Genetically Engineered Nanofiber-Like Viruses For Tissue Regenerating Materials
Anna Merzlyak, Shyam Indrakanti, and Seung-Wuk Lee*
Department of Bioengineering, UniVersity of California, Berkeley, Physical Biosciences DiVision, Lawrence Berkeley National Laboratory, Berkeley Nanoscience and Nanoengineering Institute, Berkeley, California 94720
Nano Letters 2009 Vol 9, No 2 846-852
This paper introduces a novel biomaterial composed of nanofiber-like viruses, M13 Bacteria Phage. These viruses are capable of providing an ordered extracellular matrix for cell adhesion and display signaling motif on their coat protein to direct cell behavior and growth. As shown in Figure 1, The different functionalities of viruses come from their shape, charge, peptide displayed which can be modified through chemical or genetic approaches.
RGD and IKVAV peptides were displayed at the N-terminus of viral major coat protein VIII (pVIII). RGD is a cell adhesion integrin binding motif and IKVAV is a laminin motif known to promote neural adhesion and extention. The pVIIIs are expressed 2700 times on a single phage, representing 99% of the phage surface. Peptides of up to 8 amino acids can be inserted at pVIII.
Non-toxicity of the phages was shown through CyQuant assay. CyQuant assay is based on fluorescent intensity after cyanine dye binds to nucleic acid, which is proportional to cell count. Cell culture with phage had similar CyQuant fluorescent readings in comparison to the Cells with out phage on day 1, 3 and 5.
To verified the effect of RGD and IKVAV, hippocampal neural progenitor cells (NPCs) were selected for the potential application of neural regeneration in case of spinal cord injury. Immunostainning showed RGD and IKVAV phage colocalize with the cell while the wild phage does not. This shows the specificity interaction of their engineered phage with Cell. Further microscopy studies from SEM showed NPCs on RGD or IKVAV spread along the direction of the phage film and well spread while the cell on wild phage showed aggregation instead of extension.
3D Phage fiber in agrose gel was then studied. RGD and IKVAV phage matrices were able to promote unidirectional cell spreading and elongation by 30% compare to wild phage. These results shows the phages were chemically encouraging and guides the cell to grow in specific direction. This is particular useful in neural regeneration, because neural network is well known for its directionality that provide the platform for our cognitive functions. The ability to recreate such directional guidance is significant.
Potential Improvement:
During 3D culture experiments, RGD, IKVAV, and Wild phages were compared. I will add a Laminin positive control to show how good the RGD and IKVAV peptides compare with the native Laminin matrix that NPCs grow on in vivo.
The other improvement might be the colocalization of the phage with the cell. Author suggested specific interaction. But colocalization does not mean interaction. She did not go the extra step to prove it is actually interaction. I will do an additional binding assay to quantify the interaction.
9 comments:
Very interesting paper...
"The different functionalities of viruses come fro their shape, charge, peptide displayed which can be modified through chemical or genetic approaches."
I understand that the ECM contains proteoglycans which also derive their functionalities from their shape and charge. You can say this biomaterial was bio-inspired. Another interesting example of how form fits function. This idea is prevalent all through biology.
How are the phages immobilized to create a stable surface on which the cells can grow?
Can peptides also be made to be expressed on the pIX and pIII? I'm assuming these proteins are located at the top and bottom of the phage (based on figure 1).
If there are sufficient pIX and pIII, it seems like the phage can act as a bridge connecting two different targets if each coat protein expressed different peptides.
Are the phage aligned in a particular orientation?
I have learned in bioec118 that the neighboring ammino acids to RGD sites in biology are important as well as the RGD itself. Testing the addition of ammino acids flanking the RGD might be interesting to pursue.
I found this paper to be very interesting because of the novelty of genetically engineering viruses for tissue engineering. The paper mentioned that you can modify all the proteins surrounding the M13 phage and also the shape of the phage. This would mean potential engineering strategies for other applications as well. I was wondering if they ever tested the phage with NPC's in vivo? Conditions in vivo might alter the outcome.
Michelle: I think the phages were on a substrate so they were in the form of a film. The paper also mentioned "liquid crystalline phage matrices" so I think the phage was somehow immobilized in crystal form.
Fergus: It can be expressed on PIII. but I am not sure about p9. My guess is people is trying but I have not read any paper that uses p9 extensively. there may be 5 copies of each pIII and p9 on a single phage, I think theoretically it can form a bridge...
Michelle: phages were fixed onto a glass slides first, the fixation at the same time" crystallizes" the phage, then cell were plated onto the phage film...
Andrew: since the phage is filamentous, if you have high enough density, phage will align themselves in one direction, there maybe two orientations ( one is the 180 degree flip of the other)
How was it determined that RGD and IKVAV peptides should be the ones displayed on the major coat protein? Was it strictly based on the roles they played in well-known protein interactions, or was it somehow experimentally determined?
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