This article takes you through liposomal drug delivery systems

Liposomes were first discovered and described
in 1965 by Bangham et al.
Its basic structure is a vesicle formed by a phospholipid bilamellar membrane with an aqueous core
wrapped inside.
Figure 1 shows the formation process of a single-chamber liposome and its basic structure
.

Due to liposomes' amphiphilic nature, both hydrophilic and hydrophobic drugs can be efficiently loaded into liposomes
.
Liposomes tend to encase water-soluble drugs in a central aqueous phase and fat-soluble drugs in the region
between the bimembranes.

At the same time, since the basic structure of the biofilm is also a phospholipid bilayer membrane, liposomes have good biocompatibility and biodegradability
.
After liposomes enter the human body, they act as "invaders" and activate the body's immune mechanism, engulfed by the Mononeuclear Phagocyte System (MPS), and thus targeted enrichment in tissues such as the liver, spleen, lungs and bone marrow, which is the passive targeting potential
of liposomes.

In addition, liposomes can also provide better pharmacokinetic properties, prolong the circulation time of drugs in the body, and improve the efficacy
of drugs.
Based on the above characteristics, liposomes have broad development space
as drug delivery systems.

Liposomes used for drug delivery are primarily composed of phospholipids (glycerophospholipids and sphingomylotins) that form their basic skeleton
.
Figure 2 shows the basic structure of glycerophospholipids and sphingomyelins:

Sphingomyelin and glycerophospholipids are similar in structure and have basically the same
properties.
These lipids are amphiphilic molecules, both possessing hydrophilic head and hydrophobic tail regions
.
In an aqueous environment, phospholipid molecules spontaneously arrange into liposomes
, driven by hydrophobic interactions and other intermolecular interaction forces.

With the exception of phospholipids, cholesterol is added to almost all liposomes
.
Its functions are: to promote the accumulation of lipid chains and the formation of bilayers, reduce the fluidity of bilayers, and reduce the transmembrane transport
of water-soluble drugs.
Not only that, cholesterol can also reduce the interaction of liposomes with proteins in the body, reduce the loss of phospholipids, and thus improve the stability
of liposomes.
In addition, oligosaccharides, chitosan, alginate, whey protein, etc.
can also be added to liposomes as membranes to improve their stability and regulate drug release
.

Necessary antioxidants (such as vitamin E), metal ion chelators (such as EDTA), etc.
may also be added to liposomes to prevent the oxidation
of lipids or drugs.

Special modifications on the surface of liposomes can improve their functionality, the most common being PEG modifications
.
PEG is a hydrophilic and flexible polymer, which can prevent the adsorption of opsonins to the surface of liposomes, thereby weakening the opadociotic effect, reducing the phagocytosis of the mononuclear phagocyte system (MPS), and achieving the purpose of
long cycle.

Depending on the number of membrane layers and particle size of liposomes, liposomes can be divided into the following categories, as shown
in Figure 3.

The liposome products currently on the market contain a variety of structures in the figure above, most of which are small unilamellar vesicles (SUVs).

The preparation of liposomes is mainly by using the spontaneous arrangement
of amphiphilic phospholipids in solvents.
The key point for liposome formation is temperature: it must be above
the phase transition temperature Tg of the lipid.
The particle size and bimolecular film layers, particle size and particle size distribution of the formed liposomes are affected
by the preparation method, lipid type, lipid composition, surfactant, organic solvent and ionic strength of the dispersion medium.
At present, the common liposome preparation technologies are as follows
.

1 Film hydration method

Preparation process: the film materials that form liposomes, such as phospholipids and cholesterol, are dissolved into organic solvents; The organic solvent was removed by rotary steaming to obtain a lipid film; The film is then sufficiently shaken and hydrated with a buffer to dissolve the drug to be encapsulated to form liposomes
.
The temperature of hydration should be greater than the phase transition temperature (Tm)
of the lipid.

Buffer volume and hydration time affect the structure of the formation of liposomes and the encapsulation rate
of the drug.
Liposomes prepared by film hydration are usually multilayer liposomes (MLVs).

Multilayer liposomes (MLVs) can be converted into large single-chamber liposomes (LUVs) or small single-chamber liposomes (SUVs) by applying mechanical force, such as extrusion and ultrasonication
.
The extrusion method is to filter liposomes through a film of a specific pore size, and if necessary, ensure that the extrusion temperature is greater than Tm
.
Extrusion results in more homogeneous liposomes, and the process is relatively gentle, making it suitable for unstable drugs
.
Moreover, compared with ultrasound, liposomes obtained by extrusion have better long-term stability
.
Although the ultrasound method is simple, it may cause the degradation of liposomes or drugs, and the obtained liposomes are poorly
homogeneous.

