A biocompatible betaine-functionalized polycation for coacervation.

The aqueous nature of complex coacervates provides a biologically-relevant context for various therapeutic applications. In this sense, biological applications demand a corresponding level of biocompatibility from the polyelectrolytes that participate in complex coacervation. Continued development with naturally-occurring polyelectrolytes such as heparin and chitosan underscore such aims. Herein, we design a synthetic polycation, in which betaine is conjugated to a biodegradable polyester backbone. Betaine is a naturally-occurring methylated amino acid that is ubiquitously present in human plasma. Inspired by its vast range of benefits - including but not limited to anti-inflammation, anti-cancer, anti-bacterial, anti-oxidant, protein stabilization, and cardiovascular health - we aim to impart additional functionality to a polycation for eventual use in a complex coacervate with heparin. We report on its in vitro and in vivo biocompatibility, in vitro and in vivo effect on angiogenesis, in vitro effect on microbial growth, and ability to form complex coacervates with heparin.

Pair of Functional Polyesters That Are Photo-Cross-Linkable and Electrospinnable to Engineer Elastomeric Scaffolds with Tunable Structure and Properties.

A pair of alkyne- and thiol-functionalized polyesters are designed to engineer elastomeric scaffolds with a wide range of tunable material properties (e.g., thermal, degradation, and mechanical properties) for different tissues, given their different host responses, mechanics, and regenerative capacities. The two prepolymers are quickly photo-cross-linkable through thiol-yne click chemistry to form robust elastomers with small permanent deformations. The elastic moduli can be easily tuned between 0.96 ± 0.18 and 7.5 ± 2.0 MPa, and in vitro degradation is mediated from hours up to days by adjusting the prepolymer weight ratios. These elastomers bear free hydroxyl and thiol groups with a water contact angle of less than 85.6 ± 3.58 degrees, indicating a hydrophilic nature. The elastomer is compatible with NIH/3T3 fibroblast cells with cell viability reaching 88 ± 8.7% relative to the TCPS control at 48 h incubation. Differing from prior soft elastomers, a mixture of the two prepolymers without a carrying polymer is electrospinnable and UV-cross-linkable to fabricate elastic fibrous scaffolds for soft tissues. The designed prepolymer pair can thus ease the fabrication of elastic fibrous conduits, leading to potential use as a resorbable synthetic graft. The elastomers could find use in other tissue engineering applications as well.

Synthesis and Characterization of Alkyne-Functionalized Photo-Cross-Linkable Polyesters.

An alkyne-functionalized elastomer derived from sebacic acid, 1,3-propanediol, and alkyne-functionalized serinol is synthesized via melt condensation. A low-power UV lamp triggers the cross-linking rapidly via thiol-yne click chemistry. The cross-linking behavior is studied by photorheology and NMR spectroscopy. The resultant elastomer possesses mechanical properties similar to those of human soft tissues and exhibits in vitro degradability and good cytocompatibility.

Using Solution Electrowriting to Control the Properties of Tubular Fibrous Conduits.

Multiple additive manufacturing techniques have been developed in recent years to produce structures with tunable physical, chemical, and mechanical properties and defined architecture. Solution electrospinning, although an older and more established technique, normally cannot achieve the pattern resolution and tunability of these newer manufacturing techniques. In this study, we present solution electrowriting as a method to produce fibrous conduits from various polymers with tunable patterns, dimensions, and scaffold porosity. We demonstrate the importance of solvent selection during solution electrowriting and discuss how solvent polarity and volatility can be exploited to controllably alter the structure of the resulting scaffolds. The technique can be readily implemented with equipment for conventional electrospinning and offers versatility, control, and customization that is uncommon in the solution electrospinning field.

Citrate Crosslinked Poly(Glycerol Sebacate) with Tunable Elastomeric Properties.

Poly(glycerol-sebacate) (PGS) is a biodegradable elastomer known for its mechanical properties and biocompatibility for soft tissue engineering. However, harsh thermal crosslinking conditions are needed to make PGS devices. To facilitate the thermal crosslinking, citric acid is explored as a crosslinker to form poly(glycerol sebacate citrate) (PGSC) elastomers. The effects of varying citrate contents and curing times are investigated on the mechanical properties, elasticity, degradation, and hydrophilicity. To examine the potential presence of unreacted citric acid, material acidity is monitored in relation to the citrate content and curing times. It is discovered that a low citrate content and a short curing time produce PGSC with tunable mechanical characteristics similar to PGS with enhanced elasticity. The materials demonstrate good cytocompatibility with human umbilical vein endothelial cells similar to the PGS control. The research study suggests that PGSC is a potential candidate for large-scale biomedical applications because of the quick thermal crosslink and tunable elastomeric properties.

