4-4 Progress in siRNA Delivery Using Multifunctional Nanoparticles

CreativeBiolabs is a contract research organization that supports mRNA studies. They produce podcast series related to mRNA technology. In this episode, they discuss the delivery strategies of siRNA, including physical, covalent binding, and viral and non-viral carrier strategies. Nanoparticle delivery systems have been widely used to deliver siRNA in vivo, improving stability and efficiency. Cationic copolymers can self-assemble into nanoparticles and prevent siRNA degradation. Endosomescape is key for successful siRNA delivery. Cationic polymers with pKa slightly lower than physiological pH can help endosomes escape lysosomal degradation. Histidine-containing polymers have shown effectiveness in delivering siRNA. There are methods to release siRNA without destroying the endosome. Protein transduction domain peptides can promote siRNA packaging and cell targeting. Researchers are working on vesicles composed of arginine protein transduction domains. These nanoparticles need to encapsu Welcome to CreativeBiolab Science Channel. CreativeBiolabs is a specialized contract research organization supporting mRNA studies with all-round solutions covering mRNA synthesis, modification, and mRNA therapeutics development. With an unwavering pursuit of innovation and lifelong learning, we keep on producing podcast series related to mRNA technology based on our knowledge and practical experience gained through years of exploration in this area. Subscribe to our channel and keep updated with our podcasts. Good evening, dear friends. Nice to have you here with us again. At the end of last program, we talked about a variety of targeted ligands that have been designed and applied in siRNA delivery. These include monoclonal antibodies and antibody fragments, small peptide-based targeting molecules, oligonucleotide aptamers, and some small molecules. The efficiency of these targeted ligands has been confirmed in various experiments. Today, David will detail for us when siRNA is delivered to the target cells, how is it released into the cytoplasm. Welcome to join us today, David. Thank you for your invitation. I'm very excited to be here. Let's continue with our topic on siRNAs. In this program, we have went over the delivery strategies of siRNAs a few times. They are mainly divided into physical strategies, covalent binding strategies, and viral and non-viral carrier delivery strategies. In recent years, nanoparticle delivery systems, such as nanoparticles, nanoparticles, dendrimers, and liposomes, have been widely used to deliver siRNA in vivo. They can improve the stability of small interfering RNA in vivo, enhance the targeting specificity, increase the uptake of small interfering RNA by cells or tissues, and ultimately improve the efficiency of gene silencing and reduce the side effects. Can you tell us more about the siRNA cationic copolymer nanoparticle system for in vivo delivery? Cationic copolymers can self-assemble into nanoparticles by electrostatic adsorption. They can effectively prevent siRNA from being degraded by nuclease. Cationic copolymers have been widely used in the preparation of nanoparticles loaded with siRNA because of their advantages of simple synthesis, stable storage, high gene loading rate, and low immunogenicity. Their biggest problem is the low transfection efficiency. So I think future studies can consider the use of targeted ligands to modify copolymers using receptor-mediated targeting to increase cell or tissue uptake. I understand that polyethylene imine and dendrimer are the most widely studied cationic copolymers in recent years. Under physiological conditions, one-sixth to one-fifth of nitrogen atoms in polyethylene imine molecule is protonated, which has a proton sponge effect, high charge density, and strong buffering ability, and can enhance the release of small interfering RNA nanoparticles from endosomes. Can you talk about the siRNA cytoplasm release? Sure. Nanoparticles with targeted ligands first enter cells through passive or receptor-mediated endocytosis. However, we know that siRNA is negatively charged and hydrophilic. So those trapped in the endosome cannot spontaneously cross the plasma membrane to reach the cytoplasm and are degraded by lysosomal enzymes in the process of endocytic lysosomal sorting. In this case, endosomescape is the key step for the successful delivery of siRNA. Are there any ways to help endosomes escape the fate of lysosomal degradation? A typical method is to use cationic polymers with pKa slightly lower than physiological pH. pKa is the acidity coefficient or the dissociation constant of the drug. Due to the acidity of the endosome, the cationic polymer absorbs protons and increases the osmotic pressure of the endosome chamber. This proton sponge effect results in the destruction of the plasma membrane, followed by the release of small interfering RNA into the cytoplasm. Can you give an example of a cationic polymer with a pKa slightly lower than the physiological pH value? Cationic polymers containing histidine residues belong to this range. Some studies have shown that the histidine residues with pKa of 6.4 could achieve endosome buffering and escape. And it has been reported that the expression of hepatitis B virus surface antigen and the corresponding viral titer of human liver cancer cells, oh it is also known as the Alexander cells, can be reduced by using reduced polycation transfer siRNA containing histidine monomer. In another study using histidine lysine-rich polymers to deliver small interfering RNA targeting RAF1 gene, effective downregulation of RAF1 messenger RNA was observed, leading to apoptosis in several cell lines. In addition to endosome escape, histidine can promote nucleic acid condensation and membrane fusion, which may be conducive to small interfering RNA delivery. Nucleic acid condensation refers to the formation of phosphodiester bond. But the release of siRNA by proton sponge effect seems to lead to the rupture of endosome. Is there a way to release siRNA without destroying the endosome? Yeah there are some, but these methods help the nanoparticles escape from the endosome. For example, researchers have developed a composite micelle with the separation of polyethylene glycol, which can be separated in the reduced endosome compartment by reversible disulfide bonds around the polyethylene glycol layer around the core of the particles. What is the so-called escape mechanism we are talking about here? Polyethylene glycol detachment is believed to enhance endosome escape by increasing the interaction between the exposed cationic core of nanoparticles and the intima. Due to the removal of the surrounding PEG layer, polyethylene glycol detachment also facilitates the release of siRNA from nanoparticles. As you have previously described, protein transduction domain peptides can promote small interfering RNA packaging and cell targeting. So, is it capable of helping the endosomes escape? Affirmative. Some of these peptides are particularly able to escape the endosome and enter the cytoplasm. Although the mechanism of protein transduction domain peptide penetrating cell membrane is still unclear, its effect on small interfering RNA cytoplasmic transport has been confirmed by a large number of studies. Can these protein transduction domains be integrated into complex nanoparticle structures as components? It is still challenging since at the same time, we also need to maintain their membrane permeability. Recently, researchers are working on the preparation of vesicles composed of arginine protein transduction domains. These particles can capture water-soluble substances and be processed into different sizes and scales. What is the function of protein transduction domains fragment here? There are dual functions to guide the structure of the vesicles and to provide effective vesicle delivery in the cells. This is a good example of the unique synergy between nanoscale self-assembly and inherent peptide functions when designing multifunctional nanoparticles for siRNA delivery. We again have reached the end of today's program. We have introduced nanoparticles made from synthetic polymers been developed for siRNA delivery. In order to successfully transmit small interfering RNA, these nanoparticles need to encapsulate small interfering RNA efficiently, target targets actively, and release small interfering RNA in cells. We have also reviewed recent advancements in the design and engineering of polymer nanoparticles for siRNA delivery using a multifunctional approach. New methods of multifunctional nanoparticle design and engineering continue to improve the performance of nanoparticles in preclinical and clinical research. An RNA interference method is expected to produce a new class of effective medical drugs. Thank you, David, for sharing your knowledge and insight with us. Thank you, everyone, for listening. We will see you next time.