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:

The acquired data can find application in the future fabrication of microstructured, multicomponent or functionalised protein and liposome/protein assemblies.

 

TEM micrograph


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

 

TEM micrograph


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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