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Enzymatically crosslinked hyaluronic acid microgels as a vehicle for sustained delivery of cationic proteins
European Polymer Journal 115 (2019) 234–243
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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj
Enzymatically crosslinked hyaluronic acid microgels as a vehicle for sustained delivery of cationic proteins
T
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Elaheh Jooybara, Mohammad J. Abdekhodaiea,b, , Abbas Mousavia, Bram Zoetebierc, ⁎ Pieter J. Dijkstrac, a
Department of Chemical Engineering, Sharif University of Technology, PO 11365-11155, Tehran, Iran Environmental Applied Science and Management, Ryerson University, PO 979-5000, Toronto, Canada c MIRA–Institute for Biomedical Technology and Technical Medicine and Department of Developmental BioEngineering, Faculty of Science and Technology, University of Twente, PO Box 217, 7500 AE Enschede, the Netherlands b
A R T I C LE I N FO
A B S T R A C T
Keywords: Microgel Hyaluronic acid Enzymatic crosslinking Inverse microemulsion Protein delivery
In this study, a novel biodegradable hyaluronic acid (HA) based microgel were prepared via enzymatic crosslinking of tyramine conjugated HA (HA-TA) in an inverse microemulsion. HA-TA microdroplets were crosslinked within a few seconds in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The high water content of the polymeric network and the inherent negative charge of the HA-TA microgels provided a suitable platform for encapsulation of cationic proteins like lysozyme and TGF-β1 growth factor. The results demonstrated that lysozyme was released, after an initial burst release, in a suitable sustained manner over a period of four weeks. Both diffusion and degradation due to microgel hydrolysis controlled the release rate. In vitro cytotoxicity of microgels using human mesenchymal stem cells revealed microgels nontoxic. This study demonstrates that the developed injectable HA-TA microgels prepared by enzymatic crosslinking are a promising vehicle for delivery of cationic proteins including some important growth factors.
1. Introduction Microgels are physically or chemically crosslinked hydrophilic polymer networks in micrometer size, which can be used as delivery vehicles for controlled delivery of proteins because of their large surface area, high water content, injectability, and their tunable size [1–6]. Delivery of therapeutic proteins, including growth factors, hormones, enzymes, and antibodies remains still a challenge because of their short half-life in vivo [4,7,8]. Therefore, there is a need to develop carriers for the protection and release of proteins in a spatial and temporal manner [4,6,9]. To this end, microgels may play a significant role by incorporating the biological molecules in their interior polymeric network and modulating the release rate [10]. Microgels are prepared from both synthetic and natural polymers [7]. Polysaccharide-based microgels have received a great deal of interest due to their biocompatibility, biodegradability, and absence of immunogenicity. The presence of functional groups like hydroxyl and carboxyl groups on their molecular chain provides anchors for various chemical modifications [11–15]. Among those, hyaluronic acid (HA),
which is a linear natural glycosaminoglycan present in many tissues, has been widely investigated because of its excellent biocompatibility, biodegradability, and low immunological risks [3,16–19]. Microgels can be fabricated using an inverse emulsion technique. The water-in-oil gelation involves emulsification of a polymer precursor solution in a continuous organic phase with subsequent crosslinking of aqueous micro-droplets [20]. In general, the network of the microgels is formed by physical or electrostatic interactions (e.g. ionic crosslinking) or covalent chemical crosslinking (e.g. free radical reaction). One major concern regarding chemical crosslinking is the presence of cytotoxic reagents or exposure to harsh experimental conditions which may affect the bioactivity of encapsulated proteins [7,21]. To avoid this, enzymatic crosslinking in hydrogel formation has been extensively explored in biomaterial fields [22–24], tissue engineering [24–26] and drug delivery [21,27]. The crosslinked network can be formed under mild conditions in the presence of enzymes, like peroxidases and low concentrations of hydrogen peroxide (H2O2). By changing the amount of the enzyme and H2O2, and also degree of substitution of crosslinkable groups on the polymer backbone, the gelation time may be tuned
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Corresponding authors at: Department of Chemical Engineering, Sharif University of Technology, PO 11365-11155, Tehran, Iran (M.J. Abdekhodaie). MIRA – Institute for Biomedical Technology and Technical Medicine and Department of Developmental BioEngineering, Faculty of Science and Technology, University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands (P.J. Dijkstra). E-mail addresses: [email protected] (M.J. Abdekhodaie), [email protected] (P.J. Dijkstra). https://doi.org/10.1016/j.eurpolymj.2019.03.032 Received 2 October 2018; Received in revised form 25 February 2019; Accepted 18 March 2019 Available online 19 March 2019 0014-3057/ © 2019 Elsevier Ltd. All rights reserved.
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The polymer with NHS activated COOH groups (1.0 g) was dissolved in 30 mL of MES buffer (0.1 M, pH 6.1). Tyramine (385 mg, 2.8 mmol) was dissolved in 4 mL of DMF and added to the solution. After overnight stirring, the resulting solution was precipitated in cold ethanol, filtered and washed with ethanol and finally diethyl ether. The resulting white powder was dissolved in MilliQ-water and dialyzed (Pre-wetted RC tubing, Spectrum Labs, MWCO 1000), first against 100 mM sodium chloride solution for 2 days, followed by a mixture of distilled water and ethanol (5:1) for one day and MilliQ-water for another day. The HA-TA conjugate was obtained after lyophilization. The degree of substitution (DS), which is defined as the number of substituted groups per 100 disaccharide units, was calculated from 1H NMR (D2O) spectral data by comparing the integral values of the aromatic protons of tyramine (6.86 and 7.17 ppm) and the methyl protons of HA (1.9 ppm). 2.3. HA-TA microgels fabrication Hyaluronic acid tyramine conjugate (HA-TA) was used to prepare microgels using a water in oil emulsion method. In a typical experiment, 2 mL of HA-TA solution was mixed with a solution of HRP in PBS to give a mg HRP/mmol TA ratio of 0.6 (final concentration of HRP was 1.5 U/ml). The solution was added dropwise to 25 mL of isooctane containing 2.5% v/v Span 80 and 0.25% v/v Tween 80 as surfactants, and the resulting mixture was at 0 °C homogenized using an UltraTurrax (T25, IKA Works Inc., USA) at a predetermined speed. Subsequently, 20 µL of a H2O2 solution in PBS was added to the microemulsion to give a H2O2/TA molar ratio of 0.5. After 10 min homogenization, the mixture was magnetically stirred for 30 min to complete the crosslinking of the microgels. Particles were collected by precipitation in ice-cold acetone, followed by centrifugation at 8000 rpm for 5 min. Particles were subsequently washed with acetone and ethanol and were dried under reduced pressure at room temperature. The production yield was determined as the mass ratio of the produced dried microgel to the initial solid polymer used.
Scheme 1. Schematic representation of synthesizing HA-TA microgels via the HRP catalyzed crosslinking of an inverse microemulsion. A HA-TA solution with HRP was added to isooctane and homogenized·H2O2 was added to the microemulsion to crosslink the microdroplets.
[28–30]. Recently, the enzymatic crosslinking was used to fabricate cell-laden microgels and the results indicated that cells remained viable and functional [31,32]. In this study, a new method was developed to synthesize microgels for protein delivery by enzymatic crosslinking of tyramine conjugated HA (Scheme 1). The microgels were made in the presence of horseradish peroxidase and H2O2 in an inverse microemulsion. Protein loading was investigated by either post-fabrication encapsulation or loading during fabrication and prior to gelation. Also, the effects of different factors on the protein loading capacity and the release rate from fabricated microgels were studied and discussed. Finally, the cytotoxicity of the microgels was determined by the Prestoblue and live/ dead assays.
