Bi-functionalized aminoguanidine-PEGylated periodic mesoporous organosilica nanoparticles: a promising nanocarrier for delivery of Cas9-sgRNA ribonucleoproteine

Abstract

Background There is a great interest in the efficient intracellular delivery of Cas9-sgRNA ribonucleoprotein complex (RNP) and its possible applications for in vivo CRISPR-based gene editing. In this study, a nanoporous mediated gene-editing approach has been successfully performed using a bi-functionalized aminoguanidine-PEGylated periodic mesoporous organosilica (PMO) nanoparticles (RNP@AGu@PEG1500-PMO) as a potent and biocompatible nanocarrier for RNP delivery. Results The bi-functionalized MSN-based nanomaterials have been fully characterized using electron microscopy (TEM and SEM), nitrogen adsorption measurements, thermogravimetric analysis (TGA), X-ray powder diffraction (XRD), Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), and dynamic light scattering (DLS). The results confirm that AGu@PEG1500-PMO can be applied for gene-editing with an efficiency of about 40% as measured by GFP gene knockdown of HT1080-GFP cells with no notable change in the morphology of the cells. Conclusions Due to the high stability and biocompatibility, simple synthesis, and cost-effectiveness, the developed bi-functionalized PMO-based nano-network introduces a tailored nanocarrier that has remarkable potential as a promising trajectory for biomedical and RNP delivery applications.

Recent advances in stimuli-responsive polymeric carriers for controllable CRISPR/Cas9 gene editing system delivery

“…This temporal control enables on-demand and transient editing of genomes, whereas quantitative control only targets genome editing of specific organs or tissues, reducing off-target effects and increasing immunogenicity due to long-term expression. 23,34 For instance, temperature-sensitive polymer delivery systems have been reported to achieve temporal and spatial control by physiological temperature stimulation triggering the polymer to form a gel and followed by a slow release of CRISPR/Cas9, suggesting that stimulus-triggered polymer carriers can enhance cellular uptake, lysosomal escape, intracellular precision targeting, and efficiency in delivery for CRISPR/Cas9 genomic editing systems. 35,36 In addition, it is noteworthy that stimulus-responsive polymers with a high loading capacity have recently been reported to form nanoparticles or hydrogels that induce stronger and longer-lasting immune cell responses, outperforming other non-viral carriers, with promising applications in immunotherapy of cancer and genetic diseases.…”

“…14,15 Non-viral delivery vectors (inorganic nanoparticles, liposomes and polymers) have significant advantages over physical methods (electroporation, microinjection and microfluidics), [16][17][18][19] viral vectors (adenoviral vectors (AV), adeno-associated viruses (AAV), retroviral and lentiviral vectors (LV)), and cationic liposomal amines for delivery of the gene editing systems of CRISPR/ Cas9. [20][21][22][23][24][25] Polymer based delivery vectors could be modified specifically to target cell-or tissue-specific responses (i.e., pH, redox, and enzymes) to enhance the organ/tissue specificity through temporally and spatially controlled CRISPR/ Cas9 genome editing, which not only has low immunogenicity and good biocompatibility, but also overcomes the packaging limitations of viral vectors, reduces the oncogenic risk, and facilitates large-scale production. [26][27][28][29] Accordingly, stimuli-responsive polymer delivery systems that are efficient and reliable as well as possess excellent targeting and superior encapsulation capabilities are currently among the major advances in genome editing systems with CRISPR/Cas9.…”

Design and fabrication of intracellular therapeutic cargo delivery systems based on nanomaterials: current status and future perspectives

“…More recently, editing genetic information in mammalian cells using CRISPR/Cas9 has shown great potential in developing a new generation of protein therapeutics for the treatment of genetic diseases. 259 Similarly, Chang et al screened several major nanoparticle formulations based on a combinatorial library of cationic lipid for intracellular protein delivery. 260 In this Account, the researcher optimized the chemical structure of lipids to control lipid degradation and release of intracellular proteins for delivery of CRISPR/Cas9 genome editing proteins.…”

Nonviral In Vivo Delivery of CRISPR-Cas9 Using Protein-Agnostic, High-Loading Porous Silicon and Polymer Nanoparticles

“…For example, Waggoner et al improved loading of a therapeutic protein by >50-fold 13% w/w (∼50% v/v) in PSiNPs vs <1% w/w (<1% v/v) in PLGA] . Although no one has yet demonstrated Cas9 RNP delivery with PSiNPs, silica nanoparticles have been demonstrated for gene editing. − While the route of synthesis, geometry, and chemical structure are very different between PSi and silica, both can achieve high drug loading (∼10% w/w).…”