Recombinant Spider Silk Hydrogels for Sustained Release of

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Controlled Release and Delivery Systems

Multifunctional recombinant spider silk hydrogels for sustained release of biologicals Sushma Kumari, Hendrik Bargel, Mette Anby, Davide Lafargue, and Thomas Scheibel ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00382 • Publication Date (Web): 03 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

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ACS Biomaterials Science & Engineering

Recombinant spider silk hydrogels for sustained release of biologicals Sushma Kumari,† Hendrik Bargel,† Mette U. Anby,‡,ɸ David Lafargue,‡ and Thomas Scheibel*,†,§,¥,#,┴,Θ †Lehrstuhl Biomaterialien, Fakultät für Ingenieurwissenschaften, Universität Bayreuth, Universitätsstraße 30, 95440 Bayreuth, Germany §

Bayreuther Zentrum für Kolloide und Grenzflächen (BZKG), ¥Bayerisches Polymerinstitut

(BPI), #Bayreuther Zentrum für Bio-Makromoleküle (bio-mac), ┴Bayreuther Zentrum für Molekulare Biowissenschaften (BZMB), ΘBayreuther Materialzentrum (BayMAT), Universität Bayreuth, Universitätsstraße 30, 95440 Bayreuth, Germany §

Corresponding author: [email protected]

‡

Technologie Servier, 25/27 rue Eugène Vignat, 45000 ORLEANS, France

ɸ

H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark

ACS Paragon Plus Environment

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ABSTRACT

Therapeutic biologics (i.e. proteins) have been widely recognized for the treatment, prevention and cure of a variety of human diseases and syndromes. However, design of novel protein delivery systems to achieve a non-toxic, constant and efficient delivery with minimal doses of therapeutic biologics is still challenging. Here, recombinant spider silk-based materials are employed as a delivery system for the administration of therapeutic biologicals. Hydrogels made of the recombinant spider silk protein eADF4(C16) were used to encapsulate the model biologicals BSA, HRP and LYS by direct loading or through diffusion, and their release was studied. Release of model biologicals from eADF4(C16) hydrogels is in part dependent on the electrostatic interaction between the biological and the recombinant spider silk protein variant used. In addition, tailoring the pore sizes of eADF4(C16) hydrogels strongly influenced the release kinetics. In a second approach a particles-in-hydrogel system was used, showing a prolonged release in comparison to that of plain hydrogels (from days to week). The particleenforced spider silk hydrogels are injectable and can be 3D printed. These initial studies indicate the potential of recombinant spider silk proteins to design novel injectable hydrogels that are suitable for delivering therapeutic biologics.

KEYWORDS: protein delivery, biologics, recombinant spider silk proteins, hydrogels, particles


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INTRODUCTION Proteins are fundamental components of virtually all biological systems.1,2 Several proteins can be used as potential biotherapeutics in the treatment of miscellaneous human diseases3 including cancer therapy,4 vaccination5 and autoimmune diseases.6 Therapeutic biologics have advantages over low molecular weight drugs in performing highly specific and complex functions, minimal interference with normal biological processes and in lowering the immune response.1,7 However, the efficacy of therapeutic biologics is impeded by deliveryrelated issues such as in vitro instability, immunogenicity and shorter half-lives.2 Therefore, in recent years drug delivery systems and depot materials for biologics have attracted a great deal of research interest in the pharmaceutical and health industry.8-10 Formulation systems, such as micro and nanoparticles,11 lipid-based assemblies,12 as well as implantable hydrogels13 and polymer matrix delivery systems14 are another means to help and overcome the current limitations of applying protein therapeutics. In particular, injectable hydrogels are promising site-specific protein carrier for sustained release and have gained intensive attention, as their application reduces the administration time and side effects.15-17 Therapeutic proteins mixed with hydrogels can be easily injected into the subcutaneous tissue or target site and act as therapeutic protein release depot. Hydrogels are three-dimensional polymer networks with high water content over 95%, soft consistency and insolubility due to the physical or chemical crosslinking of individual polymer chains. Moreover, the water-rich environment in hydrogels is favorable in protecting the biomolecules from premature degradation, enhances their solubility, and improves their therapeutic efficacy with least possible side effects, thus avoiding frequent administration to lower the overall drug dosage.13 Properties of hydrogels can be easily tailored to influence the


