Mimicking the Biology of Engineered Protein and mRNA Nanoparticle Delivery Using a Versatile Microfluidic Platform

Abstract

To investigate the delivery of next-generation macromolecular drugs, such as engineered proteins and mRNA-containing nanoparticles, there is an increasing push towards the use of physiologically relevant disease models that incorporate human cells and do not face ethical dilemmas associated with animal use. Here, we illustrate the versatility and ease of use of a microfluidic platform for studying drug delivery using high-resolution microscopy in 3D. Using this microfluidic platform, we successfully demonstrate the specific targeting of carbonic anhydrase IX (CAIX) on cells overexpressing the protein in a tumor-mimicking chip system using affibodies, with CAIX-negative cells and non-binding affibodies as controls. Furthermore, we demonstrate this system’s feasibility for testing mRNA-containing biomaterials designed to regenerate bone defects. To this end, peptide- and lipid-based mRNA formulations were successfully mixed with colloidal gelatin in microfluidic devices, while translational activity was studied by the expression of a green fluorescent protein. This microfluidic platform enables the testing of mRNA delivery from colloidal biomaterials of relatively high densities, which represents a first important step towards a bone-on-a-chip platform. Collectively, by illustrating the ease of adaptation of our microfluidic platform towards use in distinct applications, we show that our microfluidic chip represents a powerful and flexible way to investigate drug delivery in 3D disease-mimicking culture systems that recapitulate key parameters associated with in vivo drug application.

Deciphering Structural Determinants Distinguishing Active from Inactive Cell-Penetrating Peptides for Cytosolic mRNA Delivery

“…We also included Lipofectamine MessengerMAX (LMM) as a further reference. Compared to PF14, mRNA fluorescence was homogeneously distributed throughout the cytosol with no signals present in nuclei, consistent with previous observations . This result demonstrates that also for PF14, a significant fraction of material remains associated with vesicular structures.…”

“…Peptide-mRNA polyplexes were formulated as previously described. , In short, two separate stock solutions of mRNA and peptide were prepared in MQ and simultaneously dispensed with electronically dispensing pipettes (E4 Electronic Pipette, LTS E4-100XLS+, Mettler-Toledo Rainin, LLC, Oakland, CA, USA) at a flow rate of 11 mL min –1 . All polyplexes were formed at a concentration of at least 10-fold the final intended concentration for transfections.…”

Chemical and Structural Engineering of Gelatin-Based Delivery Systems for Therapeutic Applications: A Review

“…Palacio-Castanẽda et al studied the drug delivery based on gelatin in 3D on-chip tissue systems. 124 They illustrated affinity-mediated on-chip targeting of Carbonic anhydrase IX (CAIX) in the context of bone regeneration biomaterial development, and gelatin nanoparticle-mediated delivery of mRNA that could express reporter protein eGFP (Figure 10c). Through the use of CAIX-positive renal cancer cells (SK-RC-52), nonbinding affibodies, and CAIX-negative cells (SK-RC-17), the researchers confirmed the specificity of CAIX targeting by utilizing anti-CAIX conjugates coupled with mCherry fluorescent protein.…”

“…Gelatin used for nanomedicine in human bodies is normally functionalized to reduce macrophage uptake and prolong the half-life of circulation, as well as the targeting effects. Palacio-Castañeda et al studied the drug delivery based on gelatin in 3D on-chip tissue systems . They illustrated affinity-mediated on-chip targeting of Carbonic anhydrase IX (CAIX) in the context of bone regeneration biomaterial development, and gelatin nanoparticle-mediated delivery of mRNA that could express reporter protein eGFP (Figure c).…”

“…Illustration showing the fusion of an affibody with mCherry and a schematic representation of the microfluidic chip. Reprinted or adapted with permission under a Creative Commons CC-BY 4.0 from ref . Copyright 2021 Molecular Diversity Preservation International.…”