Genetic diseases are still a global challenge, as they are caused by mutations in genes that affect the biological functions, metabolic processes, or production of certain essential proteins of individuals, leading to various inherited disorders. Gene therapy drugs are a type of therapeutic intervention that targets specific genes, aiming to repair or modify them, and provide new instructions for cells to produce functional proteins or intervene in the gene defects that cause diseases.
The common gene drugs mainly include DNA and RNA, where DNA can be gene fragments or gene sequences with therapeutic effects. These DNA are delivered to cells to replace or supplement the defective or missing gene functions. RNA can be messenger RNA (mRNA), small interfering RNA (siRNA), or antisense oligonucleotides (ASO), etc., which can be used to modify, inhibit the expression of harmful genes, or restore the function of specific genes.
However, both DNA and RNA face many challenges from entering the human body to reaching the target cells. Since gene sequences are unstable in the gastrointestinal tract, they are usually administered by intravenous or subcutaneous injection, but even so, most of the gene sequences are still degraded by the abundant nucleases in the human body.
When a small amount of residual gene drugs reaches the outside of the target cells, they cannot enter the cells because gene sequences are large molecules with phosphate groups carrying negative charges, while cell membranes also carry negative charges. When two negative charges meet, genes are repelled and cannot enter the cells. Therefore, we need to develop specific gene drug delivery methods to prevent their degradation in vivo and improve the efficiency of cell membrane penetration, and deliver DNA or RNA sequences to the cells smoothly.
Delivery Methods for Gene Drugs
The current common gene delivery methods are mainly divided into two categories: viral vector delivery and non-viral vector delivery. Viral vector delivery mainly includes adeno-associated virus (AAV) and lentivirus, etc., which can simulate the process of virus infection of host cells and easily cross cell barriers, thus achieving high delivery efficiency. However, viral vector delivery also has problems: first, it has limited packaging capacity, such as AAV's maximum packaging capacity is 4.7kb, which cannot deliver longer gene sequences; secondly, viral vectors may cause serious immune reactions, including cellular immunity and humoral immunity, posing risks for clinical applications. Compared with that, non-viral vectors have huge advantages in safety and drug loading capacity.
The current commonly used non-viral vectors mainly include liposomes, polymers, nanoparticles, exosomes, etc., among which liposomes are vesicles formed by amphiphilic molecules with diameters ranging from 100nm to 1um. They are mainly composed of phospholipids, cholesterol, PEG lipids and cationic lipids. The phospholipid bilayer structure is similar to cell membranes and can enter cells smoothly through endocytosis. In addition, liposomes can also modify PEG or specific antibodies on their surfaces to escape the immune clearance of the body while targeting specific tissues or cells. Cationic liposomes are liposomes that contain positively charged quaternary ammonium salt lipids, which can better bind with negatively charged nucleic acid drugs and maintain the stability of the delivery system. Therefore, liposomes are one of the common non-viral vector delivery methods for gene drugs.
However, traditional liposomes also have some problems: first of all, due to the large span of liposome diameters, although this can ensure sufficient drug loading capacity, when liposomes pass through the vascular endothelial gap (100-200nm), too large size will hinder this process. In addition, after liposomes enter cells through endocytosis and form endosome-lysosome system, traditional liposomes cannot complete lysosomal escape in time, resulting in most gene drugs being degraded by lysosomes and unable to exert their effects. Based on these shortcomings of traditional liposomes in delivering gene drugs, lipid nanoparticles (LNP) have been developed and become an important delivery method especially suitable for delivering nucleic acids to target cells and tissues.
Introduction to LNP Carrier System
Lipid nanoparticles (LNP) are nanoparticles composed of lipid-like substances and a diameter generally between 10-200nm. Compared with traditional liposomes, they are smaller and easier to pass through vascular endothelial gaps. In addition, LNP usually consist of cationic/ionizable lipids, auxiliary phospholipids, polyethylene glycol (PEG) lipids and cholesterol. Among them, auxiliary phospholipids and cholesterol can increase the stability of LNP; PEG lipids help increase the circulation time of LNP in vivo and reduce immune reactions. These components have similar functions as traditional liposomes. The difference lies in the cationic/ionizable lipids, which are also the main area of patent protection for LNP.
