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Purpose: To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii., Methods: A. castellanii trophozoites and cysts were grown in the presence of pɛK solution (0-2.17 mM), pɛK or pɛK+ hydrogels, or commercial hydrogel contact lens (CL) for 24 hours or 7 days in PBS or Peptone-Yeast-Glucose (PYG) media (nutrient-deplete or nutrient-replete cultures, respectively). Toxicity was determined using propidium iodide and imaged using fluorescence microscopy. Ex vivo porcine corneas were inoculated with A. castellanii trophozoites ± pɛK, pɛK+ hydrogels or commercial hydrogel CL for 7 days. Corneal infection was assessed by periodic acid-Schiff staining and histologic analysis. Regrowth of A. castellanii from hydrogel lenses and corneal discs at 7 days was assessed using microscopy and enumeration., Results: The toxicity of pɛK+ hydrogels resulted in the death of 98.52% or 83.31% of the trophozoites at 24 hours or 7 days, respectively. The toxicity of pɛK+ hydrogels resulted in the death of 70.59% or 82.32% of the cysts in PBS at 24 hours or 7 days, respectively. Cysts exposed to pɛK+ hydrogels in PYG medium resulted in 75.37% and 87.14% death at 24 hours and 7 days. Ex vivo corneas infected with trophozoites and incubated with pɛK+ hydrogels showed the absence of A. castellanii in the stroma, with no regrowth from corneas or pɛK+ hydrogel, compared with infected-only corneas and those incubated in presence of commercial hydrogel CL., Conclusions: pɛK+ hydrogels demonstrated pronounced amoebicidal and cysticidal activity against A. castellanii. pɛK+ hydrogels have the potential for use as CLs that could minimize the risk of CL-associated Acanthamoeba keratitis.

Corneal opacities are the 4th leading cause of blindness, and the only current treatment method is the replacement of damaged tissue with a donor cornea. The worldwide shortage of donor eye bank tissue has influenced research into biomaterial substrates for both partial and full thickness corneal implantation. Here, polymer hydrogels based on natural peptides, poly-ɛ-lysine and gellan gum, can be manufactured using reactive inkjet printing (RIJ). The inks used for printing were optimised based on their rheological properties. Printing alternating layers of ink forms a unique surface pattern, based on the immediate formation of ionic bonds between polymers of opposing charges. This surface pattern resembles a repeating honeycomb-like structure, visible by both optical and scanning electron microscopy. The structure of the printed hydrogels can be modified to include pores, a feature of interest for the tissue engineering of full thickness corneal constructs. Printed poly-ɛ-lysine/gellan gum hydrogels demonstrated a transparency of 80% and cyto-compatibility with both corneal epithelial and endothelial cells. Both corneal cell types demonstrated cell attachment across the surface of the printed hydrogel arrays, displaying their typical cell morphology. This gives confidence of the cyto-compatibility of these hydrogels in vitro. Reactive inkjet printing can produce 3D structures with a high resolution, producing printed tracks in the micron range. Additionally, RIJ demonstrates versatility, as constructs can be tailored to meet various dimension and thickness requirements. Furthermore, this work demonstrates for the first time that reactive inkjet printing can been used to produce hydrogel constructs based on these two inks, with the aim of producing constructs for corneal tissue engineering., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A family of poly-ε-lysine hydrogels can be synthesized by crosslinking with bis-carboxylic acids using carbodiimide chemistry. In addition to creating hydrogels using a simple cast method, a fragmented method is used to introduce increased porosity within the hydrogel structure. Both methods have created tunable characteristics ranging in their mechanical properties, transparency, and water content, which is of interest to corneal tissue engineering and can be tailored to specific cellular needs and applications. With a worldwide shortage of cornea donor tissue available for transplant and limitations including rejection and potential infection, a synthetic material that can be used as a graft, or a partial thickness corneal replacement, would be an advantageous treatment method. These hydrogels can be tuned to have similar mechanical and transparency properties to the human cornea. They also support the attachment and growth of corneal epithelial cells and the integration of corneal stromal cells., (© 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)

