Role of Electrostatic Interactions on the Transport of Druglike

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Role of Electrostatic Interactions on the Transport of Drug-Like Molecules in Hydrogel-Based Articular Cartilage Mimics: Implications for Drug Delivery Fengbin Ye, Stefania Baldursdottir, Soren Hvidt, Henrik Jensen, Susan Weng Larsen, Anan Yaghmur, Claus Larsen, and Jesper Ostergaard Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00725 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on January 26, 2016

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Molecular Pharmaceutics

Role of Electrostatic Interactions on the Transport of Drug-Like Molecules in Hydrogel-Based Articular Cartilage Mimics: Implications for Drug Delivery Fengbin Ye,1,† Stefania Baldursdottir,1 Søren Hvidt,2 Henrik Jensen,1 Susan W. Larsen,1 Anan Yaghmur,1 Claus Larsen,1 & Jesper Østergaard 1,* 1

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen,

Universitetsparken 2, DK-2100 Copenhagen, Denmark 2

Department of Chemistry-NSM, Roskilde University, Universitetsvej 1, DK-4000 Roskilde,

Denmark

Corresponding Author *E-mail: [email protected] Phone: +45 35336138, Fax: +45 35336030 Present Address †

Coloplast A/S, Holtedam 1, DK-3050 Humlebæk, Denmark

1 ACS Paragon Plus Environment


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TOC

ABSTRACT In the field of drug delivery to the articular cartilage, it is advantageous to apply artificial tissue models as surrogates of cartilage for investigating drug transport and release properties. In this study, artificial cartilage models consisting of 0.5% (w/v) agarose gel containing 0.5% (w/v) chondroitin sulfate or 0.5% (w/v) hyaluronic acid were developed, and their rheological and morphological properties were characterized. UV imaging was utilized to quantify the transport properties of the following 4 model compounds in the agarose gel and in the developed artificial cartilage models: H-Ala-β-naphthylamide, H-Lys-Lys-β-naphthylamide, lysozyme, and αlactalbumin. The obtained results showed that the incorporation of the polyelectrolytes chondroitin sulfate or hyaluronic acid into agarose gel induced a significant reduction in the apparent diffusivities of the cationic model compounds as compared to the pure agarose gel. The decrease in apparent diffusivity of the cationic compounds was not caused by a change in the gel structure since a similar reduction in apparent diffusivity was not observed for the net negatively charged protein α-lactalbumin. The apparent diffusivity of the cationic compounds in the negatively charged hydrogels was highly dependent on the ionic strength pointing out the importance of electrostatic interactions between the diffusant and the polyelectrolytes. Solution based affinity studies between


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the model compounds and the two investigated polyelectrolytes further confirmed the electrostatic nature of their interactions. The results obtained from the UV imaging diffusion studies are important for understanding the effect of drug physicochemical properties on the transport in articular cartilage. The extracted information may be useful in the development of hydrogels for in

vitro release testing having features resembling the articular cartilage.

KEYWORDS Cartilage mimic, Chondroitin sulfate, Diffusion, Drug delivery, Electrostatic interactions, Hyaluronic acid, Hydrogel, Transport, UV imaging

INTRODUCTION Transport of a solute from the synovial cavity into the avascular articular cartilage can take place by at least two mechanisms: (1) diffusion due to a solute concentration gradient, and (2) the fluid flow due to a hydraulic pressure gradient.1 Compared to the convection contribution, diffusion plays the primary role in providing the necessary nutrients and removing metabolites and degradation products.2-5 Insufficient supply of nutrients to the articular cartilage, as well as inadequate removal of waste, has been suggested to be a major cause of osteoarthritis.2 The extracellular matrix (ECM) of cartilage consists of an organized water-filled collagen network entrapping the negatively charged glycosaminoglycans (GAGs) in the extrafibrillar space.3, 5 This densely packed anionic network may constitute a physical barrier by interacting with the diffusing molecules to slow down their transport in cartilage ECM. Previous reports have demonstrated that the highly charged GAGs are vital for the barrier function and filter effect of ECM.6-7 It has been speculated that the diffusion rate in spontaneously degenerated and enzymatically degraded cartilage is faster than in healthy



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tissue.4 In the development of efficient drug delivery carriers to the cartilage, it is crucial to understand the diffusion of drugs and biomacromolecules in ECM.

