(T1430-08-47) Engineering 3D Printed Microfluidic Chips for the Fabrication of Nanomedicines

Purpose: Nanomedicine manufacturing is still costly and difficult to scale up, limiting industrial adoption. Thus, continuous and controlled manufacturing processes are urgently needed. Microfluidic manufacturing is a novel and adaptable solution to these issues. Still, most chips are made with polydimethylsiloxane (PDMS) and soft mask lithography, which is tedious, difficult to customise, and requires specialised equipment and expertise. In addition, 3D printed chips can provide microfluidic chips for quick pilot studies towards the manufacture of nanomedicines under controlled conditions, allowing easy scale-up and shorter development times. 3D printed chips must have printing channels with sufficient resolution to produce the required geometry to be successful as an alternative. Finally, we used commercially available solvent resistant filaments and resin to create microfluidic chips with the appropriate geometry and channel characteristics to manufacture polymeric nanoparticles based on polymethacrylate polymers encapsulating high concentrations of a BCS class II drug (nifedipine, NFD).

Methods: Chips were designed in Tinkercad® (Autodesk®) and measured 8.2 cm in length, 3.5 cm in width, and 0.7 cm in height. The channel length was 44 cm and the diameter was 1 mm. 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, 10 mm/s printers and 30 mm/s travel speed with cyclic olefin copolymer filament. For stereolithography, the Anycubic Photon Mono X (LCD-based SLA printer with 405 nm light source and 0.01 mm Z resolution) was used. 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 -50^oC. Formulations were characterized in terms of drug loading, particle size, zeta potential and morphology and the channels were imaged with light microscopy, and scanning electron microscopy while the surface roughness was measured with profilometry. Solid-state characterisation of lyophilized particles was also undertaken. The release of NFD from nanoparticles was assessed using a type II dissolution apparatus (Erweka DT 80, Heusenstamm, Germany) under simulated gastric and intestinal media.

Results: The chip geometry produced was in close accordance with the .stl file sent for printing. 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), closer to the loading reported by solvent evaporation. NFD nanoparticles manufactured using SLA chips were significantly smaller than those 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. Lyophilised nanoparticles showed similar FTIR, pXRD, and DSC patterns obtained from SLA and FDM chips.

NFD release was hampered in acidic media ( < 20% at 1 hour), but the near-complete release 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 a controlled release in simulated intestinal media (p < 0.05, One-way Anova). NFD localisation within the particles produced with different 3D printed chips due to differences in surface roughness and drug-polymer interactions contributes to these findings. In addition, the smoother channels of SLA chips lead to a more homogenous loading process, where NFD is located within the core of the polymeric nanoparticles, further supported by the smaller particle size and controlled release profile in acidic media where NFD is more likely to be soluble.

Conclusion: 3D printed microfluidic chips with 1 mm diameter channels have been successfully designed and manufactured and are capable of engineering 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, easily industrialised nanomedicine manufacturing process.

References: Ayyoubi, S., et al., 3D printed spherical mini-tablets: Geometry versus composition effects in controlling dissolution from personalised solid dosage forms. International Journal of Pharmaceutics, 2021. 597: p. 120336.

Acknowledgements:

Funding: This work has been partially funded by the Research group (Formulation and bioavaila-bility of novel medicines) from Universidad Complutense de Madrid (Ref. 910939). We thank Ga-lenicus Group Madrid for their support in performing this work. We thank the Centros Asociados a la Investigación (CAI) from Universidad Complutense for their support in performing the solid state characterization analysis (DSC, XRD, FTIR, TEM). Athina Vassiliadou was supported by an Erasmus+ mobility grant.


Figure 1. Schematic representation and 3D printed chips. Key: a) Geometrical design of the 3D printed microfluidic chip (Tinkercad); b) Photograph of 3D printed microfluidic chip by SLA; c) Photograph of 3D printed microfluidic chip by FDM. Scale: 10 mm.


Figure 2. TEM micrographs of NFD polymeric nanoparticles stained with 1% uranyl acetate. Key: (a) NFD nanoparticles prepared using FDM printed microfluidic chips, (b) NFD nanoparticles prepared using SLA printed microfluidic chips, (c) NFD nanoparticles prepared by conventional method. Scale bars: 200 nm.


Figure 3. NFD release from polymeric nanoparticulate formulations in simulated gastric fluid (pH 1.2 over 2 hours) and simulated intestinal fluid (pH 6.8 over remaining time). Key: (-●-) Polymeric nanoparticles prepared with SLA printed microfluidic chip, (-■-) Polymeric nanoparticles prepared with FDM printed microfluidic chip, (-▲-) Polymeric nanoparticles prepared with the conventional method. A repeated-measures ANOVA was undertaken (GraphPad 9) using a Tukey’s post-hoc test and indicated that release from FDM microfluidically prepared polymeric nanoparticles are different (p < 0.05) from both SLA microfluidically prepared polymeric nanoparticles as well as those prepared with solvent evaporation.