Dynamic Biomaterials
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Living bacterial therapeutics represent an exciting frontier for achieving controlled drug release within the body. However, genetic modules require improvement to control the production and release of therapeutic biomolecules in medically relevant strains. Model probiotic strains like E. coli Nissle 1917 have extensive genetic toolkits but still lack rapidly responsive and stringent genetic switches to regulate drug release. On the other hand, probiotic bacteria from the Lactobacilli family have broader applicability in the body but remain as non-model strains with restrictive genetic programmability. This thesis addresses both these limitations. Firstly, I developed a strategy to achieve strict control over the release of an enzymatically synthesized antibiotic (darobactin) from E. coli Nissle 1917. By combining parts from pre-established genetic switches, I created a thermo-amplifier circuit that released darobactin at pathogen inhibitory levels within a few hours. Secondly, I expanded the genetic toolbox of the probiotic Lactiplantibacillus plantarum WCFS1 strain with two genetic parts - a strong constitutive promoter (PtlpA) and several type II toxin-antitoxin (TA)-based plasmid retention systems. The performance of these genetic modules in recombinant plasmids was verified using reporter proteins such as mCherry and Staphylococcal nuclease without the need for antibiotic-based selection pressure.
Photocrosslinkable formulations based on the radical thiol-ene reaction are considered better alternatives than methacrylated counterparts for light-based fabrication processes. This study quantifies differences between thiol-ene and methacrylated crosslinked hydrogels in terms of precursors stability, the control of the crosslinking process, and the resolution of printed features particularized for hyaluronic acid (HA) inks at concentrations relevant for bioprinting. First, the synthesis of HA functionalized with norbornene, allyl ether, or methacrylate groups with the same molecular weight and comparable degrees of functionalization is presented. The thiol-ene hydrogel precursors show storage stability over 15 months, 3.8 times higher than the methacrylated derivative. Photorheology experiments demonstrate up to 4.7-times faster photocrosslinking. Network formation in photoinitiated thiol-ene HA crosslinking allows higher temporal control than in methacrylated HA, which shows long post-illumination hardening. Using digital light processing, 4% w/v HA hydrogels crosslinked with a dithiol allowed printing of 13.5 × 4 × 1 mm3 layers with holes of 100 µm resolution within 2 s. This is the smallest feature size demonstrated in DLP printing with HA-based thiol-ene hydrogels. The results are important to estimate the extent to which the synthetic effort of introducing –ene functions can pay off in the printing step.
Side-emitting optical fibers allow light to be deliberately outcoupled along the fiber. Introducing a customized side-emission profile requires modulation of the guiding and emitting properties along the fiber length, which is a particular challenge in continuous processing of soft waveguides. In this work, it is demonstrated that multimaterial extrusion printing can generate hydrogel optical fibers with tailored segments for light-side emission. The fibers are based on diacrylated Pluronic F-127 (PluDA). 1 mm diameter fibers are printed with segments of different optical properties by switching between a PluDA waveguiding ink and a PluDA scattering ink containing nanoparticles. The method allows the fabrication of fibers with segment lengths below 500 microns in a continuous process. The length of the segments is tailored by varying the switching time between inks during printing. Fibers with customized side-emission profiles along their length are presented. The functionality of the printed fibers is demonstrated by exciting fluorescence inside a surrounding 3D hydrogel. The presented technology and material combination allow unprecedented flexibility for designing soft optical fibers with customizable optical properties using simple processes and a medical material. This approach can be of interest to improve illumination inside tissues for photodynamic therapy (PDT).