A cartilage-like construct containing the components of native tissues may be useful as a diffusion medium for in vitro investigations of transport phenomena. In this context, hydrogels are increasingly employed as ECM mimics for different applications including tissue engineering,8-9 as well as artificial tissue models for drug release and transport studies.10-12 There is a strive towards using less animals in drug discovery and development, the availability of suitable animal tissue samples may be limited and significant efforts also have to be put into standardization or calibration in order to generate results that can be compared between series of experiments made on tissue samples and formulations. Artificial tissue matrices may hold advantages in terms of reproducebility and availability which can offset the potential disadvantages of not studing drug behavior and transport processes in real tissue. Among various proposed hydrogels, agarose gels possess a porosity similar to the articular cartilage and have been employed for cartilage ECM engineering.8-9, 13 In addition, agarose can function as a main polymer backbone for gelation of other biopolymers and developing matrices with a good mechanical stability.14-15 Hyaluronic acid (HA) and chondroitin sulfate (CS) are two major cartilage GAGs consisting of the repeating disaccharide units of glucuronic acid and N-acetylgalactosamine, and glucuronic acid and glucosamine, respectively (Figure 1). They display different properties as regard charge density and solution viscosity.16 Hence, an agarose gel containing HA or CS as a model of the cartilage ECM was utilized for studying the diffusional transport of the model compounds in the present work.

The transport of an analyte through hydrogels depends on both physical (e.g., obstruction effects17) and chemical (e.g., electrostatic effects18-20) interactions between the diffusant and the polymer


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network. These interactions are highly related to the analyte hydrodynamic size2, 21-22 and surface charge,18, 23-25 as well as the gel cross-linking density.17, 26 Previous studies and models have mainly focused on the geometric obstruction process describing the diffusion of neutral or charged compounds in neutral hydrogels.17, 27 The diffusive properties of charged ligands/analytes in charged hydrogels are less well characterized.

The main objective of this study was to investigate how the physicochemical properties of the analyte, in particular hydrodynamic radius and net charge, influence the transport (apparent diffusion) properties within articular cartilage mimics. Furthermore, it was an aim to improve the design of hydrogel models as surrogates of articular cartilage for in vitro drug transport investigations by incorporation of main components of the cartilage (CS or HA). The model compounds included two low-molecular-weight peptide β-naphthylamide derivatives (H-Ala-βNA and H-Lys-Lys-βNA, Figure 1) and two small proteins, α-lactalbumin (α-LA), and lysozyme. The size and structure of βNA to some degree resemble that of low-molecular weight drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs), e.g., naproxen. Drug conjugation and prodrug strategies providing affinity for ECM structures have been envisioned.28-31 To this end, derivatization of the active molecule with amino acid and peptide structures similar to those of the model compounds might constitute a feasible approach. In contrast, α-LA and lysozyme represent small proteins of similar size but having opposite net charge (Table 1). Collectively, these molecules would allow assessment of the suitability of the experimental approach and potentially provide insights into the influence of size as well as charge on the transport properties (Table 1). UV imaging was applied to quantify the transport properties of the model compounds in the applied hydrogel-based cartilage mimics. This technology provides an ability to attain temporally resolved images from absorption of UV light32-34 and has recently been introduced to monitor drug diffusion



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and release properties in hydrogel matrices.12, 35-37 In addition to drug substance transport properties, an imaging approach allows assessment of the physical integrity and fate of the delivery systems upon administration into a biomimetic matrix. Taylor Dispersion Analysis (TDA) and capillary electrophoresis-frontal analysis (CE-FA) were applied to determine the hydrodynamic radii and characterize the interactions of the model compounds with HA and CS, respectively. Rheological measurements and cryo-SEM were applied for characterizing the gel structure. In the present report, the possible relationships between the physicochemical properties of the model compounds, apparent diffusion coefficients in the gels and the affinity towards the cartilage key components are discussed.


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Figure 1. Chemical structures of the investigated peptide β-naphthylamide derivatives, H-Ala-βnaphthylamide (H-Ala-βNA) and H-Lys-Lys-β-naphthylamide (H-Lys-Lys-βNA), and the glycosaminoglycans, chondroitin sulfate (CS) and hyaluronic acid (HA).



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Table 1. The molecular weight (MW), the isoelectric point (pI), the net charge at pH 7.4, the hydrodynamic radius (Rh), and the diffusion coefficient (D) of the investigated model compounds in 67 mM phosphate buffer at pH 7.40 and I = 0.17 M. Rh and D values were determined at 25 °C.

Compounds

MW (Da)

pI

Rh (nm) Net charge D (10-10 m2/s)a at pH 7.4 TDAa Literature

H-Ala-βNA

215

+1

6.2 ± 0.2

0.40

H-Lys-Lys-βNA

400

+3

4.1 ± 0.3

0.60

Lysozyme

14200

11.038

+ 738,b

1.2 ± 0.2

2.02

2.0038

α-LA

14400

5.138

- 738,b

1.2 ± 0.1

2.00

2.0238

a

Determined by Taylor dispersion analysis (TDA).

b

Net charge at pH 7.4 as estimated from potentiometric titration experiments.38-40

MATERIALS AND METHODS Materials Agarose (type Ι), chondroitin sulfate sodium salt from bovine cartilage (MW 10-40 kDa), αlactalbumin from bovine milk (α-LA, type Ι), and 20% (w/v) technical grade solution of poly(diallyldimethylammonium chloride) (PDADMAC) were obtained from Sigma-Aldrich (St. Louis, MO). H-Ala-β-naphthylamide (bromide salt, H-Ala-βNA) and H-Lys-Lys-β-naphthylamide (acetate salt, H-Lys-Lys-βNA) were purchased from Bachem (Bubendorf, Switzerland). Hyaluronic acid sodium salt (MW 1500-1800 kDa) from Streptococcus equi was obtained from Fluka-Chemie (Buchs, Switzerland). Lysozyme from egg white, sodium hydroxide, and sodium dihydrogenphosphate monohydrate were obtained from Merck (Darmstadt, Germany). All chemicals were used as received. Purified water from a Milli-Q deionization unit (Millipore, Bedford, MA) was used throughout.


