Investigation of the Effectiveness of Photo Deprotection of Polypeptides in Solution and within the Core of Miniemulsion-Derived Nanoparticles.

Homopolymerization of ortho-nitrobenzyl (oNB)-protected l-cysteine and l-glutamic acid was systematically studied in different solvents and at different monomer to initiator ratios, revealing the best reaction control in dimethylformamide (DMF) across a range of degrees of polymerization. In the subsequent ultraviolet (UV)-cleavage studies, it was found that quantitative deprotection upon UV exposure at 365 nm was not achievable for either of the homopolypeptides as confirmed by 1H NMR and UV/visible (UV/vis) analyses. While the poly(oNB-l-cysteine) deprotected more readily with no effect of the polypeptide molecular weight, lower molecular weight poly(oNB-l-glutamate) reached maximum deprotection faster than high molecular weight samples. This was further confirmed by the pH changes of the solution. When incorporated into the core of miniemulsion-derived nanoparticles, both oNB-protected copolypeptides were successfully deprotected as evident from a color change and a pH change in the case of poly(oNB-l-glutamate). However, the removal of the deprotection byproduct nitrosobenzaldehyde proved unsuccessful, which indicates a diffusion barrier caused by the nanoparticle's surfactant. The study provides insights and guidelines for the UV deprotection of polypeptides and demonstrates the ability to selectively UV-deprotect polypeptides in the confined space of a nanoparticle dispersion.

Optimizing the Delivery of mRNA to Mesenchymal Stem Cells for Tissue Engineering Applications.

Messenger RNA (mRNA) represents a promising therapeutic tool in the field of tissue engineering for the fast and transient production of growth factors to support new tissue regeneration. However, one of the main challenges to optimizing its use is achieving efficient uptake and delivery to mesenchymal stem cells (MSCs), which have been long reported as difficult-to-transfect. The aim of this study was to systematically screen a range of nonviral vectors to identify optimal transfection conditions for mRNA delivery to MSCs. Furthermore, for the first time, we wanted to directly compare the protein expression profile from three different types of mRNA, namely, unmodified mRNA (uRNA), base-modified mRNA (modRNA), and self-amplifying mRNA (saRNA) in MSCs. A range of polymer- and lipid-based vectors were used to encapsulate mRNA and directly compared in terms of physicochemical properties as well as transfection efficiency and cytotoxicity in MSCs. We found that both lipid- and polymer-based materials were able to successfully condense and encapsulate mRNA into nanosized particles (<200 nm). The overall charge and encapsulation efficiency of the nanoparticles was dependent on the vector type as well as the vector:mRNA ratio. When screened in vitro, lipid-based vectors proved to be superior in terms of mRNA delivery to MSCs cultured in a 2D monolayer and from a 3D collagen-based scaffold with minimal effects on cell viability, thus opening the potential for scaffold-based mRNA delivery. Modified mRNA consistently showed the highest levels of protein expression in MSCs, demonstrating 1.2-fold and 5.6-fold increases versus uRNA and saRNA, respectively. In summary, we have fully optimized the nonviral delivery of mRNA to MSCs, determined the importance of careful selection of the mRNA type used, and highlighted the strong potential of mRNA for tissue engineering applications.

High Molecular Weight Polyproline as a Potential Biosourced Ice Growth Inhibitor: Synthesis, Ice Recrystallization Inhibition, and Specific Ice Face Binding.

Ice-binding proteins (IBPs) from extremophile organisms can modulate ice formation and growth. There are many (bio)technological applications of IBPs, from cryopreservation to mitigating freeze-thaw damage in concrete to frozen food texture modifiers. Extraction or expression of IBPs can be challenging to scale up, and hence polymeric biomimetics have emerged. It is, however, desirable to use biosourced monomers and heteroatom-containing backbones in polymers for in vivo or environmental applications to allow degradation. Here we investigate high molecular weight polyproline as an ice recrystallization inhibitor (IRI). Low molecular weight polyproline is known to be a weak IRI. Its activity is hypothesized to be due to the unique PPI helix it adopts, but it has not been thoroughly investigated. Here an open-to-air aqueous N-carboxyanhydride polymerization is employed to obtain polyproline with molecular weights of up to 50000 g mol-1. These polymers were found to have IRI activity down to 5 mg mL-1, unlike a control peptide of polysarcosine, which did not inhibit all ice growth at up to 40 mg mL-1. The polyprolines exhibited lower critical solution temperature behavior and assembly/aggregation observed at room temperature, which may contribute to its activity. Single ice crystal assays with polyproline led to faceting, consistent with specific ice-face binding. This work shows that non-vinyl-based polymers can be designed to inhibit ice recrystallization and may offer a more sustainable or environmentally acceptable, while synthetically scalable, route to large-scale applications.