2 Reverse evaporation method

Preparation process: the organic phase dissolved in the film material (commonly used is ether and isopropyl ether, an appropriate amount of chloroform or methanol can be added when the solubility is poor) and buffer and briefly sonicated for 2-5min until a water-in-oil emulsion is formed; The emulsion was initially reduced pressure and rotated to obtain a gel; Continue to rotate to obtain liposomes; At this point, buffer or aqueous phase can be added to continue steaming (not necessary) to remove traces of organic solvent
.

The reverse evaporation method is characterized by the preparation of liposomes with a larger aqueous-lipid ratio, that is, the volume of the central aqueous phase in which it is wrapped is larger, which is suitable for encapsulating proteins or macromolecular drugs such as DNA and RNA; The disadvantages of this method are the use of organic solvents and the possibility of lipid or drug degradation
by transient ultrasound.

3 Injection method

Preparation process: The membrane and the drug are dissolved in an organic solvent, and then the oil phase is injected into the aqueous phase (which may contain water-soluble drugs) at a uniform speed, and the continuous stirring is used, and the vortex generated by the stirring is used to promote the arrangement of lipids to form liposomes
.
Finally, the organic solvent
is removed by low-pressure evaporation, dialysis or filtration.

Organic solvents commonly used in injection methods include ether and ethanol
.
The boiling point of ether is low, and when the temperature is higher than the boiling point of ether, ether is easy to remove, mainly forming large single-chamber liposomes
.
The ethanol boiling point is higher and can be removed
by dialysis or filtration.
The injection method is suitable for the preparation of large quantities, but the resulting liposomes have a wide
particle size distribution.

4 Detergent removal method

Preparation process: the membrane (phospholipids, cholesterol) and detergents (such as bile salts, etc.
, the dosage is much higher than the critical micelle concentration) are added to the organic solvent to dissolve and dry; The resulting lipid film was added to the buffer for hydration; During the hydration process, the film and detergent will form mixed micelles (in this case, mixed micelles, pure detergent micelles, and free detergents coexist); The detergent is removed by dialysis, size exclusion chromatography or dilution method, and the detergent in the mixed micelles is continuously released during the detergent removal process, causing the hybrid micelles to fuse larger, gradually forming a curvature hybrid bilayer, and finally forming liposomes
.
The formation process is shown in
Figure 4.

The advantages of this method are that the prepared single-chamber liposomes have excellent particle size uniformity.
The disadvantage is that the use of a large amount of detergent increases the cost of preparation and may also introduce additional impurities
.

5 Freeze-thaw method

Freeze-thaw is generally used as a supplement
to liposome preparation.
Adding freeze-thaw cycles to the liposome synthesis process improves lipid accumulation and forms single-chamber liposomes
.
The basic process is to freeze the formed liposomes in liquid nitrogen, the formed ice crystals puncture the lipid film, and then during the melting of the ice crystals, the broken lipid membranes re-fuse to form new liposomes
.

6 Scale-up of liposomes

It is not difficult to prepare liposomes with different properties in the laboratory, but there are only a few technologies for industrial mass preparation, and their use is limited
by the process and the drug to be encapsulated.
Therefore, the industrial production line equipment of liposomes generally needs to be customized
according to the product.
During production, drugs are subject to mechanical or chemical pressures and are not suitable for drug molecules
that are sensitive to these pressures.
The high difficulty index of industrial production is one of the factors restricting the application of
liposomes.

The drug loading methods of liposomes mainly include passive drug delivery method and active drug delivery method
.

Passive drug loading refers to the process of liposome preparation, the drug is dissolved in the organic phase or the water phase, and the drug is loaded into the liposome at the same time as the
liposome is formed.

Active drug loading method refers to the regulation and regulation of the pH value of the internal and external aqueous phase of lipids, forming a certain pH gradient difference, and weak acid or weak base drugs follow the pH gradient and cross the phospholipid membrane in molecular form so that they are encapsulated in the internal aqueous phase in ionic form, also known as pH gradient method
.

Among them, ammonium sulfate gradient method and calcium acetate gradient method are two developed forms of pH gradient method, and the two methods are suitable for loading weak alkaline and weak acid drugs
, respectively.

1 Ammonium sulfate gradient method

First, ammonium sulfate solution was used to prepare blank liposomes for the aqueous phase, and then the ammonium sulfate in the outer aqueous phase was removed, at which time the ammonium sulfate concentration gradient was formed in the aqueous phase and the outer aqueous phase in the liposome
.
NH4+ in the inner aqueous phase is easy to decompose into NH3 and H+, NH3 is easy to overflow through the bilayer, and H+ is trapped in the inner aqueous phase, so the pH in the inner aqueous phase continues to decrease, forming an acidic environment
.
After entering the internal aqueous phase, the molecular weak alkaline drug becomes an ion, and the sulfate is salted, and the transmembrane ability is reduced, and the drug can accumulate
inside the liposome.