2.4. Microgel characterization Microgel size and morphology: The microgels size in the swollen state was determined by dispersion in water and imaging using a phase contrast microscopy (Nikon Eclipse TE300 microscope equipped with Nikon Digital Sight DS-fi1 camera). Images were analyzed by using Image J software. The morphology of the microgels was analyzed using a FEI Quanta 650 ESEM, equipped with a Peltier cooling stage for sample and humidity (gas pressure) control, using an acceleration voltage of 10 kV. Dry microgels were placed on a SEM mounting stud and hydrated by stepwise increasing the pressure at 3 °C. Once fully hydrated, the sample was dried by reducing the pressure. Microgel swelling: To 3 mg of dried microgels placed in a 2 mL Eppendorf vial, 1 mL of PBS (10 mM, pH = 7.4) was added. The particles were dispersed using a shaker and incubated overnight at either 4 °C or 37 °C with continuous shaking. Microgels were centrifuged at 7000 rpm for 10 min and the excess water was discarded and blotted from vials and microgel pellet. The swelling is expressed as the ratio of weight in the swollen state to dry weight of microgels (Ws/Wd).
2. Materials and methods 2.1. Materials Sodium hyaluronate (MW = 25 kDa) was purchased from Contipro Pharma, the Czech Republic. Tyramine (TA) (99%), anhydrous N,Ndimethylformamide (DMF) (99.8%), N-(3-dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDAC), N-hydroxysuccinimide (NHS), hydrogen peroxide (H2O2), horseradish peroxidase (HRP, 325 units/mg solid), Hyaluronidase from bovine testes (lyophilized powder, 400–1000 units/mg) and MES hemisodium salt dry powder were obtained from Sigma-Aldrich and used without further purification. Phosphate buffered saline (PBS, 10 mM, pH 7.4) was purchased from Lonza. All other solvents were purchased from Biosolve (Valkenswaard, The Netherlands) and used as received. 2,2,4-Trimethylpentane (isooctane) was purchased from Honeywell. Tween 80, Span 80, lysozyme from chicken egg white, bovine serum albumin (BSA) and immunoglobulin G (IgG), FITC- BSA, and FITC-lysozyme were purchased from Sigma-Aldrich. Human TGF-β1 was from R&D systems.
2.5. Protein loading and release Three model proteins, lysozyme (14 kDa, pI = 11.35 [33]), bovine serum albumin (BSA, 60 kDa, pI = 4.7 [34]) and immunoglobulin G (IgG, 150 kDa, pI = 7.2 [35]) were used to investigate the protein loading capacity of the HA-TA microgel. The protein uptake was determined at two different incubation temperatures, 4 °C and 37 °C. In brief, 2 mg of dry microgels were placed in a 2 mL microcentrifuge tube and incubated with 1 mL of protein solution at a concentration of 500 µg/mL. After 16 h incubation on an orbital shaker, the microgels were centrifuged (7000 rpm, 10 min) and the supernatant was removed
2.2. Hyaluronic acid tyramine (HA-TA) synthesis Typically, sodium hyaluronate with the molecular weight (MW) of 25 kDa (1.0 g, 2.5 mmol of COOH groups) was dissolved in 30 mL of PBS. EDAC (1.0 g, 5.2 mmol) and NHS (1.3 g, 11.3 mmol) were added and the solution was stirred for 2 h. The product was precipitated in cold ethanol, filtered and washed with ethanol and finally diethyl ether. 235
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Fig. 1. Hyaluronic acid tyramine (HA-TA) synthesis. (A) Synthetic scheme used in the conjugation of tyramine to hyaluronic acid. First, HA carboxylic acid groups were activated by conversion to NHS esters. In the second step, NHS esters were reacted with tyramine, (B) 1H NMR spectra of hyaluronic acid and HA-TA (solvent D2O).
was calculated as the amount of protein determined after microgel degradation to initial amount of protein added to the polymer solution * 100. The release of model proteins into PBS (10 mM, pH = 7.4) containing 0.05% w/v sodium azide (to prevent bacterial growth) was monitored over a period of 28 days. At selected time points, the microgels were collected, centrifuged at 7000 rpm for 10 min, the supernatant was removed and replaced with an equal amount of fresh medium. The release samples were stored at −20 °C for further analysis on protein content using a Bradford assay.
and analyzed for its protein content using a Bradford Assay (Coomassie protein assay kit, Thermo scientific). The absorbance was measured at 595 nm. Unloaded microgels were used as a negative control. To further investigate the protein uptake of the microgels, 2 mg of dry microgels were incubated with 1 mL of lysozyme solution with concentrations ranging from 250 to 4000 µg/mL and subsequent steps were performed as mentioned above. To evaluate the uptake of TGF-β1 (25 kDa, pI = 8.83 [36]), as a representative growth factor, another experiment was performed. To 5 mg of HA-TA microgels, 1 mL of human TGF-β1 solution (100 ng/ml containing 0.1% (w/v) BSA) was added and the resulting suspension was incubated overnight. BSA was added to prevent the unwanted attachment of the growth factor to the wall of the vial. The microgels were centrifuged and the amount of remaining TGF-β1 in the supernatant was measured using a Quantikine ELISA Kit (R&D Systems, Minneapolis, MN). To evaluate protein loading the supernatant was collected and the amount of unloaded growth factor was quantified. Protein loading was also studied by dissolving the proteins in HA-TA precursor solutions prior to microgel fabrication and network formation. In a typical example, 10 mg of protein was added to 1 mL of a 5% w/v HA-TA solution and mixed (protein/HA-TA ratio is 1/5 w/w). The HA-TA microgels were prepared as described above. To determine the encapsulation efficiency, the matrix was degraded overnight using 1 mL of a hyaluronidase solution (200 U/ml) at 37 °C and the protein content was determined using a Bradford assay. The encapsulation efficiency
2.6. In vitro cytotoxicity Cytotoxicity of microgels was studied using human mesenchymal stem cells (hMSCs) at passage 4. Cells were cultured at a density of 1000 cells/cm2 in a 48 well plate. Different amounts of microgels were dispersed in the culture medium (α-MEM (Gibco) supplemented with 10% fetal bovine serum, 1% L-glutamine (Gibco), 0.2 mM ascorbic acid (Gibco), 100 U/mL penicillin, 10 µg/mL streptomycin) and 0.5 mL of the suspension was added to each well. The microgels were sterilized before use using 70% ethanol followed by several washes with PBS. Cell viability was assayed by a live/dead assay Kit (Invitrogen) after one day. The cells were rinsed with PBS twice and then stained with calcein AM/ethidium homodimer according to the manufacturer’s instructions. Cells were visualized using a fluorescence microscope (EVOS FL). 236
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Living cells fluoresce green and the nuclei of dead cells red. Additionally, the metabolic activity of the cells, cultured with and without microgels, was measured using a Prestoblue assay (Invitrogen). Briefly, cells were incubated with a 10% solution of Prestoblue in culture medium for 2 h. The absorbance was measured using a microplate reader at 530 nm excitation and 590 nm emission. Data are presented as average ± SD.
HA-TA hydrogel formation by an enzyme-mediated oxidation reaction is schematically presented in Fig. 2A. Prior to emulsification and microgel formation, the gelation times of HA-TA solutions by addition of Horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) was studied. Gelation times, at a fixed H2O2/TA molar ratio of 0.5 and various HA-TA concentrations are presented in Fig. 2B. The results showed that with increasing HRP concentrations the gelation times decreased and ranged from 400 to 2 s. Microgels were prepared through crosslinking of HRP containing HA-TA microdroplets in isooctane (Scheme 1). The inverse microemulsion was stabilized with both Span 80 and Tween 80 as surfactants. To provide a fast gelation of the microdroplets, below 10 s, the ratio of mg HRP/mmol TA was fixed at 0.6. Addition of H2O2 to the microemulsion at a H2O2/TA molar ratio of 0.5, which is needed theoretically for the reaction of all tyramine groups, triggered the crosslinking of the HA-TA microdroplets. The microgels were isolated as fine powders that could easily disperse in water. The production yield of the HA-TA microgels was almost 78%.
