Cytoplasm Targeted Nano Drug Carrier

  • The cytoplasm is the general term for all translucent, colloidal, granular materials except the nuclear area surrounded by the cytoplasmic membrane, and is composed of the cytoplasmic matrix, endomembrane system, cytoskeleton, and inclusions. Among them, the cytoplasmic matrix, also known as cytosol, is a homogeneous and translucent colloid in the cytoplasm, which is filled between other tangible structures;  its main function is to provide an ionic environment for various organelles to maintain their normal structure and supply all substrates for various organelles to complete functional activities, and is also the venue for certain biochemical activities. The importance of cytoplasmic matrix transport is not only due to the existence of multiple drug targets in the matrix (such as glucocorticoid receptors, proteins), but also because this is the only way to reach various target organelles, so overcoming the cytoplasmic membrane barrier is also a prerequisite for targeted administration of subcellular structures. Substances taken up by endocytosis will eventually enter the late endosome lysosome, which is digested and degraded by various enzymes in the lysosome. For drugs that require a cytoplasmic matrix or specific organelles to function, escaping from lysosomes is a critical step, so effective cytoplasmic matrix transport is controlled by two factors: effective cellular uptake and endosomal escape.

    Directly into the Cytoplasm

    Cell-penetrating peptides (CPPs) are short peptides that carry macromolecular substances into cells, and their ability to penetrate membranes does not depend on classical endocytosis. These substances are all peptide fragments of different lengths of positive charge, which are rich in basic amino acid residues such as arginine and lysine, and the secondary structure has an alpha helix spatial conformation. The synthetic penetrating peptides fuse hydrophilic and hydrophobic domains from different sources to have stronger penetrating power and higher efficiency, for example, penetrating peptides Pep-1, MPG, transporter, TAT, and successfully carrying large molecules into the cell. Therefore, CPPs can significantly enhance the intracellular targeting of liposomes. TAT modified liposomes can be delivered into many different types of cells, such as Lewis lung cancer cells, human breast tumor cells BT20, rat cardiomyocytes H9C2, and mouse fibroblast NIH/3T344.

    Among them, the internalization ability of modified liposomes is related to the number of surface polypeptide molecules, and the uptake kinetic characteristics are related to the types of penetrating peptides and cell lines. The connection between the penetrating peptide and the carrier requires chemical cross-linking, which limits its application. For example, a protein modified by HIV-1 TAT must be denatured to increase its binding site to the penetrating peptide. Since Pep-1 is a synthetic amphiphilic peptide, it can load proteins and peptides into human cells without cross-linking or denaturation, so it can load proteins into cells without endocytosis.


    Endosome Escape

    In addition to directly entering the cytoplasm, nanocarriers will also be taken up by cells through receptor-mediated endocytosis and reach the endosome. If you want the drug to function in the cytoplasm, the drug must escape from the endosome before the active substance degrades. Fusion peptides are important hydrophobic fragments that cause fusion of endosomal membranes when viruses infect host cells. For example, the fusion peptides derived from influenza virus erythrocyte agglutination subunit HA-2 or synthetic fusion peptides in an environment where the pH changes from neutral (pH 7.0) to acidic (pH 5.0), its configuration can be changed from a random helix to an amphiphilic a helix, inserted into the bilayer of the membrane, and be polymerized to form a transmembrane spiral pore, making the endosome/lysosomal membrane unstable. Therefore, the fusion peptide can improve the transfection efficiency of various gene vectors.

    Polymer Nanocarrier

    The characteristics of the ideal polymer nanoparticles include: ①The material is biocompatible and degradable; ②Avoiding degradation of encapsulated drugs; ƒ Appropriate particle size that can enter cells; ④Avoiding phagocytosis by mononuclear phagocytes after administration, and targeting cells.

    (1) Cationic Polymer

    Cationic polymers are positively charged on the surface, which can neutralize the negative charge on the surface of DNA, greatly compress the huge volume of the original DNA helix structure, and also protect DNA from degradation by enzymes. At the same time, the positively charged polymer easily contacts the negatively charged cell membrane, and then enters the cell through endocytosis. Commonly used cationic polymers include poly-L-lysine (PLL), polyethyleneimine (PEI), chitosan, N, N-dimethylaminoethyl methacrylate (pDMAEMA), cationic polysaccharides, and dendrimers polymer. PLL and chitosan are biodegradable, but they cannot quickly escape from the endosome, and the transfection efficiency is not as high as PEI. If they are introduced into cells together with endosomal activity inhibitors (fusion peptides, inactivated adenovirus, chloroquine), they can promote the release of drugs or DNA into the cytoplasm. The gene transfer ability of the dendrimer on the surface of the amino group is very strong, because a large number of amino groups have a "buffer effect" or "proton sponge effect". When they reach the endosome, the acidic environment protonates the amino group, the osmotic pressure increases, a large amount of water enters the endosome, the endosome membrane ruptures, and the drug enters the cytoplasm. If the polymer is modified with ligands, the drug can also be introduced into the cytoplasm through the action of ligand receptors, such as non-saliva serum mucin, transferrin and folic acid.

    (2) pH Sensitive Polymer

    The pH-sensitive polymer mimics the pH-sensitive fusion peptide, which makes the endosomal membrane lose stability and escape in an acidic environment. The conformation of alkyl-substituted polyacrylic acid and N-isobutylacrylamide depends on pH and temperature. At 37℃, when the pH changes from 7.4 to 4.5, their conformation changes from being stretch to spherical; when the pH is between 4.9 and 5.5, the drugs they encapsulate are quickly released.


    Liposomes have advantages over viral vectors as gene carriers: ① not immunogenic; ② particle size, charge, composition and morphology can be prepared as needed; ③ protect genes from nuclease hydrolysis and improve their biological stability. Various liposomes have been prepared for gene delivery. The pH-sensitive liposomes are unstable under acidic conditions. After entering the endosome, they form an unstable form that can destroy the endosomal membrane and release the drug. The most commonly used pH-sensitive lipid is the dioleic phosphatidylethanolamine (DOPE), which forms a hexagonal phase under acidity. Stearyl isobutylene methacrylate copolymer incorporated into liposomes is stable when the pH> 6; when the pH is 5, there are fine cracks on the surface of the liposome, and when the pH is less than 5, the liposomes are completely broken.

    The composition of cationic liposomes includes cationic lipids and neutral auxiliary lipids. Commonly used cationic lipids are DOTAP, DOTMA, DC-CHOL, DDAB and so on. Commonly used zwitterionic lipids include DOPE and cholesterol. DOPE can fuse endosomal membrane and lysosomal membrane, which helps the escape of liposome contents from the endosome and also helps reduce the cytotoxicity of cationic lipids. Cationic liposomes and negatively charged genes form a nano-liposome gene complex with a certain positive charge through electrostatic action, which is adsorbed on the surface of negatively charged cells, fused with the cell membrane or engulfed into the cell, releasing the gene in the cell and further reaching the nucleus. It is worth noting that both plasma proteins and nucleases reduce the transfection efficiency of cationic lipid complexes. In addition, anionic extracellular substances (such as heparin) can be combined with cationic liposomes, and some cationic extracellular substances can be combined with DNA, allowing DNA to be released from the complex in advance.