Living Therapeutic Materials (LTMs) are a promising alternative to polymeric drug carriers for long term release of biotherapeutics. LTMs contain living drug biofactories that produce the drug using energy sources from the body fluids. To clarify their application potential, it is fundamental to adapt biocompatibility and cytotoxicity assays applied from non-living biomaterials and therapeutics to evaluate how LTMs interact with host cells. Here, we have established a first step in this direction, by developing a practical workflow to parallelize in vitro assessment of minimal safety and cytocompatibility properties of bacterial LTMs. It allows systematic monitoring and quantification of the dynamic evolution of the bacterial population (growth, metabolic activity) in parallel to quantify the response of different mammalian cells to LTM supernatants with regards to cytotoxicity and release of pro-inflammatory cytokines over a period of 7 days using a maximum of 10 samples. The protocol was tested with a Pluronic-based thin film containing ClearColi. The results show no cytotoxic effects of ClearColi containing hydrogels in three mammalian cell lines, and no induction of pro-inflammatory cytokines under the tested conditions. This workflow represents a first step in establishing a roadmap for the safety assessment of LTMs, and investigation of biocompatibility potential of future living therapeutic devices.
The role of growth factors is important to stimulate regenerative cellular changes to rejuvenate damaged cells, tissues, and organs. Growth factor engineering and delivery systems are developed quite a lot for example, emergence of small protein like chains (peptidomimetics) and matrices for controlled release of growth factors. Despite these advancements, the use of growth factors in regenerative medicine is limited because of their low stability. In this thesis, angiogenesis inducing Engineered Living Materials (ELMs) are used as the strategy to overcome the limitations associated with traditional GF delivery methods. These ELMs contain living bacteria programmed to synthesize angiogenic protein in response to light. This thesis describes challenges and successes in developing light regulated Engineered Living Material that releases angiogenic protein. The bacteria were ontogenetically engineered to synthesize and secrete a Vascular Endothelial Growth Factor (VEGF) mimetic peptide (QK) attached to a Collagen Binding Domain (CBD). To create an ELM, the engineered bacteria were safely encapsulated in a bilayer hydrogel designed to help aid survival and to prevent bacterial escape from the material. It is proven that in-situ control over production of pro-angiogenic protein can be attained with light. Secreted protein can bind to collagen and promote endothelial cell network formation which is a hallmark of angiogenesis. These results highlight the potential of this light inducible ELM to support vascularization in endothelial cells.
Engineered living materials (ELMs), which usually comprise bacteria, fungi, or animal cells entrapped in polymeric matrices, offer limitless possibilities in fields like drug delivery or biosensing. Determining the conditions that sustain ELM performance while ensuring compatibility with ELM hosts is essential before testing them in vivo. This is critical to reduce animal experimentation and can be achieved through in vitro investigations. Currently, there are no standards that ensure ELM compatibility with host tissues. Towards this goal, we designed a 96-well plate-based screening method to streamline ELM growth across culture conditions and determine their compatibility potential in vitro. We showed proliferation of three bacterial species encapsulated in hydrogels over time and screened six different cell culture media. We fabricated ELMs in bilayer and monolayer formats and tracked bacterial leakage as a measure of ELM biocontainment. After screening, an appropriate medium was selected that sustained growth of an ELM, and it was used to study cytocompatibility in vitro. ELM cytotoxicity on murine fibroblasts and human monocytes was studied by adding ELM supernatants and measuring cell membrane integrity and live/dead staining, respectively, proving ELM cytocompatibility. Our work illustrates a simple setup to streamline the screening of compatible environmental conditions of ELMs with the host.
Polyacrylamide Hydrogels as Versatile Biomimetic Platforms to Study Cell-Materials Interactions
(2024)
Polyacrylamide (PAAm) hydrogels are widely adopted as 2D-model soft substrates for investigating cell-material interactions in a controlled in vitro environment. They offer facile synthesis, tunable physico-chemical properties, diverse biofunctionalization routes, optical transparency, mouldability in a range of geometries and shapes, and compatibility with living cells. PAAm hydrogels can be engineered to reconstruct physiologically relevant biointerfaces, like cell-matrix or cell–cell interfaces, featuring biochemical, mechanical, and topographical cues present in the extracellular environment. This Review provides a materials science perspective on PAAm material properties, fabrication, and modification strategies relevant to cell studies, highlighting their versatility and potential to address a wide range of biological questions. Current routes are presented to integrate cell-instructive features, such as 2D patterns, 2.5D surface topographies, or mechanical stiffness gradients. Finally, the recent advances are emphasized toward dynamic PAAm hydrogels with on-demand control over hydrogel properties as well as electrically conductive PAAm hydrogels for bioelectronics.
