Engineering bioactive 3D printing bioinks towards targeted personalised therapies

Constructing functional scaffolds for targeted controlled drug release and tissue regeneration offers promising therapies for many diseases such as tumours, bone regeneration, wound healing, bacterial and fungal infections. 3D bioprinting is an additive manufacturing approach that utilizes a “bioink” to fabricate complex structures out of molecules, similar to assembling Lego pieces. The ideal should satisfy certain material (printability, mechanics, degradation, functionalisation) and biological requirements (biocompatibility, cytocompatilibilty, and bioactivity). Inclusion or conjugation of peptides is common within bioinks as specific regulators of cell activities or for their therapeutic potential. However, the lack of peptide stability to enzymatic degradation remains a problem in realising their potential. We have shown that peptide amphiphiles (PAs), prepared by introducing lipidic parts within peptides, are highly stable and stop the growth of brain and breast cancer cells alone or when loaded with chemotherapeutics. We aim to develop 3D printable biodegradable bioactive bio-inks prepared by cellulose based cell-friendly bioinks loaded with peptides or peptide amphiphiles that when printed into patient specific implants can elicit a specific anticancer effect and target chemotherapeutics to cancer cells remaining after surgery towards targeted and personalised treatments in cancer. In this respect, in this TRIF project, we have synthesised cellulose nanocrystals and we have used them to prepare 3D printable gels when mixed with calcium chloride. We have studied and optimised the rheological properties of cellulose nanocrystal hydrogels and characterised them in terms of particle size and morphology. Finally, we have embedded a model peptide, leucine encephalin, and studied its release from 3D printed scaffolds. We are planning to embedded an antiproliferative peptide amphiphile using similar protocols and understand its antiproliferative effects in vitro using breast and brain tumour cell lines (MCF-7, MDA-MD-231, U87MG)

Antifungal and antibacterial electrospun wound dressings for complex wounds

Introduction: Management of open fractures wounds, diabetic ulcers and military wounds frequently involve infections with gram-positive or gram-negative bacteria and in some cases invasive fungal infections which are linked to mycotic emboli and delays in reconstructive efforts or amputations. Topical antibiotics and antifungals are recommended and local delivery of antimicrobials through beads or bead pouches along with a water impermeable dressing has been shown to be beneficial 1 . Here we present a dressing prepared by alternating electrospun polymeric mats loaded with combination of antifungal (amphotericin B, AmB) and antibacterial (vancomycin) agents in clinically relevant concentrations that can be used for the treatment of complex wounds. Methods: Electrospun membranes were produced using a Spraybase 30kV electrospinning kit attached to a syringe pump (NE-1000). The collection distance was set to 12 cm and a voltage of 16.5 kV was utilised. Polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus) (8g) was dissolved in acidified methanol and AmB or vancomycin were dispersed prior to the addition of dichloromethane to elicit a suspension that was perfused at 8mL/h via the inner needle of a co-axial electrospinning needle (~900 µm, Rame-hart Instrument Co). Membranes were collected on foil after a stable “cone-jet” mode and a uniform fiber production process was achieved and stored under desiccated conditions in the fridge till further use. Release experiments were conducted in phosphate buffer (50mM, pH 7.4) and levels were quantified using a validated HPLC method 2 . Membranes were analysed using FT-IR, DSC and TGA and their morphology using SEM 3 . Disk diffusion inhibition halo assays against Candida albicans (strain) were performed as previously described 3 . Results: Amphotericin B membranes contained near 100% of AmB sprayed (1.05±0.96 mg/g) and demonstrated a fibrous morphology with higher curvature than Soluplus electrospun fibers (Figure 1). AmB electrospun dressings were amorphous and FT-IR indicated hydrogen bonding between the protonated amine of Amphotericin B and carbonyl groups of Soluplus. Near 30% release was achieved after 1 hour, while a controlled release profile was observed for the first 2 days. Released AmB was present in monomeric form (UV). Inhibition halo of AmB dressings or AmB DMSO impregnated filter papers (6mm, 10 µg) resulted in comparable halos against C. albicans (23 ± 1 mm and 24 ± 1mm respectively). Currently work is undertaken to characterise vancomycin dressings and combined dressings. Conclusions/Implications: Prepared dressings can be utilised for treatment of complex fungal infected wounds to avoid mycotic embolism

