Jirada Kaewchuchuena,
Saphia A. L. Matthewa,
Suttinee Phuagkhaopongab,
Luis M. Bimboacde and
F. Philipp Seib*afg
aStrathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow, G4 0RE, UK
bDepartment of Pharmacology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
cDepartment of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal
dCNC – Center for Neuroscience and Cell Biology, Rua Larga, University of Coimbra, 3004-504 Coimbra, Portugal
eCIBB – Center for Innovative Biomedicine and Biotechnology, Rua Larga, University of Coimbra, 3004-504 Coimbra, Portugal
fFraunhofer Institute for Molecular Biology & Applied Ecology, Branch Bioresources, Ohlebergsweg 12, 35392 Giessen, Germany
gFriedrich Schiller University Jena, Institute of Pharmacy, Lessingstr. 8, 07743 Jena, Germany. E-mail: philipp.seib@uni-jena.de; Tel: +49 3641 9 499 00
First published on 22nd January 2024
Despite many reports detailing silk hydrogels, the development of composite silk hydrogels with homotypic and heterotypic silk nanoparticles and their impact on material mechanics and biology have remained largely unexplored. We hypothesise that the inclusion of nanoparticles into silk-based hydrogels enables the formation of homotropic and heterotropic material assemblies. The aim was to explore how well these systems allow tuning of mechanics and cell adhesion to ultimately control the cell–material interface. We utilised nonporous silica nanoparticles as a standard reference and compared them to nanoparticles derived from Bombyx mori silk and Antheraea mylitta (tasar) silk (approximately 100–150 nm in size). Initially, physically cross-linked B. mori silk hydrogels were prepared containing silica, B. mori silk nanoparticles, or tasar silk nanoparticles at concentrations of either 0.05% or 0.5% (w/v). The initial modulus (stiffness) of these nanoparticle-functionalised silk hydrogels was similar. Stress relaxation was substantially faster for nanoparticle-modified silk hydrogels than for unmodified control hydrogels. Increasing the concentrations of B. mori silk and silica nanoparticles slowed stress relaxation, while the opposite trend was observed for hydrogels modified with tasar nanoparticles. Cell attachment was similar for all hydrogels, but proliferation during the initial 24 h was significantly improved with the nanoparticle-modified hydrogels. Overall, this study demonstrates the manufacture and utilisation of homotropic and heterotropic silk hydrogels.
A fundamental requirement for a biomaterial is that it supports the desired function, such as cellular organisation. To date, many different biomaterials have been explored,7 including synthetic polymers (e.g. polylactide8 and polyethylene glycol9), natural polymers (e.g. chitosan,10 collagen11 and alginate12) and cell-derived extracellular matrix.13–15 However, all of these polymers have drawbacks, including rapid degradation, shrinkage (e.g. collagen), low mechanical strength (e.g. collagen, alginate), complicated processing (e.g. polyethylene glycol, alginate, extracellular matrix), biocompatibility concerns (e.g. leaching of chemical crosslinkers) and perturbation of the microenvironment (e.g. polylactide degradation causes acidification). Silk fibroin is emerging as an interesting material that shows promise for overcoming these particular limitations.16 Chemical modification of silk further opens up new properties to create bespoke materials with novel functions.17
Silk is a natural fibrous protein spun by spiders and insects (e.g. Lepidoptera). Silk self-assembles into a hierarchal solid fibre during the spinning process by responding to processing parameters, including shear.18 The most studied silk for current biomedical applications is derived from the domesticated silkworm Bombyx mori because its silk is clinically approved for use in humans and is readily available in large quantities due to silk farming (i.e. sericulture), thereby ensuring a robust silk supply chain.19 Silk's desirable trademarks include its remarkable mechanical properties, biocompatibility, biodegradability, hemocompatibility and water and oxygen permeability.20,21 Using all-aqueous processing, the silk fibre can be unspun using reverse engineering principles to yield a liquid regenerated silk feedstock (reviewed in22). This liquid silk can then be processed into a wide range of material formats, including films, monolithic blocks, fibres, particles, scaffolds and hydrogels,23 again using all-aqueous processing without any need for chemical crosslinkers or harsh solvents. Self-assembling silk hydrogels have an excellent biocompatibility track record, that also applies to the in vivo setting (e.g. ref. 24).