The understanding of diseases and the development of personalized therapies relies on the predictive potential of 3D cell culture models. Automation of the culture steps is crucial, for which in situ crosslinked hydrogels with adequate and tunable kinetics under physiological conditions are needed to temporally replace the natural 3D matrix. This Thesis investigates hydrogels formed by tetrazol methylsulfone (TzMS) derivatized star-polyethylene glycol and thiol-crosslinkers for automated 3D cell encapsulation, including precursor synthesis scale-up, characterization of their crosslinking kinetics and mechanical properties, and their semi-automated preparation with pipetting robots. The optimization of reaction and purification conditions allowed for an upscaling to 0.6-g scale of polymer precursor. The gelation kinetics and mechanical properties of the hydrogels were studied as a function of precursors stoichiometry, crosslinker architecture, biofunctionalization degree and pH. Tunability of gelation time and stiffness at pH 7.0 – 8.0 was explored for a cell-compatible hydrogel composition including the cell-adhesive ligand RGD and the enzyme-cleavable peptide VPM as crosslinker. A semi-automated pipetting protocol was set up to prepare 5 µl hydrogels in a 384-well plate format, and statistical experimental design was used to systematically minimize variability. The results demonstrate the suitability and limitations of TzMS/thiol chemistry for in situ cell encapsulation.
Photopolymerizable formulations based on the thiol-ene crosslinking are considered better alternatives than methacrylated counterparts for light-based fabrication processes. The thiol-ene photoreaction requires lower light doses for polymerization and leads to networks with lower molecular heterogeneity. For hydrogels at low polymer concentrations as used in bioinks (<8 wt%), faster polymerization rates and higher conversion of the crosslinking reaction have been reported in thiol-ene systems. Here we quantify further differences between thiol-ene and methacrylated crosslinked hydrogels for HA functionalized with norbornene, allyl ether or methacrylate groups. We show storage stability of the thiol-ene hydrogel precursors over 15 months, 3.8 times higher than the methacrylated derivative. Photorheology experiments demonstrated up to 4.7-times faster photocrosslinking. Network formation in photoinitiated thiol-ene HA crosslinking allows higher temporal control than in methacrylated HA, which shows long post-illumination hardening. Using digital light processing, 4% w/v HA hydrogels polymerized with a dithiol crosslinker allowed printing of 13.5x4x1 mm3 layers with holes of 100 µm resolution within 2 s. Considering that ene-derivatized hydrogel precursors are less commercially available than methacrylated counterparts, our results are important to estimate the extent to which the synthetic effort of introducing –ene functions can pay off in the printing step.
Engineered living materials (ELMs), which usually comprise bacteria, fungi, or animal cells entrapped in polymeric matrices, offer limitless possibilities in fields like drug delivery or biosensing. To determine the conditions that sustain ELM performance while ensuring ELM-host compatibility is essential before testing them in vivo. This is critical to reduce animal experimentation and can be achieved through in vitro investigations. Towards this goal, we designed a 96-well plate-based screening method to streamline ELM growth across culture conditions and determine their compatibility potential in vitro. We showed proliferation of three bacterial species encapsulated in hydrogels over time and screened six different cell culture media. We fabricated ELMs in bilayer and monolayer formats and tracked bacterial leakage. After screening, an appropriate medium was selected that sustained growth of an ELM, and it was used to study cytocompatibility in vitro. ELM cytotoxicity on murine fibroblasts and human monocytes was studied by adding ELM supernatants and measuring cell membrane integrity and live/dead staining, respectively, proving ELM cytocompatibility. Our work illustrates a simple setup to streamline the screening of compatible environmental conditions of ELMs with the host.