Slow degrading poly(glycerol sebacate) derivatives improve vascular graft remodeling in a rat carotid artery interposition model.

Porous synthetic grafts made of poly (glycerol sebacate) (PGS) can transform into autologous vascular conduits in vivo upon degradation of PGS. A long-held doctrine in tissue engineering is the necessity to match degradation of the scaffolds to tissue regeneration. Here, we tested the impact of degradation of PGS and its derivative in an interposition model of rat common carotid artery (CCA). Previous work indicates a complete degradation of PGS within approximately 2 weeks, likely at the fast end of the spectrum. Thus, the derivation of PGS focuses on delay degradation by conjugating the free hydroxy groups in PGS with a long chain carboxylic acid: palmitic acid, one of the most common lipid components. We evaluated two of the resultant palmitate-PGS (PPGS) in this study: one containing 9% palmitate (9-PPGS) and the other16% palmitate (16-PPGS). 16-PPGS grafts had the highest patency. Ultrasound imaging showed that the lumens of 16-PPGS grafts were similar to CCA and smaller than 9-PPGS and PGS grafts 12 weeks post-operation. Immunohistological and histological examination showed an endothelialized lumens in all three types of grafts within 4 weeks. Inflammatory responses to 16-PPGS grafts were limited to the adventitial space in contrast to a more diffusive infiltration in 9-PPGS and PGS grafts in week 4. Examination of calponin+ and αSMA+ cells revealed that 16-PPGS grafts remodeled into a distinctive bi-layered wall, while the walls of 9-PPGS grafts and PGS grafts only had one thick layer of smooth muscle-like cells. Correspondingly, the expression of collagen III and elastin displayed an identical layered structure in the remodeled 16-PPGS grafts, in contrast to a more spread distribution in 9-PPGS and PGS grafts. All the three types of grafts exhibited the same collagen content and burst pressure after 12 weeks of host remodeling. However, the compliance and elastin content of 16-PPGS grafts in week 12 were closest to those of CCA. Overall, placing the degradation of PGS derived elastomer to a window of 4-12 weeks results in vascular conduits closer to arteries in a rat carotid artery interposition model over a 12-week observation period.

Control the Mechanical Properties and Degradation of Poly(Glycerol Sebacate) by Substitution of the Hydroxyl Groups with Palmitates.

Mechanical properties and degradation profile are important parameters for the applications of biodegradable polyester such as poly(glycerol sebacate) in biomedical engineering. Here, a strategy is reported to make palmitate functionalized poly(glycerol sebacate) (PPGS) to alter the polymer hydrophobicity, crystallinity, microstructures and thermal properties. The changes of these intrinsic properties impart tunable degradation profiles and mechanical properties to the resultant elastomers depending on the palmitate contents. When the palmitates reach up to 16 mol%, the elastic modulus is tuned from initially 838 ± 55 kPa for the PGS to 333 ± 21 kPa for the PPGS under the same crosslinking conditions. The elastomer undergoes reversible elastic deformations for at least 1000 cycles within 20% strain without failure and shows enhanced elasticity. The polymer degradation is simultaneously inhibited because of the increased hydrophobicity. This strategy is different with other PGS modifications which could form a softer elastomer with less crosslinks but typically lead to a quicker degradation. Because the materials are made from endogenous molecules, they possess good cytocompatibility similar to the PGS control. Although these materials are designed specifically for small arteries, it is expected that they will be useful for other soft tissues too.

Chelation Crosslinking of Biodegradable Elastomers.