The Bombyx mori silk fibroin consists of a heavy chain of approximately 391 kDa and a light chain of approximately 26 kDa, which are linked by a single disulphide bond at the C-terminus.25 The C- and N-termini of the Bombyx mori silk heavy chain consist entirely of nonrepeating amino acid sequences and are believed to aid in the storage and self-assembly process. However, the mechanical properties of silk fibroin arise from the amino acid sequences of the silk-heavy chain that assemble into beta sheets. The copolymer-like arrangement of the silk-heavy chain contains two main motifs, namely, repetitive hydrophilic amino acid sequences and hydrophobic stretches, which result in a copolymer-like arrangement containing 11 hydrophilic and 12 hydrophobic blocks.26 The hydrophobic region is dominated by glycine–X repeats, where X is alanine, serine, or tyrosine. B. mori silk fibroin lacks the tripeptide sequence arginine, glycine, and glutamic acid (RGD) that is typically exploited by cells to mediate cell–substrate attachment via integrin engagement. Instead, the N terminal of the silk-heavy chain contains a fibroblast growth-promoting peptide.27
Lepidoptera silks share several structural features, including light and heavy chains, with conservation of the positions and spacing of cysteine residues that covalently crosslink the light and heavy chains. These silks also have an amphiphilic structure. Nevertheless, sequence specificity exists between different silkworm silks; for example, the Indian non-mulberry tasar silkworm (Antheraea mylitta) contains RGD sequences that are absent in B. mori silk, while the silk from Antheraea assama (golden silk) has substantial polyalanine stretches that are associated with better mechanics and thermal stability than is observed with B. mori silk.28 Therefore, possibilities exist for the creation of new materials with desired properties by blending different liquid silk types. For example, blending Antheraea assama with B. mori liquid silks triggered the solution–gel transition within 40 min of mixing. These hydrogels were physically crosslinked via their beta sheets and showed promising in vivo wound-healing properties.19
The present study exploits silk self-assembly using sonication as a solution–gel transition trigger.16 This assembly strategy has been widely used in the past because the process is simple and eliminates the need for solvents or chemical crosslinkers. Examples where these silk hydrogels have been used include soft29 and hard30 tissue engineering (e.g. tissue fillers, bone engineering). For example, silk hydrogels modified with several nanoparticle types, such as triphasic ceramic (Mg2SIO4, Si3Sr5, and MgO),31 silica,32 iron, silver and gold nanoparticles,33,34 have shown promise for a spectrum of applications, including magnetic field actuation,35 antibacterial functions34 and bone tissue engineering.31,32 Physically crosslinked silk hydrogels reinforced with native-like B. mori fibres36 and chemically crosslinked silk hydrogels doped with amorphous silk fibroin nanofibers37 have also been reported, demonstrating the possibility of tuning the silk secondary structure and fibre format. Unmodified38 and nanoparticle-modified silk hydrogels39 have also been explored for drug delivery. For example, Keiji Numata and co-workers developed the first-generation B. mori drug release system that incorporated silk nanoparticles within physically crosslinked silk hydrogels,39 while others have advanced this concept further and used these systems to release multiple drugs in vivo.40 An emerging research avenue is to use silk hydrogels as an in vitro tissue model that includes the tumour microenvironment.41
