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4-1 Progress in siRNA Delivery Using Multifunctional Nanoparticle-I
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Welcome to Creative BioLabs, a contract research organization based in New York. They specialize in antibody discovery, engineering, and biomanufacturing solutions. The focus of the discussion is on small interfering RNA (siRNA) and its role in RNA interference technology. siRNAs have the potential to treat a wide range of diseases, including cancer and genetic diseases. However, the delivery of siRNAs to target cells is a challenge. Scientists have explored viral delivery systems but face limitations such as side effects and high cost. Synthetic polymer nanoparticles have emerged as an alternative for siRNA delivery. These nanoparticles need to encapsulate siRNA, protect it from degradation, and be functionalized with targeted ligands to improve delivery efficiency. The nanoparticles enter cells through receptor-mediated endocytosis but need mechanisms to escape the endosomal compartment and reach the cytoplasm for the siRNA to be functional. Polymers are important in the development Welcome to Creative BioLabs, 100% of the effort, 100% of the service. As a dynamic contract research organization, we are based in New York and serve the whole world. Our seasoned scientists are skilled in antibody discovery, antibody engineering, and biomanufacturing solutions. Good evening, dear friends. In the past few decades, we have learned so much about the structure and function of DNA. Everything about DNA has been thoroughly analyzed. And when we think of ribonucleic acid or RNA, which seemed to be less important back then, so much still remains unclear, and it has become a topic of interest for researchers recently. The small interfering RNA, or more commonly known as the siRNAs, one of the members of the RNA group, takes an important share of the popularity in the field of life science, if not the most. Its role in RNA interference technology is indispensable. Today, we invite our researchers David to our program to tell us what he knows about siRNAs. So to start today's program, David, can you educate us on the relationship between RNA interference technology and small interfering RNAs? Thank you for inviting me. I'm very excited to be here. I must say that RNA interference is one of the most important discoveries in life science in recent years. It refers to the phenomenon that small double-stranded RNA can specifically degrade or inhibit the expression of homologous messenger RNA, inhibiting or shutting down the expression of specific genes. As long as we know the pathogenic gene of a certain disease, we can design small interfering RNA for the messenger RNA of the gene to inhibit or block the expression of the pathogenic gene, so as to achieve the purpose of disease treatment. What disease treatment are we speaking of here? Is it just for genetic diseases? In theory, almost all diseases can be treated by small interfering RNA, including cancer, infectious diseases, genetic diseases, and so on. And it is for this reason that RNA interference has received widespread attention in the scientific community. Not only is it the most popular, but it is also the most promising field of new drug development in the future. Synthetic double-stranded small interfering RNA has been found to induce sequence-specific and post-transcriptional gene silencing through the RNA interference pathway. It really sounds to be a very hopeful discovery. Aren't we all glad that the therapeutic potential of small interfering RNA is widely recognized? But if small interfering RNA is to be used as a new drug to treat diseases, how do we go about it? It is. Synthetic small interfering RNA has become a potential drug candidate. We just need to deliver it to the target in a consistent and correct way. But we already have natural small interfering RNA, don't we? What are the advantages of synthetic small interfering RNA over natural ones? The synthetic ones can be chemically modified and produced on a large scale. In various disease indications, it was the use of synthetic small interfering RNA that initially established the therapeutic effect of siRNA in vivo. However, some scientists think that free small interfering RNA is not ideal in producing effective and predictable therapeutic effects. Why is that? In clinical trials, it was observed that most of the injected small interfering RNA was removed from circulation by liver and kidney clearance within a few minutes after intravenous injection. As a result, only a small proportion was left for target cells or tissues. To make things worse, this small fraction of small interfering RNA is also degraded by nonspecific nucleuses in the blood. How about their efficiency entering target cells? If the entrance is passive, then the efficiency is very low. But we were talking about the intravenous injection. What about other routes of administration? Can they be given locally to achieve higher efficiency? Local administration is desirable in some cases. It can avoid some limitations faced by intravenous administration. But in many cases, drugs are not easy to reach the target cells or tissues, or the scope of the disease is too large for local treatment. And to overcome these difficulties, it is necessary to develop efficient small interfering RNA delivery systems. What are some ways, or is there any way to do that? Yeah. Scientists have tried several ways. Early work on small interfering RNA delivery focused on viral delivery systems, such as those developed for gene delivery. Over millions of years, viruses have evolved complex replication mechanisms to deliver their gene loads to host cells. Many virus delivery systems have been explored for small interfering RNA delivery. The delivery efficiency of the viral delivery systems can be very high. However, they face serious challenges. For example, some gene therapy trials based on viral delivery systems have had side effects. In addition, a high cost of producing large viral stocks for clinical use, and the limited quantity of nucleic acids that can be packaged for treatment, also limit the application of viral delivery systems. Is it possible to overcome these difficulties by modifying the virus carriers? Although the virus transmission system is further designed to overcome these limitations, I believe it is better to develop non-virus alternatives, such as synthetic polymer nanoparticles. These have been developed to deliver various therapeutic drugs. Multifunctional polymeric nanoparticles for small interfering RNA delivery are one of them. They have recently become alternatives to viral vectors to deliver siRNA. Can you elaborate on the role of nanoparticles in the delivery of siRNA? Polymer nanoparticles need to encapsulate small interfering RNA and protect them from being removed or degraded. Ideally, they need to be functionalized with targeted ligands, which may improve the specificity and efficiency of delivery. Can you tell us the general process of this transmission? What are