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release kinetics of the encapsulated biologics, for example adjusting the hydrogel pore size.18,19 Bio-as well as synthetic polymers have been used to prepare hydrogels.13 However, biopolymers such as polysaccharides and protein-based hydrogels are in the focus of research because of their bioavailability, enhanced biocompatibility, highly porous structure, good hydrophilicity, and controllable biodegradability with low toxicity.20,23 Despite the intense research conducted on myriad biopolymers, only a limited number of protein carrier are available. It is unlikely that a single system can accomplish all the challenges of protein delivery, and therefore the exploration of new biomaterials for safe and effective protein delivery systems is necessary.13 In previous studies, the recombinant spider silk protein eADF4(C16) has been established as a promising biopolymer for the preparation of injectable hydrogels and bioinks.24-26 Hydrogels made of recombinant spider silk proteins are physically cross-linked through β-sheet structures, hydrophobic interactions, and physical entanglement. In addition, the morphology and average pore size of spider silk hydrogels can be modulated by simply varying the protein concentration prior to gelation and by other means of protein functionalization.24 Furthermore, eADF4(C16) hydrogels have been thoroughly investigated for proteolytic degradation in the presence collagenase IA (CHC) and protease mix XIV (PXIV), with 1300 x higher protease concentration than the naturally occurring wound proteases. The results show that 91 % of hydrogel degraded in the presence of PXIV within 4 days, whereby 53 % of the hydrogel was degraded with CHC in 15 days.27 The polyanionic eADF4(C16) is a recombinantly produced spider silk protein consisting of 16 repeats of a module named C (sequence: GSSAAAAAAAASGPGGYGPENQGPSGPGG YGPGGP) mimicking the consensus sequence of the repetitive part of the dragline silk protein ADF4 of the European garden spider (Araneus diadematus).28,29 To date, materials made of


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eADF4(C16) have been explored as promising candidates for biomedical applications,30-32 due to their excellent biocompatibility, mechanical flexibility, absence of toxicity and low/non-immune reactivity. Furthermore, materials based on recombinant spider silk proteins such as films,33,34 non-woven mats,35 hydrogels,24 capsules,36,37 and particles38,39 and their use as biomaterials have been studied in detail. In the context of drug delivery, eADF4(C16) particles which have anionic surface charges have been used as carrier for positively and neutrally charged drugs.40-43 In contrast, particles made of a polycationic variant, eADF4(κ16), were used for loading negatively charged drugs or oligonucleotides.43 Such spider silk particles can be up-taken by cells for intracellular drug release.44,45 Herein, we developed a new protein carrier system based on eADF4(C16) hydrogels which offer interesting advantages, such as (i) encapsulation of proteins under mild formulation conditions, (ii) exclusively preparation in aqueous media, (iii) evasion of toxic organic solvents and crosslinking agents (iv) and no need of elevated temperature or physical pressure. This set up helps to preserve the native structure of the loaded protein and its activity. A range of model biologicals such as bovine serum albumin (BSA), horseradish peroxidase (HRP) and lysozyme (LYS) with different molecular weights (MW) and isoelectric points (pI) were chosen, and encapsulated directly or by diffusion in eADF4(C16) hydrogels, and further their release profiles were examined. To confirm the released protein structure and function, model proteins were analyzed by using circular dichroism (CD) and fluorescent spectroscopy before and after release. Bioactivity assays were performed to indicate that the structural integrity and bioactivity of encapsulated proteins are maintained and preserved by the hydrogels. Furthermore, eADF4(C16) hydrogels prepared with different pore sizes have a strong influence on the release kinetics of encapsulated biologicals. To further extend the biological release, spider silk particles were


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loaded with biologicals and implemented in eADF4(C16) hydrogels. Finally, the versatility of such particle-loaded eADF4(C16) hydrogels was explored by 3D printing.

MATERIALS AND METHODS Biologicals: Lysozyme (LYS) from chicken egg-white, bovine serum albumin (BSA) and horseradish peroxidase (HRP) were obtained from Sigma-Aldrich. eADF4(C16) was purchased from AMSilk GmbH (Planegg/ München, Germany). eADF4(κ16) was produced and purified as described previously.28,43 Micrococcus luteus (No. 20030) was obtained from DSMZ, Braunschweig, Germany. All other chemicals and solvents were purchased from Roth (Karlsruhe, Germany) in analytical grade and were used as received without further purification. In all the experiments, ultrapure water from a Milli-Q-system (Merck Millipore, Darmstadt, Germany) was used. Preparation of hydrogels: Lyophilized eADF4(C16) was dissolved in 6 M guanidinium thiocyanate (GdmSCN) at 5 mg/mL and dialyzed against 10 mM Tris/HCl, pH 7.5 overnight at room temperature using dialysis membranes with a molecular weight cut-off of 6000-8000 Da. Subsequent dialysis against 25% w/v poly(ethylene glycol) (PEG, 20,000 g/mol), 10mM Tris/HCl, pH 7.5 at a volume ratio of PEG/eADF4(C16) of 100:1 was used to remove water by osmotic stress and to prepare high concentration protein solutions with 30-50 mg/mL (3-5 % w/v) as described previously.24 Direct loading method: eADF4(C16) hydrogel loading with BSA, HRP and LYS was done by mixing the respective biologicals with a spider silk solution followed by silk self-assembly. Solutions at a concentration of 4% w/v eADF4(C16) were gently mixed with biologicals at the


