The distribution of passively targeted microparticles in vivo after intravenous injection first depends on the particle size. Nanocapsules or nanospheres smaller than 100 nm can slowly accumulate in the bone marrow; those smaller than 3 μm are generally taken up by macrophages in the liver and spleen. ; Particles larger than 7 μm are usually retained by mechanical filtration by the smallest capillary bed of the lung and taken up by monocytes into the lung tissue or air bubbles. The surface properties of the particles play an important role in the distribution.
Natural targeting preparations, which are the in vivo distribution characteristics of drug-loaded particles entering the body, that is, the natural tendency of macrophages to be phagocytosed as foreign foreign bodies. This type of targeted formulation uses lipids, lipids, proteins, and biodegradable macromolecules as carriers to encapsulate or embed drugs into microparticle drug delivery systems.
Such targeted preparations mainly include liposomes, microspheres, nanocapsules and nanospheres.
Liposomes refer to microvesicles formed by encapsulating drugs in lipid bilayers. With a cell membrane-like structure, it can be recognized as a foreign body by the reticuloendothelial system in vivo, and its phagocytosis is mainly distributed in tissues and organs such as liver, spleen, lung and bone marrow, thereby improving the therapeutic index of the drug.
In the late 1960s, llahman et al. first applied liposomes as drug carriers. In recent years, with the continuous development of biotechnology, the preparation process of liposomes has been gradually improved; the mechanism of action of liposomes has been further elucidated, and liposomes are suitable for in vivo degradation, non-toxicity and non-immunogenicity, especially a large number of experimental data prove that liposomes As a drug carrier, plastids can improve the therapeutic index of drugs, reduce drug toxicity, reduce drug side effects, and reduce drug doses. Therefore, in recent years, more and more attention has been paid to the research of liposomes as drug carriers, and the research progress in this area is very rapid.
(1) Classification of liposomes
Liposomes are classified into unilamellar liposomes and multilamellar liposomes according to the number of lipid bilayers they contain. Vesicles containing a single bilayer are called unilamellar vesicles (singer unilamelar vesicles, SUV), with a particle size of about 0.02-0.08μm;
Vesicles containing multi-layered bilayers are called multilamellar vesicles (MLVs), and the particle size is between 1-5 μm; large unilamellar vesicles (large unilamellar vesicles LUVs) are large unilamellar vesicles. The particle size is between 0.1-1μm. Water-soluble drugs are encapsulated in the interlayer of hydrophilic groups of vesicles, while lipid-soluble drugs are dispersed in the interlayer of hydrophobic groups of vesicles. Large unilamellar liposomes can encapsulate 10-fold, or even tens of times, more drug than unicompartmental liposomes.
(2) The composition and structure of liposomes
The composition and structure of liposomes are different from micelles composed of surfactants. Liposomes are composed of bilayers, while micelles are composed of monolayers. The composition of liposomes is composed of lipids (such as phospholipids and cholesterol) as membrane materials and additives. The phospholipid structure contains a phosphoric acid group and a quaternary ammonium salt group, both of which are hydrophilic groups, and two longer hydrocarbon groups are hydrophobic chains. Cholesterol is also an amphiphilic substance, and its structure also has two groups, hydrophobic and hydrophilic, and its hydrophobicity is stronger than hydrophilicity.
When a phospholipid molecule forms a liposome, it has two hydrophobic chains pointing inward, and the hydrophilic group is on the inner and outer surfaces of the membrane. The phospholipid bilayer forms a closed cell, and the interior contains an aqueous solution. , the phospholipid bi-chamber forms vesicles which are separated by the aqueous medium. Liposomes can be unilamellar closed bilayers or multilamellar closed bilayers. Under the electron microscope, the shapes of liposomes are usually spherical, elliptical, etc., with diameters ranging from tens of nanometers to several micrometers.
(3) Materials of liposomes
The membrane material of liposomes is mainly composed of phospholipids and cholesterol. The two components form a bilayer structure of liposomes, similar to “artificial biological membranes”, which are easily digested and decomposed by the body.
Phospholipids, including lecithin, cephalin, soybean phospholipid and other synthetic phospholipids, can be used as the basic material of liposome bilayer. Lecithin is prepared from egg yolk lecithin and extracted with chloroform as solvent, but the chloroform in the product is not easy to remove, and the cost is higher than that of soybean lecithin. The composition of soybean lecithin is a mixture of lecithin and a small amount of cephalin. Soybean lecithin for injection must be further refined to remove substances that cause heat, sensitization and hypotension.
Cholesterol and phospholipids are the basic substances that together constitute cell membranes and liposomes. Cholesterol has the effect of regulating membrane fluidity, so it can be called the “fluidity buffer” of liposomes. It has been reported in the literature that cholesterol can increase the encapsulation efficiency and drug loading of liposomes. When the temperature is lower than the phase transition temperature, cholesterol can reduce the ordered arrangement of the membrane and increase the fluidity.
(4) Physical and chemical properties of liposomes
1. Phase transition temperature The physical properties of liposomes are closely related to the temperature of the medium. When the temperature is raised, the hydrophobic chains in the liposome bilayer can change from ordered arrangement to disordered arrangement, resulting in a series of changes, such as The thickness of the film decreases, the fluidity increases, etc. The temperature at which the transition occurs is called the phase transition temperature, and it depends on the type of phospholipid. The liposome membrane can be composed of two or more phospholipids, each of which has a specific phase transition temperature, and can exist in different phases at the same time under certain conditions.
