Combined Chemisorption and Complexation Generate siRNA Nanocarriers with Biophysics Optimized for Efficient Gene Knockdown and Air-Blood Barrier Crossing.

Alexander Taschauer,Martina Anton,Slavica Metz,Simon Decker,Hermann Bloß,Stefan Pöschl,Manfred Ogris,Julia Scholda,Haider Sami,Nathalie Tepe,Julia Maier,Thilo Hofmann,Norbert Cyran,Wolfram Polzer

doi:10.1021/acsami.0c06608

Abstract

Current nucleic acid (NA) nanotherapeutic approaches face challenges because of shortcomings such as limited control on loading efficiency, complex formulation procedure involving purification steps, low load of NA cargo per nanoparticle, endosomal trapping, and hampered release inside the cell. When combined, these factors significantly limit the amount of biologically active NA delivered per cell in vitro, delivered dosages in vivo for a prolonged biological effect, and the upscalability potential, thereby warranting early consideration in the design and developmental phase. Here, we report a versatile nanotherapeutic platform, termed auropolyplexes, for improved and efficient delivery of small interfering RNA (siRNA). Semitelechelic, thiolated linear polyethylenimine (PEI) was chemisorbed onto gold nanoparticles to endow them with positive charge. A simple two-step complexation method offers tunable loading of siRNA at concentrations relevant for in vivo studies and the flexibility for inclusion of multiple functionalities without any purification steps. SiRNA was electrostatically complexed with these cationic gold nanoparticles and further condensed with polycation or polyethyleneglycol–polycation conjugates. The resulting auropolyplexes ensured complete complexation of siRNA into nanoparticles with a high load of ∼15,500 siRNA molecules/nanoparticle. After efficient internalization into the tumor cell, an 80% knockdown of the luciferase reporter gene was achieved. Auropolyplexes were applied intratracheally in Balb/c mice for pulmonary delivery, and their biodistribution were studied spatio-temporally and quantitatively by optical tomography. Auropolyplexes were well tolerated with ∼25% of the siRNA dose remaining in the lungs after 24 h. Importantly, siRNA was released from auropolyplexes in vivo and a fraction also crossed the air–blood barrier, which was then excreted via kidneys, whereas >97% of gold nanoparticles were retained in the lung. Linear PEI-based auropolyplexes offer a combination of successful endosomal escape and better biocompatibility profile in vivo. Taken together, combined chemisorption and complexation endow auropolyplexes with crucial biophysical attributes, enabling a versatile and upscalable nanogold-based platform for siRNA delivery in vitro and in vivo.

Highlights

Nanotechnology has the potential to overcome current challenges associated with nucleic acid-based therapeutics and is an active area of nanomedicine application
UV−vis spectroscopy of AuNPs demonstrated the presence of characteristic surface plasmon resonance (SPR) with an absorption wavelength maximum of around 530 nm (Figure S1), which matches with the reported data.[9]
Tunability of short interfering RNA (siRNA) loading is demonstrated by preparing auropolyplexes at siRNA concentrations of 10 and 133 μg/mL for in vitro and in vivo experiments, respectively

Summary

Introduction

Nanotechnology has the potential to overcome current challenges associated with nucleic acid-based therapeutics and is an active area of nanomedicine application. Preclinical and clinical translation of nucleic acid-based nanomedicines is decisively dependent on factors, including, but not limited to, loading efficiency, yield and scale-up, effective delivery to target organ/cells, and successful endosomal escape.[1−3] For instance, high and tunable loading efficiency is crucial when considered in the context of the “total nanoparticle load” needed to deliver the desired dosage in vivo for an efficacious/ prolonged biological effect[3−5] and the toxicity associated with high nanoparticle loads in vivo. Bottom: in vivo biodistribution of the near-infrared dye (AF750)-labeled siRNA-loaded ALSL10 auropolyplexes after microspray-based intratracheal administration was studied noninvasively and spatio-temporally by fluorescence imaging tomography (FLIT)/X-ray absorption computed tomography followed by AF750-siRNA and gold quantification by fluorescence-based absolute quantification and inductively coupled plasma mass spectrometry, respectively

Methods

Results

Conclusion