Two-Photon Polymerization: Fundamentals, Materials, and Chemical Modification Strategies.

Two-photon polymerization (TPP) has become a premier state-of-the-art method for microscale fabrication of bespoke polymeric devices and surfaces. With applications ranging from the production of optical, drug delivery, tissue engineering, and microfluidic devices, TPP has grown immensely in the past two decades. Significantly, the field has expanded from standard acrylate- and epoxy-based photoresists to custom formulated monomers designed to change the hydrophilicity, surface chemistry, mechanical properties, and more of the resulting structures. This review explains the essentials of TPP, from its initial conception through to standard operating principles and advanced chemical modification strategies for TPP materials. At the outset, the fundamental chemistries of radical and cationic polymerization are described, along with strategies used to tailor mechanical and functional properties. This review then describes TPP systems and introduces an array of commonly used photoresists including hard polyacrylic resins, soft hydrogel acrylic esters, epoxides, and organic/inorganic hybrid materials. Specific examples of each class-including chemically modified photoresists-are described to inform the understanding of their applications to the fields of tissue-engineering scaffolds, micromedical, optical, and drug delivery devices.

Modified poly(L-lysine)-based structures as novel antimicrobials for diabetic foot infections, an in-vitro study.

Background: Wound infections occur as sequelae to skin trauma and cause significant hospitalizations, morbidity and mortality. Skin traumas arise more frequently in those with diabetes or cardiovascular disease and in these settings, may be chronic with poorer outcomes including lower limb amputation. Treatment of chronic wound infection is challenging due to antibiotic resistance and biofilm formation by bacteria including S. aureus and P. aeruginosa, which are among the most frequent causative pathogens. Managing these challenging infections requires new molecules and modalities. Methods: We evaluated antimicrobial and anti-biofilm activity of star-shaped poly(L-lysine) (PLL) polymers against S. aureus and P. aeruginosa strains and clinical isolates recovered from wounds including diabetic foot wounds (DFW) in a Dublin Hospital in 2019. A star-shaped PLL polypeptide series, specifically G2(8)PLL 20, G3(16)PLL 10, G4(32)PLL 5 with variation in polypeptide chain length and arm-multiplicity, were compared to a linear peptide, PLL 160 with equivalent number of lysine residues. Results: All PLLs, including the linear polypeptide, were bactericidal at 1µM against S. aureus 25923 and P. aeruginosa PAO1, with log reduction in colony forming units/ml between 2.7-3.6. PLL 160 demonstrated similar killing potency against 20 S. aureus and five P. aeruginosa clinical isolates from DFW, mean log reductions: 3.04 ± 0.16 and 3.96 ± 0.82 respectively after 1 hour incubation. Potent anti-biofilm activity was demonstrated against S. aureus 25923 but for clinical isolates, low to moderate loss of biofilm viability was shown using PLL 160 and G3(16)PLL 10 at 50 µM ( S. aureus) and 200 µM ( P. aeruginosa) with high inter-isolate variability . In the star-shaped architecture, antimicrobial activity was retained with incorporation of 5-mer hydrophobic amino-acid modifications to the arms of the polypeptides (series G3(16)PLL 20-coPLT 5, G3(16)PLL 20-coPLI 5, G3(16)PLL 20-coPLP 5). Conclusion: These polypeptides offer structural flexibility for clinical applications and have potential for further development, particularly in the setting of diabetic foot and other chronic wound infections.