2.7. Statistical analysis Each experiment was performed in triplicate. The results are presented as the mean ± standard deviation (SD). Experimental data were analyzed for statistical significance using a one-way analysis of variance (ANOVA) with Turkey’s post hoc analysis. Statistical significance was set to *p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001. 3. Results and discussion 3.1. Hyaluronic acid tyramine synthesis and microgel fabrication
3.2. Microgel size and distribution HA-TA conjugates were synthesized in a two-step procedure (Fig. 1A). First, part of the carboxylic acid groups of the hyaluronic acid were activated by conversion to NHS esters. The degree of substitution of carboxylic acid groups as determined from the 1H NMR spectrum was 24%. In the second step, the NHS esters were reacted with tyramine to provide the HA-TA. According to the 1H NMR spectral data, the degree of substitution calculated from the integral ratio of the tyramine phenolic protons and the hyaluronic acid methyl protons was 17% (Fig. 1B).
To determine the size of the microgels in the swollen state, they were dispersed in the PBS (10 mM, pH = 7.4) and imaged by phase contrast microscopy (Fig. 3). The crosslinked microgels were stable in an aqueous solution. The size distribution was analyzed using Image J software and the histograms of different microgels, are shown in Fig. 3. The average microgels size and polydispersity of the microgels are presented in Table 1. By decreasing the polymer concentration in the initial solution from 10% to 5% w/v, the microgels average diameter
Fig. 2. Gelation of HA-TA solution. (A) Tyramine conjugated hyaluronic acid is crosslinked by Horseradish peroxidase (HRP) in the presence of H2O2. (B) Gelation times of HA-TA solutions in PBS at different concentrations (% w/v) and mgHRP/mmolTA ratios. The H2O2/TA molar ratio was 0.5. 237
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Fig. 3. Microscopic images and size distribution of HA-TA microgels. (A) M25-5, (B) M25-10 microgels. Scale bars are 100 µm.
centrifugation from the initial amount of protein. It appeared that lysozyme was loaded in the microgel with a good efficiency (59.9% ± 1.1%). In contrast, IgG and BSA were not significantly depleted from the feed solution and low loading efficiencies of 6.5% ± 1.1% and 5.4% ± 2.9%, respectively, were found (Fig. 5A). At pH 7.4, the lysozyme is positively charged (pI = 11.35) providing electrostatic interaction with the negatively charged carboxylic acid groups of the HA-TA (HA pI = 2.5 [37]). Conversely, BSA as a negatively charged protein could not penetrate into the microgel network because of the repulsive forces between the protein and microgel. Because IgG is a neutral protein at the physiological condition, the repulsive force is not the reason for its inefficient loading, and therefore, the low protein uptake is possibly due to the protein size (r = 5.29 nm, 150 kDa) [38]. Similar experiments performed with M25-10 microgels afforded the same results. The role of the electrostatic interaction on the protein uptake into the microgels was also shown by Nguyen et al. [39] and Pepe at al. [40]. To further investigate the protein loading of the microgels a series of experiments were performed. To study the effect of the initial protein concentration on the loading efficiency, at an uptake temperature of 4 °C, 2 mg of M25-5 microgels was incubated in 1 mL of a lysozyme solution at various concentrations ranging from 250 to 4000 µg/mL. After protein loading, the microgels were separated by centrifugation and washed with 1 mL of PBS to remove adsorbed proteins from the surface of the microgels. The loading efficiency of lysozyme in the HATA microgels decreased from 70% to 50% by increasing the solution
decreased from 8.8 µm to 4.7 µm. Moreover, the polydispersity index (PDI) value of the M25-10 microgels (0.73) was much higher compared to the M25-5 sample (0.13). This can be attributed to the viscosity of the aqueous solution as higher concentrations in the feed increase the viscosity of microemulsions which leads to a less efficient emulsification. The effect of the polymer concentration and thus the solution viscosity on the microgel size was also shown by Jia et al. [3]. 3.3. Morphological analysis and equilibrium swelling ratio SEM micrographs of M25-5 and M25-10 microgels showed a perfectly spherical shape and a smooth surface (Fig. 4A). Only the surface of large microgels present in the M25-10 sample appeared somewhat irregular which could be related to shrinkage upon drying for imaging. The equilibrium swelling ratio of microgels incubated at pH 7.4 was lower for microgels prepared at a higher HA-TA concentration because of more densely crosslinked network formed (Fig. 4B). Additionally, the microgels swelling at 37 °C was significantly higher than that at 4 °C. 3.4. Protein loading capacity of HA-TA microgels Lysozyme, BSA, and IgG were used as model proteins to investigate the protein uptake efficiency of M25-5 microgels. In preliminary experiments, 2 mg of HA-TA microgels were incubated in a solution containing 500 µg of the protein. The amount of loaded proteins was determined by subtracting the protein content in the supernatant after Table 1 Average diameters and polydispersity of HA-TA microgels. Sample
Concentration % w/v
Stirring speed rpm
Surfactanta (Span 80) % v/v
Diameter µm
PDIb
M25-5 M25-10
5 10
13,500 13,500
2.5 2.5
4.7 ± 1.7 8.8 ± 7.5
0.13 0.73
a b
The ratio of Span 80 to Tween 80 was 10:1 v/v. The Polydispersity Index was calculated as Standard deviation2/Average2. 238
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Fig. 4. Microgels characteristics. (A) Scanning electron micrographs of M25-5 and M25-10 microgels (scale bar is 20 µm), (B) Swelling ratio of M25-5 and M25-10 microgels at 4 and 37 °C. Error bars are means ± SD (n = 3) and *p < 0.05, **p < 0.01.
lysozyme concentrations up to 2000 µg/mg and the loading leveled off at higher concentrations (Fig. 5C). Moreover, loading appeared to be efficient, and about 500 µg of lysozyme could be encapsulated per mg of microgels after washing. Different amounts of dry microgels were
concentration from 250 µg/ml to 4000 µg/ml (Fig. 5B). However, the amount of the loaded protein increased as the protein concentration in the loading solution increased. A linear relation between the loading capacity of M25-5 microgels and lysozyme concentration was found at
Fig. 5. Protein loading capacity of HA-TA microgels. (A) Loading efficiency of HA-TA microgels for lysozyme, IgG, BSA, and TGF-β1. (B) Loading efficiency and (C) amount of protein loaded per mg of microgels at different lysozyme concentrations. A linear relation between µg of encapsulated lysozyme per mg of microgels and initial protein concentration is shown by a dashed line. (D) Loading efficiency of lysozyme loaded at 4 and 37 °C. Error bars were means ± SD (n = 3), **p < 0.01, and ***p < 0.001. 239
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were loaded at 4 °C in the release medium (37 °C) may cause the larger initial burst release. Similar release profiles of lysozyme were obtained from M25-10 microgels (Fig. S2). The release profiles of lysozyme from microgels loaded by absorption (post-fabrication encapsulation) or during fabrication were shown in Fig. 7C. The initial release after one day of incubation was almost 15% higher for the microgels loaded by absorption. The sustained release observed for lysozyme loaded microgels reveals these anionic microgels are an efficient system for the release of cationic proteins over longer periods of time. Although a higher degree of crosslinking might influence the protein uptake and release rates, the degree of substitution of tyramine groups to the hyaluronic acid and polysaccharide conjugate concentrations used are on the upper limit. Moreover, comparing the release profiles of lysozyme and BSA from M25-5 microgels, while they were loaded during microgels fabrication, showed that almost 90% of the BSA, a negatively charged protein, was already released within seven days (Fig. 7D). In the system investigated, the release of lysozyme is mainly controlled by diffusion, especially in the first stages. When the release mechanism is diffusion controlled, the initial burst release is unavoidable. Comparing the release profile of lysozyme to BSA shows that the electrostatic interaction retained the lysozyme within the microgels matrix, and thus provided a sustained release of the cationic model protein. Optical microscopy of M25-5 microgels dispersed in PBS and incubated at 37 oC showed that the microgels started to swell after two weeks and were fully degraded after four weeks (Fig. 7E). Therefore, degradation also played a role and affected the release rate. Swelling of the microgels, as a result of degradation, compensated the effect of the decrease in the concentration gradient and helped to release the entrapped proteins. Considering all these parameters, the encapsulated proteins were release continuously and completely with a steady rate. The present study has focused on developing a proper carrier for delivery of therapeutic molecules. Efficient encapsulation and sustained delivery of proteins are important factors of designing such systems [28]. The ability of hyaluronic acid to swell and its inherent negative charge are the main factors for the high loading of cationic proteins. Moreover, it was shown that the uptake temperature, initial protein concentration, and also the method of protein encapsulation affect significantly the loading and thus the amount of proteins released during time. The HA-TA microgels can be used alone or embedded in scaffolds to increase the multifunctionality of the construct by providing specific proteins like growth factors for tissue engineering applications.