Engineering 3D printed microfluidic chips for the fabrication of nanomedicines

1.Purpose -- Nanomedicine manufacture remains expensive and difficult to scale-up which limits the uptake of nano-enabled technologies by industry. Thus, there is an urgent unmet need for continuous and controlled manufacturing processes. Microfluidic manufacture has emerged as a novel and easily adaptable strategy to overcome these challenges, but majority of chips used are fabricated using polydimethulsiloxane (PDMS) and soft mask lithography that remains tedious, not easily customizable and requires specialized equipment and expertise for their production. 3D printed chips are a novel and easily adaptable cost-effective alternative able to provide microfluidic chips for enabling quick pilot studies towards the manufacture of nanomedicines under controlled conditions with optimal and controlled characteristics enabling easy scale-up and shorter development times. However, for 3D printed chips to be successful as an alternative, 3D printing channels with adequate resolution to produce the required geometry needs to be demonstrated. In this work, we utilized the most easily accessible 3D printing techniques (fused deposition modelling (FDM) and sterolithography (SLA)) and commercially available solvent resistant filaments and resin to produce designed microfluidic chips with appropriate geometry and channel characteristics to allow for the manufacture of polymeric nanoparticles based on polymethacrylate polymers encapsulating high concentration of a BCS class II drug (nifedipine, NFD). 2.Method -- Chips were designed in Tinkercad® (Autodesk®) and measured 8.2 cm in length, 3.5 cm in width, and 0.7 cm in height. Channel length was 44 cm and the diameter was 1 mm. Chip designed was exported into a standard tessellation language (.stl) digital file. An Anycubic Mega Zero FDM printer printed 70 layers of the microfluidic chip at 245°C, with a 0.4 mm diameter nozzle, 0.1 mm layer height, and 10 mm/s printer and 30 mm/s travel speed with cyclic olefin copolymer filament. The Anycubic Photon Mono X (LCD-based SLA printer with 405 nm light source and 0.01 mm Z resolution) was used for stereolithography. Anycubic® UV sensitive resin (transparent yellow) was photopolymerized at 405 nm. The print settings were 0.05 mm layer height, 60 s bottom exposure, 3 s normal exposure, 1 s off-time, and 140 layers. Polymeric nifedipine loaded nanoparticles were prepared using solvent evaporation and microfluidically. For the latter, the aqueous phase (8 ml) consisting of Tween 80 in deionized water (0.25 % w/v) and the organic phase consisting of Eudragit L100-55 (30 mg) and NFD (10 mg) dissolved in ethanol (2 ml) were loaded into two 10 ml syringes. Using two syringe pumps (New Era Pump Systems, NY, USA), the organic phase was flown at a rate of 0.5 ml min-1 and the aqueous phase at 2 ml min-1. The eluate was rota-evaporated for 10 minutes at 150 rpm and 60 °C to remove the ethanol and centrifuged at 5,000 rpm for 5 minutes to remove any free NFD. Part of the supernatant was lyophilized for 24 hours under 0.2 mbar pressure at -50oC. Formulations were characterized in terms of drug loading, particle size, zeta potential and morphology and the channels were imaged with light microscopy, scanning electron microscopy while the surface roughness was measured with profilometry. Solid state characterisation of lyophilized particles were also undertaken. Release of NFD from nanoparticles was assessed using a type II dissolution apparatus (Ewerka DT 80, Heusenstamm, Germany) under simulated gastric and intestinal media (Ayyoubi S.). 3.Results --  The chip geometry produced was in close accordance to the .stl file sent for printing (Fig. 1a). The channel diameter ranged from 985 – 1015 µm. SLA-printed chips exhibited channels with a smoother surface (10.5-fold) than FDM chips. NFD nanoparticles showed a 7% greater drug encapsulation when manufactured by SLA than with FDM chips (one-way ANOVA, p < 0.05) which was closer to the loading reported by solvent evaporation. NFD nanoparticles manufactured using SLA chips were significantly smaller than those particles obtained from FDM chips, 68 ± 1 nm versus 75 ± 1 nm, respectively (one-way ANOVA, p < 0.05), which was closer to the particle size obtained by solvent evaporation (Fig. 2). Lyophilised nanoparticles showed similar FTIR, pXRD, and DSC patterns obtained from both SLA and FDM chips. NFD release was hampered in acidic media (<20% at 1 hour), but near complete released was achieved when the pH was raised to 6.8 within 6 hours, which was similar to that obtained for particles prepared with solvent evaluation (Fig. 3). However, NFD particles produced with FDM showed a burst release in acidic media (~40%) followed by controlled release in simulated intestinal media (p<0.05, One-way Anova). NFD localization within the particles produced with different 3D printed chips due differences in surface roughness and drug–polymer interactions are contributing to these findings. The smoother channels of SLA chips lead to a more homogenous loading process, where NFD is located within the core of the polymeric nanoparticles, which is further supported by the smaller particle size and controlled release profile in acidic media where NFD is more likely to be soluble.  4. Conclusion -- 3D printed microfluidic chips with 1 mm diameter channels have been successfully designed and manufactured and are capable to engineer polymeric nanoparticles with good encapsulation efficiencies and particle sizes of ~100 nm, like nanoparticles obtained by solvent evaporation. 3D printed microfluidic chips control the process and convert discontinuous methods into a continuous nanomedicine manufacturing process that are easily industrialized