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molar ratio of 3:1 (silk protein / biological), BSA ( ̴1.9 mg / 100 µL silk solution), HRP ( ̴1.2 mg / 100 µL silk solution) and LYS ( ̴0.4 mg / 100 µL silk solution). The hydrogels self-assembled upon incubation overnight at 37 °C.24 Diffusion method: eADF4(C16) hydrogels were loaded with LYS through diffusion into preformed spider silk hydrogels. 4% w/v eADF4(C16) hydrogels (100 µL) were incubated with biological at the molar ratio of 1:2 (silk protein / biological), LYS ( ̴2.4 mg/mL, 10 mM Tris/HCl, pH 7.5) at room temperature for 24 h. The concentration of LYS was determined in the supernatant using UV/Vis spectroscopy. The loading efficiency was calculated using Eq. (1).

Loading efficiency % =

Amount of biological in hydrogel x 100 Amount of biological added

Preparation of spider silk particles: eADF4(C16) or eADF4(κ16) spider silk protein solutions (20 nmol) in Tris buffer were mixed at a 1:1 ratio with 2 M potassium phosphate, pH 7.5 and incubated for 30 min at 25 °C followed by centrifugation at 17000 g for 2 min to obtain protein particles. The spider silk particles were washed three times with Milli-Q water.39 Loading of eADF4(C16) and eADF4(κ16) particles with biologicals: eADF4(C16) or eADF4(κ16) spider silk particles were loaded with fluorescein labeled LYS and BSA by diffusion depending on their charge-charge interaction. Here, fluorescein labeled biologicals were used for higher sensitivity. Due to the net negative charge of eADF4(C16) particles, positively charged LYS could be efficiently loaded. The positively charged eADF4(κ16) was loaded with BSA. Loading efficiencies were determined using Eq. (1). Release study of model biologicals from spider silk hydrogels and particles: To analyze the release profile of loaded biologicals, eADF4(C16) hydrogels and particles (500 µL) were


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incubated in 1 mL of release buffer for defined time intervals (30 min, 1 h) in a shaker (Thermomixer compact; Eppendorf, Germany) with 50 rpm at 37 °C. Spider silk hydrogels with encapsulated biological-loaded spider silk particles were incubated for 24 h to examine the prolonged release profile. Supernatants were carefully removed, and hydrogels were resuspended in 1 mL of fresh release buffer. The removed supernatants were analyzed using UV/Vis spectroscopy at 280 nm to determine the protein concentration of released biologicals. Similarly, control experiments were performed using eADF4(C16) hydrogels without encapsulated biological, confirming that apparently no spider silk proteins were released during these experiments. Circular dichroism (CD) spectroscopy: Far-ultraviolet (UV) CD spectra were recorded between 200-260 nm on a Jasco J815 spectropolarimeter (Jasco, Groβ-Umstadt, Germany) using a 0.1 cm pathway quartz cuvette at 20 °C. CD spectra of proteins released from the hydrogel were compared with same concentration of freshly prepared solutions of native protein. Protein concentrations were adjusted using 10 mM potassium phosphate buffer (PB), pH 7.5. The scan speed was 100 nm min−1, response time 1 s, acquisition interval 0.1 nm, and bandwidth 1 nm. The spectra were calculated as an average of five scans and were subsequently smoothed by applying a Savitzky−Golay filter. Fluorescence Spectroscopy: Tryptophan fluorescence spectra were recorded on a Jasco FP- 6500 spectrofluorometer. The excitation wavelength was 295 nm for selective excitation of tryptophan residues. Emission spectra were recorded between 300 to 450 nm. The emission spectra were measured with a bandwidth of 5 nm, response time of 1 s, scan rate of 100 nm/min and data pitch of 0.1 nm at 25 °C. Sample conditions were identical to those described for the CD


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measurements. All spectra were corrected for the background emission of PB (10 mM K-Phos, pH 7.5). Protein Functionality Assays. Specific activity of BSA: Esterase activity of BSA was determined by following the hydrolysis of the substrate p-nitrophenylacetate (pNPA) to p-nitrophenol (pNP)46 at 400 nm using a CaryBio 50 UV Vis (Beckmann) spectrometer with 1 cm cuvettes. The assay contained 1 mM pNPA and 20 mM of the protein in PB (10 mM K-Phos, pH 7.4), at 37 °C. Concentration of released BSA was adjusted using PB. Progression of the hydrolysis was monitored by continuous UV/Vis measurements, and spectra were recorded at regular intervals of 0.3 min. Initial rates were calculated by linear least-squares analysis of time courses using the molar extinction coefficient of pNP, (ε414 = 17700 M-1 cm-1). Enzyme activity in units (U) was defined as the amount of enzyme catalyzing the reaction of 1 µmol of substrate per minute. Specific activity of HRP: The enzyme activity of the released HRP was determined using the ABTS assay for peroxidases. HRP catalyzes 2, 2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) in the presence of hydrogen peroxide (H2O2) to form an oxidized ABTS.47 The reaction mixture contained 10 nM HRP, 10 mM H2O2 and 20 mM ABTS in a 1 cm cuvette. The reaction was initiated upon the addition of HRP to a sample mixture of ABTS and H2O2 in 1 ml of PB (10 mM K-Phos, pH 7.0), at 37 °C. Concentration of released HRP was adjusted using PB. Interconversion of ABTS (λ max = 340 nm) to its radical cation (λmax = 414 nm) was monitored by taking UV–VIS spectra of reaction mixture at every 20 sec for 10 min. Enzyme activity in units (U) was defined as the amount of enzyme catalyzing the reaction of 1 µmol of the substrate per


