Digital Light Processing of Thermoresponsive Hydrogels from Polyproline-Based Star Polypeptides.

The first report of star poly(L-proline) crosslinkers is disclosed for digital light processing 3D printing of thermoresponsive hydrogels. Through chain end functionalization of star poly(L-proline)s with methacryloyl groups, access to high-resolution defined 3D hydrogel structures via digital light processing is achieved through photoinitiated free radical polymerization. Changing the poly(L-proline) molecular weight has a direct influence on both thermoresponsiveness and printability, while shape-morphing behavior can be induced thermally.

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.

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.

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.

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.

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.

Bioinspired Star-Shaped Poly(l-lysine) Polypeptides: Efficient Polymeric Nanocarriers for the Delivery of DNA to Mesenchymal Stem Cells.

The field of tissue engineering is increasingly recognizing that gene therapy can be employed for modulating in vivo cellular response thereby guiding tissue regeneration. However, the field lacks a versatile and biocompatible gene delivery platform capable of efficiently delivering transgenes to mesenchymal stem cells (MSCs), a cell type often refractory to transfection. Herein, we describe the extensive and systematic exploration of three architectural variations of star-shaped poly(l-lysine) polypeptide (star-PLL) with varying number and length of poly(l-lysine) arms as potential nonviral gene delivery vectors for MSCs. We demonstrate that star-PLL vectors are capable of self-assembling with pDNA to form stable, cationic nanomedicines. Utilizing high content screening, live cell imaging, and mechanistic uptake studies we confirm the intracellular delivery of pDNA by star-PLLs to MSCs is a rapid process, which likely proceeds via a clathrin-independent mechanism. We identify a star-PLL composition with 64 poly(l-lysine) arms and five l-lysine subunits per arm as a particularly efficient vector that is capable of delivering both reporter genes and the therapeutic transgenes bone morphogenetic protein-2 and vascular endothelial growth factor to MSCs. This composition facilitated a 1000-fold increase in transgene expression in MSCs compared to its linear analogue, linear poly(l-lysine). Furthermore, it demonstrated comparable transgene expression to the widely used vector polyethylenimine using a lower pDNA dose with significantly less cytotoxicity. Overall, this study illustrates the ability of the star-PLL vectors to facilitate efficient, nontoxic nucleic acid delivery to MSCs thereby functioning as an innovative nanomedicine platform for tissue engineering applications.

Chemo-Enzymatic Synthesis of Poly(4-piperidine lactone- b-ω-pentadecalactone) Block Copolymers as Biomaterials with Antibacterial Properties.

With increasing troubles in bacterial contamination and antibiotic-resistance, new materials possessing both biocompatibility and antimicrobial efficacy are supposed to be developed for future biomedical application. Herein, we demonstrated a chemo-enzymatic ring opening polymerization (ROP) approach for block copolyester, that is, poly(4-benzyl formate piperidine lactone- b-ω-pentadecalactone) (PNPIL- b-PPDL), in a one-pot two-step process. Afterward, cationic poly(4-piperidine lactone- b-ω-pentadecalactone) (PPIL- b-PPDL) with pendent secondary amino groups was obtained via acidic hydrolysis of PNPIL- b-PPDL. The resulting cationic block copolyester exhibited high antibacterial activity against Gram negative E. coli and Gram positive S. aureus, while showed low toxicity toward NIH-3T3 cells. Moreover, the antibacterial property, cytotoxicity and degradation behavior could be tuned simply by variation of PPIL content. Therefore, we anticipate that such cationic block copolymers could potentially be applied as biomaterials for medicine or implants.

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.

Polypeptide Polymer Brushes by Light-Induced Surface Polymerization of Amino Acid N-Carboxyanhydrides.

Silicon wafers are decorated with photoamine generator 4,5-dimethoxy-2-nitrobenzyl 3-(triethoxysilyl)propyl carbamate. UV-irradiation in the presence of benzyl-l-glutamate N-carboxyanhydride is carried out, resulting in the release of the surface-bound primary amines, making them viable N-carboxyanhydride (NCA) polymerization initiators. Successful polypeptide grafting is confirmed by water contact angle measurements as well as by ellipsometry, revealing a poly(benzyl-l-glutamate) (PBLG) layer of ≈3 nm. X-ray photoelectron spectroscopy confirms the presence of amide groups in the grafted PBLG while time-of-flight secondary ion mass spectroscopy provides additional evidence for the presence of PBLG on the surface. Evaluation of negative control samples confirms successful UV surface grafting. The approach is thus established as a viable general method for light exposure directable polypeptide functionalization of silicon surfaces.

Synthesis and gelation of copolypept(o)ides with random and block structure.