2 Calcium acetate gradient method

The principle is similar to the ammonium sulfate gradient method, the transmembrane ability of acetic acid is much greater than that of calcium ions, acetic acid brings protons out of liposomes, forming an alkaline inner aqueous phase, and weakly acidic drugs form ions and accumulate
after entering liposomes.

The stability of liposomes is a major factor
limiting their role as a drug delivery system.
The stability
of liposomes will be introduced from three perspectives: physics, chemistry, and biology.

1 Physical stability

Liposomes are spherical sacs, and in order to maintain their structural integrity, the balance of various interacting forces within and between liposomes must be maintained, such as: polar heads, solvent systems, electrostatic interactions between components contained in liposomes must be balanced with hydrophobic interactions between hydrocarbon chains and van der Waals forces
.

Liposomes are a thermodynamically unstable colloidal system, and nanoscale sacs have a tendency to condense and flocculate, and surface charges will affect the aggregation behavior
of liposomes.
In general, neutral liposomes are more likely to aggregate or even fuse, while charged liposomes remain dispersed
under the influence of static electricity.
Drug load, phospholipid types, sterols, and polymer encapsulation all affect
the physical stability of liposomes.

2 Chemical stability

The main chemical stability problems faced by liposomes are mainly oxidation and hydrolysis
.
The backbone of liposomes, phospholipids, contains a large number of functional groups sensitive to oxidation and hydrolysis, such as unsaturated double bonds and ester groups
.

In general, the unsaturated fraction of phospholipids is more easily oxidized
than the saturated fraction.
Therefore, saturated phospholipids offer better stability and higher glass transition temperatures
than unsaturated phospholipids.
If the prepared liposome must have unsaturated chains, the lower the unsaturation, the better, and in general, monounsaturated acids such as octadecenoic acid can meet the demand
.
The oxidation of fatty acids is a free radical chain reaction that does not require a specific oxidant, so it is triggered whenever trace metal ions are present or hydrogen peroxide is formed by the oxygen present
.
In order to avoid the occurrence of oxidation, metal ions or hydrogen peroxide in raw materials should be avoided, stored at low temperatures, and light
.
The addition of antioxidants such as vitamin E or BHT can also slow down the oxidation
of liposomes.

Ester bonds in liposomes are also prone to hydrolysis, and in order to prevent hydrolysis, liquid liposomes can generally be stored
in solid form by freeze-drying.
The addition of cholesterol also slows the hydrolysis of liposomes (mainly steric hindrance).

In addition, temperature, pH, ionic strength, buffer type, aggregation state, head group, and alkane chain length all affect the hydrolysis of liposomes and require special investigation
.

3 Biological stability

The biological stability of liposomes is affected by a variety of factors, including route of administration, carrier molecules, preparation methods, lipid composition, surface properties, morphology, drug loading methods, and other physicochemical properties
.
Some research surfaces have shown that adding an appropriate amount of sphingomyrosin as a skeleton to liposomes can significantly improve the stability of liposomes and prolong the cycle time
.
PEGation is also a way to improve the cycle time of liposomes: it is generally believed that PEG adsorbed on the surface of nanoparticles can combine with water to form a hydration layer, which hinders PEG removal
by MPS.

Since the first liposome product, Doxil?, was launched in 1995, FDA and EMA approval of liposome products has remained limited, mainly due to some shortcomings that limit their use
.
For example, some drugs have low drug load, poor stability, high production costs, and potential toxic side effects
.

The use of liposomes as drug delivery platforms must consider the balance between expenditure and revenue, and products that can be made into ordinary injections must not be prepared into liposomes unless the therapeutic index
of the drug can be significantly improved.
Therefore, this is about the fit between liposomes and drugs
.
With the rise of biological drugs, the need for delivering proteins and nucleic acid molecules is increasing, and there will be more and more
scenarios for this "fit".
Because these macromolecules are particularly unstable in the body, they will be quickly degraded and removed
.
Using liposomes as a carrier can significantly extend the circulation time of drugs in vivo, improve the delivery of drugs to cells, and improve efficacy
.
Therefore, we have reason to believe that the combination of liposomes and biopharmaceuticals will bring more new possibilities
to drug development.

References:

       1.
Liu P, Chen G, Zhang J.
A review of liposomes as a drug delivery system: current status of approved products, regulatory environments, and future perspectives[J].
Molecules, 2022, 27(4): 1372.

       2.
Large D E, Abdelmessih R G, Fink E A, et al.
Liposome composition in drug delivery design, synthesis, characterization, and clinical application[J].
Advanced Drug Delivery Reviews, 2021, 176: 113851.

       3.
Maritim S, Boulas P, Lin Y.
Comprehensive analysis of liposome formulation parameters and their influence on encapsulation, stability and drug release in glibenclamide liposomes[J].
International journal of pharmaceutics, 2021, 592: 120051.