(W0930-07-43) 3D-Printed Microfluidic vs Conventional Fabrication of P(DMAEMA-co-SMA)-Based Lipopolyplexes: A Comparative Study of Stability, Morphology and Cytotoxicity

Purpose: Lipopolyplexes have demonstrated several advantages for nucleic acid delivery, including enhanced in vitro transfection efficiency and improved physicochemical stability during prolonged storage compared to polyplexes. However, the lack of conventional methods to precisely control self-assembly, combined with the inherent cytotoxicity of polyethylenimine (PEI), the gold standard in non-viral gene delivery, limits their clinical application. To address these limitations, we present Digital Light Processing (DLP) 3D-printing technology to fabricate microfluidic chips with embedded microstructures, enabling reproducible nanoparticle preparation and fine-tuning of the physicochemical properties. In this context, limited research has been conducted on lipopolyplexes consisting of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), despite its potential as a promising alternative to PEI, offering approximately 90% of the transfection efficiency of branched PEI, and lower cytotoxicity. Therefore, the pH-responsive cationic copolymer P(DMAEMA-co-SMA) was synthesized and formulated into lipopolyplex nanocomplexes. The aim of the present study was to evaluate the reproducibility and morphology of lipopolyplexes prepared using the 3D-printed microfluidic chip compared to conventional thin film hydration method, and to assess the potential of the synthesized copolymer for safe and effective gene delivery.

Methods: The pH responsive cationic copolymer P(DMAEMA-co-SMA) was synthesized by RAFT polymerization and characterized using ¹H-NMR spectroscopy, and size-exclusion chromatography (SEC), whereas its proton buffering capacity was determined using the acid-base titration method within the pH range of 7.4-5. DLP 3D-printing technology was used to fabricate a 3-port microfluidic chip with embedded fin-like microstructures. Scanning Electron Microscopy (SEM) was used to evaluate the printing accuracy of DLP technology in fabricating microfluidic devices for nanoparticle preparation (Figure 1). The microfluidic chip with 3-inlets was used to prepare lipopolyplexes at N/P 16 directly by adjusting the flow rate ratio (FRR 5:2:3) and total flow rate (TFR 10 mL/min). Moreover, lipopolyplexes at N/P 16 were developed by conventional thin film hydration followed by extrusion through polycarbonate membranes. In this method, polyplexes at N/P 16 were coated with specific lipids in a post-processing step. The storage stability of the resulting nanocomplexes at 4^oC was assessed by measuring  physicochemical properties (hydrodynamic diameter, polydispersity index, z-potential) at predetermined time points for 28 days by using dynamic and electrophoretic light scattering. The colloidal stability of the nanocomplexes was assessed in biorelevant conditions by measuring physicochemical properties at pH 6.5, and 5.5, corresponding to the environments of early, and late endosomes, as well as under cell culture conditions. The morphology of the developed nanocomplexes was determined using cryogenic transmission electron microscopy (cryo-TEM). Moreover, in vitro cytotoxicity of the prepared nanosystems was evaluated at a wide range of concentrations (1-50 μg/mL) in HeLa cells using the MTT assay with PEI 50kDa and dextran as controls. Subsequently, hemolysis assay was performed spectrophotometrically at the same nanoparticle concentration range following incubation in fresh rat red blood cells (RBC) for initial assessment of cellular toxicity.

Results: The chemical structure and composition of the copolymer were identified by ¹H-NMR spectroscopy, whereas its molecular weight (Mw 54kDa) and molecular weight distribution (Mw/Mn 1.16) were determined by SEC. Lipopolyplexes prepared by both 3D-printed microfluidic chip and conventional thin film hydration method exhibited superior colloidal stability for 28 days. Specifically, nanocomplexes with hydrodynamic diameter of approximately 90 nm were prepared by the 3D-printed microfluidic chip with reproducible results, while nanocomplexes prepared by thin film hydration had a higher hydrodynamic diameter (230 nm) and greater batch-to-batch variability (Figure 2). These results indicate that the microfluidic chip with embedded fin-like microstructures enables continuous, efficient, and more uniform mixing between the lipids, copolymer and DNA compared to conventional thin film hydration method. The polydispersity index of the developed nanocomplexes was approximately 0.2, indicating great homogeneity for both preparation methods. The results from cryo-TEM revealed multiple coexisting nanostructures including onion-like nanocomplexes and amorphous particles. The physicochemical properties of the resulting lipopolyplexes were assessed in biorelevant conditions, showing a pH-responsive behavior, with a decrease in the hydrodynamic diameter under acidic conditions, which can be attributed to enhanced protonation of the copolymer, leading to stronger electrostatic interactions with DNA. These results combined with the determined buffering capacity of the copolymer suggest that the nanosystems have the potential for endosomal escape. Furthermore, colloidal stability was maintained in cell culture medium. The in vitro cytotoxicity of lipopolyplexes in HeLa cells demonstrate that the nanocomplexes were well tolerated within the examined concentration range (1-50 μg/mL), while PEI showed concentration-dependent cytotoxicity and constantly lower cell viability compared to lipopolyplexes. Subsequently, hemolysis assay performed on fresh rat RBC showed low hemolytic activity for the developed lipopolyplexes at all concentrations after 1 h of incubation, while PEI induced a higher rate of hemolysis.

Conclusion: This study presents a novel 3D-printed microfluidic chip with 3 inlets for the reproducible fabrication of P(DMAEMA-co-SMA)-based lipopolyplexes. Compared to conventional thin film hydration/extrusion method, microfluidic development produced structurally uniform nanocomplexes with enhanced colloidal stability, reduced cytotoxicity, and low hemolysis, demonstrating its potential for scalable, and safe non-viral gene delivery.

References: M. Rezaee, R. K. Oskuee, H. Nassirli, and B. Malaekeh-Nikouei, ‘Progress in the development of lipopolyplexes as efficient non-viral gene delivery systems’, Journal of Controlled Release, vol. 236, pp. 1–14, Aug. 2016.

Acknowledgements: This work was supported by the programme "Greek-British Cooperation on Short-Term Scholarships”, which is delivered by the State Scholarships Foundation (IKY), the British Embassy Athens and the British Council Greece.


The fabricated 3D-printed microfluidic chip with three inlets (left), and scanning electron microscopy (SEM) image (right) showing the internal fin-like microstructures within the main channel.


The hydrodynamic diameter of P(DMAEMA-co-SMA)-based lipopolyplexes prepared via microfluidics (blue) and conventional thin film hydration/extrusion (red) methods at N/P 16 ratio over 28 days. Data represent mean ± SD (n = 3). Significant differences between methods at all time points (****p < 0.0001).