Copolypept(o)ides of polysarcosine (PSar) and poly(N-isopropyl-L-glutamine) (PIGA) with random and block sequence structures were synthesized by ring-opening polymerization (ROP) of sarcosine N-carboxyanhydrides (Sar-NCA) and γ-benzyl-l-glutamate N-carboxyanhydrides (BLG-NCA) and post modification. With different distribution of Sar along the main chain, H-bonding pattern and secondary structure of polypeptides were turned, as well as aggregation and gelation behavior. Both copolypept(o)ides formed hydrogels above their critical gelation concentrations (CGCs) without thermo-sensitivity, which was normally reserved for PEG copolypeptides (eg, PEG-b-PIGA). In particular, a different mechanism from previously reported micellar percolation or fibrillar entanglement was suggested for gelation of the random copolypept(o)ide. Therefore, hydrogels from copolymers of PSar and PIGA represented a new approach to construct easy-handling, biocompatible, biodegradable and thermo-stable gels that could potentially be applied in biomedical fields.

Block-Sequence-Specific Glycopolypeptides with Selective Lectin Binding Properties.

Glycopolypeptides with defined block sequences were prepared by sequential addition of two different N-carboxyanhydrides (NCAs), followed by selective deprotection and functionalization of predefined positions within the polypeptide backbone. The sequential arrangement of the galactose units and the block-sequence length have been systematically varied. All the glycopolypeptides have been obtained with a similar overall composition and comparable molecular weights. Circular dichroism measurements revealed some dependence of the secondary structure on the primary composition of the glycopolypeptides at physiological pH. While statistical, diblock, and tetrablock glycopolypeptides adopted a random coil conformation, the octablock glycopolypeptide was mostly α-helical. The ability to selectively bind to lectins was investigated by turbidity measurements as well as surface plasmon resonance (SPR) studies. It was found that the extent of binding was dependent on the position of the galactose units and thus the primary glycopolypeptide structure. The octablock glycopolypeptide favored interaction with lectin RCA120 while the tetrablock glycopolypeptide demonstrated the strongest binding activity to Galectin-3. The results suggest that different lectins are very sensitive to glyco coding and that precise control of carbohydrate units in synthetic polymeric glycopeptides will remain important.

Direct UV-Triggered Thiol-ene Cross-Linking of Electrospun Polyester Fibers from Unsaturated Poly(macrolactone)s and Their Drug Loading by Solvent Swelling.

Electrospinning is considered a relatively simple and versatile technique to form high porosity porous scaffolds with micron to nanoscale fibers for biomedical applications. Here, electrospinning of unsaturated aliphatic polyglobalide (PGl) into well-defined fibers with an average diameter of 9 µm is demonstrated. Addition of a dithiol cross-linker and a photoinitiator to the polymer solution enabled the UV-triggered intracross-linking of the fibers during the spinning process. The in situ cross-linking of the fibers resulted in amorphous material able to swell up to 14% in tetrahydrofurane (THF) without losing the fiber morphology. Seeding mesenchymal stem cells (MSCs) onto both cross-linked and non-cross-linked PGl fibers proved their compatibility with MSCs and suitability as scaffolds for cell growth and proliferation of MSCs. Moreover, the ability to directly load cross-linked PGl with hydrophobic molecules by soaking the fiber mesh in solution is shown with Rhodamine B and Indomethacin, a hydrophobic anti-inflammatory drug. This marks an advantage over conventional aliphatic polyesters and opens opportunities for the design of drug loaded polyester scaffolds for biomedical applications or tissue engineering.

Rational design of polymeric core shell ratiometric oxygen-sensing nanostructures.

A new approach for the fabrication of luminescent ratiometric sensing nanosensors is described using core-shell nanoparticles in which the probe and reference are spatially separated into the shell and core of the nanostructure respectively. The isolation of the reference in the core of the particle ensures a stable emission reference signal unaffected by the external environment. The core shell structure was prepared by engineering structurally well-defined Ru-conjugated block copolymers which acted as emulsifiers in the miniemulsion polymerisation of BODIPY loaded styrene nanoparticles. The resulting particles are highly stable and show excellent size monodispersity. The nanosensors exhibit dual emission under a single excitation wavelength with a reversible and quantitative ratiometric response to the O2 content in aqueous media. In the presence of a low concentration of CTAB, the particles cross the cell membrane and the particles show negligible cytotoxicity. Such an approach to sensor nanoparticles should be of value across a range of applications where a stable ratiometric signal in diverse environments is required.

Star-Shaped Polypeptides: Synthesis and Opportunities for Delivery of Therapeutics.

Significant advances in the synthesis of polypeptides by N-carboxyanhydride (NCA) polymerisation over the last decade have enabled the design of advanced polypeptide architectures such as star-shaped polypeptides. These materials combine the functionality offered by amino acids with the flexibility of creating stable nanoparticles with adjustable cargo space for therapeutic delivery. This review highlights recent advances in the synthesis of star polypeptides by NCA polymerisation followed by a critical review of the applications of this class of polymer in the delivery of therapeutic agents. This includes examples of traditional small-molecule drugs as well as the emerging class of biologics such as genetic therapeutics (gene delivery).

