Project Topics
Get Complete Project Material File(s) Now! »
Encapsulation yield and preparation conditions
Several methods for the encapsulation of molecules in liposomes have been proposed. Some are compatible with the manipulation of proteins, while others not due to the use of solvents or inadequate temperatures (see section 2.4.4 Other procedures below). One important parameter is the encapsulation yield. As mentioned in the introduction, the processed molecules may include proteins produced in low amounts (≤ 2 mg) and/or difficult to concentrate in aqueous solution (≥ g/L). Preparation procedures of large unilamellar vesicles will be reviewed with this constraint of a need for a high encapsulation yield in mind.
The film hydration method
The film hydration method relies on the hydration of a preformed lipid film with the solution to encapsulate. The film is made by the evaporation of a solution of lipids in organic solvents, like methanol or chloroform. While these solvents may be harmful, extensive drying of immersed in a solution containing the molecule to encapsulate. Spontaneously or mechanically, the film detaches to form vesicles, entrapping the surrounding solution. the film gets rid of all of them. The dried film is hydrated with a solution of the molecule to encapsulate. The concentration of the lipid is generally in the range of 1–10 g/L. The steps involved in film hydration may include suspension shearing, freeze/thaw cycles, sonication, heating and extrusion on a syringe filter [105–107]. The techniques relying on freeze/thawing, sonication and heating are not adapted to our application due to the risks of protein denaturation.
As illustrated on figure 2.33, not all the engaged solution is encapsulated in the vesicles, leading to a limited encapsulation yield. In a typical experiment, where 200 nm sized DOPC liposomes are prepared, with lipid concentration of 10 g/L, the encapsulation yield is at best 8 % (see appendix C.4.1, p. 200). This technique has however the main advantage of being compatible with mild handling conditions.
An encapsulation yield as high as 50 % can be reached under certain conditions. If the molecule to encapsulate can be made to have some affinity for the lipid membrane, up to half the initial amount of protein may stay in the liposome, while the other half will be attached onto the outer leaflet and removed during the liposome isolation step. This technique has been applied to the encapsulation of superoxide dismutase in liposomes made of DPPC:cholesterol [108] or trypsin in DPPC:cholesterol:dicetyl phosphate liposomes [109]. Depending on how the protein is retained on the interface, the encapsulation yield may depend on the pH or the ionic strength of the solution, e.g. in the case of electrostatic interactions.
Release mechanisms and associated timescales
In this chapter, we presented light-sensitive liposomes with various release properties. Concerning the release time, a broad range is covered and ranges from the microsecond to several hours. We are interested in fast releasing systems, with a characteristic time below the second. Only two systems amongst the reported ones reach this criterion: the first one is based on the permeabilization of the membrane and the other one on the disruption of the membrane.
The first system family relies on the increase of permeability of the lipid bilayer. It is possible to release small hydrophilic molecules, but nothing has been shown on the release of proteins. In fact, they may not be able to cross the membrane at all, due to their size, their ionization state and their hydrophilicity. Slightly hydrophobic molecules have been reported to stay stuck in the lipid bilayer. Therefore, this system is not adequate to the release of proteins. For these objects to escape, nanometer sized holes must be created in the lipid bilayer.
The second system family relies on a physical disruption of the membrane, which leads to release of the entrapped content regardless of its molecular weight. This method has successfully been applied to the release of active proteins in a cell culture medium. However, the fast release relies on high-power laser irradiation. Several configurations have been reported to this aim: either bare liposomes irradiated with pulsed UV light or liposomes sensitized with dyes or nanoparticles. The mechanism of release is not always specified, but the temperature elevation induced by the laser irradiation is predominant. Specific lipids formulations – in particular the ones containing lysolipids – have been developed to respond violently to a slight increase of temperature. In fact, the vesicles can be completely tight at 37 ℃, but made permeable and released their content above 41 ℃. The increase of temperature is thus neither harmful for the cells, if it ever reach them, nor to the encapsulated material. Coupled to a dye or light-absorbing nanoparticles, these vesicles can be used for a fast light-triggered release of proteins. One precaution should be taken when used dye-sensitized liposomes, in particular with the ones inserted inside the lipid bilayer. As cells and liposomes are similar by their membrane composition, the dyes can also attach to the cell membrane; depending on the dye concentration, this may perturb or even kill the cell.