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minute. Initial rates were calculated by linear least-squares analysis of time courses using the molar extinction coefficient of radical-cation ABTS•+, (ε414 = 36000 M-1 cm-1). Specific activity of lysozyme: Normalized lysozyme activity (substrate units per mg of enzyme) was determined by using an assay that is based on the hydrolysis of the outer cell membrane of Micrococcus lysodeikticus.48 Samples of native and hydrogel-released lysozyme were mixed with the M. lysodeikticus suspension in PB (100 mM K-Phos, pH 6.2), and the decrease in turbidity was measured for 10 min at 450 nm. The activity of the hydrogel-released lysozyme was compared to that of native lysozyme to confirm functionality of the released protein. The obtained A450 nm values were plotted as a function of time. The activity of LYS was calculated from the slope of the time course by linear regression of data points. A unit of enzyme was defined in this study, as the quantity of enzyme causing a decrease in absorbance of 0.001 min-1, and the following equation was adopted for the specific activity calculation:

Units/mg =

!"#$ / min %&'% − !"#$ / min )*+, 0 0.001 x mg enzyme in reaction mixture

Scanning Electron Microscopy (SEM) Imaging: To analyze the morphological structure using via SEM, hydrogels were lyophilized and fixed with carbon black solution. Before measurements, samples were sputter coated with 2 nm platinum (Sputter Coater 208 HR with MTM 20, Cressington, Watford, UK) and were imaged at an accelerating voltage of 2.5 kV, using a scanning electron microscope Zeiss Sigma 300 (Zeiss, Oberkochen, Germany). Rheological Properties: Stress–strain curves of different eADF4(C16) hydrogel preparations were analyzed according to a protocol established previously.24-26 Briefly, hydrogels were measured for 10 min at a constant shear rate of 3.0×10−3 s−1 using the Rheometer AR-G2 (TA


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Instruments, New Castle, DE, USA) with 25 mm plate-plate geometry and a 0.5 mm gap at a hydrogel volume of 600 µL at room temperature. Here, the shear rate was increased from 0.1 to 100 s-1. A solvent trap with a wet sponge was used to minimize evaporation. The apparent yield stress of hydrogels was obtained by stress-strain curves. Three samples (n = 3) were measured for each experimental group 3D Robotic dispense plotting: A 3D Discovery (RegenHU, Villaz-Saint-Pierre, Switzerland) was operated according to manufacturer’s instructions with the direct dispensing print-head using a needle with an inner diameter of 0.33 mm. Scaffolds were printed out of a 3 cc cartridge by applying 1-2 bar of pressure and a print-head velocity of 40 mm/s.

RESULTS AND DISCUSSION Direct loading of biologicals Aqueous solutions of the recombinant spider silk protein eADF4(C16) with a concentration range of 3-7% w/v, spontaneously self-assembled into hydrogels.24 Previously, it has been shown that 4% w/v eADF4(C16) hydrogels had a higher storage and loss modulus than 3% w/v eADF4(C16) hydrogels. Moreover, 4% w/v eADF4(C16) hydrogels provides an optimum mechanical stiffness for handling and therefore, represents an ideal system for the development of a protein carrier. To test the potential of eADF4(C16) hydrogels for protein delivery applications, we studied the encapsulation and release of diverse biologicals including BSA, HRP and LYS, with different physicochemical properties (pI 4.9, 7.0 and 10.5, respectively), different molecular weights (14.3, 44 and 66.5 kDa, respectively) and different hydrodynamic dimensions as presented in (Table S1). Direct loading to encapsulate biologicals was successfully performed by dissolving the biologicals into a 4% w/v eADF4(C16) solution


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under static conditions, followed by gelation of the silk at 37 °C (Figure 1a). Direct loading allowed biologicals to be easily and uniformly loaded into the silk hydrogel in situ. Amongst the three tested model biologicals, eADF4(C16) solutions with LYS turned milky and nontransparent due to strong electrostatic interactions between the positively charged LYS and the negatively charged eADF4(C16). However, all biologicals did not interfere with the general gelation process of eADF4(C16),25 and stable spider silk hydrogels could be prepared in the presence of all biologicals. The absolute amount of biologicals in the respective formulations is summarized in (Table S2). The release profiles of the three tested biologicals (BSA, HRP and LYS) from eADF4(C16) hydrogels are shown in (Figure 1b-d). The release profile of LYS was slow and constant in comparison to that of BSA and HRP. After 10 hours, 87%, 90% and 12% of BSA, HRP and LYS, respectively were released in Tris/HCl buffer (10 mM, pH 7.5). The slow release profile of LYS is related to the mentioned strong charge-charge interactions between the positively charged LYS and the negatively charged eADF4(C16).