also incubated with a similar concentration of lysozyme (1 mL, 500 µg/ mL). The protein loading efficiency increased from 18% to 80% with the addition of higher amounts (0.5 to 5 mg) of microgels (Fig. S1). The effect of the temperature on the loading efficiency was studied by incubating HA-TA microgels in 1 mL of lysozyme solution (500 µg/ mL) at 4 and 37 °C. The results revealed that the uptake efficiency increased by 10% at a higher temperature which can be ascribed to the higher degree of swelling and a larger diffusion coefficient of the protein [41,42] (Fig. 5D). Zhang et al. [42] also showed the effect of the incubation temperature on the microgel swelling and thus the protein uptake. The other possible reason is that as microgels swell more at higher temperature, the spaces between the polymer chains will increase that means the structure has larger pore sizes which may affect the protein uptake. Based on the results, protein uptake by HA-TA microgels depends on the charge and size of the proteins. The results indicated that the HA-TA microgels efficiently absorb lysozyme as a cationic protein (pI = 11.35) with a MW of 14 kDa and hydrodynamic radius (r) of 1.9 nm [43]. In addition, the uptake of the recombinant human TGF-β1 (25 kDa, pI = 8.83, r = 2.8 nm [14,36]) was evaluated and a loading efficiency of 97% was afforded. Comparing the charge and size of different growth factors like PDGF-BB (24 kDa, pI = 9.8, r = 2.5 nm [44,45]), VEGF (27 kDa, pI = 8.5, r = 3 nm [45,46]), and BMP-2 (32 kDa, pI = 8.4, r = 2.3 nm [47]) to TGF-β1 and lysozyme suggests that HA-TA microgels could be a potential carrier for delivery of such a cationic proteins. 3.5. Protein loading during HA-TA microgels fabrication Protein loading of microgels upon emulsification and subsequent gelation was investigated as an alternative loading method. Lysozyme or BSA was added to a HA-TA (5% w/v) solution with a protein to HATA ratio of 1/5 w/w. The loading efficiency was determined by degradation of the microgels using a hyaluronidase solution (200 U/ml) at 37 °C. The loading efficiency of lysozyme and BSA was 83.7% ± 5.2% and 77.4% ± 9.4%, respectively. To compare this loading method with the previous (incubation method), 5 mg of dry HA-TA microgels were incubated with 1 mL of lysozyme and BSA solution at a concentration of 1 mg/ml overnight (16 h, 37 °C), separately. In the incubation method, loading efficiency of 88.1% ± 2.9% and 4.2% ± 2.1% for lysozyme and BSA was afforded, respectively. The results revealed that both methods gave similar loading efficiencies for lysozyme. However, for BSA the loading efficiency was much higher when the protein was incorporated in the polymer network during the microgel fabrication. The size of the microgels did not change significantly by incorporating the proteins in their structure according to the optical microscopy and Image J analysis (Fig. 6A–C). In independent experiments, FITC-lysozyme and FITC-BSA were loaded in HA-TA microgels during fabrication and the microgels were imaged by an EVOS digital microscope (Invitrogen, Netherlands). A homogeneous distribution of FITC-labled proteins in the microgels was observed (Fig. 6D).
3.7. Cytotoxicity of HA-TA microgels In the enzymatic crosslinking, remaining H2O2 may cause toxic effects to cells when used for implantation [48]. In the present study, the H2O2/TA molar ratio was set to 0.5 which is the theoretical ratio needed for complete conversion of tyramine groups and leading to crosslinking. In previous papers we have shown that such a concentration used imposes no cytotoxic effects on cells. Moreover, any low amounts remaining are likely removed from the microgels in the precipitation and washing steps. In vitro cytotoxicity of HA-TA microgels was investigated by incubation of microgels at different concentrations with human mesenchymal stem cells (hMSCs). As shown by the live-dead assay, hMSCs were approximately 100% viable after 1 day of exposure to microgels (Fig. 8B). Moreover, no significant changes were observed in the metabolic activity of hMSCs cultured at different concentrations of microgels over a period of 7 days in comparison to the control condition in which no micrpgels were used (Fig. 8C).
3.6. In vitro release study The cumulative release profiles from microgels loaded with different amounts of lysozyme (uptake temperature of 4 °C) are shown in Fig. 7A. The overall release trend shows an initial burst release followed by a sustained release over a period of four weeks. The microgels loaded with a larger amount of lysozyme, showed a higher burst release. The release profile of lysozyme from HA-TA microgels at uptake temperatures of 4 °C and 37 °C are shown in Fig. 7B. Protein loading of the microgels were 150 and 180 µg lysozyme per mg of dry microgels at 4 °C and 37 °C, respectively. The release experiments revealed a larger initial burst release of the lysozyme from HA-TA microgels when they were loaded at 4 °C. This shows that lysozyme loading at 37 °C provides easier diffusion into the inner part of the microgels resulting in a lower initial burst release. Moreover, the swelling of the microgels which
4. Conclusion In this study, a novel method was developed to synthesize polysaccharide (HA) based microgels as a platform for protein delivery. 240
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Fig. 6. Microscopic images of loaded and unloaded M25-5 microgels. (A) Unloaded microgels, (B) Lysozyme loaded microgels, and (C) BSA loaded microgels. Scale bares are 100 µm. (D) Distribution of FITCLysozyme and FITC-BSA in HA-TA microgels. The proteins were encapsulated during the microgel fabrication process. Scale bars are 200 µm.
were taken up by the microgels due to electrostatic interactions with the negatively charged microgel, while anionic (BSA) and neutral (IgG) proteins as payload models were not able to adequately diffuse into the microgels. At the higher temperature, the swelling and thereby the
Spherical microgels were prepared by enzymatic crosslinking of HA-TA microdroplets using Span80/Tween80/isooctane as a continuous phase. The resulting microgels were readily dispersible in aqueous solution without aggregation. Cationic proteins such as lysozyme and TGF-β1
Fig. 7. Release kinetics of proteins from M25-5 microgels. (A) Cumulative release of lysozyme from microgels at different protein loadings. (B) Cumulative release of lysozyme from microgels at uptake temperature of 4 °C and 37 °C. (C) Comparison of cumulative release profiles of lysozyme encapsulated by absorption or during microgel fabrication. (D) BSA and lysozyme release profiles. Both proteins were encapsulated during fabrication of the microgels. (E) Degradation of M25-5 microgels incubated at 37 °C. 241
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Fig. 8. Cytotoxicity of HA-TA microgels. (A) Representative images of hMSCs cultured with microgels, (B) Live-dead assay of hMSCs, (C) Metabolic activity of hMSCs cultured with medium containing 1, 5 and 10 mg/mL HA-TA microgels. Scale bar 400 µm.
loading efficiency of the microgels significantly increased. The proteins can be loaded in the microgels by absorption after microgel fabrication or during microgel reparation. The mild crosslinking condition of HATA droplets minimizes damages to loaded proteins. The mild crosslinking conditions, high loading capacity of the cationic proteins and sustained release of the loaded proteins besides the microgel degradability make this system a promising carrier for delivery of the positively charged proteins, such as growth factors, to be used for disease treatment or tissue engineering approaches.
