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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Molecular environment and reactivity in gels and colloidal solutions under identical conditions.

A PEG-Tyr block copolymer forms a kinetically stable colloidal solution in water at room temperature which undergoes an irreversible conversion to a gel phase upon heating. A micellar solution and a gel can therefore be studied under identical experimental conditions. This made it possible to compare physical properties and chemical reactivity of micelles and gels in identical chemical environments and under identical conditions. EPR spectra of the spin-labelled copolymer showed that tyrosine mobility in gels was slightly reduced compared to micelles. Chemical reactivity was studied using photochemical degradation of tyrosine and tyrosine dimerization, in the absence and in the presence of an Fe(iii) salt. The reactivity trends were explained by reduced tyrosine mobility in the gel environment. The largest reactivity difference in gels and micelles was observed for bimolecular dityrosine formation which was also attributed to the reduction in molecular mobility.

Amphiphilic Star Polypept(o)ides as Nanomeric Vectors in Mucosal Drug Delivery.

Mucosal delivery across the gastrointestinal (GI) tract, airways, and buccal epithelia is an attractive mode of therapeutic administration, but the challenge is to overcome the mucus and epithelial barriers. Here, we present degradable star polypept(o)ides capable of permeating both barriers as a promising biomaterial platform for mucosal delivery. Star polypept(o)ides were obtained by the initiation of benzyl-l-glutamate N-carboxyanhydride (NCA) from an 8-arm poly(propyleneimine) (PPI) dendrimer, with subsequent chain extension with sarcosine NCA. The hydrophobic poly(benzyl-l-glutamate) (PBLG) block length was maintained at 20 monomers, while the length of the hydrophilic poly(sarcosine) (PSar) block ranged from 20-640 monomers to produce star polypept(o)ides with increasing hydrophilic: hydrophobic ratios. Transmission electron microscopy (TEM) images revealed elongated particles of ∼120 nm length, while dynamic light scattering (DLS) provided evidence of a decrease in the size of polymer aggregates in water with increasing poly(sarcosine) block length, with the smallest size obtained for the star PBLG20-b-PSar640. Fluorescein isothiocyanate (FITC)-conjugated PBLG20-b-PSar640 permeated artificial mucus and isolated rat mucus, as well as rat intestinal jejunal tissue mounted in Franz diffusion chambers. An apparent permeability coefficient (Papp) of 15.4 ± 3.1 ×10-6 cm/s for FITC-PBLG20-b-PSar640 was calculated from the transepithelial flux obtained with the apical-side addition of 7.5 mg polypept(o)ide to jejunal tissue over 2 h. This Papp could not be accounted for by flux of unconjugated FITC. Resistance to trypsin demonstrated the stability of FITC-labeled polypept(o)ide over 2 h, but enzymatic degradation at the mucus-epithelial interface or during flux could not be ruled out as contributing to the Papp. The absence of any histological damage to the jejunal tissue during the 2 h exposure suggests that the flux was not associated with overt toxicity.

Development of a nanomedicine-loaded hydrogel for sustained delivery of an angiogenic growth factor to the ischaemic myocardium.

The 5-year mortality rate for heart failure borders on 50%. The main cause is an ischaemic cardiac event where blood supply to the tissue is lost and cell death occurs. Over time, this damage spreads and the heart is no longer able to pump efficiently. Increasing vascularisation of the affected area has been shown to reduce patient symptoms. The growth factors required to do this have short half-lives making development of an efficacious therapy difficult. Herein, the angiogenic growth factor Vascular Endothelial Growth Factor (VEGF) is complexed electrostatically with star-shaped or linear polyglutamic acid (PGA) polypeptides. Optimised PGA-VEGF nanomedicines provide VEGF encapsulation of > 99% and facilitate sustained release of VEGF for up to 28 days in vitro. The star-PGA-VEGF nanomedicines are loaded into a percutaneous delivery compliant hyaluronic acid hydrogel. Sustained release of VEGF from the composite nano-in-gel system is evident for up to 35 days and the released VEGF has comparable bioactivity to free, fresh VEGF when tested on both Matrigel® and scratch assays. The final star-PGA-VEGF nanomedicine-loaded hydrogel is biocompatible and provides sustained release of bioactive VEGF. Therefore, we report the development of novel, self-assembling PGA-VEGF nanomedicines and their incorporation into a hyaluronic acid hydrogel that is compatible with medical devices to enable minimally invasive delivery to the heart. The final star-PGA-VEGF nanomedicine-loaded hydrogel is biocompatible and provides sustained release of bioactive VEGF. This formulation provides the basis for optimal spatiotemporal delivery of an angiogenic growth factor to the ischaemic myocardium.

Rapid healing of a critical-sized bone defect using a collagen-hydroxyapatite scaffold to facilitate low dose, combinatorial growth factor delivery.