some necessary steps? Cationic polymers first need to encapsulate anionic siRNA by self-assembly. Then, the nanoparticle is coated with siRNA and target ligand. Next, the nanoparticles target cell surface receptors. After that, nanoparticles are ingested through receptor-mediated endocytosis. Finally, endosome disruption and small interfering RNA release in cells. When an siRNA enters the cells via nanoparticle delivery, it just reaches the target gene. Not yet. Because of its strong hydrophilic and negative charge, the release of a siRNA cannot be spread spontaneously through the membrane. After nanoparticles enter the cell through ligand-mediated endocytosis, instead, they may be trapped in the endocytosis or lysosomal compartment and degraded by lysosomes. So to efficiently deliver siRNAs, nanoparticles should contain some mechanisms for them to escape from the endosomal gap and enter the cytoplasm. If siRNA cannot get into the cytoplasm, it cannot be functional, can it? It cannot. Only when it enters the cytoplasm can siRNA have biological activity. And effectively silence the gene. And its biological activity also needs to be activated. In order to meet the standard of effective delivery of small interfering RNAs, multiple functions may need to be integrated into a single nanoparticle. As we are speaking of polymeric nanoparticles, I just suddenly feel that polymers are so important in this field. You are absolutely correct. Polymers are attractive in the development of multifunctional nanoparticles as they allow advanced synthesis and conjugation chemistry. In addition, by blending copolymers, the fine engineering of nanoparticles can be realized. But why is that polymer can uniquely accomplish this task? Polymers with hydrophobic and hydrophilic segments can self-assemble in an aqueous solution and spontaneously form stable particles on the nanoscale. For siRNA delivery, these polymer particles may include cationic segments so that siRNA can be effectively encapsulated into the particle core. Do polymer nanoparticles need adjuvants? Generally speaking, adjuvants are needed. They are usually used to keep nanoparticles stable. For example, polyethylene glycol is usually a hydrophilic blocking agent to stabilize nanoparticles and prevent an immune response. OK, so when is the surface functionalization of targeted ligands formed? The surface functionalization of target ligands can be carried out before and after the formation of nanoparticles by conjugation chemistry. In addition to encapsulation, small interfering RNA can be directly coupled with polymers or polymer conjugates for subsequent delivery. Although polymer nanoparticles and polymer conjugates have different structures, they are designed and engineered to integrate the key functions of small interfering RNA into the cytoplasm. I see. With much research going on around gene delivery and therapy, especially DNA encapsulation and cytoplasmic delivery, do we know the difference between gene delivery and siRNA delivery? Right. As we all know, small interfering RNA and double-stranded DNA have many common properties, but there are significant differences in delivery. For example, in terms of stability, DNA is more stable than RNA because it contains deoxynucleotides. In contrast, the hydroxyl group of small interfering RNA is easy to degrade. When it comes to molecular topology and complex size, compared with a rigid rod of small interfering RNA, double-stranded DNA is compressible so that its complex size is smaller. In addition, double-stranded DNA tends to be activated in the host nucleus while small interfering RNA is activated in the cytoplasm. Furthermore, the duration of their treatment effect is also different. Double-stranded DNA can be transient, long-term or permanent transgene expression, while small interfering RNA therapy usually lasts for three to seven days. And with all these differences, does it mean that the delivery mechanism of one cannot be applied to the other? Pretty much. Because of these differences, the knowledge gained from gene delivery research is not always directly applied to small interfering RNA delivery without verification by various experiments. Do the topological structure and the size of the complex between double-stranded DNA and small interfering RNA determine the differences in their delivery process and effects? Exactly. The difference of molecular structure between the two, not only affects the interaction between double-stranded DNA and carrier, but also leads to the potency change of specific transfection vector. Can you share with us an example? Let's say double-stranded DNA is a larger molecule, but can condense into nanoscale particles. Although the molecular size of siRNA is much smaller, it behaves like a stiff rod. There is a hypothesis that the molecular weight of small interfering RNA is too low to interact with polymer effectively. Therefore, they tend to form disordered and loose complexes with polymers, resulting in less stable particle formulations and reduced delivery efficiency. Is there any strategy to improve the efficiency of siRNA delivery? Some researchers show that by adding short complementary overhangs, small interfering RNA molecules showed stronger binding and formed a large genotype structure. This structure has stronger stability, improved the protection of ribonucleus and better gene silencing. Similarly, other researchers also found that double-stranded DNA can promote the encapsulation of small interfering RNA. They integrate high molecular weight carrier DNA into nanoparticles. Compared with the formula without carrier DNA, the particle size of the formula with carrier DNA is reduced by 10-30%, and the delivery efficiency is increased by 20-80%. As mentioned above, we can say that packaging, specific targeting and intracellular release of small interfering RNA are essential requirements for designing nanoparticles for small interfering RNA delivery. Yes, that's right. Nanoparticles made of synthetic polymers have been developed for siRNA delivery. These nanoparticles need to encapsulate siRNA efficiently, actively target and release siRNA in cells to make sure a successful delivery. With that, we are concluding today's program. We have talked about small interfering RNA or siRNA today. It is a specific and efficient method to inhibit gene expression. At present, RNAi technology has been used in tumor, cancer, diabetes and gene detection. However, siRNA can be easily and rapidly hydrolyzed by nuclease in vivo, which has weak transmembrane transportability, short half-life and low gene silencing efficiency in vivo. Therefore, CIRNA needs a suitable, safe and effective in vivo delivery system to play a good therapeutic role. At present, the delivery strategies of siRNA are mainly divided into physical strategies, covalent binding strategies and viral and non-viral vector delivery strategies. Thank you, David, for sharing your knowledge with us. Thank you everyone for listening. We will continue our discussion next time. See you then.
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