Fig. 1. General structure of liposome vaccines. The liposome vaccines consist of a two-layer structure with added cholesterol phospholipids, as well as pH-sensitive materials and (polyethylene glycol) PEG modifications for versatility. The ways in which liposomes carry antigens include encapsulation, absorption or admixed. Lipid A is an adjuvant added in a liposome vaccine which is a lipid layer.
Preparation of liposome carriers. The properties of liposomes are closely related to the preparation liquid. In the past, liposome preparation techniques had similar general steps: drying from volatile solvents, dispersion, purification, and synthesis in aqueous medium, and finally liposomes were obtained [20].
Thin film hydration is the earliest liposome preparation technology, which mainly includes dissolving phospholipids in organic solvents, removing phospholipids by evaporation, and finally pouring them into aqueous buffer solution, stirring to make the dried lipid membranes hydrate successfully [21]. However, this method will produce many heterogeneous paleosecular liposomes. The reverse phase evaporation method is to reisolate the phospholipid molecules in the organic phase, and then adding aqueous phase buffer to a mixed solution to decompress distillation [22]. The two-phase system was ultrasonic treated to transparent and uniform single-phase dispersion, and it was observed that the solution state did not change for at least 30 minutes, then the organic solvent was removed by rotary evaporation. The liposomes prepared by reversed-phase evaporation method have unique advantages in encapsulating water-soluble substances, but may have drug residue [23]. The solvent injection method does not need to be helpful in ultrasonic waves, simply dissolve lipids in the organic phase which is generally diethyl ether or ethanol, and then injects the lipid solution into the aqueous solution, the particle diameter is obtained by narrowing a smaller-distribution [24]. With using ethanol, it can remain stable at higher temperature, but it is difficult to remove all the solvent, and the possibility of biological deactivation is high. Compared with ethanol, ether injection can completely remove the solvent and form liposomes with high entrapment efficiency, but due to the low boiling point of ether, it will lead to uneven aggregation and dissolution at high temperature [25]. The bubble method is a method without the use of organic solvents, in which inert gas bubbles are introduced into the lipid mixture to prepare liposomes with mild preparation conditions and high entrapment efficiency [26]. The uniqueness of supercritical fluids is that there is no difference in liquid and gaseous. The researchers found that the properties of liposomes obtained by supercritical fluids were better than those prepared by conventional solvents [27]. Supercritical carbon dioxide has become a substitute for organic solvents because of its low critical temperature and pressure, similar solubility to non-polar solvents, low application cost, and non-toxicity [28].
Conventional preparation techniques generally lack the precise regulation of liposome particle size on macro levels, batch reproducibility and quality repetitive control. Microfluidics refers to the precise control of fluid behaviors in micro-channels or designing different microfluidic channels to realize the mixed reaction of different liquids. Delivery carriers with small particle size distribution, high repeatability between batches and significantly improved drug loading rate can be prepared by microfluidic technology [29]. Microfluidic techniques can overcome the disadvantages of conventional methods, a highly stabilizing liposomes that can accurately control physical parameters can be obtained by designing flow rate, flow rate ratio, mixing speed, and lipid concentration. In order to be able to scale production, the preparation steps should be as simple as possible. For human health, liposomes should avoid using hazardous chemicals or solvents when prepared. The residual solution is potentially toxic to human body and causes great environmental pollution, so it should be eliminated in the preparation process. Therefore, when preparing liposomes, we should consider comprehensively and choose the most suitable preparation method to achieve the most beneficial results.
Relationship between the properties and immunogenicity of liposome carrier. Since Allison first reported in 1974, liposomes mainly exert immunity through the ”pool effect” and immune stimulation, and gradually become the research object of vaccines as adjuvants [30, 31]. A mass of liposomes containing antigens are ingested by macrophages through natural channels, they can be stored in macrophages for slow release, thus introducing antigens to APC, promoting downstream cells to secrete a series of cytokines, and enabling the body to maintain high-valence antibodies (Fig. 2). Although vaccine carriers based on other materials, such as metal nanoparticles, inorganic (such as silica) nanoparticles, and polymer nanoparticles [32, 33] have also shown advantages over traditional preparations in achieving delivery and adjuvant functions, most vaccine vectors are still in the preclinical development stage, and their feasibility needs to be further explored. Also, based on the studies of existing tumor-specific antigen delivery, we can tell liposome carriers show better properties than non-liposomes [34].
Several key parameters mentioned above, such as surface charge, physical size, and degree of PEG modification, play a very important role in regulating the biological behavior of liposome vaccine vectors. The interaction of various factors finally plays a comprehensive role in the immunostimulatory activity of the whole carrier. In addition, the loaded way of antigen is also very important. Mixing the carrier with the antigen and using mild adsorption may be more beneficial to maintain the relative activity of the antigen, but the surface adsorption also means a higher antigen release rate, resulting in low efficiency of antigen uptake. Encapsulating antigens in liposome carriers can achieve sustained release of antigens or drugs, resulting in a more effective cross-presentation process and a better lysosome escape [35].