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Hydrogel Preparation A weighed amount of agarose powder was suspended at a prescribed concentration in phosphate buffers (pH 7.4) with various phosphate concentrations (Ionic strength (I) of 0.025, 0.08, 0.14, or 0.17 M). The resulting agarose suspensions were heated to the boiling temperature to allow complete dissolution of the polymer. For the preparation of composite gels, a stock solution of CS or HA was added to the preheated agarose solution to obtain a final CS and HA concentration of 0.5% (w/v). Then the temperature of agarose solutions was lowered to ~60 °C, and the model compound was added to achieve a final concentration of 0.03, 0.03, 0.06, 0.08 mM for H-Ala-βNA, H-Lys-Lys-βNA, lysozyme, and α-LA, respectively. Samples used for the diffusion measurements were transferred into quartz cells (7.0 × 3.0 × 63 mm3) using a pipette, and the gelation occurred at room temperature. The final concentration of agarose was 0.5% (w/v) in all investigated hydrogels.

Characterization of Hydrogels

Rheological analysis The rheological properties of the hydrogels were studied by performing oscillatory shear experiments using a TA AR-G2 rheometer (TA Instruments, Waters, USA) using a cone-plate geometry with a diameter of 60 mm and 1̊ cone angle. After the polymers were dissolved at the boiling temperature, an appropriate amount of hot solution was transferred to the pre-heated plate (70 °C). Temperature scanning from 50 to 20 °C was conducted with one degree intervals at constant strain amplitude of 0.1%, at which frequency sweeps were performed from 0.1 to 10 Hz. The evolution of the storage modulus G' and the loss modulus G" was monitored. At 20 °C, stress amplitude sweeps were carried out to ensure that the measurements were within the linear



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viscoelastic region. To prevent solvent evaporation, the outside of samples was covered with low viscosity silicone oil. All samples were investigated in triplicate.

Cryo-Scanning Electron Microscopy (cryo-SEM) Hydrogel samples were attached to the sample stubs and plunge-frozen in slushed liquid nitrogen at -210 °C. The frozen samples were then transferred to the cryo-preparation chamber attached to the Quanta 3D FEG (FEI, Hillsboro, OR, USA) under continuous vacuum. Samples were fractured using a cold knife to expose the interior gel structure. The sample temperature was raised to -65 °C for 3 min to sublime any condensed ice from the surface gained/exposed during transfer. The temperature was then lowered to -130 °C and the samples sputter-coated with platinum for 160 s. The coated samples were passed through the transfer lock to the FIB-SEM cryo-stage which was maintained at -125 °C. Imaging was performed at an acceleration voltage of 5 kV. Pore size of the hydrogels was determined as a mean value of randomly selected 40 pores from different regions of the SEM images using the Image J program (National Institutes of Health, Bethesda, MD).

Determination of Aqueous Diffusion Coefficient by Taylor Dispersion Analysis The aqueous diffusivities were determined by Taylor dispersion analysis (TDA) conducted using an Agilent 3DCE instrument (Agilent Technologies, Waldbronn, Germany) coupled to an Actipix D100 UV imaging detector (Paraytec Ltd., York, UK) that provides two detector points along the capillary as previously detailed.41 Uncoated fused silica capillary (Polymicro Technologies, Phoenix, AZ, USA) was cut into 90 cm in length (75 µm ID) with 30 cm and 50 cm to the center of the first and the second detection window, respectively. A dynamically coated capillary was used for the positively charged compounds H-Ala-βNA, H-Lys-Lys-βNA, and lysozyme, whereas the uncoated fused silica capillary was used for negatively charged protein α-LA. Coating of the


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capillary was performed by flushing the capillary with 1 M NaOH for 30 min, Milli-Q water for 15 min, 1% (w/v) PDADMAC for 30 min, and then phosphate buffer for 15 min. Between measurements, the uncoated capillary was flushed for 2 min each with 0.1 M NaOH and the dispersion buffer, and the PDADMAC-coated capillary was flushed for 2 min each with 1% (w/v) PDADMAC and the dispersion buffer. A pressure of 50 mbar was applied for 5 s for sample injection and subsequently to force the sample plug through the capillary. The dispersion profiles were recorded at 280 nm. The analyte concentrations used for TDA analysis were 0.3, 0.3, 0.6, and 0.8 mM for H-Ala-βNA, H-Lys-Lys-βNA, lysozyme, and α-LA, respectively. Each sample was analyzed 6 times. The experimental temperature in the vicinity of the capillary was measured using a Fluke temperature probe (American Fork, UT, USA). The diffusivity (D) was determined using Actipix control software (version 1.4) by analyzing the difference in peak residence time (t) and temporal variance (σ2) of the first and the second Gaussian shaped peaks.41 Then the diffusion coefficient was recalculated and given at 25 °C. The hydrodynamic radius (Rh) of the model compounds was calculated according to the Stokes-Einstein equation: Rh =

k BT 6πηD

(1)

where kB is the Boltzmann’s constant, and ߟ is the solvent viscosity.