Previous studies have used silk fibroin hydrogels to assess their baseline performance as a tumour microenvironment, including the capacity to support cell migration.42,43 The ultimate goal of these studies is to recapitulate specific biological processes and behaviours that are dictated by the material design.44 In the present study we have used the human prostate cancer cell line DU145. These cells are not hormone sensitive and are moderately metastatic making them an ideal starting point for developing in vitro tumour models. Important factors to consider when designing these living tissue systems are the elastic and viscoelastic moduli of the extracellular mimetic matrix.45 For example, during solid tumour progression, the mechanical properties of the extracellular matrix change (thereby assisting disease diagnosis of solid tumours, for example). In the context of solid tumours, the substrate stiffness is accompanied by changes in flow characteristics (i.e. stress relaxation). Therefore, when developing extracellular matrix models, hydrogel performance is often assessed against cell function (e.g. cell migration, differentiation, proliferation etc.). Physically crosslinked silk hydrogels can mimic the three-dimensional structure of native extracellular matrix.16 For example, self-assembled silk hydrogels with a solid silk content of 4% w/v show viscoelasticity, which in turn impacts the cell biology.46
Despite many reports detailing silk hydrogels, the development of composite silk hydrogels containing homotypic and heterotypic silk nanoparticles and their impact on material mechanics and biology, has remained largely unexplored. A caveat when working with B. mori hydrogels is the lack of arginine–glycine–aspartic acid (RGD) sequences in this silk, as these sequences are necessary for integrin-mediated cell adhesion.21 For this reason, silks from non-mulberry tasar silkworms (e.g., Antheraea mylitta) are more promising because they contain the RGD sequence. However, how well these silks allow tuning of the mechanics and cell adhesion that ultimately control the cell–material interface is unknown. Therefore, the aim of this research was to create hydrogels functionalised with nanoparticles derived from blends of B. mori and tasar silk to probe cell responses and to compare them to hydrogels prepared using silica nanoparticles as a reference. This work reports the manufacture of nonporous Stober silica, B. mori and tasar silk nanoparticles and their addition at low (0.05% w/v) and high (0.5% w/v) concentrations to 3% w/v B. mori silk undergoing solution–gel transition. The resulting nanoparticle-functionalised silk hydrogels had similar stiffnesses but exhibited substantial differences in stress relaxation when compared to unmodified control hydrogels. Cell attachment was similar for all the tested hydrogels.
Antheraea mylitta silkworm cocoons were prepared based on previous work by others.48 Briefly, dried cocoons were cut into 5 × 5 mm pieces and 5 g samples were degummed with 2 L of 0.025 M Na2CO3 for 60 min, followed by 60 min in 2 L of 0.0125 mM Na2CO3. The silk fibres were then washed three times with 1 L of distilled water for 20 min and then dried in a fume hood overnight. The dried silk fibroin was dissolved in 1 N NaOH at a silk to NaOH ratio of 1 g to 25 mL. The samples were kept at 25 °C for up to 16 h under constant stirring at 250 rpm. Insoluble material was removed by centrifugation for 20 min at 9500 × g. The supernatant was transferred to a dialysis cassette (molecular weight cut-off 3500 Da; Thermo Fisher Scientific Inc., Waltham, MA, USA) and dialysed against distilled water, with four water changes over the 24 h dialysis period. The dialysed silk fibroin solution was collected and centrifuged twice at 9500 × g for 20 min to remove any remaining aggregates. Samples were freeze dried and reconstituted to 4% (w/v) and stored at 4 °C until use. The tasar silk fibroin concentration was calculated using the bicinchoninic acid assay protein assay and bovine serum albumin as a protein standard (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific).
