(512f) A Simple Mixing Method for Cytosolic Delivery of Protein, siRNA, and Plasmid DNA Using Hydrophobic Ion Pairing for Use As a Non-Toxic and Inexpensive Transfection Agent | AIChE

(512f) A Simple Mixing Method for Cytosolic Delivery of Protein, siRNA, and Plasmid DNA Using Hydrophobic Ion Pairing for Use As a Non-Toxic and Inexpensive Transfection Agent

Authors 

Seeler, M., Georgia Institute of Technology
De Janon, A., Georgia Institute of Technology
Panoskaltsis, N., Northwick Park Hospital
Mantalaris, A., Imperial College London
Hydrophilic biomacromolecule therapeutics are difficult to deliver because of poor encapsulation and inability to cross the cell membrane. However, proteins and nucleic acids are often more specific and possess less toxicity than small molecules. Existing transfection agents, whether for in vivo or in vitro use, face high cost, low efficacy, and toxicity. In this work, simple mixing of a hydrophobic counterion with a charged biomacromolecule formed a hydrophobic ion complex capable of intracellular delivery without toxicity by entering through endocytosis and releasing de-complexed cargo into the cytosol following endosomal membrane disruption. Hydrophobic ion pairing has been utilized to improve encapsulation efficiency and endosomal escape in various nanoparticle systems. All counterions used are generally regarded as safe and part of everyday use as laxatives, medicines, or sunflower oil. To ensure that positively and negatively charged counterions were non-toxic, we delivered two charge variants of super folder green fluorescent protein. We demonstrated functional cytosolic protein delivery by delivering Cytochrome C, a protein that induces caspase pathway mediated cell apoptosis only when delivered to mitochondria in the cytosol. Anionic counterions, sodium docusate and oleic acid, resulted in effective cytosolic delivery to HeLa cells and subsequent loss in viability. Cytochrome C HIP complexes were also delivered into a 3D polyurethane scaffold hosting K562 leukemia cells. HIP complexes yielded a 76.1 ± 0.2% reduction in cell viability in the acute myeloid leukemia organoid after 24 hours, demonstrating the potential of HIP at characterizing organoid responses to new protein therapeutics with intracellular targets. To investigate nucleic acid cargo delivery, we delivered anti-GFP small interfering RNA (siRNA) and found 74.5 ± 5.8% gene silencing in NIH3T3 GFP expressing cells - comparable to lipofectamine RNAiMax but without any toxicity. Lastly, we delivered plasmid DNA encoding hemagglutinin stalk, an influenza antigen inducing broadly protective immunity, into HEK293 mammalian protein expression cells using HIP and measured delivery success by confirming the protein expression using a western blot. Altogether, HIP delivery demonstrates broad use across protein and nucleic acid cargos and cell type, the cost of HIP delivery is significantly lower than commercial reagents and the complexes are stable without requiring refrigeration, which will undoubtably expediate the discovery of new therapeutics.