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Acknowledgments This project has received funding from Iranian National Science Foundation (INSF) under grant number 94004082, and the European Union's Horizon 2020 research and innovation program under grant agreement No. 691128. Conflict of interest The authors declare no conflict of interest. Data availability Data will be made available on request. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.eurpolymj.2019.03.032. References [1] S.C. Leeuwenburgh, J. Jo, H. Wang, M. Yamamoto, J.A. Jansen, Y. Tabata, Mineralization, biodegradation, and drug release behavior of gelatin/apatite composite microspheres for bone regeneration, Biomacromolecules 11 (10) (2010) 2653–2659. [2] J.K. Oh, D.I. Lee, J.M. Park, Biopolymer-based microgels/nanogels for drug delivery applications, Prog. Polym. Sci. 34 (12) (2009) 1261–1282.
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Contents lists available at ScienceDirect
European Polymer Journal journal homepage: www.elsevier.com/locate/europolj
Enzymatically crosslinked hyaluronic acid microgels as a vehicle for sustained delivery of cationic proteins
T
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Elaheh Jooybara, Mohammad J. Abdekhodaiea,b, , Abbas Mousavia, Bram Zoetebierc, ⁎ Pieter J. Dijkstrac, a
Department of Chemical Engineering, Sharif University of Technology, PO 11365-11155, Tehran, Iran Environmental Applied Science and Management, Ryerson University, PO 979-5000, Toronto, Canada c MIRA–Institute for Biomedical Technology and Technical Medicine and Department of Developmental BioEngineering, Faculty of Science and Technology, University of Twente, PO Box 217, 7500 AE Enschede, the Netherlands b
A R T I C LE I N FO
A B S T R A C T
Keywords: Microgel Hyaluronic acid Enzymatic crosslinking Inverse microemulsion Protein delivery
In this study, a novel biodegradable hyaluronic acid (HA) based microgel were prepared via enzymatic crosslinking of tyramine conjugated HA (HA-TA) in an inverse microemulsion. HA-TA microdroplets were crosslinked within a few seconds in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The high water content of the polymeric network and the inherent negative charge of the HA-TA microgels provided a suitable platform for encapsulation of cationic proteins like lysozyme and TGF-β1 growth factor. The results demonstrated that lysozyme was released, after an initial burst release, in a suitable sustained manner over a period of four weeks. Both diffusion and degradation due to microgel hydrolysis controlled the release rate. In vitro cytotoxicity of microgels using human mesenchymal stem cells revealed microgels nontoxic. This study demonstrates that the developed injectable HA-TA microgels prepared by enzymatic crosslinking are a promising vehicle for delivery of cationic proteins including some important growth factors.
1. Introduction Microgels are physically or chemically crosslinked hydrophilic polymer networks in micrometer size, which can be used as delivery vehicles for controlled delivery of proteins because of their large surface area, high water content, injectability, and their tunable size [1–6]. Delivery of therapeutic proteins, including growth factors, hormones, enzymes, and antibodies remains still a challenge because of their short half-life in vivo [4,7,8]. Therefore, there is a need to develop carriers for the protection and release of proteins in a spatial and temporal manner [4,6,9]. To this end, microgels may play a significant role by incorporating the biological molecules in their interior polymeric network and modulating the release rate [10]. Microgels are prepared from both synthetic and natural polymers [7]. Polysaccharide-based microgels have received a great deal of interest due to their biocompatibility, biodegradability, and absence of immunogenicity. The presence of functional groups like hydroxyl and carboxyl groups on their molecular chain provides anchors for various chemical modifications [11–15]. Among those, hyaluronic acid (HA),
which is a linear natural glycosaminoglycan present in many tissues, has been widely investigated because of its excellent biocompatibility, biodegradability, and low immunological risks [3,16–19]. Microgels can be fabricated using an inverse emulsion technique. The water-in-oil gelation involves emulsification of a polymer precursor solution in a continuous organic phase with subsequent crosslinking of aqueous micro-droplets [20]. In general, the network of the microgels is formed by physical or electrostatic interactions (e.g. ionic crosslinking) or covalent chemical crosslinking (e.g. free radical reaction). One major concern regarding chemical crosslinking is the presence of cytotoxic reagents or exposure to harsh experimental conditions which may affect the bioactivity of encapsulated proteins [7,21]. To avoid this, enzymatic crosslinking in hydrogel formation has been extensively explored in biomaterial fields [22–24], tissue engineering [24–26] and drug delivery [21,27]. The crosslinked network can be formed under mild conditions in the presence of enzymes, like peroxidases and low concentrations of hydrogen peroxide (H2O2). By changing the amount of the enzyme and H2O2, and also degree of substitution of crosslinkable groups on the polymer backbone, the gelation time may be tuned
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Corresponding authors at: Department of Chemical Engineering, Sharif University of Technology, PO 11365-11155, Tehran, Iran (M.J. Abdekhodaie). MIRA – Institute for Biomedical Technology and Technical Medicine and Department of Developmental BioEngineering, Faculty of Science and Technology, University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands (P.J. Dijkstra). E-mail addresses: [email protected] (M.J. Abdekhodaie), [email protected] (P.J. Dijkstra). https://doi.org/10.1016/j.eurpolymj.2019.03.032 Received 2 October 2018; Received in revised form 25 February 2019; Accepted 18 March 2019 Available online 19 March 2019 0014-3057/ © 2019 Elsevier Ltd. All rights reserved.
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The polymer with NHS activated COOH groups (1.0 g) was dissolved in 30 mL of MES buffer (0.1 M, pH 6.1). Tyramine (385 mg, 2.8 mmol) was dissolved in 4 mL of DMF and added to the solution. After overnight stirring, the resulting solution was precipitated in cold ethanol, filtered and washed with ethanol and finally diethyl ether. The resulting white powder was dissolved in MilliQ-water and dialyzed (Pre-wetted RC tubing, Spectrum Labs, MWCO 1000), first against 100 mM sodium chloride solution for 2 days, followed by a mixture of distilled water and ethanol (5:1) for one day and MilliQ-water for another day. The HA-TA conjugate was obtained after lyophilization. The degree of substitution (DS), which is defined as the number of substituted groups per 100 disaccharide units, was calculated from 1H NMR (D2O) spectral data by comparing the integral values of the aromatic protons of tyramine (6.86 and 7.17 ppm) and the methyl protons of HA (1.9 ppm). 2.3. HA-TA microgels fabrication Hyaluronic acid tyramine conjugate (HA-TA) was used to prepare microgels using a water in oil emulsion method. In a typical experiment, 2 mL of HA-TA solution was mixed with a solution of HRP in PBS to give a mg HRP/mmol TA ratio of 0.6 (final concentration of HRP was 1.5 U/ml). The solution was added dropwise to 25 mL of isooctane containing 2.5% v/v Span 80 and 0.25% v/v Tween 80 as surfactants, and the resulting mixture was at 0 °C homogenized using an UltraTurrax (T25, IKA Works Inc., USA) at a predetermined speed. Subsequently, 20 µL of a H2O2 solution in PBS was added to the microemulsion to give a H2O2/TA molar ratio of 0.5. After 10 min homogenization, the mixture was magnetically stirred for 30 min to complete the crosslinking of the microgels. Particles were collected by precipitation in ice-cold acetone, followed by centrifugation at 8000 rpm for 5 min. Particles were subsequently washed with acetone and ethanol and were dried under reduced pressure at room temperature. The production yield was determined as the mass ratio of the produced dried microgel to the initial solid polymer used.
Scheme 1. Schematic representation of synthesizing HA-TA microgels via the HRP catalyzed crosslinking of an inverse microemulsion. A HA-TA solution with HRP was added to isooctane and homogenized·H2O2 was added to the microemulsion to crosslink the microdroplets.
[28–30]. Recently, the enzymatic crosslinking was used to fabricate cell-laden microgels and the results indicated that cells remained viable and functional [31,32]. In this study, a new method was developed to synthesize microgels for protein delivery by enzymatic crosslinking of tyramine conjugated HA (Scheme 1). The microgels were made in the presence of horseradish peroxidase and H2O2 in an inverse microemulsion. Protein loading was investigated by either post-fabrication encapsulation or loading during fabrication and prior to gelation. Also, the effects of different factors on the protein loading capacity and the release rate from fabricated microgels were studied and discussed. Finally, the cytotoxicity of the microgels was determined by the Prestoblue and live/ dead assays.