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Figure 1. Schematic representation of a direct loading method to encapsulate biologicals into recombinant eADF4(C16) hydrogels (a). Time dependent release profile of BSA (b), HRP (c) and LYS (d) from 4% w/v eADF4(C16) hydrogels in different release buffers: Tris/HCl (10 mM, pH 7.5), PB (25 mM K-Phos, pH 7.5) and PBS (25 mM K-Phos, 100 mM NaCl, pH 7.5).

Next, release of biologicals was investigated in physiological phosphate buffer and at different ionic strengths. In case of BSA and HRP, the presence of salt had only a slight impact on the release kinetics, due to the low surface charge of these two biologicals. Since, LYS is bound to eADF4(C16) hydrogels by strong electrostatic interactions, the addition of salt in this case had a substantial effect on the release kinetics, with a significant increase in the presence of 25 mM potassium phosphate buffer (PB), pH 7.5 and a further increase in 100 mM NaCl (PBS) (Figure 1d). Here, the increased local ionic strength of chloride anions resulted in the weakening of the electrostatic interactions between eADF4(C16) and LYS, and thus the release of LYS was facilitated. These results indicate that release kinetics of the biologicals from eADF4(C16) hydrogels are influenced by the physicochemical characteristics of both the silk protein used and the biologicals. Conformational Properties of Released Proteins. For any therapeutic application, it is important that the encapsulated proteins are not misfolded or aggregated during encapsulation, and also that they maintain their activity. To ascertain the conformational state of the released proteins, far-UV CD and fluorescence spectroscopy were used to examine the secondary and tertiary structural characteristics, respectively.


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Using CD spectropolarimetry, secondary structure of all released proteins from the hydrogels was compared to that of the native proteins prepared under identical conditions. CD spectra showed that the secondary structure of the released proteins were indistinguishable to those of the proteins in the native state, reflecting the spectra reported in literature for BSA,49 HRP,50 and LYS,51 respectively (Figure 2a-c). This suggested that hydrogels did not hampered the secondary structure of proteins upon encapsulation and release.

Figure 2. Conformational stability of the proteins released from eADF4(C16) hydrogels. Far-UV CD spectra of native protein (red line) and released protein (blue line) through the hydrogels (ac). Normalized fluorescence emission spectra of the native (red line) and released proteins (blue line) through the eADF4(C16) hydrogels (d-e); excitation wavelength was 295 nm. All the spectra were recorded at room temperature in PB (25 mM K-Phos, pH 7.5).

Fluorescence emission spectra were recorded with excitation at 295 nm to excite the tryptophan residues, which give insights into the tertiary structure of the protein, since Trp fluorescence is highly sensitive to the tryptophan microenvironment within the 3D structure of


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the protein. The fluorescence spectra of the proteins studied in this work were in line with literature reports recorded at the same experimental conditions for BSA,49 HRP,50 and LYS51 respectively (Figure 2d-e). The emission spectra of all released proteins were similar to those of the native proteins with respect to both the emission maximum and fluorescence intensity after normalization for the concentration. Functionality Assays of Released Protein BSA shows esterase-like activity and the specific activity evaluation was done by monitoring the hydrolysis of 4-nitrophenyl acetate (NPA), in which formation of p-nitrophenol is followed by measuring the absorbance at 400 nm.46 We tested the NPA hydrolysis activity of native BSA and released BSA under identical conditions. BSA released from hydrogel showed 94% of esterase-like activity of native BSA, suggesting that the encapsulation into hydrogels did not significantly reduce its activity, (Figure S1). Similarly, the catalytic activity of native and released HRP from hydrogels was determined using the well known peroxidase-catalyzed reaction of one electron oxidation of ABTS to the corresponding radical-cation ABTS•+ in the presence of hydrogen peroxide,47 which has an absorption maximum at 414 nm. The experimental results demonstrated that the released HRP retained its specific activity of 96% (Figure S2). The enzymatic activity of lysozyme was determined using the lysis of the outer membrane of M. lysodeikticus,48 which results in the solubilization of the affected bacteria and consequent decrease in turbidity at 450 nm. The percent enzymatic activity was comparing between the released LYS and freshly prepared native lysozyme solution (0.1 mg/ml). Released lysozyme retained its specific activity of 94% (Figure S3), yielded a specific activity of ≈102