UV imaging of Diffusion in Hydrogels The diffusion of the model compounds in hydrogels was studied using a previously developed experimental setup utilizing the Actipix SDI300 dissolution imaging system (Paraytec Ltd, York, UK).12 Briefly, the analyte/model compound-loaded gel was placed in a quartz cell (7.0 × 3.0 × 63 mm3) in contact with a blank gel (Figure 2a). Absorbance images (9 × 7 mm2) in the hydrogels were collected at a rate of ~0.2 images per s by monitoring the intensity of UV light passing through the sample cell. UV imaging was performed at 280 nm at ambient temperature (21-24 °C) for up to 10



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h. Conversion of the pixel intensities into the absorbance values was performed using the Actipix software (version 1.4). During the conversion, the UV absorbance of the background (blank gel) was subtracted from sample images. The absorbance-distance profiles at given time points were exported and loaded into Microsoft Excel for subsequent data analysis. The relationship between the absorbance and the analyte concentration was maintained in the linear range of Beer’s law. All experiments were performed in triplicate.

Figure 2. (a) Diffusion of the model compounds (black curled structures) in agarose gel containing CS or HA. (b) UV absorbance maps of the lysozyme transport in 0.5% (w/v) agarose gel containing


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0.5% (w/v) chondroitin sulfate (CS) at 280 nm at ambient temperature (21-24 °C). Intense red color represents high UV absorbance (analyte concentration) and the contour lines are iso-absorbance lines. (c) The absorbance-distance profiles along the line indicated in Figure 2b at different time points. The solid curves represent fits of the concentration profiles to eq 3.

Due to the geometry of the diffusion cell and the placement of the gel, the diffusion of the analyte in the gel was considered in one dimension only according to Fick’s second law:42 ∂C ∂ 2C =D 2 ∂t ∂x

(2)

where x is the distance, t is the time, C refers to the analyte concentration, and D is the (apparent) analyte diffusivity. The boundary interface between the analyte-loaded and the blank gel phases was located at x = x0. Initially, the model compound was homogeneously distributed in the donor gel (x < x0) at a concentration of C0; whereas the analyte concentration C is 0 in the blank gel (x > x0). Eq 2 was solved using the infinite approximation for the gel phase.42 The duration of the experiments was selected to maintain the validity of the infinite model: tD 50 °C) ensuring that CS and HA molecules were homogenously distributed within the agarose networks. The gelation process of agarose has been proposed to occur through the formation of agarose double helical structures followed by their aggregation to form the gel.14-15 Upon cooling the samples from 50 to 20 °C, gel formation proceeded as indicated by the steadily increasing value of the storage moduli G' from 35 oC (Figure 3a). G' is a measure of the material’s mechanical rigidity. Addition of 0.5% (w/v) CS and HA led to a significant decrease of 24% and 30% in G', respectively, as compared to that of the agarose gel at 20 °C. However, the addition did not influence the temperature where the gel formation was initiated. The decrease in G' is probably due to the fact that the presence of the embedded polyelectrolytes hindered the formation of agarose chain aggregates, thus inducing the formation of a mechanically looser gel matrix with slightly lower modulus but with a similar gelation temperature. Similar observations have been reported for xanthan gum-containing agarose gels.14 For the three hydrogels, G' was two orders of magnitude larger than G" (Figure 3b), which is a typical behavior of strong elastic hydrogels. Overall, the rheological measurements demonstrated


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that the incorporation of CS or HA into the gel structure slightly decreased the gelation ability of agarose but still allowed the formation of rigid and elastic hydrogels. In comparison to articular cartilage, it should be mentioned that the stiffness of the hydrogels is orders of magnitude less than that of cartilage.46-47

Figure 3. Viscoelastic properties of 0.5% (w/v) agarose (agarose), 0.5% (w/v) agarose with 0.5% (w/v) CS (CS-agarose), and 0.5% (w/v) agarose with 0.5% (w/v) HA (HA-agarose). (a) Temperature dependence of the gel elastic storage modulus (G') at the frequency of 1 Hz. (b) Frequency dependence of the elastic storage modulus and the loss modulus (G") at 20 °C. All samples were prepared using a 67 mM phosphate buffer (pH 7.4).