Nucleid-acid based hydrogels
Recently a hydrogel encapsulation system made of plasmids crosslinked with ethylene glycol diglycidyl ether (EDGE, a di-epoxide) was reported. The gel self-disassembles upon blue light irradiation (400 nm, unknown power), which releases both entrapped material and the plasmid itself. The mesh size of the system has been characterized experimentally and is in the range of 30–40 Å. The release of the plasmid occurs within 500 hours, with the entrapped material, such as BSA, lysozyme or FITC-dextran (77 kg/mol). The timescales associated with the release depends on the entrapped molecules 200 hours are reported for BSA and dextran, while 48 hours are for lysozyme [143, 144]. The hydrodynamic radius of the lysozyme is ≈ 16 Å, smaller than the mesh size, while the radius of BSA is ≈ 35 Å and the one of the dextran 55 Å; this explains why the release of lysozyme is faster than the other molecules. However, the comparison with the other systems is not obvious, as the gel particles are macroscopic (millimetric). The mechanism of degradation suggested by the authors is the self degradation of ethers in water under light irradiation. Linked to nucleic acid, the wavelength of maximum degradation efficiency is 400 nm.
Table of contents :
Introduction
1. Sensitivity of cells to uneven distributions of peptides and proteins
1.1. Representative proteins of interest
1.2. Characteristic scales: concentrations, time and lengths
1.3. Summary and conclusion
I. Light-controlled release, a review
2. Light-sensitive liposomes
2.1. Light-induced increase of permeability
2.2. Light-triggered pore formation
2.3. Membrane disruption
2.4. Encapsulation yield and preparation conditions
2.5. Summary on light-sensitive vesicles
3. Micro- and nanometer-sized hydrogels
3.1. Photodegradable hydrogels
3.2. Light-triggered gel collapse
3.3. Encapsulation yield and loading constraints
4. Aqueous core—polymer shell capsules
4.1. Layer-by-layer assemblies
4.2. Polymersomes
4.3. Capsules made of solid polymers
4.4. Hybrid membranes
4.5. Role of the stimulation wavelength
Summary
II. Light-sensitive liposomes
5. Surfactant sensitized vesicles
Introduction
5.1. Liposome solubilization by the azoTAB surfactant
Conclusion
6. Azobenzene-based polymer sensitizer
Introduction
6.1. Experimental conditions
6.2. Interpretation of typical photo-triggered release patterns
6.3. Data analysis
Conclusion
III. Temperature-sensitive capsules made by the reverse emulsion method
Introduction
7. PolyNIPAM-based millimeter sized capsules
7.1. Adaptation of published protocols
7.2. PolyNIPAM-based capsules
7.3. Effect of mixed membrane composition
8. PolyNIPAM-based nanocapsules
8.1. Description of the synthesis of nanometer sized capsules
8.2. PolyNIPAM nanocapsules characterization
9. UCST polymer-based millimeter-sized capsules
9.1. Characterization of the UCST polymer
9.2. Adaptation of the capsule preparation procedure to the UCST polymers .
9.3. Characterization of capsules made from UCST polymers and ovalbumin
Conclusions & perspectives
Liposomes
Polymer capsules
IV. Appendices
A. Role of molecular gradients on biological systems
B. Currently used gradient generators
C. Materials & Methods
D. Experimental techniques
E. Preparation and characterization of liposomes
F. Preparation and characterization of polymer capsules
G. Quantitative and real-time FRAP measurements
Bibliography