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substrate units/mg of lysozyme in the reaction solution and is nearly identical to native lysozyme solution with ≈108 substrate units/mg. Loading of biologicals through diffusion into pre-formed hydrogels

For diffusion-based loading, 4% w/v eADF4(C16) hydrogels were incubated with biologicals in a 1:2 molar ratio (Figure 3a). For the positively charged LYS, a high encapsulation efficiency (95%)

Figure 3. Schematic representation of eADF4(C16) hydrogels loading with biologicals by a diffusion method (a). Time dependent release profile of LYS (b) from 4% w/v eADF4(C16) hydrogels in different release buffers: Tris/HCl (10 mM, pH 7.5), PB (25 mM K-Phos, pH 7.5) and PBS (25 mM K-Phos, 100 mM NaCl, pH 7.5).

was achieved, while the encapsulation of negatively/neutral charged proteins (BSA and HRP) was limited due to electrostatic repulsion. The release of LYS was investigated in different


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buffers. It can be clearly seen from the release kinetics (Figure 3b) that LYS encapsulated in eADF4(C16) hydrogels by diffusion showed slow release kinetics. At low ionic strength, the initial release rate of LYS was very low, and only 7% of the encapsulated protein was released in Tris buffer (10 mM, pH 7.5). The release increased to 20% in PB buffer (25 mM, pH 7.5), and a further increase to 38% was observed, when PBS (25 mM, pH 7.5) was employed. The results suggest that a sustained release of positively charged biologicals is possible upon encapsulation in eADF4(C16) hydrogels. Impact of pore size of the hydrogels on the release profile One important aspect concerning the release profile of biologicals is the pore size of the hydrogel used, which is in case of spider silk hydrogels dependent on the protein concentration. To demonstrate this, eADF4(C16) hydrogels prepared at 3 wt %, 4 wt % and 5 wt % were loaded with the same amount of biologicals (BSA, HRP and LYS), and the release kinetics were spectroscopically examined in PBS buffer. Notably, hydrogels with higher concentrations had slower release rates (Figure 4a-c), presumably due to decreased pore sizes at increased eADF4(C16) concentration, from around 30 ± 3 µm (3 wt %) to 7 ± 0.8 µm (4 wt %) and to 3 ± 0.7 µm (5 wt %) (Figure 4d-f).


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Figure 4. Time dependent release profile of BSA (a), HRP (b) and LYS (c) from different eADF4(C16) hydrogels varying in silk concentration. Release buffer: PBS (25 mM K-Phos, 100 mM NaCl, pH 7.5). SEM images of eADF4(C16) hydrogels prepared at 3 wt % (d), 4 wt % (e), and 5 wt % (f). Scale bars = 10 µm.

Encapsulation of biological-loaded spider silk particles in spider silk hydrogels Next, biological-loaded spider silk particles were encapsulated in eADF4(C16) hydrogels to extend and better control the time of release (Figure 5a). Spider silk particles were analyzed by scanning electron microscopy (SEM) (Figure 5b, c) verifying the average size of eADF4(C16) and eADF4(κ16) particles to be 320 ± 40 nm and 540 ± 35 nm, respectively. Spider silk particles show a hierarchical structure with three independent regimes: a solid core determining the mechanical response, a fuzzy interfacial layer of protruding protein strands with an extension of about 30-50 nm and an external charge layer. The variability of the interfacial layer allows a transition of a primarily hard particle (with a thin layer) to an increasingly soft particle permeable for ions or biologicals. Importantly, higher molecular weight biologicals


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(BSA, HRP and LYS) will unlikely protrude into the core but will solely interact with the fuzzy polymer brush and the charged surface layer of the particles. The overall interaction forces are based on two contributions, namely electrostatic and steric forces. In particular, negatively charged eADF4(C16) particles were used to encapsulate positively charged lysozyme, and negatively charged BSA was efficiently encapsulated within positively charged eADF4(κ16) particles. Unfortunately, it was difficult to load HRP because of its neutral net charge at pH 7.5.


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Figure 5. Schematic representation of the encapsulation of biological-loaded particles into 4% w/v eADF4(C16) hydrogels (a). SEM image of spider silk particles: eADF4(C16) particles with an average size of 320 ± 40 nm (b); eADF4(κ16) particles with an average size of 540 ± 35 nm (c); internal structure of a freeze-dried hydrogel (4% w/v eADF4(C16)) supplemented with 1% w/v eADF4(C16) particles (d), (e) is the higher magnification image of (d). The arrows indicate encapsulated particles within the hydrogel.