Ion-Triggered Hydrogels Self-Assembled from Statistical Copolypeptides.

Statistical copolypeptides comprising lysine and tyrosine with unprecedented ion-induced gelation behavior are reported. Copolypeptides are obtained by one-step N-carboxyanhydride (NCA) ring-opening polymerization. The gelation mechanism is studied by in situ SAXS analyses, in addition to optical spectroscopy and transmission electron microscopy (TEM). It is found that the gelation of these statistically polymerized polypeptides is due to the formation of stable intermolecular ß-sheet secondary structures induced by the presence of salt ions as well as the aggregation of an α-helix between the copolypeptides. This behavior is unique to the statistical lysine/tyrosine copolypeptides and was not observed in any other amino acid combination or arrangement. Furthermore, the diffusion and mechanical properties of these hydrogels can be tuned through tailoring the polypeptide chain length and ion strength.

Systematic Study of Enzymatic Degradation and Plasmid DNA Complexation of Mucus Penetrating Star-Shaped Lysine/Sarcosine Polypept(o)ides with Different Block Arrangements.

8-Arm star polypep(o)ides comprising cationic polylysine and hydrophilic polysarcosine blocks with a degree of polymerization (DP) of 30 per block are synthesized. Two different block sequences with polylysine as the inner and polysarcosine as the outer block and vice versa are obtained in addition to a statistical copolymer. Analysis of the enzymatic hydrolysis by the proteolytic enzyme trypsin demonstrates a strong dependence on structural arrangements. While polypept(o)ide disintegration is detectible after 24 h by Size Exclusion Chromatography (SEC), significant hydrolysis of the lysine blocks is only monitored after 48 h by fluorescamine labeling of the produced lysine and clearly accelerated in structures with more accessible polylysine blocks. All structures are capable of complexing plasmid DNA and form gene nanomedicines at sizes around or below 200 nm as determined by Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), and Transition Electron Microscopy (TEM). The polyplex formation is slightly enhanced for both block structures over the random copolypept(o)ide. Moreover, it is demonstrated that the polyplexes can transport through mucus. The results highlight the importance of structural control in compartmentalized polymeric gene vector candidates with hydrophilic domains for potential mucosal delivery.

Versatility of unsaturated polyesters from electrospun macrolactones: RGD immobilization to increase cell attachment.

Poly(globalide) (PGl), an aliphatic polyester derived from unsaturated macrocylic lactone, can be cross-linked during electrospinning and drug-loaded for regenerative medicine applications. However, it lacks intrinsic recognition sites for cell adhesion and proliferation. In order to improve their cell adhesiveness, and therefore their therapeutic potential, we aimed to functionalize electrospun PGl fibers with RGD sequence generating a biomimetic scaffold. First, an amine compound was attached to the surface double bonds of the PGl fibers. Subsequently, the amino groups were coupled with RGD sequences. X-ray photoelectron spectroscopy (XPS) analysis confirmed the functionalization. The obtained fibers were more hydrophilic, as observed by contact angle analysis, and presented smaller Young's modulus, although similar tensile strength compared with non-functionalized cross-linked fibers. In addition, the functionalization process did not significantly alter fibers morphology, as observed by scanning electron microscopy (SEM). Finally, in vitro analysis evidenced the increase in human mesenchymal stromal cells (hMSC) adhesion (9.88 times higher DNA content after 1 day of culture) and proliferation (3.57 times higher DNA content after 8 days of culture) compared with non-functionalized non-cross-linked fibers. This is the first report demonstrating the functionalization of PGl fibers with RGD sequence, improving PGl therapeutic potential and further corroborating the use of this highly versatile material toward regenerative medicine applications.

Ru(ii)/BODIPY core co-encapsulated ratiometric nanotools for intracellular O2 sensing in live cancer cells.

