(T1030-02-10) Synthesis a Cationic-Polymer Incorporating Carboxylesterase Active Ester for the Delivery of CRISPR/Cas9 to Ameliorate Hyperlipidemia

Purpose: Gene editing technologies have become powerful tools to perform efficient gene modification in specific cells of multiple organs, including the liver. The broad application of CRISPR)/Cas9 genome editing tools is hindered by factors such as poor deliverability. Hyperlipidemia is caused by gene mutations encoding low-density lipoprotein receptors (LDLR). We aim to deliver CRISPR/Cas9 by hybrid lipid-polymeric nanoparticles to liver cells that can precisely edit the E207X point mutation of LDLR. The cationic lipid-polymeric nanoparticles can self-assemble with nucleotides, indicating the potential of nanoparticles as a delivery carrier. Complexes of the carrier system - a novel carboxylesterase-responsive cationic lipid-polymer hybrid nanoparticles and gene materials including Cas9-mRNA and sgRNA-targeted LDLR for hyperlipidemia amelioration are developed. Carboxylesterase (CES) is an esterase enzyme hydrolyzes carboxyl esters. CES is abundantly expressed in the liver where it is heavily involved in the metabolism of drugs. We hypothesize that incorporating a CES-active property would achieve the liver specific target effect. The nanoparticle vehicle can efficiently release CRISPR/Cas9 in response to CES for genome editing.

Methods: Polymer design and synthesis: 2-Guanidinopropanol was synthesized by adding 3-aminopropanol to 2-ethyl-2-thiopseudourea hydrobromide solution. Then, water was removed using rotary evaporation. Thin layer chromatography (TLC) technique was performed to analyze a chemical reaction. The 1H Nuclear Magnetic Resonance (NMR) spectra of the obtained sample in D2O was recorded at room temperature using Bruker NEO 400 400MHz spectrometer (Bruker, Billerica, MA, USA). The NMR analysis was conducted to identify and confirm the chemical structure of 2-Guanidinopropanol.2-guanidinoethyl methacrylate was synthesized by dissolving 2-Guanidinopropanol and methacryloyl chloride in acetonitrile. Then, acetonitrile was removed using rotary evaporation. TLC was performed to analyze the chemical reaction. The 1H NMR spectra of the obtained sample in D2O was recorded at room temperature using Bruker NEO 400 400MHz spectrometer (Bruker, Billerica, MA, USA). The NMR analysis was performed to identify and confirm the chemical structure of the 2-guanidinoethyl methacrylate. Column chromatography was used to purify the product. Molecular docking analysis was performed to evaluate the CES activation of 2-guaninidoethyl methacrylate (ligand). The polymeric nanoparticles were prepared by polymerizing guadinium group(s), ester group(s), methacrylic acid, and polyethylene glycol (PEG) by reversible addition-fragmentation chain transfer polymerization process. Polymethacrylate is a polymer that binds with DNA due to its inherent cationic charge. These polymers have been used in gene delivery in vitro as well as in vivo models. In the preliminary study, we synthesized a polymethacrylate polymer. Poly-L-arginine hydrochloride is a positively charged synthetic poly amino acid that we consider using in comparison to polymethacrylate. PEG-copolymerization is a way for adding hydrophilic force to reduce the toxicity of the cationic particles and help when hybrids are assembled. Methoxy-PEG and its derivatives are taken into consideration as useful modifications of compounds. Preparation of lipid nanoparticles for RNA encapsulation - LIPOID PC 18:0/18:0 (1,2-distearoyl-sn-glycero-3-phosphocholine) (DSPC) was a kind gift from Lipoid (Ludwigshafen, Germany). Cholesterol was purchased from Sigma Aldrich (St. Louis, MO, USA). Liposomes were fabricated using DSPC and cholesterol at different ratios by thin film hydration method using a rotary evaporator. Lipid vesicles are prepared by thin film hydration method as described with the optimal ratios of lipids, organic solvent, and the addition of surfactant. The particle size can be controlled by further subjecting liposomes to extrusion, sonication, homogenization, and microfluidic methods. The lipid-polymeric hybrid nanoparticles are prepared by hydrating the lipid film in the polymer and nucleic acids mixture.

Results: 2-Guanidinopropanol, 2-guanidinoethyl methacrylate, and the polymer was synthesized. Molecular docking analysis identified the conformational space of CES binding pockets with 2-guaninidoethyl methacrylate. The binding affinity of 2-guaninidoethyl methacrylate towards binding sites of CES was determined to be very strong (-44.73). Lipid vesicles were prepared, and optimized. Blank liposomes (lipid vesicles) with a DSPC to cholesterol (w/w) ratio of 3: 2 were prepared, the liposomes size average size was 191 ± 35.03 nm with negative zeta potential (-24.3 ± 0.05). A zeta potential value of < –30 mV or >30 mV is generally sufficient to maintain particle suspension stability. The lipid-polymeric hybrid nanoparticles are prepared by hydrating the lipid film in the polymer and nucleic acids mixture. The particle size can be controlled by further subjecting liposomes to extrusion, sonication, homogenization, and microfluidic methods. Lipid-polymeric hybrid nanoparticles can be prepared by vigorously mixing lipid ingredients in organic solvent with nucleic acids in polymer using microfluidic technology. 

Conclusion: We propose a novel lipid-polymeric hybrid nanoparticle system which merges the features of both polymeric and lipid nanoparticles. Lipid-polymeric hybrid nanoparticles consist of 1) aqueous polymeric nanoparticle suspension and 2) lipid vesicles. The cationic mixture can self-assemble encapsulating Cas9‐mRNA and sgRNA). We have successfully prepared the cationic lipid-polymeric hybrid nanoparticles which have high binding affinity towards CES in the liver. Further studies include characterization of lipid-polymeric hybrid nanoparticles, RNA loading capacity and RNA release studies, Intracellular delivery study on liver cells and in vitro evaluation of gene editing performance

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Acknowledgements: Figure "Preparation of lipid-polymeric hybrid nanoparticles" was created with BioRender.com (accessed on 7 September 2023). The research was supported by the National Institutes of Health (Grants: R01 AA030486 and R01 AI172959).

Preparation of lipid-polymeric hybrid nanoparticles

Molecular docking result of 2-guanidinoethyl methacrylate monomer binding toward CES

Particle size (nm) and zeta potential (mV) of lipid vessels (blank liposomes) as a function of DSPC/cholesterol ratio; and particle size (nm) and zeta potential (mV) of mixtures of cationic polymer and lipid vessels as a function of polymer/liposomes ratio