Loading efficiencies of eADF4(C16) and eADF4(κ16) particles were tested at different biological/silk protein ratios (1, 2 and 4 eq). Particles were made of 20 nmol eADF4(C16) or 20 nmol eADF4(κ16), and the respective biological concentration was adjusted accordingly. The maximum loading efficiency of BSA was 61% (12 nmol) when using a 1:1 molar ratio of BSA / eADF4(κ16). For LYS, the maximum loading efficiency was 83% (36 nmol) when using a 1:2 molar ratio of eADF4(C16) / LYS. Loaded eADF4(C16)/eADF4(κ16) particles (1% w/v) were gently mixed with 4% w/v eADF4(C16) solution, before gelation was initiated. The hydrogels were formed by incubation overnight at 37 °C as schematically shown in (Figure 5a). The morphology of the freeze-dried eADF4(C16) hydrogel/particle composites was then investigated by SEM (Figure 5d-e). The hydrogels showed sheet-like morphologies with an interconnected, highly porous structure. The in vitro release of LYS and BSA from eADF4(C16) / eADF4(κ16) particles was investigated in PBS. The release profiles showed a rapid burst release in both cases (Figure 6a, b). Release was almost completed (97%) within 2 h - 3 h. However, encapsulating biologicalloaded eADF4(C16) and eADF4(κ16) particles within the molecular network of eADF4(C16) hydrogels extended the release kinetics significantly, with a release of BSA and LYS of 19% and 14%, respectively, in PBS within 6-7 h (Figure 6a, b). Further, the prolonged release profile of


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biologicals from eADF4(C16) hydrogels in combination with particles was studied over a period of 7 days resulting in release of BSA and LYS of 40% and 28%, respectively, in PBS (Figure 6a, b).

Figure 6. Release behaviour of BSA from eADF4(κ16) particles directly or after encapsulation within ADF4(C16) hydrogels (a), release of LYS from eADF4(C16) particles directly or after encapsulation within eADF4(C16) hydrogels (b). Release buffer: PBS (25 mM K-Phos, 100 mM NaCl, pH 7.5).

Rheological properties and 3D printing of spider silk particle-hydrogel composites The mechanical properties of hydrogels are important considerations for specific applications especially for an injectable formulation. In previous studies, it has been shown that in eADF4(C16) hydrogels elastic behavior dominates over viscous behavior in combination with a low-viscosity flow behavior.24-26 To analyze that the loaded proteins and particles did not significantly influence the apparent yield stress of eADF4(C16) hydrogels, all hydrogels were


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analyzed by stress-strain curves. As expected, eADF4(C16) hydrogels showed similar viscoelastic behavior, with stress changing proportionally to linearly increasing strain. Further, eADF4(C16) particle-enforced hydrogels had roughly double the apparent yield stress in comparison to plain eADF4(C16) hydrogels, while loading the hydrogels with just biologicals (exemplarily LYS) had only a slight influence on the mechanical behavior (Figure 7a). However, all hydrogels could be

Figure 7. Stress-strain curves of 4 % eADF4(C16) hydrogels and particle-enforced hydrogels: the particles were loaded with LYS (a). eADF4(C16) hydrogel with encapsulated spider silk particles after extrusion from a syringe (b); 3D printing of spider silk hydrogels by robotic


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dispensing: digital images of 3D printed 2-layer constructs of eADF4(C16) hydrogels (c), and eADF4(C16) particle-enforced hydrogels (d). Scale bars (c-d): 2 mm.

easily used as an injectable formulation for biomedical applications. To confirm the injectability, we showed that particle-enforced hydrogels could be easily hand-extruded from a syringe (Figure 7b).

3D printing is an attractive fabrication technique to formulate therapeutic proteins in 3D constructs of hydrogels for tissue engineering and regenerative medicine. 3D printing enables the rapid production of constructs with a well-defined architecture, which mimics the complex biological and functional organization of native tissues and which easily can be combined with cells. In recent studies, eADF4(C16) hydrogels were successfully demonstrated as a promising bioink for 3D bioprinting.25,26 As a step towards 3D-fabrication of scaffolds including therapeutic proteins, 4% w/v eADF4(C16) hydrogels with 1% w/v encapsulated spider silk particles were assessed regarding their 3D-printability. As expected, spider silk constructs could be 3D printed by robotic dispensing using a print head with an electromagnetic valve. The hydrogels were process-compatible and had high shape fidelity without the use of postprocessing, crosslinking or the addition of thickeners (Figure 7c-d). Printability of these hydrogels has been attributed to its well-characterized shear thinning behavior as well as recovery after deformation.24-26 This allows the incorporation of biologically active agents in 3D printed constructs which can be utilized in personalized and programmable medicine.