In order to further investigate the effect of CS or HA on the hydrogels, cryo-SEM was used to study the hydrogel structures in their fully hydrated state. Figure 4 shows representative cryo-SEM



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images of the interior networks of the pure agarose gel and the composite hydrogels. For both agarose and CS-agarose composite hydrogels, similar pore structures were observed. The HAcontaining agarose had a lower degree of network formation as compared to that formed from the pure agarose gel (Figure 4). The HA-agarose composite had a more lumpy-like local structure which may be attributed to a higher water binding capability of HA. SEM images of human articular cartilage can be found in the literature, e.g., Lim et al.48 These are fundamentally different because of the presence of collagen which is the dominating structural element in cartilage. To this end the, it should be noted that the architecture of the applied gels is significantly different from the organized structure in cartilage where proteoglycans are confined within the collagen scaffold.

From the cryo-SEM images, the determined pore sizes were 5.1 ± 2.0 and 2.2 ± 0.8 µm for 0.5% (w/v) agarose, 0.5% (w/v) agarose containing 0.5% (w/v) CS, respectively. The pore size of 0.5% (w/v) agarose gel containing 0.5% (w/v) HA was not determined due to its less porous structure. Xiong et al. reported that the pore size of a 0.5% (w/v) agarose gel was in the range of 200-500 nm using the turbidity method and gel electrophoresis.49 It has to be noted that SEM images can only provide a rough estimation of pore size of hydrogels, here the main purpose was to assess whether the pore sizes were in the same size range as the diffusants or significantly different (vide infra). A quantitative comparison of the data from these different techniques is not relevant due to the heterogeneous nature of the interior gel structure and the different measuring principles. Also, specimen preparation artifacts, such as ice crystals, may potentially affect the gel pore size in cryoSEM experiments.


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Figure 4. Cryo-SEM images showing the hydrogel structures of 0.5% (w/v) agarose, 0.5% (w/v) agarose containing 0.5% (w/v) chondroitin sulfate (CS), and 0.5% (w/v) agarose containing 0.5% (w/v) hyaluronic acid (HA). All hydrogel samples were prepared using 67 mM phosphate buffer (pH 7.4).

Diffusion in Aqueous Solution The diffusion coefficients of the 4 model compounds in phosphate buffer solution were determined by TDA.41 The analyte dispersion due to the pressure driven flow was observed as a decrease in the peak height and a broadening of the second peak as compared to the first peak (Figure 5). The extent of the dispersion is dependent on the analyte diffusivity (size) with smaller molecules leading to relatively narrow peaks due to increased cross sectional diffusion as compared to larger molecules. The diffusivities were determined from the difference in the residence time and in the temporal variance of the peak at the two detection windows.41 The obtained diffusion coefficients and the corresponding hydrodynamic radii, calculated according to the Stokes-Einstein equation (eq 1), are given in Table 1. The obtained molecular radii show a good agreement with the previously reported values for α-LA and lysozyme (Table 1).



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Figure 5. Taylor dispersion analysis of H-Ala-β-naphthylamide (H-Ala-βNA) and lysozyme. Dispersion profiles of 0.3 mM H-Ala-βNA and 0.6 mM lysozyme were obtained in 67 mM phosphate buffer (pH 7.4) using two detection windows at 280 nm. The injected sample (22 nL) was forced through the capillary tube by a pressure drop of 50 mbar. The total length of the 75 µm i.d. capillary was 90 cm, with a distance of 30 and 50 cm to the first and the second detection window, respectively.

Diffusion in Hydrogels The hydrodynamic radius and the charge state of a diffusant are two important factors affecting diffusion in a charged polymer network.20, 50 The diffusive transport of the 4 model compounds in the pure agarose and the composite hydrogels was studied using UV imaging. Figure 2b shows the UV absorbance maps of lysozyme transport in negatively charged CS-agarose composite hydrogel as a function of time. The intense red color represents high UV absorbance (lysozyme concentration) and the contour lines are iso-absorbance lines. Lysozyme diffusion along the concentration gradient was apparent as the spaces between contour lines increased over time. The CS- and HA-agarose composite gels loaded with lysozyme became turbid after exposure to UV light for periods longer than one hour. Therefore, the lysozyme transport experiments in CS- or HA-