B. mori silk nanoparticles were manufactured as described previously (Matthew et al., 2020). Briefly, silk nanoparticles were generated using semi-automated nanoprecipitation by controlling the silk and solvent flow with a syringe pump (Harvard Apparatus 22, Holliston, MA) (Fig. 1). The system was equipped with a syringe and blunt needle (23 G × 0.25′′) and operated at room temperature. Isopropanol was contained in a short-neck round-bottom flask and the ratio of isopropanol to silk was set at 5:1 (v/v). A 3% (w/v) B. mori silk solution was added dropwise at a rate of 1 mL min−1. The resulting suspension was transferred to polypropylene ultracentrifugation tubes, the volume was made up to 43 mL with distilled water and the tubes were centrifuged at 48400 g for 2 h at 4 °C (Beckman Coulter Avanti J-E equipped with a JA-20 rotor). The supernatant was discarded and the pellet was resuspended in 20 mL distilled water and sonicated twice for 30 s at 30% amplitude with a Sonopuls HD 2070 sonicator (ultrasonic homogeniser, Bandelin, Berlin, Germany) fitted with a 23 cm long sonication tip (0.3 cm diameter tip and tapered over 8 cm). Next, 23 mL of distilled water was added and the sonicated material was centrifuged. This washing and resuspension of the pellet was repeated twice. The final pellet was collected and resuspended in 2–3 mL water. This final silk nanoparticle suspension was stored at 4 °C until use.
Fig. 1 Flow diagram of nanoparticle manufacture. Silica, B. mori and tasar silk nanoparticle synthesis. |
Tasar silk nanoparticles were synthesised using a NanoAssemblr microfluidic system (NanoAssemblr™ Benchtop Instrument version 1.5, Canada). Prefilled syringes containing 2% (w/v) silk solution and acetone were dispensed into a microfluidic cartridge and mixed at a 1:4 ratio (v/v) of silk solution to acetone at a total flow rate of 1 mL min−1 (Fig. 2). The precipitated silk nanoparticles were collected and centrifuged at 48400 × g for 2 h, the supernatant was aspirated, and the pellet was resuspended in distilled water, vortexed and subsequently sonicated twice for 30 s sonication cycles at a 30% amplitude. The washing steps were repeated at least twice more. The silk particles were then stored at 4 °C until use.
One limitation when working with B. mori silk fibroin is its lack of the RGD sequence necessary for integrin-mediated cell adhesion.19 For this reason, B. mori silk hydrogels can be viewed as ‘blank slates’ that require modification to maximise their potential. Tasar silk does contain the RGD sequence, but no examples currently exist in the literature of B. mori hydrogels functionalised with tasar silk nanoparticles. Therefore, the present work closes a critical knowledge gap.
The use of tasar silk nanoparticles to functionalise B. mori silk hydrogels is ideal because it permits spatial control of RGD functionalisation. In the present work, nonporous silica nanoparticles synthesised using the Stober method49 were used as a reference. The B. mori silk nanoparticles were manufactured using the Matthew semi-batch set-up57 and tasar silk nanoparticles were synthesised using microfluidic-assisted antisolvent precipitation.48 All three nanoparticle types were characterised according to their size, polydispersity index and zeta potential using dynamic light scattering and zeta potential measurements, as our aim was to work with particles that were of similar size and surface charge. We therefore tuned the silica particle size by adjusting the NH4OH concentration (data not shown), because increasing ammonium hydroxide concentration is known to increase the nanoparticle size.50 Ultimately, all three nanoparticle types had a size range of 120 to 150 nm, a zeta potential between −33 and 39 mV and similar polydispersity indices that were not statistically different. Specifically, B. mori silk nanoparticles had an average size of 117 ± 5.31 nm, a polydispersity index of 0.14 ± 0.02 and a zeta potential of −38.6 ± 2.23 mV (Fig. 2A), the Antheraea mylitta (tasar) silk nanoparticles had an average size of 146 ± 8.64 nm, a polydispersity index of 0.34 ± 0.04 and a zeta potential of −32.8 ± 2.64 mV, and the silica nanoparticles had an average size of 122 ± 22.68 nm, a polydispersity index of 0.31 ± 0.17 and a zeta potential of −37.8 ± 2.98 mV (Fig. 2A). These silk nanoparticle results are consistent with previous work using B. mori57–59 and tasar48 silk stocks.