2. Liposomes of electrically acidic lipids such as phosphatidic acid (PA) and phosphatidylserine (PS) are negatively charged, and liposomes containing base (amino) lipids such as octadecylamine are positively charged, not Liposomes containing ions are electrically neutral. The surface electrical properties of liposomes are related to their encapsulation efficiency, stability, target organ distribution and effect on target cells.
(5) Characteristics of liposomes
Liposomes can encapsulate both fat-soluble drugs and water-soluble drugs. After the drugs are encapsulated by liposomes, the main features are as follows:
Liposomes can be phagocytosed by macrophages as external foreign bodies in the human body, and are mainly phagocytosed and taken up by macrophages of the monocyte-macrophage system, forming passive targeting of the reticuloendothelial system such as the liver and spleen. Liposomes can be used to treat liver tumors and prevent tumor spread and metastasis, as well as liver parasitic diseases, leishmaniasis and other monocyte-macrophage system diseases. For example, after the anti-hepatic leishmania drug meglumine antimonate is encapsulated by liposomes, the concentration of the drug in the liver is increased by 200-700 times. After intramuscular, subcutaneous or intraperitoneal injection, liposomes can first enter the regional lymph nodes.
2. Cell affinity and histocompatibility
Because liposome is a structure similar to biological membrane structure, it has no damage or inhibitory effect on normal cells and tissues, has cell affinity and histocompatibility, and can be adsorbed around target cells for a long time, so that drugs can be fully targeted Cell target tissue penetration, liposomes can also enter cells through fusion, and release drugs through lysosome digestion. If anti-tuberculosis drugs are encapsulated in liposomes, the drugs can be loaded into cells to kill tuberculosis bacteria and improve the curative effect.
3. Slow release effect
Many drugs have a short duration of action due to rapid metabolism or excretion in the body. Encapsulating the drug into liposomes can reduce renal excretion and metabolism, prolong the residence time of the drug in the blood, and release the drug slowly in the body, thereby prolonging the action time of the drug.
4. Reduce drug toxicity
After the drug is encapsulated by liposomes, it is effectively concentrated in organs with rich mononuclear-macrophages such as the liver, spleen and bone marrow, so that the accumulation of the drug in the heart and kidney is lower than that of free drug, and it has adverse effects on the heart and kidney. Toxic drugs or anticancer drugs that are toxic to normal cells are encapsulated in liposomes, which can significantly reduce the toxicity of the drugs. For example, amphotericin B, which is more toxic to most mammals, can be made into amphotericin B liposomes, which can greatly reduce the toxicity without affecting the antifungal activity.
5. Protect the drug to improve the stability
After some unstable drugs are encapsulated by liposomes, they can be protected by the liposome bilayer membrane. For example, penicillin G salt is unstable to acid, and is easily destroyed by gastric acid when taken orally. Making it into liposome can improve its stability and the effect of oral absorption.
(6) Preparation of liposomes
There are many preparation methods for liposomes, the commonly used ones are:
1. Injection method
Co-dissolve phospholipids, cholesterol and other lipids and lipid-soluble drugs in an organic solvent (usually diethyl ether is used), and then slowly inject this drug solution into phosphate buffer heated to 50-60 °C (with magnetic stirring) through a syringe In the liquid (which may contain water-soluble drugs), after adding, continue stirring until the ether is completely removed, that is, liposomes are obtained, and their particle size is large, which is not suitable for intravenous injection. The liposome suspension was then passed through a high-pressure homogenizer twice, and the obtained products were mostly unilamellar liposomes, a few were multilamellar liposomes, and most of the particle sizes were below 2 μm.
2. Thin film dispersion method
Dissolve lipids such as phospholipids, cholesterol, and fat-soluble drugs in chloroform (or other organic solvents), and then rotate the chloroform solution in a glass bottle to form a film on the inner wall of the flask; dissolve water-soluble drugs in phosphate The buffer solution was added to the flask with constant stirring to obtain liposomes.
3. Ultrasonic dispersion method
Dissolve water-soluble drugs in phosphate buffer, add a solution of phospholipids, cholesterol and fat-soluble drugs co-dissolved in an organic solvent, stir and evaporate to remove the organic solvent, the residual liquid is treated with ultrasonic waves, and then the liposomes are separated, and then suspended in In phosphate buffered saline, it is prepared as a liposome suspension injection. Most of the liposome suspensions dispersed by ultrasonic are unilamellar liposomes. Multilamellar liposomes can also obtain fairly uniform unilamellar liposomes as long as they are sonicated.
4. Reverse-phase evaporation method
Membrane materials such as phospholipids are dissolved in organic solvents, such as chloroform and ether, and an aqueous solution of the drug to be encapsulated (the amount of organic solvent is 3-6 times that of the aqueous solution) is added for short-time ultrasonic treatment until a stable W/O type is formed. Emulsion, and then evaporated under reduced pressure to remove the organic solvent, after reaching a colloidal state, drop the buffer solution, rotate to make the gel on the wall fall off, and continue to evaporate under reduced pressure to obtain an aqueous suspension, which is subjected to gel chromatography or By ultracentrifugation, the unencapsulated drug is removed to obtain large unilamellar liposomes. The characteristics of this method are that the amount of encapsulated drugs is large, and the volume encapsulation rate can be 30 times greater than that of the ultrasonic dispersion method. It is suitable for encapsulating water-soluble drugs and macromolecular biologically active substances, such as various antibiotics, insulin, immunoglobulin, alkali phospholipase, nucleic acid, etc.
5. Freeze drying
The drug is highly dispersed in buffered saline solution, freeze-dried after adding cryoprotectant (such as mannitol, dextran, alginic acid, etc.) plastid. This method is suitable for encapsulating heat-sensitive drugs.