Assembly of Protein Structures onto Liposomes by Using Non-specific and Specific Interactions
Published in the Special Issue on Advances in Biophysics, vol. 34, 139-157 (1997)
SUMMARY
We investigated different schemes for fabrication of nanometer sized assemblies that consist of a liposome core, over which a shell of ferritin is attached. Three distinct interactions were used for this assembly:
- Electrostatic attraction. The liposomes are charged by the presence of cationic surfactant (HTAB) and at an appropriate pH collect the ferritin molecules into 2D-ordered ferritin shell. The protein shells can be fixed by glutaraldehyde. Next, the liposomes can be removed by solubilisation,
leaving behind ordered ferritin clusters (Figure 1).
- Specific avidin-biotin or streptavidin-biotin binding. The ferritin molecules are conjugated to avidin or streptavidin and the liposomes incorporate biotinylated lipid. We found out that the specific binding can be completely impaired by unfavourable electrostatic repulsion. To adjust the appropriate liposome charge we include cationic surfactant in the lipid layer. Thus, to accomplish the assembly process, we need to design and
modify both the specific and non-specific colloid interactions in the system – see Table I below.
The result is liposomes heavily coated with strongly and specifically attached ferritin layer (Figure 2a,b).
- Specific polysaccharide/lectin binding. The liposomes are first coated with cholesterol-anchored mannan layer. The ferritin molecules are conjugated with Concanavalin A that binds to the polysaccharide.
A smooth and dense coating with ferritin is obtained - Figure 2c,d.
The acquired data can find application in the future fabrication of microstructured, multicomponent or functionalised protein and liposome/protein assemblies.
Figure 1.
Ferritin structures assembled by electrostatic interaction and cross-linking. The pictures are obtained by electron microscopy of negatively stained samples.
Each ferritin molecule is seen as a small dark circle, surrounded by a brighter ring.
Bar = 100 nm.
a) Vesicle with adherent non-fixed ferritin array. The liposomes are positively charged by incorporating some amount of the cationic surfactant hexadecyltrimethyl ammonium bromide (HTAB). As the environment is sustained at pH=6, the ferritin molecules are above their pI point of 4.8 and possess a negative charge. The ionic strength is sustained at 0.1 M;
b) Ferritin shell cross-linked over a liposome. The cross-linking is accomplished by glutaraldehyde;
c) Ferritin aggregate obtained after the liposomes are dissolved by solubilisation with non-ionic surfactant (Tween 20).
Table I. Summary of the interplay of specific and electrostatic interactions in ferritin attachment to biotinylated vesicles. The data was confirmed by using both ferritin-avidin or ferritin-streptavidin conjugates.
|
Ferritin-Avidin or |
System |
|
Streptavidin Conjugate at |
Negatively Charged Vesicles |
Slightly Positively Charged |
Ferritin Pre-treated with Free Biotin |
|
pH = 7 |
N # |
C * |
N o |
|
pH = 4 |
C * |
LC # |
N o |
Key:
C = coating, LC = low coating, N = no coating or agglutination
*
Favourable electrostatics,
#
Unfavourable electrostatics (repulsion),
o
No specific binding
Figure 2.
Liposome/ferritin assemblies obtained by "lock-and-key" binding.
a) Schematics of the avidin-biotin or streptavidin-biotin assembly;
b) Electron micrographs of vesicles covered with ferritin by avidin-biotin binding. The vesicles incorporate 10 mol% of biotinylated lipid (Biotin-LC-DPPE) and approximately equimolar amount of HTAB.;
c) Schematics of the assembly based on Concanavalin A-mannan binding. The liposomes are first coated with cholesterol-anchored polysaccharide layer;
d) Electron micrograph of vesicle whose ferritin shell is attached by Concanavalin A-mannan binding.
Concluding Remarks
Our study demonstrates, that complex microstructured particles could be assembled from liposomes and ferritin by using colloid interaction schemes including either non-specific or a combination between non-specific and specific interactions. While the microstructured supraparticles presented above are only model systems, suitable for observation by TEM, further development in the area could bring as closer to the fabrication of functional nanometer-sized assemblies, that may include combinations of enzymes and other biologically active molecules. The knowledge, control, and multi-step modification of the colloid interactions in the system appear to be
the real tool for "smart" particle assembly.
Acknowledgement. This study was performed in collaboration with M. Ohta. The author is very thankful to Prof. K. Nagayama, Dr. S. Ebina and Dr. H. Yoshimura for their help and guidance.
Reprint requests welcome: velev@che.udel.edu
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