CONCLUSIONS


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eADF4(C16) hydrogels show a great potential in overcoming the formulation challenges of biotherapeutics, related to biologics stability, encapsulation efficiency, loading, and sustained release. In this study, we have successfully prepared injectable hydrogel-protein depots by encapsulating biologicals into spider silk hydrogels and further demonstrated their potential for localized therapies and sustained release applications. Proteins with different molecular weights and with significantly different isoelectric points were easily encapsulated within eADF4(C16) hydrogels through direct loading, involving simple mixing of the biological with the silk solution before initiating the gelation process. In vitro release testing showed a correlation between protein release and electrostatic interactions with the hydrogel. Release of model biologicals (BSA and HRP) from eADF4(C16) hydrogels was fast (finished within a day) in case no electrostatic interaction between the biological and the hydrogel existed. However, release of the positively charged model LYS which electrostatically interacts with the negatively charged eADF4(C16) hydrogel showed much prolonged release. At low ionic strength, release of LYS from hydrogels was very low ≈12% which increased to ≈48% with increasing ionic strength. eADF4(C16) hydrogels also allow the encapsulation of protein via diffusion, and positively charged LYS was encapsulated efficiently within preformed eADF4(C16) hydrogels. The release profile could be modestly controlled by varying the pore sizes of eADF4(C16) hydrogels. We prepared eADF4(C16) hydrogels with three different pore sizes by varying the silk concentration. Examining the release profile, all the proteins (BSA, HRP and LYS) were observed to be released much slower from 5% w/v eADF4(C16) hydrogels having smaller pore in comparison to 3% w/v eADF4(C16) hydrogels.


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Furthermore, we designed a combined protein release system using spider silk particles loaded with the biologicals which were then embedded within the silk hydrogels. This set up yielded significantly prolonged release (from hours to week). eADF4(C16) particle-hydrogel composites showed also an excellent viscoelastic behavior and 3D printability, which is highly appreciated for biomedical applications and for rapid prototyping. An efficient controlled release system should yield biologically active protein. The protein structure of all encapsulated proteins as well as their activity was maintained upon encapsulation and release. Injectable spider silk hydrogels can be used as depots for biologicals with high molecular weights and designed to show a tunable release profile by simply varying the pore size of the hydrogel or by embedding biological loaded particles. These promising results lay the foundation for eADF4(C16) hydrogels and hydrogel–particle composites as injectable systems for delivering therapeutic biologics in a sustainable and highly efficient way.

Supporting Information The following files are available free of charge: Physical properties of encapsulated proteins and their absolute amount in eADF4(C16) hydrogels; Table S1 and Table S2, respectively. Specific activity data of BSA, HRP and LYS; Figure S1−S3.

AUTHOR INFORMATION

Corresponding Author *Thomas Scheibel: [email protected] Author Contributions


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S.K. and T.S. wrote the manuscript. All authors discussed the results, provided critical feedback and have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We gratefully thank Elise DeSimone for help in 3D printing and proof reading.

ABBREVIATIONS eADF4, engineered Araneus diadematus fibroin 4; BSA, bovine serum albumin; HRP, horseradish peroxidase; LYS, lysozyme; M. lysodeikticus, Micrococcus lysodeikticus; w/v, weight/volume; Tris/HCl, tris(hydroxymethyl)aminomethane hydrochloride; PB, phosphate buffer; PBS, phosphate buffered saline; K-Phos, potassium phosphate; NaCl, sodium chloride; PEG,

polyethylene

glycol;

pNPA,

p-nitrophenylacetate;

ABTS,

2,

2′-azino-bis(3-

ethylbenzthiazoline-6-sulfonic acid); SEM, scanning electron microscopy.

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For Table of Contents Use Only Recombinant spider silk hydrogels for sustained release of biologicals Sushma Kumari,† Hendrik Bargel,† Mette U. Anby,‡,ɸ David Lafargue,‡ and Thomas Scheibel*,†,§,¥,#,┴,Θ †Lehrstuhl Biomaterialien, Fakultät für Ingenieurwissenschaften, Universität Bayreuth, Universitätsstraße 30, 95440 Bayreuth, Germany §

Bayreuther Zentrum für Kolloide und Grenzflächen (BZKG), ¥Bayerisches Polymerinstitut

(BPI), #Bayreuther Zentrum für Bio-Makromoleküle (bio-mac), ┴Bayreuther Zentrum für Molekulare Biowissenschaften (BZMB), ΘBayreuther Materialzentrum (BayMAT), Universität Bayreuth, Universitätsstraße 30, 95440 Bayreuth, Germany


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§

Corresponding author: [email protected]

‡

Technologie Servier, 25/27 rue Eugène Vignat, 45000 ORLEANS, France

ɸ

Page 32 of 33

H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark

For Table of Contents Use Only Recombinant spider silk hydrogels for sustained release of biologicals Sushma Kumari,† Hendrik Bargel,† Mette U. Anby,‡,ɸ David Lafargue,‡ and Thomas Scheibel*,†,§,¥,#,┴,Θ


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Recombinant Spider Silk Hydrogels for Sustained Release of

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