Lipid Nanoparticles for Nucleic Acid Delivery

Nucleic acids, as a new generation of biopharmaceuticals, not only have the potential to treat diseases at the genetic level but also exhibit significant platform characteristics in both technology and production, offering broad application prospects in the medical field. However, the poor stability and low delivery efficiency of nucleic acids both in vivo and in vitro greatly limit their drug development potential. In recent years, lipid nanoparticles (LNPs) based on ionizable lipids have demonstrated good clinical application potential and have been validated in nucleic acid COVID-19 vaccines. With their unique structural and physicochemical properties, LNPs show high delivery efficiency and good safety in vivo, providing more possibilities for the future clinical application of nucleic acid drugs.

Nucleic Acid Definition

Nucleic acids are core biomolecules in living organisms that store and transmit genetic information, widely found in cells. Chemically, nucleic acids are classified into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is primarily responsible for storing genetic information, while RNA participates in the transcription and translation of genetic information, driving important biological processes such as protein synthesis. The basic structural unit of nucleic acids is the nucleotide, composed of a five-carbon sugar, a nitrogenous base, and a phosphate group. The five-carbon sugar in DNA is deoxyribose, while in RNA, it is ribose; the nitrogenous bases include purines (adenine A and guanine G) and pyrimidines (thymine T, uracil U, and cytosine C), where DNA contains thymine and RNA contains uracil. The phosphate group connects nucleotides to form the backbone via phosphodiester bonds. Nucleic acids possess a multi-level structure, including a primary structure of linear nucleotide sequences, a secondary structure such as the double helix (in DNA) or complex folding (in RNA), and higher-level structures such as the cloverleaf configuration of tRNA. The specificity and stability of nucleic acid molecules provide the critical foundation for performing their genetic functions.

Fig. 1. Nucleic acid structure.

Nucleic Acid Function

Nucleic acids, including DNA and RNA, are core biological molecules responsible for storing, transmitting, and expressing genetic information. DNA is the primary genetic blueprint, carrying the instructions necessary for the development, function, and reproduction of all living organisms. These instructions exist in the form of genes, which are transcribed into RNA and then translated into proteins. RNA not only serves as the messenger of information but also performs various functions. Messenger RNA (mRNA) conveys genetic information from DNA to the ribosome for protein synthesis; transfer RNA (tRNA) and ribosomal RNA (rRNA) play critical roles during translation, ensuring the accurate assembly of proteins. In addition to these functions, nucleic acids are involved in the regulation of gene expression. For instance, non-coding RNAs such as microRNAs and small interfering RNAs regulate gene expression by modulating the stability or translation of mRNA. Furthermore, nucleic acids play important roles in cellular defense mechanisms, such as the CRISPR system in prokaryotes, which provides immunity against invading genetic elements. Additionally, synthetic nucleic acids are widely used in therapeutic, diagnostic, and biotechnological applications, driving innovations like gene editing, RNA vaccines, and precision medicine. The multifunctionality of nucleic acids makes them indispensable molecules in biological and cutting-edge biomedical research.

Nucleic Acid Therapeutics

Nucleic acid therapeutics is an emerging technology that treats diseases by regulating gene expression or correcting genetic defects. In recent years, with the rapid development of technologies such as gene editing, RNA interference (RNAi), and mRNA vaccines, nucleic acid therapeutics has become a critical area in precision medicine. Its advantages include high specificity, with the ability to precisely identify target sequences and minimize interference with non-target genes; broad applicability, capable of targeting many drug-resistant or difficult-to-drug targets; and a fast development cycle, making it more efficient than traditional drugs. These characteristics give nucleic acid therapeutics significant potential in treating genetic disorders, cancer, and infectious diseases.

Table. 1. Categories of nucleic acid therapies.

Nucleic Acid Delivery

The delivery of nucleic acid drugs is a critical aspect of their development and application, directly influencing therapeutic efficacy and safety. Due to the large molecular size, negative charge, and susceptibility to enzymatic degradation of nucleic acid molecules, their delivery in vivo faces several challenges. Nucleic acid drugs need to overcome cellular membrane barriers, avoid degradation in the bloodstream, and successfully reach specific subcellular regions (such as the nucleus or cytoplasm) within target cells. Therefore, designing effective delivery systems has become a central focus in the development of nucleic acid drugs. Current delivery strategies typically fall into two main categories: viral vectors and non-viral vectors. Viral vectors, such as adenoviruses and adeno-associated viruses, offer high delivery efficiency but may cause immune reactions or genomic integration issues. In contrast, non-viral vectors provide greater safety and controllability, including cationic polymers, inorganic nanoparticles, and lipid nanoparticles.

Fig. 2. Lipid nanoparticles for nucleic acid delivery (Adv Drug Deliv Rev. 2020, 154-155: 37-63).

LNPs have become a leading technology in the field of nucleic acid drug delivery. LNPs encapsulate nucleic acid molecules, protecting them from enzymatic degradation in the body, and utilize the biocompatibility of lipids to promote cellular uptake and release. In recent years, LNPs have been successfully used for the delivery of mRNA vaccines (such as COVID-19 vaccines) and siRNA drugs (such as Patisiran), opening a new chapter for the clinical application of nucleic acid drugs. The stability, modifiability, and delivery efficiency of LNPs make them an ideal choice for nucleic acid drug delivery, driving the rapid development of this field.

What are Lipid Nanoparticles?