Widely present in nature and in manufactured goods, elastomers are network polymers typically crosslinked by strong covalent bonds. Elastomers crosslinked by weak bonds usually exhibit more plastic deformation. Here, chelation as a mechanism to produce biodegradable elastomers is reported. Polycondensation of sebacic acid, 1,3-propanediol, and a Schiff-base (2-[[(2-hydroxyphenyl) methylene]amino]-1,3-propanediol) forms a block copolymer that binds several biologically relevant metal ions. Chelation offers a unique advantage unseen in conventional elastomer design because one ligand binds multiple metal ions, yielding bonds of different strengths. Therefore, one polymeric ligand coordinated with different metal ions produces elastomers with vastly different characteristics. Mixing different metal ions in one polymer offers another degree of control on material properties. The density of the ligands in the block copolymer further regulates the mechanical properties. Moreover, a murine model reveals that Fe3+ crosslinked foam displays higher compatibility with subcutaneous tissues than the widely used biomaterial-polycaprolactone. The implantation sites restore to their normal architecture with little fibrosis upon degradation of the implants. The versatility of chelation-based design has already shown promise in hydrogels and highly stretchy nondegradable polymers. The biodegradable elastomers reported here would enable new materials and new possibilities in biomedicine and beyond.

Three-Dimensional Printing of Poly(glycerol sebacate) Acrylate Scaffolds via Digital Light Processing.

Digital light processing (DLP)-based three-dimensional (3D) printing offers large improvements in fabrication throughput and spatial resolution when compared to various other additive manufacturing techniques. Both properties are highly desirable when fabricating biomaterial scaffolds that require design precision. Poly(glycerol sebacate) acrylate (PGSA) is a degradable, biocompatible, and photocurable elastomer. In this work, PGSA ink was developed for DLP 3D printing of porous tubular structures. Ink formulations with varying prepolymer concentrations (10-60 wt %), diluent (dimethyl sulfoxide (DMSO), 2-butoxyethyl acetate (EGBEA), and 1:1 DMSO/EGBEA), and degree of PGSA acrylation (17-75%) were studied to optimize printing efficiency and bulk properties of the printed scaffolds. Prepolymer inks with viscosity (<5 Pa·s) and photopolymerization kinetics (exposure time <10 s) appropriate for DLP were developed. Photocrosslinked PGSA scaffolds were further exposed to postfabrication treatments including additional UV exposure or thermal curing (150 °C) to demonstrate tunability in scaffold degradation kinetics and mechanical properties. Complementary to this effort, a 3D model-generation tool was developed to enable user-friendly customization of tubular scaffold design by controlling the pore and strut size of the volumetric mesh. The resulting DLP-printed PGSA scaffolds present high mimicry to complex 3D models with a minimum feature thickness of 80 µm. The tunable properties of PGSA coupled with enhanced precision in microstructure geometry provide a fabrication platform for a variety of tissue regeneration applications.

Azido-Functionalized Polyurethane Designed for Making Tunable Elastomers by Click Chemistry.

Polyurethane is an important biomaterial with wide applications in biomedical engineering. Here, we report a new method to make an azido-functionalized polyurethane prepolymer with no need of postmodification. This prepolymer can easily form stable porous elastomers through click chemistry for cross-linking, instead of using a toxic polyisocyanate. The mechanical properties can be modulated by simply adjusting either the prepolymer concentrations or azido/alkyne ratios for cross-linking. Young's modulus therefore varies from 0.52 to 2.02 MPa for the porous elastomers. When the azido-functionalized polyurethane elastomer is made with a compact structure, Young's modulus increases up to 28.8 MPa at 0-15% strain. The strain at break reaches 150% that is comparable to the commercially resourced Nylon-12. Both the porous and compact elastomers could undergo reversible elastic deformations for at least 200 and 1000 cycles, respectively, within 20% strain without failure. The material showed a considerable stability against erosion in a basic solution. In vivo biocompatibility study demonstrated no degradation by subcutaneous implantation in mice over 2 months. The implant induced only a mild inflammatory response and fibrotic capsule. This material might be useful to make elastomeric components of biomedical devices.

Imidazoquinoline-Conjugated Degradable Coacervate Conjugate for Local Cancer Immunotherapy.

Strategies that can reduce the harmful side effects of potent immunomodulatory drugs are in high demand to facilitate clinical translation of the newest generation of immunotherapy. Indeed, uncontrolled triggering of the immune system can lead to life-threatening cascade reactions, such as e.g. cytokine storm. In particular, drug formulations that combine simplicity and degradability are of formidable relevance. Imidazoquinolines are an excellent example of such small molecule immunomodulatory drugs that exhibit in unformulated form a highly undesirable pharmacokinetic profile. Imidazoquinolines are potent inducers of type I interferons that are of great interest in the context of anticancer and antiviral therapy through triggering of Toll like receptors 7 and 8. In this work we aimed to alter the pharmacokinetic profile of imidazoquinolines using a simple, yet efficient, strategy that holds high potential for clinical translation. Hereto, we conjugated an imidazoquinoline to the backbone of poly(aspartate) and further formulated this into a degradable coacervate through complex coacervation with a nontoxic degradable polycation. The intrinsic TLR activity of the imidazoquinoline was well preserved and our formulation strategy offered spatial control over its biological activity in vivo.