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agarose composite gels were only exposed to the UV light at a few selected time points. The mathematical model (eq 3) derived from Fick’s second law was applied to describe the lysozyme concentration-distance profiles from which the apparent diffusion coefficient was estimated (Figure 2c). Table 2 lists the obtained apparent diffusion coefficients of the 4 analytes in the investigated hydrogel systems. A priori, the diffusivities in agarose gel were expected to be similar or lower than the respective values in buffer determined by TDA. For the model compounds H-Lys-Lys-βNA, lysozyme and α-lactalbumin, the diffusion coefficient in pure phosphate buffered agarose network was similar to or slightly higher than those observed in aqueous buffer with pH 7.4 (Tables 1 and 2) taking into account the variability normally associated with diffusion experiments and the use of different methods. The reason that the diffusivities tend to be slightly higher in the agarose gel as compared to the buffer solution (TDA measurements) is most likely caused by electronics heating of the UV imaging sensor head leading to a temperature rise of ~5 oC above the surroundings in the gel, as identified recently.35 The cause of the relatively larger diffusion coefficient observed for HAla-βNA in agarose as compared to the buffer solution is not known. Overall, the results were consistent with previous findings also indicating that the diffusion coefficients in low-concentration agarose gels are similar to those in buffer.10, 35, 51 When the average gel pore size is much larger than the radius of the diffusant and significant interactions between the diffusant and the agarose matrix do not occur, the apparent diffusion coefficient in the gel may approach the value in aqueous solution. In our study, the pore size of 0.5% (w/v) agarose gel (5.1 µm) was approximately 1000 times larger than the diameter of the model compounds (Table 1).

Incorporation of the negatively charged CS or HA into agarose gel significantly retarded the transport of the cationic model compounds (Table 2). This decrease in apparent diffusivity was strongly affected by the net charge of the investigated model compounds, being more pronounced



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for the positively charged species with higher net charge. For example, the apparent diffusivities in the CS-agarose composite hydrogel decreased by 15%, 32%, and 52% for the H-Ala-βNA (+ 1), HLys-Lys-βNA (+ 3), and lysozyme (+ 7), respectively, as compared to the diffusion coefficients observed in the agarose gel. Incorporation of CS or HA into an agarose gel introduced immobilized negative charges within the networks. As discussed previously for carrageenan-agarose gels,51 the polyelectrolytes CS and HA are because of their sizes expected to entangle with the agarose, hereby further decreasing their already low diffusivities relative to the apparent diffusion coefficients of the model compounds. Due to the size distribution of the CS applied (10 – 40 kDa) a small diffusive contribution for model substances bound to CS might take place. However, it was assumed that the diffusion of the polyelectrolytes CS and HA was negligible in the gel matrix. Since the apparent diffusivity of the anionic α-LA (-7) was not affected by the structural change of the gel matrix (Table 2), it is likely that the decreased diffusivities of the cationic compounds only to a lesser extent were affected by a change in gel pore size.

The decrease in apparent diffusivity of the cationic compounds was most likely a consequence of the non-specific electrostatic interactions between the diffusants and the polyelectrolytes. Electrostatic effects on the transport of electrolytes have previously been observed in charged hydrogels,18,44 as well as in articular cartilage.28-31, 52 The repulsive electrostatic interactions between the overall anionic α-LA and the negatively charged CS and HA did not show a significant effect on its diffusivity in the investigated hydrogels.


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Table 2. Apparent diffusion coefficients of the model compounds in 0.5% (w/v) pure agarose, 0.5% (w/v) agarose containing 0.5% (w/v) chondroitin sulfate (CS), and 0.5% (w/v) agarose containing 0.5% (w/v) hyaluronic acid (HA) determined by UV imaging at pH 7.40 (ionic strength of 0.17 M) and 21-24 °C. Agarose

CS-agarose

HA-agarose

Compounds

(× 10-10 m2/s)

(× 10-10 m2/s)

(× 10-10 m2/s)

H-Ala-βNA

7.7 ± 0.5

6.5 ± 0.2*

6.5 ± 0.4*

H-Lys-Lys-βNA

4.7 ± 0.2

3.2 ± 0.2*

3.4 ± 0.5*

Lysozyme

1.2 ± 0.1

0.6 ± 0.03*

0.8 ± 0.06*#

α-LA

1.3 ± 0.1

1.2 ± 0.1

1.1 ± 0.2

All values represent a mean ± SD (n = 3 × 4). *The obtained diffusion coeffcients in CS-agarose and HA-agarose gel represent significant difference (p < 0.05) as compared to the diffusion coefficient in agarose gel. #

The obtained diffusion coeffcient in HA-agarose gel represents significant difference (p < 0.05) as

compared to the diffusion coefficient in CS-agarose gel.

HA carries one carboxylate group per repeating disaccharide unit; whereas CS has an additional sulfate group (Figure 1). A concentration of 0.5% (w/v) of CS and HA in the composite hydrogels corresponds to 0.019 and 0.012 mEq/mL, respectively. The interactions between the cations and CS are expected to be favored due to the high charge density of CS as compared with HA. As shown in Table 2, the diffusion coefficient of lysozyme in CS-agarose composite gel was 25% smaller than that obtained in the HA-agarose hydrogel pointing to the importance of electrostatic interactions between the cationic diffusants and the negatively charged polyelectrolytes in modulating the diffusion. A similar trend, but not statistically significant, was also observed for the low-molecular-



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weight cationic compound H-Lys-Lys-βNA. Electrostatic interactions of the model compounds may be even stronger in natural cartilage since the reported charge density of cartilage ECM was in a range of 0.1-0.3 mEq/mL.1 Here primarily the role of charges is assessed. Future studies should seek to evaluate the importance of the special cartilage matrix architecture which is significantly more complex than the architecture of the composite gels applied here where some of the key components are present but potentially in non-native forms.

