A study of complexation and biological fate of polyethyleneimine-siRNA polyplexes in vitro and in vivo by fluorescence correlation spectroscopy and positron emission tomography imaging.

A deeper knowledge on the formation and biological fate of polymer based gene vectors is needed for their translation into therapy. Here, polyplexes of polyethyleneimine (PEI) and silencing RNA (siRNA) are formed with theoretical N/P ratios of 2, 4 and 12. Fluorescence correlation spectroscopy (FCS) is used to study the formation of polyplexes from fluorescently labelled PEI and siRNA. FCS proves the presence of free PEI. From the analysis of the autocorrelation functions it was possible to determine the actual stoichiometry of polyplexes. FCS and fluorescence cross correlation spectroscopy (FCCS) are used to follow the fate of the polyplexes intracellularly. Polyplexes disassemble after 1 day inside cells. Positron emission tomography (PET) studies are conducted with radiolabelled polyplexes prepared with siRNA or PEI labelled with 2,3,5,6-tetrafluorophenyl 6-[18F]-fluoronicotinate ([18F]F-PyTFP). PET studies in healthy mice show that [18F]siRNA/PEI and siRNA/[18F]PEI polyplexes show similar biodistribution patterns with limited circulation in the bloodstream and accumulation in the liver. Higher activity for [18F]PEI in the kidney and bladder suggests the presence of free PEI.

Positron Emission Tomography Studies of the Biodistribution, Translocation, and Fate of Poly Allyl Amine-Based Carriers for Sirna Delivery by Systemic and Intratumoral Administration.

Polyamine-based vectors offer many advantages for gene therapy, but they are hampered by a limited knowledge on their biological fate and efficacy for nucleic acid delivery. The 18 F radiolabeled siRNA is complexed with poly(allyl amine) hydrochloride (PAH), PEGylated PAH (PAHPEG ), or oleic acid-modified PAH (PAHOleic ) to form polyplexes, and injected them intravenously into healthy rodents. The biodistribution patterns obtained by positron emission tomography (PET) imaging vary according to the polymer used for complexation. Free siRNA is quickly eliminated through the bladder. PAH and oleic acid modify PAH polyplexes accumulate in the lungs and liver. No elimination through the bladder is observed for PAH and PAHOleic within 2 h after administration. PAHPEG polyplexes accumulate in kidneys and are eliminated through the bladder. Polyplexes prepared with 18 F-labeled oleic acid-modified PAH and non-labeled siRNA show similar biodistribution to those prepared with labeled siRNA, but with more accumulation in the lungs due to the presence of non-complexed polymer. Intravenous administration of PAHOleic polyplexes in tumor models results in a limited availability of siRNA. When PAHOleic polyplexes are administered intratumorally in tumor bearing rodents, ≈40% of the radioactivity is retained in the tumor after 180 min while free siRNA is completely eliminated.

Self-Assembled Oleic Acid-Modified Polyallylamines for Improved siRNA Transfection Efficiency and Lower Cytotoxicity.

Small interference RNA (siRNA) is a tool for gene modulation, which can silence any gene involved in genetic disorders. The potential of this therapeutic tool is hampered by RNA instability in the blood stream and difficulties to reach the cytosol. Polyamine-based nanoparticles play an important role in gene delivery. Polyallylamine hydrochloride (PAH) is a polycation displaying primary amines that can be easily chemically modified to match the balance between cell viability and siRNA transfection. In this work, PAH has been covalently functionalized with oleic acid at different molar ratios by carbodiimide chemistry. The substituted polymers form polyplexes that keep positive surface charge and fully encapsulate siRNA. Oleic acid substitution improves cell viability in the pulmonary cell line A549. Moreover, 6 and 14% of oleic acid substitution show an improvement in siRNA transfection efficiency. CD47 is a ubiquitous protein which acts as "don't eat me signal." SIRPα protein of macrophages recognizes CD47, leading to tumor cell phagocytosis by macrophages. By knocking down CD47 with siRNA, cancer cells become vulnerable to be eliminated by the immune system. PAH-oleic acid substitutes show high efficacy in silencing the CD47 protein, making them a potential candidate for immunotherapy.

Neutralization of ionic interactions by dextran-based single-chain nanoparticles improves tobramycin diffusion into a mature biofilm.

The extracellular matrix protects biofilm cells by reducing diffusion of antimicrobials. Tobramycin is an antibiotic used extensively to treat P. aeruginosa biofilms, but it is sequestered in the biofilm periphery by the extracellular negative charge matrix and loses its efficacy significantly. Dispersal of the biofilm extracellular matrix with enzymes such as DNase I is another promising therapy that enhances antibiotic diffusion into the biofilm. Here, we combine the charge neutralization of tobramycin provided by dextran-based single-chain polymer nanoparticles (SCPNs) together with DNase I to break the biofilm matrix. Our study demonstrates that the SCPNs improve the activity of tobramycin and DNase I by neutralizing the ionic interactions that keep this antibiotic in the biofilm periphery. Moreover, the detailed effects and interactions of nanoformulations with extracellular matrix components were revealed through time-lapse imaging of the P. aeruginosa biofilms by laser scanning confocal microscopy with specific labeling of the different biofilm components.

Self-assembly of poly(allylamine)/siRNA nanoparticles, their intracellular fate and siRNA delivery.

Silencing RNA (siRNA) technologies attract significant interest as a therapeutic tool for a large number of diseases. However, the medical translation of this technology is hampered by the lack of effective delivery vehicles for siRNAs in cytosol that prevent their degradation in the bloodstream. The use of molecular complexes based on polyamines have great potential for siRNA delivery as polyamines can protect the siRNA during circulation and at the same time favor siRNA translocation in cytosol. Here, nanoparticles are prepared by complexation of poly(allylamine hydrochloride) (PAH) and siRNA varying the ratio of nitrogen groups from PAH to phosphate groups from siRNA (N/P ratio). Nanoparticles are characterized by transmission electron microscopy and dynamic light scattering. The stability of complexes of green rhodamine labelled PAH (G-PAH) and Cy5 labelled siRNA (R-siRNA) at different pHs and in cell media is studied by fluorescence cross-correlation spectroscopy (FCCS). FCCS studies show that the nanoparticles are stable at physiological pH and in cell media but they disassemble at acidic pH. An optimal N/P ratio of 2 is identified in terms of stability in media, degradation at endosomal pH and toxicity. The intracellular fate of the complexes is studied following uptake in A549 cells. The cross-correlation between G-PAH and R-siRNA decreases substantially 24 h after uptake, while diffusion times of siRNA decrease indicating that the complexes disassemble, liberating the siRNAs. The release of siRNAs into the cytosol is confirmed with parallel confocal laser scanning microscopy. Flow cytometry studies show that PAH/siRNA nanoparticles are effective at silencing green fluorescent protein expression at low N/P ratios at which polyethylenimine/siRNA shows no significant silencing.