Stimuli responsive synthetic polypeptides derived from N-carboxyanhydride (NCA) polymerisation.

The progress in NCA polymerisation combined with advanced orthogonal functionalization techniques as well as the integration with other controlled polymerisation techniques significantly widened the scope of polypeptide building blocks in a variety of material designs. Well-defined synthetic stimuli-responsive polypeptides ("smart" polypeptides) with incorporated different functionalities have been extensively explored over the past decades. Their significant potential lies in the fact that they combine natural and synthetic elements both contributing to their properties. These novel materials have potential applications in biomedicine and biotechnology including tissue engineering, drug delivery and biodiagnostics. Responsive polypeptides are capable of undergoing conformational changes and phase transition accompanied by variations in the chemical and physical changes of the polypeptides in response to an external stimulus such as biologically relevant species (i.e. biomolecules), the environment (i.e. temperature, pH), irradiation with light or exposure to a magnetic field. In this review, the recent developments including synthetic strategies and applications of synthetic stimuli-responsive homo- and block polypeptides are reviewed.

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.

Antimicrobial spectrum against wound pathogens and cytotoxicity of star-arranged poly-l-lysine-based antimicrobial peptide polymers.

Introduction. As growing numbers of patients are at higher risk of infection, novel topical broad-spectrum antimicrobials are urgently required for wound infection management. Robust pre-clinical studies should support the development of such novel antimicrobials.Gap statement. To date, evidence of robust investigation of the cytotoxicity and antimicrobial spectrum of activity of antimicrobial peptides (AMP)s is lacking in published literature. Using a more clinical lens, we address this gap in experimental approach, building on our experience with poly-l-lysine (PLL)-based AMP polymers.Aim. To evaluate the in vitro bactericidal activity and cytotoxicity of a PLL-based 16-armed star AMP polymer, designated 16-PLL10, as a novel candidate antimicrobial.Methods. Antimicrobial susceptibilities of clinical isolates and reference strains of ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) pathogens, to 16-PLL10 were investigated. Human erythrocyte haemolysis and keratinocyte viability assays were used to assess toxicity. Modifications were made to 16-PLL10 and re-evaluated for improvement.Results. Minimum bactericidal concentration of 16-PLL10 ranged from 1.25 µM to ≥25 µM. At 2.5 µM, 16-PLL10 was broadly bactericidal against ESKAPE strains/wound isolates. Log-reduction in colony forming units (c.f.u.) per millilitre after 1 h, ranged from 0.3 (E. cloacae) to 5.6 (K. pneumoniae). At bactericidal concentrations, 16-PLL10 was toxic to human keratinocyte and erythrocytes. Conjugates of 16-PLL10, Trifluoroacetylated (TFA)-16-PLL10, and Poly-ethylene glycol (PEG)ylated 16-PLL10, synthesised to address toxicity, only moderately reduced cytotoxicity and haemolysis.Conclusions. Due to poor selectivity indices, further development of 16-PLL10 is unlikely warranted. However, considering the unmet need for novel topical antimicrobials, the ease of AMP polymer synthesises/modification is attractive. To support more rational development, prioritising clinically relevant pathogens and human cells, to establish selective toxicity profiles in vitro, is critical. Further characterisation and discovery utilising artificial intelligence and computational screening approaches can accelerate future AMP nanomaterial development.

Thermoresponsiveness Across the Physiologically Accessible Range: Effect of Surfactant, Cross-Linker, and Initiator Content on Size, Structure, and Transition Temperature of Poly(N-isopropylmethacrylamide) Microgels.

The influence of surfactant, cross-linker, and initiator on the final structure and thermoresponse of poly(N-isopropylmethacrylamide) (pNIPMAM) microgels was evaluated. The goals were to control particle size (into the nanorange) and transition temperature (across the physiologically accessible range). The concentration of the reactants used in the synthesis was varied, except for the monomer, which was kept constant. The thermoresponsive suspensions formed were characterized by dynamic light scattering, small-angle X-ray scattering, atomic force microscopy, and rheology. Increasing surfactant, sodium dodecyl sulfate content, produced smaller microgels, as expected, into the nanorange and with greater internal entanglement, but with no change in phase transition temperature (LCST), which is contrary to previous reports. Increasing cross-linker, N,N-methylenebis acrylamide, content had no impact on particle size but reduced particle deformability and, again contrary to previous reports of decreases, progressively increased the LCST from 39 to 46 °C. The unusual LCST trends were confirmed using different rheological techniques. Initiator, potassium persulfate, content was found to weakly influence the outcomes. An optimized content was identified that provides functional nanogels in the 100 nm (swollen) size range with controlled LCST, just above physiological temperature. The study contributes chemistry-derived design rules for thermally responsive colloidal particles with physiologically accessible LCST for a variety of biomedical and soft robotics applications.