A biodegradable synthetic graft for small arteries matches the performance of autologous vein in rat carotid arteries.

Autologous veins are the most widely used grafts for bypassing small arteries in coronary and peripheral arterial occlusive diseases. However, they have limited availability and cause donor-site morbidity. Here, we report a direct comparison of acellular biodegradable synthetic grafts and autologous veins as interposition grafts of rat carotid arteries, which is a good model for clinically relevant small arteries. Notably, extensive but transient infiltration of circulating monocytes at day 14 in synthetic grafts leads to a quickly-resolved inflammation and arterial-like tissue remodeling. The vein graft exhibits a similar inflammation phase except the prolonged presence of inflammatory monocytes. The walls of the remodeled synthetic graft contain many circumferentially aligned contractile non-proliferative smooth muscle cells (SMCs), collagen and elastin. In contrast, the walls of the vein grafts contain disorganized proliferating SMCs and thicken over time, suggesting the onset of stenosis. At 3 months, both grafts have a similar patency, extracellular matrix composition, and mechanical properties. Furthermore, synthetic grafts exhibit recruitment and re-orientation of newly synthesized collagen fibers upon mechanical loading. To our knowledge, this is the first demonstration of a biodegradable synthetic vascular graft with a performance similar to an autologous vein in small artery grafting.

Weak Bond-Based Injectable and Stimuli Responsive Hydrogels for Biomedical Applications.

Here we define hydrogels crosslinked by weak bonds as physical hydrogels. They possess unique features including reversible bonding, shear thinning and stimuli-responsiveness. Unlike covalently crosslinked hydrogels, physical hydrogels do not require triggers to initiate chemical reactions for in situ gelation. The drug can be fully loaded in a pre-formed hydrogel for delivery with minimal cargo leakage during injection. These benefits make physical hydrogels useful as delivery vehicles for applications in biomedical engineering. This review focuses on recent advances of physical hydrogels crosslinked by weak bonds: hydrogen bonds, ionic interactions, host-guest chemistry, hydrophobic interactions, coordination bonds and π-π stacking interactions. Understanding the principles and the state of the art of gels with these dynamic bonds may give rise to breakthroughs in many biomedical research areas including drug delivery and tissue engineering.

Tyramine functionalization of poly(glycerol sebacate) increases the elasticity of the polymer.

Poly(glycerol sebacate) (PGS) is an elastomer used widely in tissue engineering studies due to good biocompatibility. Hereby we report a tyramine functionalized PGS called PGS-TA. Tyramine adds a stronger physical bonding capability to PGS-TA. Tensile tests showed that the softness and toughness of the material were similar to PGS. However, PGS-TA demonstrated 16-folds increase of elastic deformations compared to PGS processed under identical conditions. The in vitro studies demonstrated that the viability, and metabolic activity of baboon smooth muscle cells were the same as those on tissue culture polystyrene. Porous subcutaneous implants of PGS-TA substantially degraded in vivo over two weeks, showing good biodegradability and biocompatibility. We expect PGS-TA to be useful for applications in tissues and organs that are subjected to large reversible mechanical deformations.

Dual physical dynamic bond-based injectable and biodegradable hydrogel for tissue regeneration.

A biodegradable and injectable hydrogel was designed using dual physical dynamic bonds based on host-guest chemistry and electrostatic interactions to build up the network structure. The material was synthesized by simultaneously coupling mono-carboxylic acid terminated poly(ethylene glycol) and arginine to poly(ethylene aspartate diglyceride) to yield a mPEG-grafted poly(ethylene argininylaspartate diglyceride) (mPEG-g-PEAD). When mixing this polymer with α-cyclodextrin and a natural polyanion (heparin), the supramolecular network was formed in a quick gelation with shear thinning properties. The in vitro cytotoxicity was evaluated using primary baboon arterial smooth muscle cells (BaSMCs) and the results showed that cell membrane integrity, viability and metabolism were not compromised by this synthetic polycation at concentrations as high as 10 mg mL-1, a 1000-fold lower toxicity than commercial PEI. The in vitro biocompatibility of the as-made hydrogel was also evaluated using BaSMCs. Neither the hydrogel nor the hydrogel components altered cell behavior in the assays. Fibroblast growth factor 2 was incorporated into the hydrogel and sustainably released at a nearly stable rate up to 16 days without initial burst release, suggesting potential applications in wound healing and ischemic tissue regeneration, among others.