2.4. Microgel characterization Microgel size and morphology: The microgels size in the swollen state was determined by dispersion in water and imaging using a phase contrast microscopy (Nikon Eclipse TE300 microscope equipped with Nikon Digital Sight DS-fi1 camera). Images were analyzed by using Image J software. The morphology of the microgels was analyzed using a FEI Quanta 650 ESEM, equipped with a Peltier cooling stage for sample and humidity (gas pressure) control, using an acceleration voltage of 10 kV. Dry microgels were placed on a SEM mounting stud and hydrated by stepwise increasing the pressure at 3 °C. Once fully hydrated, the sample was dried by reducing the pressure. Microgel swelling: To 3 mg of dried microgels placed in a 2 mL Eppendorf vial, 1 mL of PBS (10 mM, pH = 7.4) was added. The particles were dispersed using a shaker and incubated overnight at either 4 °C or 37 °C with continuous shaking. Microgels were centrifuged at 7000 rpm for 10 min and the excess water was discarded and blotted from vials and microgel pellet. The swelling is expressed as the ratio of weight in the swollen state to dry weight of microgels (Ws/Wd).
2. Materials and methods 2.1. Materials Sodium hyaluronate (MW = 25 kDa) was purchased from Contipro Pharma, the Czech Republic. Tyramine (TA) (99%), anhydrous N,Ndimethylformamide (DMF) (99.8%), N-(3-dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDAC), N-hydroxysuccinimide (NHS), hydrogen peroxide (H2O2), horseradish peroxidase (HRP, 325 units/mg solid), Hyaluronidase from bovine testes (lyophilized powder, 400–1000 units/mg) and MES hemisodium salt dry powder were obtained from Sigma-Aldrich and used without further purification. Phosphate buffered saline (PBS, 10 mM, pH 7.4) was purchased from Lonza. All other solvents were purchased from Biosolve (Valkenswaard, The Netherlands) and used as received. 2,2,4-Trimethylpentane (isooctane) was purchased from Honeywell. Tween 80, Span 80, lysozyme from chicken egg white, bovine serum albumin (BSA) and immunoglobulin G (IgG), FITC- BSA, and FITC-lysozyme were purchased from Sigma-Aldrich. Human TGF-β1 was from R&D systems.
2.5. Protein loading and release Three model proteins, lysozyme (14 kDa, pI = 11.35 [33]), bovine serum albumin (BSA, 60 kDa, pI = 4.7 [34]) and immunoglobulin G (IgG, 150 kDa, pI = 7.2 [35]) were used to investigate the protein loading capacity of the HA-TA microgel. The protein uptake was determined at two different incubation temperatures, 4 °C and 37 °C. In brief, 2 mg of dry microgels were placed in a 2 mL microcentrifuge tube and incubated with 1 mL of protein solution at a concentration of 500 µg/mL. After 16 h incubation on an orbital shaker, the microgels were centrifuged (7000 rpm, 10 min) and the supernatant was removed
2.2. Hyaluronic acid tyramine (HA-TA) synthesis Typically, sodium hyaluronate with the molecular weight (MW) of 25 kDa (1.0 g, 2.5 mmol of COOH groups) was dissolved in 30 mL of PBS. EDAC (1.0 g, 5.2 mmol) and NHS (1.3 g, 11.3 mmol) were added and the solution was stirred for 2 h. The product was precipitated in cold ethanol, filtered and washed with ethanol and finally diethyl ether. 235
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Fig. 1. Hyaluronic acid tyramine (HA-TA) synthesis. (A) Synthetic scheme used in the conjugation of tyramine to hyaluronic acid. First, HA carboxylic acid groups were activated by conversion to NHS esters. In the second step, NHS esters were reacted with tyramine, (B) 1H NMR spectra of hyaluronic acid and HA-TA (solvent D2O).
was calculated as the amount of protein determined after microgel degradation to initial amount of protein added to the polymer solution * 100. The release of model proteins into PBS (10 mM, pH = 7.4) containing 0.05% w/v sodium azide (to prevent bacterial growth) was monitored over a period of 28 days. At selected time points, the microgels were collected, centrifuged at 7000 rpm for 10 min, the supernatant was removed and replaced with an equal amount of fresh medium. The release samples were stored at −20 °C for further analysis on protein content using a Bradford assay.
and analyzed for its protein content using a Bradford Assay (Coomassie protein assay kit, Thermo scientific). The absorbance was measured at 595 nm. Unloaded microgels were used as a negative control. To further investigate the protein uptake of the microgels, 2 mg of dry microgels were incubated with 1 mL of lysozyme solution with concentrations ranging from 250 to 4000 µg/mL and subsequent steps were performed as mentioned above. To evaluate the uptake of TGF-β1 (25 kDa, pI = 8.83 [36]), as a representative growth factor, another experiment was performed. To 5 mg of HA-TA microgels, 1 mL of human TGF-β1 solution (100 ng/ml containing 0.1% (w/v) BSA) was added and the resulting suspension was incubated overnight. BSA was added to prevent the unwanted attachment of the growth factor to the wall of the vial. The microgels were centrifuged and the amount of remaining TGF-β1 in the supernatant was measured using a Quantikine ELISA Kit (R&D Systems, Minneapolis, MN). To evaluate protein loading the supernatant was collected and the amount of unloaded growth factor was quantified. Protein loading was also studied by dissolving the proteins in HA-TA precursor solutions prior to microgel fabrication and network formation. In a typical example, 10 mg of protein was added to 1 mL of a 5% w/v HA-TA solution and mixed (protein/HA-TA ratio is 1/5 w/w). The HA-TA microgels were prepared as described above. To determine the encapsulation efficiency, the matrix was degraded overnight using 1 mL of a hyaluronidase solution (200 U/ml) at 37 °C and the protein content was determined using a Bradford assay. The encapsulation efficiency
2.6. In vitro cytotoxicity Cytotoxicity of microgels was studied using human mesenchymal stem cells (hMSCs) at passage 4. Cells were cultured at a density of 1000 cells/cm2 in a 48 well plate. Different amounts of microgels were dispersed in the culture medium (α-MEM (Gibco) supplemented with 10% fetal bovine serum, 1% L-glutamine (Gibco), 0.2 mM ascorbic acid (Gibco), 100 U/mL penicillin, 10 µg/mL streptomycin) and 0.5 mL of the suspension was added to each well. The microgels were sterilized before use using 70% ethanol followed by several washes with PBS. Cell viability was assayed by a live/dead assay Kit (Invitrogen) after one day. The cells were rinsed with PBS twice and then stained with calcein AM/ethidium homodimer according to the manufacturer’s instructions. Cells were visualized using a fluorescence microscope (EVOS FL). 236
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Living cells fluoresce green and the nuclei of dead cells red. Additionally, the metabolic activity of the cells, cultured with and without microgels, was measured using a Prestoblue assay (Invitrogen). Briefly, cells were incubated with a 10% solution of Prestoblue in culture medium for 2 h. The absorbance was measured using a microplate reader at 530 nm excitation and 590 nm emission. Data are presented as average ± SD.
HA-TA hydrogel formation by an enzyme-mediated oxidation reaction is schematically presented in Fig. 2A. Prior to emulsification and microgel formation, the gelation times of HA-TA solutions by addition of Horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) was studied. Gelation times, at a fixed H2O2/TA molar ratio of 0.5 and various HA-TA concentrations are presented in Fig. 2B. The results showed that with increasing HRP concentrations the gelation times decreased and ranged from 400 to 2 s. Microgels were prepared through crosslinking of HRP containing HA-TA microdroplets in isooctane (Scheme 1). The inverse microemulsion was stabilized with both Span 80 and Tween 80 as surfactants. To provide a fast gelation of the microdroplets, below 10 s, the ratio of mg HRP/mmol TA was fixed at 0.6. Addition of H2O2 to the microemulsion at a H2O2/TA molar ratio of 0.5, which is needed theoretically for the reaction of all tyramine groups, triggered the crosslinking of the HA-TA microdroplets. The microgels were isolated as fine powders that could easily disperse in water. The production yield of the HA-TA microgels was almost 78%.