The pore volumes and surface areas of the nanoparticles were determined using nitrogen adsorption. The single-point Brunauer–Emmett–Teller analysis showed that B. mori and tasar silk nanoparticles had pore volumes of 0.023 and 0.094 cm3 g−1, respectively. This pore volume determination may indicate particle aggregation60 because electron microscopy studies have suggested that silk nanoparticles are solid. The surface areas of the B. mori and tasar silk nanoparticles were determined to be 5.02 and 33.65 m2 g−1, respectively (Fig. 2B).
Our silica nanoparticles were classified by a type II isotherm, indicating that they were nonporous, as would be expected for Stober silica nanoparticles60 The presence of a hysteresis loop classified the material as an H1 type, indicative of nanoparticle aggregation (rather than porosity). Both the single-point Brunauer–Emmett–Teller and Barrett–Joyner–Halenda measurements revealed that the silica nanoparticles had a pore volume of 0.350 cm3 g−1 and an average surface area of 62.77 m2 g−1 (Fig. 2B). These types of values are typical for Stober silica nanoparticles, as reported in the reference literature.60
Overall, based on IUPAC guidelines,61 the silica, B. mori and tasar silk nanoparticles were non-porous and classified as type II materials because their isotherm graphs showed a monolayer adsorption up to high P/Po. We then assessed the morphology of the nanoparticles by scanning electron microscopy. Both B. mori and tasar silk nanoparticles had spherical shapes with sizes similar to those determined by dynamic light scattering (Fig. 2C). These results correlated well with previous reports (e.g. ref. 48 and 57–59). The Stober silica nanoparticles also had a spherical shape and an average size similar to that determined by dynamic light scattering, again in agreement with previous reports (e.g. ref. 49 and 50).
FTIR spectroscopy was used to determine the silk secondary structure and the different functional groups present in the silica nanoparticles. The Stober silica nanoparticles showed a typical absorption peak of Si–O–Si (797.45 cm−1 and 1062.79 cm−1) and Si–OH asymmetric stretching vibration (945.81 cm−1) (data not shown), in agreement with previous reports.62 The secondary structure of the silk nanoparticles and hydrogels indicated extensive β-sheets in our self-assembled silk hydrogels and particles (Fig. 3), consistent with other literature (e.g. ref. 57 and 63). The FTIR results showed an amide I absorption peak at 1600–1700 cm−1 for all silk samples (Fig. 3A). Comparison of the spectra from B. mori and tasar silk nanoparticles and hydrogel composite nanoparticles to the spectra from air-dried silk films (negative control) and silk films treated with 70% v/v ethanol/distilled water (positive control) revealed the highest β-sheet content in the positive control silk films (Fig. 3B). The β-sheet contents of B. mori and tasar silk nanoparticles were 62% and 58%, respectively, whereas the β-sheet content of the silk hydrogels and air-dried silk films (negative control) were 32% and 23%, respectively (Fig. 3B). Overall, these data correlated well with our own studies57 and previous studies by others.48,64
We also assessed the impact of the incorporation of homo- and heterotypic nanoparticles on hydrogel mechanics. We speculated that the formation of these composites would result in different mechanics than those observed in the pristine silk hydrogels. This speculation was based on the ability of nanoparticles to orchestrate hydrogel interactions, including the formation of dynamic nanoparticle–hydrogel structures or hydrogels with adhesive surface properties. These systems typically exploit polymer–nanoparticle interactions that impact the overall bulk properties mediated by surface adsorption, such as those occurring between silica nanoparticles and polyethylene glycol (PEG),65 between hydrophobically modified cellulose derivatives and PEGylated polylactide nanoparticles66 or between TM50 silica and polyacrylamide hydrogels.67
