Transport nanodevice immobilized specific biomolecules

We developed new polymer nanoparticles embedding quantum dots with artificial cell membrane-biointerface as a highly sensitive bioimaging probe. These nanoparticles were prepared by assembling phospholipid polymer as a platform and oligopeptide as a bioaffinity moiety on the surface of the nanoparticles. They showed high resistance to non-specific cellular uptake from HeLa cells due to the nature of phospholipid polymer with phosphorylcholine groups. On the other hand, when arginine octapeptide was immobilized on their surface, they could permeate the membrane of HeLa cells effectively and good fluorescence based on quantum dots could be observed. Thus we obtained stable fluorescent polymer nanoparticles covered with artificial cell membrane, which are useful as an excellent bioimaging probe evaluation for biomolecular function in the target cells.

To confirm the selectivity of uptake to cells, effect of chemical structure of octapeptide was examined. Many research groups reported that arginine-rich peptide is useful as a cell penetrating peptide. However, the abilities of other oligopeptides to penetrate the cell membrane have not been well characterized. Thus, we evaluated the function of various octapeptides as a cell penetrating peptide by using our PMBN/PLA/QD as an analyzing tool. We selected the octapeptides having simple sequences constructed from just one kind of amino acid, tyrosin (Y, hydrophobic), asparagine (N, hydrophilic), glutamic acid (E, hydrophilic and anionic), histidine (H, hydrophilic and weakly cationic), lysine (K, hydrophilic and cationic), or arginine (R, hydrophobic and cationic). Only K8 and R8 conjugated PMBN/PLA/QD could internalize in HeLa cells. It is well known that R8 and K8 work as a cell penetrating peptide. This result indicated that the hydrophilic and cationic nature of oligopeptides play a key role in the cell membrane permeation. In order to understand the mechanism of cell penetration induced by these hydrophilic and cationic oligopeptides, it will be required to investigate the relationship between surface cationic density on the nanoparticles and cell membrane permeation.

It is important to understand the effect of sequence of octapeptide composed of inert and active amino residue for internalization to cells. We prepared a series of octapeptide by glycine(G) and arginine(R) as G8, GGGGGGRR(G8R2), GGGGRRRR(G4R4), GGRRRRRR(G2R8), and R8. Although the surface-potential of original PNBN/PLA/QD was slightly negative, it altered by immobilization of octapeptide on the surface. Image of cells examined with fluorescence microscopy is shown. After applying the various nanoparticles, they started to internalize into the cells. However, we observed that only R8-immobilized nanoparticles could internalize into cells.

The PMBN/PLA/QD is good probe for evaluation of biomolecules to understand their performance. And R8-peptide is effective to induce cell internalization. Moreover, R8-PMBN/PLA/QD is a most suitable material for conducting the kinetic analysis of cell membrane permeation.

Publications

S. Mieda, Y. Amemiya, T. Kihara, T Okada, T. Sato, K. Fukazawa, K. Ishihara, N. Nakamura, J. Miyake, C. Nakamura, Mechanical force-based probing of intracellular proteins from living cells using antibody-immobilized nanoneedles, Biosensors Bioelectron. 15(1), 323-329 (2012).
T. Aikawa, T. Konno, M. Takai, K. Ishihara, Continuous preparation of a spherical phospholipid polymer hydrogel for cell encapsulation using a flow-focusing microfluidic channel device, Langmuir 28(4), 2145-2150 (2012).