Lipid nanoparticles (LNPs) are nanoparticles composed of lipids, which are organic molecules that can dissolve in fats. They are a novel drug delivery system capable of safely and effectively delivering nucleic acids (such as DNA or RNA) to target cells. LNPs are typically spherical in shape, with an average diameter ranging from 10 to 1000 nm. They consist of a lipid core that can dissolve lipophilic molecules and a surfactant layer that stabilizes the particles and protects the nucleic acid payload. The lipid core can be either solid or liquid, depending on the type and composition of the lipids used. The surfactant layer can be composed of various biomembrane lipids, such as phospholipids, cholesterol, bile salts, or sterols. Selectively, LNPs can also have targeting molecules, such as antibodies or peptides, attached to their surface to enhance their specificity and uptake by certain cells.

Lipid Nanoparticle Manufacturing

The manufacturing of lipid nanoparticles typically involves techniques such as solvent evaporation, ultrasonic fragmentation, or high-pressure homogenization, which allow for precise control over particle size, drug loading, and release characteristics. Based on this, BOC Sciences offers a variety of lipid materials, including phospholipids, PEG-lipids, cholesterol derivatives, and other specialized functional lipids, for the synthesis of lipid nanoparticles. Additionally, we provide custom development services for lipid nanoparticles, helping clients optimize the design and performance of drug delivery systems to meet diverse research and industrial application needs.

Lipid Nanoparticle Delivery of Nucleic Acids

LNPs have become one of the preferred carriers for delivering nucleic acid drugs due to their excellent biocompatibility and low immunogenicity. LNPs exhibit excellent in vivo distribution characteristics, effectively protecting nucleic acid molecules from degradation while promoting their entry into cells and release into specific intracellular regions. The following sections provide a detailed overview of LNPs in delivering different types of nucleic acids, such as siRNA, mRNA, oligonucleotides, and DNA.

LNPs for siRNA Delivery

Small interfering RNA (siRNA) is a double-stranded RNA molecule that silences specific gene expression through the RNA interference (RNAi) mechanism. However, siRNA molecules typically have poor stability and are prone to degradation by nucleases in the body. LNPs are ideal carriers for siRNA delivery because they can encapsulate and protect siRNA from degradation while enhancing its cellular uptake. The mechanism of LNPs delivering siRNA primarily relies on the interaction between the positively charged lipid layer on the surface of LNPs and the negatively charged cell membrane, promoting fusion between the lipid bilayer and the cell membrane. In this way, LNPs efficiently transport siRNA into the cell and facilitate its entry into the cytoplasm via endocytosis. Once inside the cell, LNPs release siRNA into the cytoplasm to exert RNAi effects and silence target gene expression. In recent years, LNPs have been applied in various clinical studies, particularly in the fields of cancer and viral infections, demonstrating significant therapeutic efficacy.

LNPs for mRNA Delivery

mRNA vaccines have been a major breakthrough in the medical field in recent years, especially during the COVID-19 pandemic. The successful application of mRNA vaccines has proven their enormous potential in infectious disease prevention and treatment. mRNA vaccines require effective delivery of mRNA into cells to induce immune responses, thereby generating antibodies and cellular immunity. LNPs have become an important carrier for mRNA vaccine research and applications due to their good biocompatibility and high delivery efficiency. LNPs protect mRNA molecules from nuclease degradation in vivo and deliver the mRNA into cells through lipid bilayer fusion with the cell membrane. Once mRNA enters the cell, it is translated into the target protein, triggering an immune response. The advantage of LNPs in mRNA delivery lies in their ability to efficiently deliver large RNA molecules while reducing the immune system's response to exogenous mRNA. The success of mRNA vaccines not only opens a new field for nucleic acid vaccines but also provides new ideas for the treatment of other diseases.

LNPs for DNA Delivery

DNA molecules typically need to be delivered to the cell nucleus to initiate gene expression. However, DNA molecules themselves have a large molecular weight and negative charge, which creates barriers for penetrating the cell membrane. LNPs can effectively address this issue as carriers for DNA delivery. LNPs can bind to DNA molecules, forming stable nanoparticles that enhance the stability of DNA molecules and promote their entry into cells. After LNPs bind to the cell membrane, they enter the cytoplasm through endocytosis. Once inside the cell, LNPs release DNA through the lysosomal pathway and deliver it to the cell nucleus via the nuclear membrane to initiate transcription of the target gene. The application of LNPs in DNA delivery holds great potential in gene therapy, particularly in genetic diseases and cancer immunotherapy.

LNPs for Oligonucleotide Delivery

Oligonucleotides are increasingly used in therapy, especially in cancer treatment, gene modification, and antiviral fields. Oligonucleotides typically have smaller molecular weights and higher negative charges, making it difficult for them to traverse the cell membrane and increasing their susceptibility to degradation in the body. The introduction of LNPs provides an effective solution to this problem. LNPs can encapsulate oligonucleotide molecules, protecting them from nuclease degradation. Oligonucleotides, especially antisense oligonucleotides and modified RNAs, are prone to degradation in the bloodstream, and these molecules need to remain stable in vivo to achieve therapeutic effects. LNPs provide a closed nanoscale environment that ensures the biological stability of oligonucleotides and prolongs their half-life. Moreover, LNPs improve the effective concentration of oligonucleotides in the body by preventing contact with degradation enzymes both in vivo and in vitro. Clinical applications of LNPs in oligonucleotide delivery have made significant progress. For example, ASO therapy has been used to treat genetic diseases such as Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). LNPs as carriers of ASOs have effectively enhanced the stability and delivery efficiency of ASOs in vivo, enabling them to target and repair defective genes.

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Reference

1. Samaridou, E. et al. Lipid nanoparticles for nucleic acid delivery: Current perspectives. Adv Drug Deliv Rev. 2020, 154-155: 37-63.