Nanoparticle shape also affects hydrogel performance.68 Previous work has shown that silk reinforcement with silica increased bulk stiffness.32 However, studies on silk hydrogels containing nanoparticles are lacking, so little is known regarding their mechanics or cellular responses. In the present study, the flow behaviour of silk hydrogels in the presence of nanoparticles is reported by comparing the stiffness of a 3% w/v silk hydrogel with similar silk hydrogels containing B. mori silk, tasar silk and non-porous silica nanoparticles (Fig. 4A). However, the initial elastic moduli did not differ for any of the tested hydrogels. For example, the stiffness was 1.23 kPa for the hydrogel containing 0.05% w/v B. mori silk (this was the highest measured value) and 1.10 kPa for the hydrogel containing 0.05% w/v tasar silk nanoparticles. By contrast, the stress relaxation and the half stress-relaxation time showed some particle dependence, as silk hydrogels containing 0.5% w/v B. mori nanoparticles had the lowest value (97 s) (Fig. 4B), while pure silk hydrogel had the highest value (312 s). These values were 163 s for silk hydrogels containing 0.05% (w/v) Stober silica nanoparticles and 146 s for silk hydrogels containing 0.5% w/v B. mori silk nanoparticles (Fig. 4B). Overall, these trends indicated that the interactions between the nanoparticles and the silk hydrogel did not significantly alter their behaviours. Possibly, the formation of physical crosslinks between the silk molecules that are responsible for the formation of the silk hydrogel was only slightly influenced by the nanoparticle doping. Perhaps the use of sequence-coded nanoparticles will provide better control over beta-sheet bulk assembly.
The preliminary biological responses of the nanoparticle-doped silk hydrogels were also assessed. Prior work showed that embedding silica nanoparticles within silk fibroin hydrogels enhanced the bulk mechanical properties of the hydrogel while promoting mesenchymal stem cell adhesion, proliferation, and osteogenic differentiation.32 In the present study, the addition of nanoparticles typically showed no significant differences for DU145 cell attachment when compared to unmodified silk hydrogels at matched timepoints (Fig. 5). However, within groups statistically significant differences were observed indicating improved cell attachment for 0.05% w/v tasar and silica nanoparticles functionalized hydrogels as well as for both 0.05 and 0.5% w/v B. mori nanoparticle hydrogels. However, the largest increase in DU145 cell numbers within 24 h was observed using tissue culture–treated polystyrene, which outcompeted all the silk substrates. This trend continued into days 6 and 9 of culture (data not shown). Therefore, the tasar nanoparticles used here apparently had little impact on cell attachment. This was surprising because the presence of the RGD motif in this silk had been expected to improve cell–material interaction via integrin engagement. This lack of improvement in cell attachment could have several reasons, including restricted accessibility of the tasar nanoparticles for integrin receptor engagement. Focal adhesion organisation shows a high sensitivity to ligand spacing, with a nanoscale average RGD spacing of 44 nm needed to form lipid raft domains at focal adhesion sites.69 This spacing mimics the RGD spacing found in the fibronectin.70 Therefore, further work is needed to improve the nanoparticle placement and spacing in our silk hydrogels. Subsequent studies can then monitor integrin engagement that will ultimately help to further characterise these substrate–cell interactions. Notably, cell viability was maintained on the silk hydrogels spiked with B. mori, tasar silk and silica nanoparticles throughout the culture period (Fig. 5). The present study used DU145 cells only and there is now scope to expand this work. For example, the use of prostate cancer cells that are hormone responsive or those that show a greater metastatic potential than DU145 cells13 would help to assess, and potentially unlock, the fully potential of these culture systems. Furthermore, the use of other cell types including reference cell lines (e.g. L929 fibroblasts71), mesenchymal stem cells15 or keratinocytes72 would further broaden the impact of this work and potentially uncover cell type specific effects. However, more work is needed to fine-tune the cell material interactions for further improvement of the biological response. We speculate that the use of sequence coded nanoparticles will enhance their interactions with the hydrogel via beta sheet engagement ultimately providing greater control over particle presentation. Also the use of larger particles (e.g. 500 nm, 1 μm and 5 μm) would enable more integrin engagement by overcoming the critical RGD spacing. The present study used 2D cultures. However, our system is readily adapted to 3D cell cultures enabling greater probing of the cell–material interface and the subsequent biological response.
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