3D reactive inkjet printing of poly-ɛ-lysine/gellan gum hydrogels for potential corneal constructs.

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.

Solid-Phase Synthesis of Difficult Purine-Rich PNAs through Selective Hmb Incorporation: Application to the Total Synthesis of Cell Penetrating Peptide-PNAs.

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.

Development of a Poly-ε-Lysine Contact Lens as a Drug Delivery Device for the Treatment of Fungal Keratitis.

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.