The healing of large, critically sized bone defects remains an unmet clinical need in modern orthopaedic medicine. The tissue engineering field is increasingly using biomaterial scaffolds as 3D templates to guide the regenerative process, which can be further augmented via the incorporation of recombinant growth factors. Typically, this necessitates supraphysiological doses of growth factor to facilitate an adequate therapeutic response. Herein, we describe a cell-free, biomaterial implant which is functionalised with a low dose, combinatorial growth factor therapy that is capable of rapidly regenerating vascularised bone tissue within a critical-sized rodent calvarial defect. Specifically, we demonstrate that the dual delivery of the growth factors bone morphogenetic protein-2 (osteogenic) and vascular endothelial growth factor (angiogenic) at a low dose (5 µg/scaffold) on an osteoconductive collagen-hydroxyapatite scaffold is highly effective in healing these critical-sized bone defects. The high affinity between the hydroxyapatite component of this biomimetic scaffold and the growth factors functions to sequester them locally at the defect site. Using this growth factor-loaded scaffold, we show complete bridging of a critical-sized calvarial defect in all specimens at a very early time point of 4 weeks, with a 28-fold increase in new bone volume and seven-fold increase in new bone area compared with a growth factor-free scaffold. Overall, this study demonstrates that a collagen-hydroxyapatite scaffold can be used to locally harness the synergistic relationship between osteogenic and angiogenic growth factors to rapidly regenerate bone tissue without the need for more complex controlled delivery vehicles or high total growth factor doses.

Polypeptide Nanoparticles Obtained from Emulsion Polymerization of Amino Acid N-Carboxyanhydrides.

Polypeptide nanoparticles were obtained by the miniemulsion polymerization of S-(o-nitrobenzyl)-l-cysteine (NBC) N-carboxyanhydride (NCA). Through process optimization, reaction conditions were identified that allowed the polymerization of the water sensitive NCA to yield nanoparticles of about 220 nm size. Subsequent UV-irradiation of the nanoparticle emulsions caused the in situ removal of the nitrobenzyl group and particle cross-linking through disulfide bond formation accompanied by the shrinkage of the particles.

Transfection of autologous host cells in vivo using gene activated collagen scaffolds incorporating star-polypeptides.

It is increasingly being recognised within the field of tissue engineering that the regenerative capacity of biomaterial scaffolds can be augmented via the incorporation of gene therapeutics. However, the field still lacks a biocompatible gene delivery vector which is capable of functionalizing scaffolds for tailored nucleic acid delivery. Herein, we describe a versatile, collagen based, gene-activated scaffold platform which can transfect autologous host cells in vivo via incorporation of star-shaped poly(˪-lysine) polypeptides (star-PLLs) and a plasmid DNA (pDNA) cargo. Two star-PLL vectors with varying number and length of poly(˪-lysine) arms were assessed. In vitro, the functionalization of a range of collagen based scaffolds containing either glycosaminoglycans (chondroitin sulfate or hyaluronic acid) or ceramics (hydroxyapatite or nano-hydroxyapatite) with star-PLL-pDNA nanomedicines facilitated prolonged, non-toxic transgene expression by mesenchymal stem cells (MSCs). We demonstrate that the star-PLL structure confers enhanced spatiotemporal control of nanomedicine release from functionalized scaffolds over a 28-day period compared to naked pDNA. Furthermore, we identify a star-PLL composition with 64 poly(˪-lysine) arms and 5 (˪-lysine) subunits per arm as a particularly effective vector, capable of facilitating a 2-fold increase in reporter transgene expression compared to the widely used vector polyethylenimine (PEI), a 44-fold increase compared to a 32 poly(˪-lysine) armed star-PLL and a 130-fold increase compared to its linear analogue, linear poly(˪-lysine) (L-PLL) from a collagen-chondroitin sulfate gene activated scaffold. In an in vivo subcutaneous implant model, star-PLL-pDNA gene activated scaffolds which were implanted cell-free exhibited extensive infiltration of autologous host cells, nanomedicine retention within the implanted construct and successful host cell transfection at the very early time point of just seven days. Overall, this article illustrates for the first time the significant ability of the star-PLL polymeric structure to transfect autologous host cells in vivo from an implanted biomaterial scaffold thereby forming a versatile platform with potential in numerous tissue engineering applications.

Degradable 3D-Printed Hydrogels Based on Star-Shaped Copolypeptides.

We present a star copolypeptide-based hydrogel ink capable of structural microfabrication using 3D extrusion printing. The material comprises an amphiphilic block copolymer structure of poly(benzyl-l-glutamate)- b-oligo(l-valine), which spontaneously forms hydrogels through hydrophobic interactions. The chemical design allows the bulk phase of the hydrogel to remain intact after application of shear due to its self-recovery behavior. It is demonstrated that the composition of the materials is ideally suited for 3D printing with scaffolds capable of maintaining structural cohesion after extrusion. Post extrusion UV-triggered fixation of the printed structures is carried out, resulting in stable hydrogel constructs. The constructs were found to be degradable, exhibited favorable release of encapsulated molecular cargo, and do not appear to affect the metabolic health of the commonly used fibroblastic cell line Balb/3T3 in the absence of the reactive diluent N, N'-methylenebis(acrylamide). The star copolypeptide inks allow for rapid prototyping enabling the fabrication of defined intricate microstructures, providing a platform for complex scaffold development that would otherwise be unattainable with other processing techniques such as molding or casting.