2.7. Statistical analysis Each experiment was performed in triplicate. The results are presented as the mean ± standard deviation (SD). Experimental data were analyzed for statistical significance using a one-way analysis of variance (ANOVA) with Turkey’s post hoc analysis. Statistical significance was set to *p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001. 3. Results and discussion 3.1. Hyaluronic acid tyramine synthesis and microgel fabrication
3.2. Microgel size and distribution HA-TA conjugates were synthesized in a two-step procedure (Fig. 1A). First, part of the carboxylic acid groups of the hyaluronic acid were activated by conversion to NHS esters. The degree of substitution of carboxylic acid groups as determined from the 1H NMR spectrum was 24%. In the second step, the NHS esters were reacted with tyramine to provide the HA-TA. According to the 1H NMR spectral data, the degree of substitution calculated from the integral ratio of the tyramine phenolic protons and the hyaluronic acid methyl protons was 17% (Fig. 1B).
To determine the size of the microgels in the swollen state, they were dispersed in the PBS (10 mM, pH = 7.4) and imaged by phase contrast microscopy (Fig. 3). The crosslinked microgels were stable in an aqueous solution. The size distribution was analyzed using Image J software and the histograms of different microgels, are shown in Fig. 3. The average microgels size and polydispersity of the microgels are presented in Table 1. By decreasing the polymer concentration in the initial solution from 10% to 5% w/v, the microgels average diameter
Fig. 2. Gelation of HA-TA solution. (A) Tyramine conjugated hyaluronic acid is crosslinked by Horseradish peroxidase (HRP) in the presence of H2O2. (B) Gelation times of HA-TA solutions in PBS at different concentrations (% w/v) and mgHRP/mmolTA ratios. The H2O2/TA molar ratio was 0.5. 237
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Fig. 3. Microscopic images and size distribution of HA-TA microgels. (A) M25-5, (B) M25-10 microgels. Scale bars are 100 µm.
centrifugation from the initial amount of protein. It appeared that lysozyme was loaded in the microgel with a good efficiency (59.9% ± 1.1%). In contrast, IgG and BSA were not significantly depleted from the feed solution and low loading efficiencies of 6.5% ± 1.1% and 5.4% ± 2.9%, respectively, were found (Fig. 5A). At pH 7.4, the lysozyme is positively charged (pI = 11.35) providing electrostatic interaction with the negatively charged carboxylic acid groups of the HA-TA (HA pI = 2.5 [37]). Conversely, BSA as a negatively charged protein could not penetrate into the microgel network because of the repulsive forces between the protein and microgel. Because IgG is a neutral protein at the physiological condition, the repulsive force is not the reason for its inefficient loading, and therefore, the low protein uptake is possibly due to the protein size (r = 5.29 nm, 150 kDa) [38]. Similar experiments performed with M25-10 microgels afforded the same results. The role of the electrostatic interaction on the protein uptake into the microgels was also shown by Nguyen et al. [39] and Pepe at al. [40]. To further investigate the protein loading of the microgels a series of experiments were performed. To study the effect of the initial protein concentration on the loading efficiency, at an uptake temperature of 4 °C, 2 mg of M25-5 microgels was incubated in 1 mL of a lysozyme solution at various concentrations ranging from 250 to 4000 µg/mL. After protein loading, the microgels were separated by centrifugation and washed with 1 mL of PBS to remove adsorbed proteins from the surface of the microgels. The loading efficiency of lysozyme in the HATA microgels decreased from 70% to 50% by increasing the solution
decreased from 8.8 µm to 4.7 µm. Moreover, the polydispersity index (PDI) value of the M25-10 microgels (0.73) was much higher compared to the M25-5 sample (0.13). This can be attributed to the viscosity of the aqueous solution as higher concentrations in the feed increase the viscosity of microemulsions which leads to a less efficient emulsification. The effect of the polymer concentration and thus the solution viscosity on the microgel size was also shown by Jia et al. [3]. 3.3. Morphological analysis and equilibrium swelling ratio SEM micrographs of M25-5 and M25-10 microgels showed a perfectly spherical shape and a smooth surface (Fig. 4A). Only the surface of large microgels present in the M25-10 sample appeared somewhat irregular which could be related to shrinkage upon drying for imaging. The equilibrium swelling ratio of microgels incubated at pH 7.4 was lower for microgels prepared at a higher HA-TA concentration because of more densely crosslinked network formed (Fig. 4B). Additionally, the microgels swelling at 37 °C was significantly higher than that at 4 °C. 3.4. Protein loading capacity of HA-TA microgels Lysozyme, BSA, and IgG were used as model proteins to investigate the protein uptake efficiency of M25-5 microgels. In preliminary experiments, 2 mg of HA-TA microgels were incubated in a solution containing 500 µg of the protein. The amount of loaded proteins was determined by subtracting the protein content in the supernatant after Table 1 Average diameters and polydispersity of HA-TA microgels. Sample
Concentration % w/v
Stirring speed rpm
Surfactanta (Span 80) % v/v
Diameter µm
PDIb
M25-5 M25-10
5 10
13,500 13,500
2.5 2.5
4.7 ± 1.7 8.8 ± 7.5
0.13 0.73
a b
The ratio of Span 80 to Tween 80 was 10:1 v/v. The Polydispersity Index was calculated as Standard deviation2/Average2. 238
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Fig. 4. Microgels characteristics. (A) Scanning electron micrographs of M25-5 and M25-10 microgels (scale bar is 20 µm), (B) Swelling ratio of M25-5 and M25-10 microgels at 4 and 37 °C. Error bars are means ± SD (n = 3) and *p < 0.05, **p < 0.01.
lysozyme concentrations up to 2000 µg/mg and the loading leveled off at higher concentrations (Fig. 5C). Moreover, loading appeared to be efficient, and about 500 µg of lysozyme could be encapsulated per mg of microgels after washing. Different amounts of dry microgels were
concentration from 250 µg/ml to 4000 µg/ml (Fig. 5B). However, the amount of the loaded protein increased as the protein concentration in the loading solution increased. A linear relation between the loading capacity of M25-5 microgels and lysozyme concentration was found at
Fig. 5. Protein loading capacity of HA-TA microgels. (A) Loading efficiency of HA-TA microgels for lysozyme, IgG, BSA, and TGF-β1. (B) Loading efficiency and (C) amount of protein loaded per mg of microgels at different lysozyme concentrations. A linear relation between µg of encapsulated lysozyme per mg of microgels and initial protein concentration is shown by a dashed line. (D) Loading efficiency of lysozyme loaded at 4 and 37 °C. Error bars were means ± SD (n = 3), **p < 0.01, and ***p < 0.001. 239
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were loaded at 4 °C in the release medium (37 °C) may cause the larger initial burst release. Similar release profiles of lysozyme were obtained from M25-10 microgels (Fig. S2). The release profiles of lysozyme from microgels loaded by absorption (post-fabrication encapsulation) or during fabrication were shown in Fig. 7C. The initial release after one day of incubation was almost 15% higher for the microgels loaded by absorption. The sustained release observed for lysozyme loaded microgels reveals these anionic microgels are an efficient system for the release of cationic proteins over longer periods of time. Although a higher degree of crosslinking might influence the protein uptake and release rates, the degree of substitution of tyramine groups to the hyaluronic acid and polysaccharide conjugate concentrations used are on the upper limit. Moreover, comparing the release profiles of lysozyme and BSA from M25-5 microgels, while they were loaded during microgels fabrication, showed that almost 90% of the BSA, a negatively charged protein, was already released within seven days (Fig. 7D). In the system investigated, the release of lysozyme is mainly controlled by diffusion, especially in the first stages. When the release mechanism is diffusion controlled, the initial burst release is unavoidable. Comparing the release profile of lysozyme to BSA shows that the electrostatic interaction retained the lysozyme within the microgels matrix, and thus provided a sustained release of the cationic model protein. Optical microscopy of M25-5 microgels dispersed in PBS and incubated at 37 oC showed that the microgels started to swell after two weeks and were fully degraded after four weeks (Fig. 7E). Therefore, degradation also played a role and affected the release rate. Swelling of the microgels, as a result of degradation, compensated the effect of the decrease in the concentration gradient and helped to release the entrapped proteins. Considering all these parameters, the encapsulated proteins were release continuously and completely with a steady rate. The present study has focused on developing a proper carrier for delivery of therapeutic molecules. Efficient encapsulation and sustained delivery of proteins are important factors of designing such systems [28]. The ability of hyaluronic acid to swell and its inherent negative charge are the main factors for the high loading of cationic proteins. Moreover, it was shown that the uptake temperature, initial protein concentration, and also the method of protein encapsulation affect significantly the loading and thus the amount of proteins released during time. The HA-TA microgels can be used alone or embedded in scaffolds to increase the multifunctionality of the construct by providing specific proteins like growth factors for tissue engineering applications.
