Investigating the Cytosolic Delivery of Proteins by Lipid Nanoparticles Using the Chloroalkane Penetration Assay

Abstract

Protein therapeutics are an expanding area for research and drug development, and lipid nanoparticles (LNPs) are the most prominent nonviral vehicles for protein delivery. The most common methods for assessing protein delivery by LNPs include assays that measure the total amount of protein taken up by cells and assays that measure the phenotypic changes associated with protein delivery. However, assays for total cellular uptake include large amounts of protein that are trapped in endosomes or are otherwise nonfunctional. Assays for functional delivery are important, but the readouts are indirect and amplified, limiting the quantitative interpretation. Here, we apply an assay for cytosolic delivery, the chloroalkane penetration assay (CAPA), to measure the cytosolic delivery of a (−30) green fluorescent protein (GFP) fused to Cre recombinase (Cre(−30)GFP) fusion protein by LNPs. We compare these data to the data from total cellular uptake and functional delivery assays to provide a richer analysis of uptake and endosomal escape for LNP-mediated protein delivery. We also use CAPA for a screen of a small library of lipidoids, identifying those with a promising ability to deliver Cre(−30)GFP to the cytosol of mammalian cells. With careful controls and optimized conditions, we expect that CAPA will be a useful tool for investigating the rate, efficiency, and mechanisms of LNPmediated delivery of therapeutic proteins.

Comparing cell penetration of biotherapeutics across human cell lines

“…This raises the possibility that part of the CAPA signal derives from degraded material, and ct-SAHB and ct-nusinersen are more protected from degradation in the presence of chloroquine. While the CAPA data for these compounds do not resemble cases in which we have documented degradation artifacts, 45,58 it remains a possibility that cannot be ruled out. Another explanation is that chloroquine has been shown to inhibit clathrin-mediated endocytosis, 33,113 which our data (Figure 5, SI Figure 14) indicate is particularly important for cell penetration of polycationic peptides and nusinersen.…”

“…CAPA has been broadly adopted in industry and academia, and it has been successfully applied to peptides, 15,[47][48][49][50] antisense oligonucleotides and siRNAs with different lengths and chemical modifications, 45,51,52 cyclotides, 53 peptidomimetic macrocycles, [54][55][56] PROTACs, 57 and lipid nanoparticle-mediated delivery of proteins. 58 It has even been adapted for 3D tissue models to measure deep penetration of biotherapeutics, 59 and for bacterial cells to evaluate the bacterial penetration of small molecules. 60 We recently reported the ability to use adeno-associated virus to deliver HaloTag to different cellular compartments and different cell types.…”

Comparing Cell Penetration of Biotherapeutics across Human Cell Lines

“…This raises the possibility that part of the CAPA signal derives from degraded material, and ct-SAHB and ct-nusinersen are more protected from degradation in the presence of chloroquine. While the CAPA data for these compounds do not resemble cases in which we have documented degradation artifacts, , it remains a possibility that cannot be completely ruled out. Another explanation is that chloroquine has been shown to inhibit clathrin-mediated endocytosis, , which our data (Figure and SI Figure 14) indicate is particularly important for cell penetration of polycationic peptides and nusinersen.…”

“…The smaller the CP 50 , the more cell-penetrant the ct-compound. CAPA has been broadly adopted in industry and academia, and it has been successfully applied to peptides, ,− antisense oligonucleotides, and siRNAs with different lengths and chemical modifications, ,, cyclotides, peptidomimetic macrocycles, − PROTACs, and lipid nanoparticle-mediated delivery of proteins . It has even been adapted for three-dimensional (3D) tissue models to measure deep penetration of biotherapeutics, and for bacterial cells to evaluate the bacterial penetration of small molecules …”