Oxygen is a crucial reagent in many biochemical processes within living cells and its concentration can be an effective marker in disease, particularly in cancer where tissue hypoxia has been shown to indicate tumour growth. Probes that can reflect the oxygen concentration and distribution using ratiometric signals can be applied to a range of conventional methods without the need for specialised equipment and are particularly useful. The preparation and in cellulo study of luminescent ratiometric core-shell nanoparticles are presented. Here, a new lipophilic and oxygen-responsive Ru(ii) tris-heteroleptic polypyridyl complex is co-encapsulated with a reference BODIPY dye into the core of poly-l-lysine-coated polystyrene particles. The co-core encapsulation ensures oxygen response but reduces the impact of the environment on both probes. Single wavelength excitation of the particles, suspended in aqueous buffer, at 480 nm, triggers well-resolved dual emission from both dyes with peak maxima at 515 nm and 618 nm. A robust ratiometric oxygen response is observed from water, with a linear dynamic range of 3.6-262 µM which matches well with typical biological ranges. The uptake of RuBDP NPs was found to be cell-line dependent, but in cancerous cell lines, the particles were strongly permeable with late endosomal and partial lysosomal co-staining observed within 3 to 4 hours, eventually leading to extensive staining of the cytoplasm. The co-localisation of the ruthenium and BODIPY emission confirms that the particles remain intact in cellulo with no indication of dye leaching. The ratiometric O2 sensing response of the particles in cellulo was demonstrated using a plate-based assay and by confocal xyλ scanning of cells exposed to hypoxic conditions.

Three-dimensionally printable shear-thinning triblock copolypeptide hydrogels with antimicrobial potency.

Through rational design, block sequence controlled triblock copolypeptides comprising cysteine and tyrosine as well as a lysine or glutamic acid central block are devised. In these copolypeptides, each block contributes a specific property to the hydrogels to render them extrusion printable and antimicrobial. Three-dimensional (3D) printing of complex hydrogel structures with high shape retention is demonstrated. Moreover, composition dependent potent antimicrobial activity in contact-killing assays is elucidated.

Gene activated scaffolds incorporating star-shaped polypeptide-pDNA nanomedicines accelerate bone tissue regeneration in vivo.

Increasingly, tissue engineering strategies such as the use of biomaterial scaffolds augmented with specific biological cues are being investigated to accelerate the regenerative process. For example, significant clinical challenges still exist in efficiently healing large bone defects which are above a critical size. Herein, we describe a cell-free, biocompatible and bioresorbable scaffold incorporating a novel star-polypeptide biomaterial as a gene vector. This gene-loaded scaffold can accelerate bone tissue repair in vivo in comparison to a scaffold alone at just four weeks post implantation in a critical sized bone defect. This is achieved via the in situ transfection of autologous host cells which migrate into the implanted collagen-based scaffold via gene-loaded, star-shaped poly(l-lysine) polypeptides (star-PLLs). In vitro, we demonstrate that star-PLL nanomaterials designed with 64 short poly(l-lysine) arms can be used to functionalise a range of collagen based scaffolds with a dual therapeutic cargo (pDual) of the bone-morphogenetic protein-2 plasmid (pBMP-2) and vascular endothelial growth factor plasmid (pVEGF). The versatility of this polymeric vector is highlighted in its ability to transfect Mesenchymal Stem Cells (MSCs) with both osteogenic and angiogenic transgenes in a 3D environment from a range of scaffolds with various macromolecular compositions. In vivo, we demonstrate that a bone-mimetic, collagen-hydroxyapatite scaffold functionalized with star-PLLs containing either 32- or 64- poly(l-lysine) arms can be used to successfully deliver this pDual cargo to autologous host cells. At the very early timepoint of just 4 weeks, we demonstrate the 64-star-PLL-pDual functionalised scaffold as a particularly efficient platform to accelerate bone tissue regeneration, with a 6-fold increase in new bone formation compared to a scaffold alone. Overall, this article describes for the first time the incorporation of novel star-polypeptide biomaterials carrying two therapeutic genes into a cell free scaffold which supports accelerated bone tissue formation in vivo.

Translational Studies on the Potential of a VEGF Nanoparticle-Loaded Hyaluronic Acid Hydrogel.

