffer 5.04
acetate buffer 5.15
5
0.55
0.68
5.06
6.18
5
0.41
0.26
5.26
6.27
3.5
N/Aa
1.25
3.36
4.98
3.5
a
N/A
1.10
3.89
5.18
3.5
N/Aa
1.04
3.41
4.90
a
S% was