Effect of Ionic Strength on Diffusion in Composite Gels

Figure 6. Effect of the ionic strength on the apparent diffusion coefficients of (●) H-Ala-βNA, (■) H-Lys-Lys-βNA, (▲) lysozyme, and (▼) α-lactalbumin in 0.5% (w/v) agarose containing 0.5% (w/v) chondroitin sulfate obtained by UV imaging at pH 7.40 and 21-24 °C. All data are represented as mean ± SD (n = 3 × 4).

To further investigate the role of electrostatic interactions, the effect of ionic strength on analyte diffusional transport in CS-agarose composite gel was investigated in an ionic strength range of 0.025-0.17 M. Figure 6 shows the plots of the measured apparent diffusion coefficients as a function of the ionic strength revealing that the apparent diffusivity decreased with decreasing ionic


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strength for the cationic compounds. The decrease in diffusivity for trivalent H-Lys-Lys-βNA was higher than for the monovalent H-Ala-βNA in the interval of the investigated ionic strength. The relative decrease in diffusivity was 20 % and 59 % for H-Ala-βNA and H-Lys-Lys-βNA, respectively, when lowering the ionic strength from 0.17 M to 0.025 M. The combination of lysozyme and CS gave a slightly turbid solution and/or produced a precipitation when the buffer ionic strength was lower than 0.12 M, and therefore the investigation was performed only at ionic strengths of 0.12 M and 0.17 M. By contrast, the diffusivity of the anionic α-LA was independent of the ionic strength investigated. The dependence of the apparent diffusivity of the positively charged species on the ionic strength further demonstrated the electrostatic nature of the interactions. The electrostatic interactions were considerable at low ionic strength and decreased with increasing ionic strength due to the screening of the charges on the polymer network. This is in agreement with previously published data.53-54

Binding of the Model Compounds to CS and HA The interactions of the model compounds with HA and CS were evaluated in phosphate buffer (pH 7.4) using CE-FA. Initial CE-FA experiments showed that the degree of H-Ala-βNA and H-LysLys-βNA binding to HA was not detectable in 67 mM phosphate buffer (pH 7.4). This is most likely due to the low bound fraction, i.e., less than 2%. Instead the ligand-HA interaction studies were performed in 33 mM phosphate buffer (pH 7.4), in which a larger fraction of bound ligand was expected due to the lower ionic strength. In the CE-FA method, the extent of binding was indicated as a fractional decrease in peak height of the free ligand in the investigated ligand/polyelectrolyte mixture as compared to the peak height of the ligand in the control sample (standard) (Supplementary Figure S1). Figure 7 shows the obtained binding density, the number of bound ligands per disaccharide unit of the polyelectrolytes, as a function of the free ligand



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concentration at 25 °C. All obtained binding isotherms were linear in the investigated ligand concentration ranges. Therefore, the binding constants were determined from the slopes of the binding isotherms. The obtained binding parameters are given in Table 3. Adsorption of lysozyme onto the capillary wall prohibited the CE-FA measurements in both uncoated and PVA-coated capillaries. Moss et al. have measured the interactions of lysozyme with CS and HA by monitoring the protein concentration in solutions before and after immerging CS- and HA-covalently immobilized agarose gels.55 They found that the lysozyme binding was strongly dependent on the ionic strength, and became negligible at ionic strengths higher than 0.15 M and 0.04 M for lysozyme-CS and lysozyme-HA interaction, respectively.

Figure 7. Binding isotherms of (●) H-Ala-βNA, (■) H-Lys-Lys-βNA, and (∇) α-lactalbumin with (a) chondroitin sulfate (CS) in 67 mM phosphate buffer (pH 7.4), and with (b) hyaluronic acid (HA)


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in 33 mM phosphate buffer (pH 7.4) obtained by capillary electrophoresis-frontal analysis at 25 °C. The solid lines were obtained by linear regression analysis.

Table 3. Binding constants (mean ± SE) and bound fraction of the model compounds with chondroitin sulfate (CS) and hyaluronic acid (HA) determined by capillary electrophoresis-frontal analysis at pH 7.4 and 25 °C. CSa

KCS (M-1) H-Ala-βNA

HAb Bound fraction KHA (M-1)

Bound fractionc

18.4 ± 0.4 0.55

13.6 ± 0.3 0.15

H-Lys-Lys-βNA 37.9 ± 0.2 0.64

26.3 ± 1.0 0.25

α-LA

ND

14.1 ± 1.1 0.48

ND

a

In 67 mM phosphate buffer (pH 7.4, I = 0.17 M).

b

In 33 mM phosphate buffer (pH 7.4, I = 0.08 M).

c

The bound fraction of model compounds ([L]bound/[L]total) in presence of CS and HA is the average

value as determined from the data in the binding isotherms. ND, binding not detectable.