Heart failure has a five-year mortality rate approaching 50%. Inducing angiogenesis following a myocardial infarction is hypothesized to reduce cardiomyocyte death and tissue damage, thereby preventing heart failure. Herein, a novel nano-in-gel delivery system for vascular endothelial growth factor (VEGF), composed of star-shaped polyglutamic acid-VEGF nanoparticles in a tyramine-modified hyaluronic acid hydrogel (nano-VEGF-HA-TA), is investigated. The ability of the nano-VEGF-HA-TA system to induce angiogenesis is assessed in vivo using a chick chorioallantoic membrane model (CAM). The formulation is then integrated with a custom-made, clinically relevant catheter suitable for minimally invasive endocardial delivery and the effect of injection on hydrogel properties is examined. Nano-VEGF-HA-TA is biocompatible on a CAM assay and significantly improves blood vessel branching (p < 0.05) and number (p < 0.05) compared to a HA-TA hydrogel without VEGF. Nano-VEGF-HA-TA is successfully injected through a 1.2 m catheter, without blocking or breaking the catheter and releases VEGF for 42 days following injection in vitro. The released VEGF retains its bioactivity, significantly improving total tubule length on a Matrigel® assay and human umbilical vein endothelial cell migration on a Transwell® migration assay. This VEGF-nano in a HA-TA hydrogel delivery system is successfully integrated with an appropriate device for clinical use, demonstrates promising angiogenic properties in vivo and is suitable for further clinical translation.

Antimicrobial and degradable triazolinedione (TAD) crosslinked polypeptide hydrogels.

Hydrogels are perfectly suited to support cell and tissue growth in advanced tissue engineering applications as well as classical wound treatment scenarios. Ideal hydrogel materials for these applications should be easy to produce, biocompatible, resorbable and antimicrobial. Here we report the fabrication of degradable covalent antimicrobial lysine and tryptophan containing copolypeptide hydrogels, whereby the hydrogel properties can be independently modulated by the copolypeptide monomer ratio and chiral composition. Well-defined statistical copolypeptides comprising different overall molecular weights as well as ratios of l- and d-lysine and tryptophan at ratios of 35 : 15, 70 : 30 and 80 : 20 were obtained by N-carboxyanhydride (NCA) polymerisation and subsequently crosslinked by the selective reaction of bifunctional triazolinedione (TAD) with tryptophan. Real-time rheology was used to monitor the crosslinking reaction recording the fastest increase and overall modulus for copolypeptides with the higher tryptophan ratio. Water uptake of cylindrical hydrogel samples was dependent on crosslinking ratio but found independent of chiral composition, while enzymatic degradation proceeded significantly faster for samples containing more l-amino acids. Antimicrobial activity on a range of hydrogels containing different polypeptide chain lengths, lysine/tryptophan composition and l/d enantiomers was tested against reference laboratory strains of Gram-negative Escherichia coli (E. coli; ATCC25922) and Gram-positive, Staphylococcus aureus (S. aureus; ATCC25923). log reductions of 2.8-3.4 were recorded for the most potent hydrogels. In vitro leachable cytotoxicity tests confirmed non-cytotoxicity as per ISO guidelines.

Exploiting a Neutral BODIPY Copolymer as an Effective Agent for Photodynamic Antimicrobial Inactivation.

We report the synthesis and photophysical properties of a neutral BODIPY photosensitizing copolymer (poly-8-(4-hydroxymethylphenyl)-4,4-difluoro-2,6-diethynyl-4-bora-3a,4a-diaza-s-indacene) containing ethynylbenzene links between the BODIPY units. The copolymer absorbs further towards the red in the UV-vis spectrum compared to the BODIPY precursor. Photolysis of the polymer produces a singlet excited state which crosses to the triplet surface in less than 300 ps. This triplet state was used to form singlet oxygen with a quantum yield of 0.34. The steps leading to population of the triplet state were studied using time-resolved spectroscopic techniques spanning the pico- to nanosecond timescales. The ability of the BODIPY polymer to generate a biocidal species for bactericidal activity in both solution- and coating-based studies was assessed. When the BODIPY copolymer was dropcast onto a surface, 4 log and 6 log reductions in colony forming units/ml representative of Gram-positive and Gram-negative bacteria, respectively, under illumination at 525 nm were observed. The potent broad-spectrum antimicrobial activity of a neutral metal-free copolymer when exposed to visible light conditions may have potential clinical applications in infection management.