also incubated with a similar concentration of lysozyme (1 mL, 500 µg/ mL). The protein loading efficiency increased from 18% to 80% with the addition of higher amounts (0.5 to 5 mg) of microgels (Fig. S1). The effect of the temperature on the loading efficiency was studied by incubating HA-TA microgels in 1 mL of lysozyme solution (500 µg/ mL) at 4 and 37 °C. The results revealed that the uptake efficiency increased by 10% at a higher temperature which can be ascribed to the higher degree of swelling and a larger diffusion coefficient of the protein [41,42] (Fig. 5D). Zhang et al. [42] also showed the effect of the incubation temperature on the microgel swelling and thus the protein uptake. The other possible reason is that as microgels swell more at higher temperature, the spaces between the polymer chains will increase that means the structure has larger pore sizes which may affect the protein uptake. Based on the results, protein uptake by HA-TA microgels depends on the charge and size of the proteins. The results indicated that the HA-TA microgels efficiently absorb lysozyme as a cationic protein (pI = 11.35) with a MW of 14 kDa and hydrodynamic radius (r) of 1.9 nm [43]. In addition, the uptake of the recombinant human TGF-β1 (25 kDa, pI = 8.83, r = 2.8 nm [14,36]) was evaluated and a loading efficiency of 97% was afforded. Comparing the charge and size of different growth factors like PDGF-BB (24 kDa, pI = 9.8, r = 2.5 nm [44,45]), VEGF (27 kDa, pI = 8.5, r = 3 nm [45,46]), and BMP-2 (32 kDa, pI = 8.4, r = 2.3 nm [47]) to TGF-β1 and lysozyme suggests that HA-TA microgels could be a potential carrier for delivery of such a cationic proteins. 3.5. Protein loading during HA-TA microgels fabrication Protein loading of microgels upon emulsification and subsequent gelation was investigated as an alternative loading method. Lysozyme or BSA was added to a HA-TA (5% w/v) solution with a protein to HATA ratio of 1/5 w/w. The loading efficiency was determined by degradation of the microgels using a hyaluronidase solution (200 U/ml) at 37 °C. The loading efficiency of lysozyme and BSA was 83.7% ± 5.2% and 77.4% ± 9.4%, respectively. To compare this loading method with the previous (incubation method), 5 mg of dry HA-TA microgels were incubated with 1 mL of lysozyme and BSA solution at a concentration of 1 mg/ml overnight (16 h, 37 °C), separately. In the incubation method, loading efficiency of 88.1% ± 2.9% and 4.2% ± 2.1% for lysozyme and BSA was afforded, respectively. The results revealed that both methods gave similar loading efficiencies for lysozyme. However, for BSA the loading efficiency was much higher when the protein was incorporated in the polymer network during the microgel fabrication. The size of the microgels did not change significantly by incorporating the proteins in their structure according to the optical microscopy and Image J analysis (Fig. 6A–C). In independent experiments, FITC-lysozyme and FITC-BSA were loaded in HA-TA microgels during fabrication and the microgels were imaged by an EVOS digital microscope (Invitrogen, Netherlands). A homogeneous distribution of FITC-labled proteins in the microgels was observed (Fig. 6D).
3.7. Cytotoxicity of HA-TA microgels In the enzymatic crosslinking, remaining H2O2 may cause toxic effects to cells when used for implantation [48]. In the present study, the H2O2/TA molar ratio was set to 0.5 which is the theoretical ratio needed for complete conversion of tyramine groups and leading to crosslinking. In previous papers we have shown that such a concentration used imposes no cytotoxic effects on cells. Moreover, any low amounts remaining are likely removed from the microgels in the precipitation and washing steps. In vitro cytotoxicity of HA-TA microgels was investigated by incubation of microgels at different concentrations with human mesenchymal stem cells (hMSCs). As shown by the live-dead assay, hMSCs were approximately 100% viable after 1 day of exposure to microgels (Fig. 8B). Moreover, no significant changes were observed in the metabolic activity of hMSCs cultured at different concentrations of microgels over a period of 7 days in comparison to the control condition in which no micrpgels were used (Fig. 8C).
3.6. In vitro release study The cumulative release profiles from microgels loaded with different amounts of lysozyme (uptake temperature of 4 °C) are shown in Fig. 7A. The overall release trend shows an initial burst release followed by a sustained release over a period of four weeks. The microgels loaded with a larger amount of lysozyme, showed a higher burst release. The release profile of lysozyme from HA-TA microgels at uptake temperatures of 4 °C and 37 °C are shown in Fig. 7B. Protein loading of the microgels were 150 and 180 µg lysozyme per mg of dry microgels at 4 °C and 37 °C, respectively. The release experiments revealed a larger initial burst release of the lysozyme from HA-TA microgels when they were loaded at 4 °C. This shows that lysozyme loading at 37 °C provides easier diffusion into the inner part of the microgels resulting in a lower initial burst release. Moreover, the swelling of the microgels which
4. Conclusion In this study, a novel method was developed to synthesize polysaccharide (HA) based microgels as a platform for protein delivery. 240
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Fig. 6. Microscopic images of loaded and unloaded M25-5 microgels. (A) Unloaded microgels, (B) Lysozyme loaded microgels, and (C) BSA loaded microgels. Scale bares are 100 µm. (D) Distribution of FITCLysozyme and FITC-BSA in HA-TA microgels. The proteins were encapsulated during the microgel fabrication process. Scale bars are 200 µm.
were taken up by the microgels due to electrostatic interactions with the negatively charged microgel, while anionic (BSA) and neutral (IgG) proteins as payload models were not able to adequately diffuse into the microgels. At the higher temperature, the swelling and thereby the
Spherical microgels were prepared by enzymatic crosslinking of HA-TA microdroplets using Span80/Tween80/isooctane as a continuous phase. The resulting microgels were readily dispersible in aqueous solution without aggregation. Cationic proteins such as lysozyme and TGF-β1
Fig. 7. Release kinetics of proteins from M25-5 microgels. (A) Cumulative release of lysozyme from microgels at different protein loadings. (B) Cumulative release of lysozyme from microgels at uptake temperature of 4 °C and 37 °C. (C) Comparison of cumulative release profiles of lysozyme encapsulated by absorption or during microgel fabrication. (D) BSA and lysozyme release profiles. Both proteins were encapsulated during fabrication of the microgels. (E) Degradation of M25-5 microgels incubated at 37 °C. 241
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Fig. 8. Cytotoxicity of HA-TA microgels. (A) Representative images of hMSCs cultured with microgels, (B) Live-dead assay of hMSCs, (C) Metabolic activity of hMSCs cultured with medium containing 1, 5 and 10 mg/mL HA-TA microgels. Scale bar 400 µm.
loading efficiency of the microgels significantly increased. The proteins can be loaded in the microgels by absorption after microgel fabrication or during microgel reparation. The mild crosslinking condition of HATA droplets minimizes damages to loaded proteins. The mild crosslinking conditions, high loading capacity of the cationic proteins and sustained release of the loaded proteins besides the microgel degradability make this system a promising carrier for delivery of the positively charged proteins, such as growth factors, to be used for disease treatment or tissue engineering approaches.
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