Purpose: To determine the antimicrobial activity of poly-epsilon-lysine (pɛK) functionalization of hydrogels against Pseudomonas aeruginosa., Methods: Antimicrobial activities of pɛK and pɛK+ hydrogels were tested against both keratitis and a laboratory strain of Paeruginosa at a range of inocula sizes, over 4 and 24 hours. The number of viable CFU on pɛK and pɛK+ hydrogels or commercial contact lenses (CL) was investigated. Ex vivo porcine corneas were inoculated with Paeruginosa PAO1 (103 CFU) and incubated with pɛK+ hydrogels or commercial hydrogel CL for 24 hours and the effects of infection determined., Results: PɛK+ hydrogels showed log reductions in viable CFU compared with pɛK hydrogels for all Paeruginosa strains, depending on inocula sizes and incubation time. After 24 hours pɛK+ hydrogels showed >5 and >7.5 log reduction in CFU compared with commercial hydrogel CL at 103 and 106 CFU, respectively. In an ex vivo porcine corneal infection model, pɛK+ hydrogels led to a significant decrease in viable PAO1 CFU and histologic analysis indicated a decreased infiltration of PAO1 into the stroma., Conclusions: PɛK+ hydrogels demonstrated enhanced antimicrobial activity versus nonfunctionalized pɛK hydrogels against clinically relevant Paeruginosa strains. PɛK+ hydrogels have the potential to be used as a bandage CL with innate antimicrobial characteristics to minimize the risk of microbial keratitis.

The limited regenerative capacity of the CNS poses formidable challenges to the repair of spinal cord injury (SCI). Two key barriers to repair are (i) the physical gap left by the injury, and (ii) the inhibitory milieu surrounding the injury, the glial scar. Biomaterial implantation into the injury site can fill the cavity, provide a substrate for cell migration, and potentially attenuate the glial scar. We investigated the biological viability of a biocompatible and biodegradable poly-ε-lysine based biomaterial, Proliferate®, in low and high cross-linked forms and when coated with IKVAV peptide, for SCI implantation. We demonstrate altered astrocyte morphology and nestin expression on Proliferate® compared to conventional glass cell coverslips suggesting a less reactive phenotype. Moreover Proliferate® supported myelination in vitro, with myelination observed sooner on IKVAV-coated constructs compared with uncoated Proliferate®, and delayed overall compared with maintenance on glass coverslips. For in vivo implantation, parallel-aligned channels were fabricated into Proliferate® to provide cell guidance cues. Extensive vascularisation and cellular infiltration were observed in constructs implanted in vivo, along with an astrocyte border and microglial response. Axonal ingrowth was observed at the construct border and inside implants in intact channels. We conclude that Proliferate® is a promising biomaterial for implantation following SCI.

Dysfunction of the corneal endothelium (CE) resulting from progressive cell loss leads to corneal oedema and significant visual impairment. Current treatments rely upon donor allogeneic tissue to replace the damaged CE. A donor cornea shortage necessitates the development of biomaterials, enabling in vitro expansion of corneal endothelial cells (CECs). This study investigated the use of a synthetic peptide hydrogel using poly-ε-lysine (pεK), cross-linked with octanedioic-acid as a potential substrate for CECs expansion and CE grafts. PεK hydrogel properties were optimised to produce a substrate which was thin, transparent, porous and robust. A human corneal endothelial cell line (HCEC-12) attached and grew on pεK hydrogels as confluent monolayers after 7 days, whereas primary porcine CECs (pCECs) detached from the pεK hydrogel. Pre-adsorption of collagen I, collagen IV and fibronectin to the pεK hydrogel increased pCEC adhesion at 24 h and confluent monolayers formed at 7 days. Minimal cell adhesion was observed with pre-adsorbed laminin, chondroitin sulphate or commercial FNC coating mix (fibronectin, collagen and albumin). Functionalisation of the pεK hydrogel with synthetic cell binding peptide H-Gly-Gly-Arg-Gly-Asp-Gly-Gly-OH (RGD) or α2β1 integrin recognition sequence H-Asp-Gly-Glu-Ala-OH (DGEA) resulted in enhanced pCEC adhesion with the RGD peptide only. pCECs grown in culture at 5 weeks on RGD pεK hydrogels showed zonula occludins 1 staining for tight junctions and expression of sodium-potassium adenosine triphosphase, suggesting a functional CE. These results demonstrate the pεK hydrogel can be tailored through covalent binding of RGD to provide a surface for CEC attachment and growth. Thus, providing a synthetic substrate with a therapeutic application for the expansion of allogenic CECs and replacement of damaged CE.

Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2'- O -methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here, we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce "on-resin" aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences.

Purpose: The purpose of this study was to develop a more efficient drug delivery device to overcome the limitations of current drop therapy for the treatment of fungal keratitis., Methods: Amphotericin B (AmpB), 0 to 30 μg/mL, was associated with a poly-ε-lysine (pεK) hydrogel. Fungicidal effect against Candida albicans was assessed at 18 and 42 hours by optical density (OD600) and growth on agar. Tear film dilution effect was mimicked by storage of AmpB pεK gels in 3.4 mL sterile PBS for 24 hours prior to fungal incubation. Drug elution over 96 hours was evaluated by HPLC, and drug stability was tested while associated with the gel by OD600 up to 48 hours. Lack of cytotoxicity toward the HCE-T corneal epithelial cell line was assessed over 7 days., Results: AmpB pεK gels show fungicidal activity in normal conditions (0.057 OD600, SD 0.003, P < 0.005) and in the presence of horse serum (0.048 OD600, SD 0.028 P < 0.005) at 18 hours. The drug release profile was above therapeutic levels (0.188 μg/mL) for up to 72 hours. Tear dilution had no significant effect at higher concentrations of AmpB (3 to 10 μg/mL). AmpB pεK gels were not cytotoxic to the HCE-T cell line., Conclusions: We demonstrated that AmpB pεK gels confer sustained therapeutic antifungal activity for at least 48 hours without corneal epithelial cell line cytotoxicity, suggesting their potential for in vivo use as an antifungal bandage contact lens. This could avoid the need for intensive topical medication in the treatment of fungal keratitis.

A peptide hydrogel with an antimicrobial activity is developed as a bandage contact lens. The antimicrobial activity is enhanced with the addition of the biomolecules penicillin G or poly-ε-lysine and is positive against Staphylococcus aureus and Escherichia coli. The lens is also noncytotoxic toward a human corneal epithelial cell line and as a consequence is of great potential as a drug-eluting bandage lens replacing conventional corneal ulcer treatment., (© 2016 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Novel non-PEG derived polyether resins, coined SLURPS (Superior Liquid Uptake Resins for Polymer-supported Synthesis), were studied for their performance in solid-phase synthesis. Novel amino functional resins, SLURPS-NH2, were prepared with a loading of up to 8.5 mmol/g and employed successfully in the solid-phase synthesis of Leu-Enkephalin. The peptide was obtained with the same purity when compared to its synthesis with commercial standard poly(dimethyl acrylamide) resins. Furthermore we show loading and cleavage of aromatic carboxylic acids in excellent yield. The advantageous solvent compatibility of our support was demonstrated through the biphasic dihydroxylation of alkenes with OsO4 in t-BuOH/water mixtures producing bound 1,2-diols and synthesis and removal of a bound oxime using ethanol/water mixtures both in excellent yields. Reactions were easily monitored by gel-phase NMR and FTIR. These results show that SLURPS are very well suited for organic transformations using highly polar solvent mixtures and reagents and at much higher loading levels than standard amphiphilic resins of similar solvent compatibility.