As mentioned earlier, cationic liposomes can effectively maintain the binding with nucleic acid drugs and improve the delivery efficiency of nucleic acid drugs. However, cationic liposomes are also prone to non-specific binding with negatively charged plasma proteins, blood cells, etc. in the body circulation, causing liposome aggregation and being quickly cleared out of the body (the half-life of cationic liposomes in vivo is only 15 min).
To solve this problem, researchers have developed a new type of lipid material called ionizable lipids. Ionizable lipids are very similar to cationic lipids in structure. Taking the common cationic lipid DOTAP and ionizable lipid DODAP as examples, the difference is that DOTAP is a quaternary ammonium salt with a positive charge, while DODAP is a tertiary amine structure. The tertiary amine structure can keep the pKa of the lipid between 6-7. When pH is lower than pKa (under acidic conditions), ionizable lipids are positively charged.
When pH is close to pKa (under neutral conditions), ionizable lipids are neutral. This design can cleverly solve the problem encountered by cationic liposomes: since the pH in blood circulation is close to neutral, ionizable lipids are also neutral, and the non-specific binding between them and plasma proteins or blood cells is significantly reduced, extending the half-life. In addition, when ionizable lipid LNP are taken up by cells and enter lysosomes, the lipids are ionized and positively charged in the lysosomal environment with pH 5-6. The positively charged lipids are easily fused with the endosome-lysosome membrane, allowing the liposomes to escape to the cytoplasm, dissociate and release nucleic acids, thus exerting their effects. In summary, designing LNP carrier system involves optimizing lipid composition, particle size, surface charge and stability to achieve efficient gene delivery.
LCMS Bioanalysis Strategy for LNP-packaged Gene Drugs
LNP-packaged gene drugs mainly consist of LNP shell and gene drug itself. The LNP shell mainly consists of lipid components (cationic/ionizable lipids account for about 30-50%, PEG lipids account for about 2%, cholesterol accounts for about 20-50%, auxiliary phospholipids account for about 10-20%), while gene drugs mainly consist of gene sequence fragments (DNA, ASO, siRNA and miRNA, etc.). According to the latest regulations of NMPA and FDA[14-15], for IND application of drug delivery system drugs such as LNP, in addition to conducting pharmacological and pharmacokinetic studies on the active ingredient (gene sequence), safety and pharmaceutical evaluation are also required for new lipid components that have not been used in marketed drugs in the drug delivery system, such as cationic/ionizable lipids and PEG lipids with special modifications in LNP.
The LCMS bioanalysis of LNP lipids focuses on cationic/ionizable lipids and PEG lipids. Due to the structural differences between them, cationic/ionizable lipid molecular weight is generally within 1 kDa, while PEG lipid molecular weight can reach 2~5 kDa after carrying modification groups, resulting in large differences in chromatographic retention and mass spectrometry response between them. Considering that the content of cationic/ionizable lipid in LNP is higher than that of PEG lipid, the response difference leads to the inability to effectively unify the pretreatment methods (the selection of extraction solvent, the determination of dilution factor) of both. In addition, due to the presence of many interfering peaks around the peak position of PEG lipid, a longer liquid phase separation gradient is often required for effective separation, which cannot guarantee high analysis efficiency. Therefore, we separately establish PEG lipid detection method in bioanalysis. At the same time, cationic/ionizable lipid and PEG lipid also have common difficulties in method development due to their lipid components, such as poor solubility in water, instability in biological matrix, protein binding leading to recovery exceeding limit, high interference and residue of PEG lipid, stability change of lipid in real sample matrix and difficulty in internal standard selection.