A shear-thinning hydrogel that extends in vivo bioactivity of FGF2.

We designed and tested a versatile hydrogel for tissue regeneration by preserving the bioactivity of growth factors. The shear-thinning gel self-assembles within 1 min from heparin and Laponite-a silicate nanoparticle, thus the name HELP gel. By not covalently modifying heparin, it should retain its natural affinity towards many proteins anchored in the extracellular matrix. In principle, HELP gel can bind any heparin-binding growth factor; we use fibroblast growth factor-2 (FGF2) in this study to demonstrate its utility. Heparin in the gel protects FGF2 from proteolytic degradation and allows it to be released over time with preserved bioactivity. FGF2 released from subcutaneously injected gel induces strong angiogenesis in a mouse model. The hydrogel degrades completely in vivo in 8 weeks with or without growth factors, eliciting mild inflammatory response but having little impacts on the surrounding tissue. The ease of preparation and scale-up makes this protein delivery platform attractive for clinical translation.

Nitro-Group Functionalization of Dopamine and its Contribution to the Viscoelastic Properties of Catechol-Containing Nanocomposite Hydrogels.

Linear polyacrylamide (PAAm) is modified with dopamine or nitrodopamine (PAAm-D and PAAm-ND, respectively) to evaluate the effect of nitro-group modification on the interfacial binding properties of polymer-bound catechol. Nanocomposite hydrogels are prepared by mixing PAAm-based polymers with Laponite and the viscoelastic properties of these materials are determined using oscillatory rheometry. The incorporation of a small amount of catechol (≈0.1 wt% in swollen hydrogel) drastically increases the shear moduli by 1-2 orders of magnitude over those of the catechol-free control. Additionally, PAAm-ND exhibits higher shear moduli values than PAAm-D across the whole pH range tested (pH 3.0-9.0). Based on the calculated effective crosslinking density, effective functionality, and molecular weight between crosslinks, nitro-group functionalization of dopamine results in a polymer network with increased crosslinking density and crosslinking points with higher functionality. Nitro-functionalization enhances the interfacial binding property of dopamine and increases its resistant to oxidation, which results in nanocomposite hydrogels with enhanced stiffness and a viscous dissipation property.

Peptide-directed self-assembly of functionalized polymeric nanoparticles. Part II: effects of nanoparticle composition on assembly behavior and multiple drug loading ability.

Peptide-functionalized polymeric nanoparticles were designed and self-assembled into continuous nanoparticle fibers and three-dimensional scaffolds via ionic complementary peptide interaction. Different nanoparticle compositions can be designed to be appropriate for each desired drug, so that the release of each drug is individually controlled and the simultaneous sustainable release of multiple drugs is achieved in a single scaffold. A self-assembled scaffold membrane was incubated with NIH3T3 fibroblast cells in a culture dish that demonstrated non-toxicity and non-inhibition on cell proliferation. This type of nanoparticle scaffold combines the advantages of peptide self-assembly and the versatility of polymeric nanoparticle controlled release systems for tissue engineering.

Peptide-directed self-assembly of functionalized polymeric nanoparticles part I: design and self-assembly of peptide-copolymer conjugates into nanoparticle fibers and 3D scaffolds.

A robust self-assembly of nanoparticles into fibers and 3D scaffolds is designed and fabricated by functionalizing a RAFT-polymerized amphiphilic triblock copolymer with designer ionic complementary peptides so that the assembled core-shell polymeric nanoparticles are directed by peptide assembly into continuous "nanoparticle fibers," ultimately leading to 3D fiber scaffolds. The assembled nanostructure is confirmed by FESEM and optical microscopy. The assembly is not hindered when a protein (insulin) is incorporated within the nanoparticles as an active ingredient. MTS cytotoxicity tests on SW-620 cell lines show that the peptides, copolymers, and peptide-copolymer conjugates are biocompatible. The methodology of self-assembled nanoparticle fibers and 3D scaffolds is intended to combine the advantages of a flexible hydrogel scaffold with the versatility of controlled release nanoparticles to offer unprecedented ability to incorporate desired drug(s) within a self-assembled scaffold system with individual control over the release of each drug.