Abstract
We previously reported that heparin/protamine particles (LHPPs) produced as nanoparticles through simple mixing of raw materials exhibit sustained protein release and can be retained in cells. In the present study, we modified LHPPs without employing any organic synthetic approach. The resulting LHPPs were re-named as improved LHPPs (i-LHPPs) and have the ability to retain cell-penetrating peptides (GRKKRRQRRRPPQ) based on electrostatic interactions. We examined whether i-LHPPs can introduce exogenous proteins (i.e., lacZ protein encoding bacterial β-galactosidase) into cultured cells in vitro, or into murine hepatocytes in vivo through intravenous injection to anesthetized mice. We found an accumulation of the transferred protein in both in vitro cultured cells and in vivo hepatocytes. To the best of our knowledge, reports of successful in vivo delivery to hepatocytes are rare. The i-LHPP-based protein delivery technique will be useful for in vivo functional genetic modification of mouse hepatocytes using Cas9 protein-mediated genome editing targeting specific genes, leading to the creation of hepatic disease animal models for research that aims to treat liver diseases.
Highlights
Introduction of genetic material is one of the representative techniques used to modify cell function
We examined whether improved low-molecular-weight heparin/protamine particles (LHPPs) (i-LHPPs) could be produced without organic synthesis, and the resulting i-LHPPs were used as protein carriers for delivery of a target protein to mouse hepatocytes via Hydrodynamics-based gene delivery (HGD)
To examine whether i-LHPP-based protein delivery is possible in vivo, we introduced the i-LHPPs/β-gal complex intravenously using the HGD method, which is known to be effective for targeted delivery of nucleic acids (NAs) to hepatocytes [9,10]
Summary
Introduction of genetic material is one of the representative techniques used to modify cell function. A foreign gene introduced into a cell can produce functional protein, via several processes, namely transcription (mRNA synthesis from the foreign gene), translation of mRNA into protein, and post-translational modifications. This cellular event is termed protein biosynthesis. Minor deposition of β-gal-reacted products was observed in the specimens derived from delivery of β-gal alone (Figure 3c), or in those from LHPPs/β-gal complexes (without using TAT) (Figure 3d), whereas the extent was less than that seen in specimens derived from delivery of the i-LHPPs/β-gal complexes (Figure 3c,d vs Figure 3e) No such deposits were noted in untreated (intact) specimens (Figure 3a), or in those that had been given i-LHPPs alone (Figure 3b). It was found that it is possible to deliver proteins into hepatocytes without loss of integrity when i-LHPPs are used as protein carriers
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