Spatiotemporally Resolved Heat Dissipation in 3D Patterned Magnetically Responsive Hydrogels.

Multifunctional nanocomposites that exhibit well-defined physical properties and encode spatiotemporally controlled responses are emerging as components for advanced responsive systems, for example, in soft robotics or drug delivery. Here an example of such a system, based on simple magnetic hydrogels composed of iron oxide magnetic nanoflowers and Pluronic F127 that generates heat upon alternating magnetic field irradiation is described. Rules for heat-induction in bulk hydrogels and the heat-dependence on particle concentration, gel volume, and gel exposed surface area are established, and the dependence on external environmental conditions in "closed" as compared to "open" (cell culture) system, with controllable heat jumps, of ∆T 0-12°C, achieved within ≤10 min and maintained described. Furthermore the use of extrusion-based 3D printing for manipulating the spatial distribution of heat in well-defined printed features with spatial resolution <150 µm, sufficiently fine to be of relevance to tissue engineering, is presented. Finally, localized heat induction in printed magnetic hydrogels is demonstrated through spatiotemporally-controlled release of molecules (in this case the dye methylene blue). The study establishes hitherto unobserved control over combined spatial and temporal induction of heat, the applications of which in developing responsive scaffold remodeling and cargo release for applications in regenerative medicine are discussed.

Synthesis of bilayer films from regenerated cellulose nanofibers and poly(globalide) for skin tissue engineering applications.

Commercial cell-based skin regenerative products are highly expensive, carry the risk of rejection and require a long cell culture period to manufacture. This work describes the synthesis of bilayer films from poly(globalide) (PGl) and regenerated cellulose nanofibers (rCNFs) and their use as a cell-free scaffold to support keratinocyte attachment and proliferation. The method is simple, eco-friendly (as the cellulose precursor is obtained from agricultural waste) and of low cost. The rCNFs were produced by acid hydrolysis and PGl was obtained via enzymatic ring-opening polymerization. The bilayer films were synthesized by layer-by-layer casting at ambient temperature. All the films showed a well-defined interface between PGl and cellulose. The produced rCNF/PGl bilayer films showed cell metabolic activity far superior in comparison with pristine PGl regarding the keratinocyte growth, which illustrates the potential use of these materials in skin tissue engineering.

A Time-Resolved Spectroscopic Investigation of a Novel BODIPY Copolymer and Its Potential Use as a Photosensitiser for Hydrogen Evolution.

A novel 4,4-difuoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) copolymer with diethynylbenzene has been synthesised, and its ability to act as a photosensitiser for the photocatalytic generation of hydrogen was investigated by time-resolved spectroscopic techniques spanning the ps- to ns-timescales. Both transient absorption and time-resolved infrared spectroscopy were used to probe the excited state dynamics of this photosensitising unit in a variety of solvents. These studies indicated how environmental factors can influence the photophysics of the BODIPY polymer. A homogeneous photocatalytic hydrogen evolution system has been developed using the BODIPY copolymer and cobaloxime which provides hydrogen evolution rates of 319 µmol h-1 g-1 after 24 h of visible irradiation.

Biocompatible polypeptide-based interpenetrating network (IPN) hydrogels with enhanced mechanical properties.