For both CS and HA, the binding constant of the model compounds increased with the number of positive charges at the experimental conditions employed (pH 7.4). For instance, the binding constants of H-Lys-Lys-βNA-polyelectrolyte complexation were approximately 2 times larger as compared to that of H-Ala-βNA (Table 3). This supported the idea that the electrostatic interactions played a predominant role in the interactions between the investigated cationic compounds and the negatively charged CS or HA. Previous studies have shown that hydrophobic interactions are also involved in the interaction of H-Ala-βNA and H-Lys-Lys-βNA with HA44 as well as the interaction



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of lysozyme and CS and HA.16,55 The model compounds H-Ala-βNA, H-Lys-Lys-βNA and α-LA all interacted to a larger extent with CS than HA, since binding with HA was not detectable at the physiological ionic strength (I = 0.17 M) using the CE-FA assay. It is of interest to note that the CEFA assay appears not to be sufficiently sensitive to detect some of the weakest interactions that manifest themselves by the reduced diffusivities in the UV imaging experiments. The binding constant between the negatively charged α-LA and the polyanion CS had a similar value as the positively charged H-Ala-βNA, which was probably a consequence of protein surface charge heterogeneity. In addition, the charge of ionizable groups is affected by the local electrostatic environments, which has been found to essentially contribute to attractive interactions between the anionic proteins and negatively charged polyelectrolytes.56-57 Similar observations have earlier been reported for the interaction between bovine serum albumin and a synthetic negatively charged polyelectrolyte.58 Comparison with the results from the transport studies, however, reveals that the observed interaction between α-LA and CS does not manifest in a significant change in diffusivity in the agarose-based mimics (Table 2).

Overall both the binding data and the results of the transport studies in the hydrogels demonstrated the importance of electrostatic interactions between the cationic model compounds and the polyelectrolytes CS and HA (Tables 2 and 3). The electrostatic interactions between the diffusing species and the negatively charged polyelectrolytes may significantly affect their binding and transport in an articular cartilage environment. This appears to be in line with literature reports suggesting that cartilage targeting may be achieved using positively charged prodrugs with affinity to cartilage.28-31 The methods applied in the current study appear capable of predicting scenarios where GAG interactions need special consideration in relation to drug transport and delivery. In addition to diffusion, convection affects drug transport in cartilage, sub-physiological levels of the


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polyelectrolytes are applied and significant structural differences relative to cartilage prevail in the current model, therefore quantitative predictions are not likely at present but rank ordering potentially useful in a drug delivery setting seems feasible. It is tempting to speculate that cationic promoieties resembling the H-Lys-Lys component of the βNA derivative might be useful for conferring affinity towards cartilage to drug candidates.

CONCLUSIONS A 0.5% agarose gel incorporating either HA or CS was constructed to mimic the cartilage extracellular matrix. The developed model was able to provide a uniform medium featuring embedded negative charges suitable for the in vitro drug transport investigations. UV imaging technology was shown to be suitable for measuring the apparent diffusion coefficients of four model compounds in an agarose gel. The incorporation of the negatively charged polyelectrolytes into agarose gel resulted in a significant reduction in the measured apparent diffusion coefficients of the cationic model compounds as compared to the pure agarose gel. This effect was most likely not due to a change in the tortuosity of the gel matrix since a similar reduction in diffusivity was not detected for the anionic compound α-LA. The apparent diffusivity of the cationic model compounds was highly dependent on the ionic strength indicating the importance of electrostatic interactions. With respect to the influence of interactions, the diffusion results were consistent with the affinity studies conducted by capillary electrophoresis. The findings obtained from these diffusion studies are relevant for understanding the drug transport in cartilage and the subsequent development of hydrogel-based cartilage mimics. In future studies, the properties of the developed cartilage model may be modified, such as the pore size and the fixed charge density, in order to better resemble the cartilage. The study may represent a first step towards an in vitro model mimicking drug transport properties within cartilage. In combination with the imaging approach taken this opens for the



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possibility of simultaneous investigating physical changes of delivery systems and drug substance transport in vitro.

ACKNOWLEDGEMENT This work was supported by the Danish Medical Research Council. Jesper Østergaard is member of the Scientific Advisor Board of Paraytec Ltd. The authors alone are responsible for the content and writing this paper.

Supporting Information. Electropherograms of free H-Lys-Lys-βNA and H-Lys-LysβNA/chondroitin sulfate mixture by CE-FA. This material is available free of charge on the ACS Publications website.

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TOC 80x44mm (150 x 150 DPI)

ACS Paragon Plus Environment


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Figure 1 93x182mm (300 x 300 DPI)


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Figure 2 110x207mm (150 x 150 DPI)



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Figure 3 80x96mm (300 x 300 DPI)


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Figure 4 144x45mm (150 x 150 DPI)



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Figure 5 164x119mm (300 x 300 DPI)


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Figure 6 101x71mm (150 x 150 DPI)



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Figure 7 244x319mm (300 x 300 DPI)


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