Hydrogels are widely used for biomedical applications such as drug delivery, tissue engineering, or wound healing owing to their mimetic properties in relation to biological tissues. The generation of peptide-based hydrogels is a topic of interest due to their potential to increase biocompatibility. However, their usages can be limited when compared to other synthetic hydrogels because of their inferior mechanical properties. Herein, we present the synthesis of novel synthetic polypeptide-based interpenetrating network (IPN) hydrogels with enhanced mechanical properties. The polypeptide single network is obtained from alkyne functional polypeptides crosslinked with di, tri and tetra azide functional PEG by copper-catalysed alkyne-azide cycloaddition (CuAAC). Interpenetrating networks were subsequently obtained by loading of the polypeptide single network with PEG-dithiol and orthogonally UV-crosslinking with varying molar ratios of pentaerythritol tetraacrylate. The characteristics, including the mechanical strength (i.e. compressive strength (UCS), fracture strain (εbreak), and Young's modulus (E)) and cell compatibility (i.e. metabolic activity and Live/Dead of human Mesenchymal Stem Cells), of each synthetic polypeptide-based IPN hydrogel were studied and evaluated in order to demonstrate their potential as mechanically robust hydrogels for use as artificial tissues. Moreover, 1H NMR diffusometry was carried out to examine the water mobility (DH2O) within the polypeptide-based hydrogels and IPNs. It was found that both the mechanical and morphological properties could be tailored concurrently with the hydrophilicity, rate of water diffusion and 'swellability'. Finally it was shown that the polypeptide-based IPN hydrogels exhibited good biocompatibility, highlighting their potential as soft tissue scaffolds.

The effect of hydrogen bonding on diffusion and permeability in UV-cured Polyacrylate-based networks for controlled release.

Polyacrylates are important polymers widely used in pharmaceutical industry such as drug coatings due to their low cost, processability and ease of functionalisation. Chemical functionalities (e.g. H-bonding) can be easily included to modulate the transport of low molecular weight drug-like entities through the network. Understanding how such microscopic structural modifications determine macroscopic diffusion is critical for designing next generation responsive polymers. In this study pulsed field gradient (PFG) 1H NMR measurements of the self-diffusion of a dye molecule (Eosin Y) in a series of polyacrylate networks with differing H-bonding strength were undertaken; it was found that the diffusion of Eosin Y is significantly reduced in networks with H-bonding. Detailed analyses by 1H NMR relaxometry and double quantum (DQ) NMR show that H-bonding can also reduce polymer chain mobility. Furthermore, DSC thermoporometry showed a significant increase in the average network mesh size potentially due to the pre-organization of H-bonding containing monomer during network curing. By introducing the H-bonding disrupter, LiClO4, it was found that the diffusivity of solute becomes positively correlated to the average mesh size across the series of networks. Hence, a modified diffusion model based on hydrodynamic theory is proposed to separate the direct (solute-network) H-bonding contribution to solute diffusion from the indirect contribution arising from monomer pre-ordering induced mesh size reduction. Finally, it is shown that the same direct and indirect contributions to microscopic diffusivity, arising from the H-bond strength of the co-monomers, also contribute significantly to the macroscopic membrane permeability which is similarly subject to H-bond disruption.

Development of a Sustained Release Nano-In-Gel Delivery System for the Chemotactic and Angiogenic Growth Factor Stromal-Derived Factor 1α.

Stromal-Derived Factor 1α (SDF) is an angiogenic, chemotactic protein with significant potential for applications in a range of clinical areas, including wound healing, myocardial infarction and orthopaedic regenerative approaches. The 26-min in vivo half-life of SDF, however, has limited its clinical translation to date. In this study, we investigate the use of star-shaped or linear poly(glutamic acid) (PGA) polypeptides to produce PGA-SDF nanoparticles, which can be incorporated into a tyramine-modified hyaluronic acid hydrogel (HA-TA) to facilitate sustained localised delivery of SDF. The physicochemical properties and biocompatibility of the PGA-SDF nanoparticle formulations were extensively characterised prior to incorporation into a HA-TA hydrogel. The biological activity of the SDF released from the nano-in-gel system was determined on Matrigel®, scratch and Transwell® migration assays. Both star-shaped and linear PGA facilitated SDF nanoparticle formation with particle sizes from 255-305 nm and almost complete SDF complexation. Star-PGA-SDF demonstrated superior biocompatibility and was incorporated into a HA-TA gel, which facilitated sustained SDF release for up to 35 days in vitro. Released SDF significantly improved gap closure on a scratch assay, produced a 2.8-fold increase in HUVEC Transwell® migration and a 1.5-fold increase in total tubule length on a Matrigel® assay at 12 h compared to untreated cells. Overall, we present a novel platform system for the sustained delivery of bioactive SDF from a nano-in-gel system which could be adapted for a range of biomedical applications.