Page 3 of 5
Journal of the American Chemical Society
importantly, the extent of guest release was found to be identical
to that without the protein from this assembly over the same
amount of time (Figure 4d and S7). These results indicate that the
guest release from the P1 assembly is indeed due to specific
ligand-protein interaction induced disassembly.
1
2
3
4
5
6
7
8
Finally, since the degradable components of this polypeptide
are considered biocompatible, we envisaged that the amphiphilic
polypeptide itself might be biocompatible, i.e. not cytotoxic. To
test this, we studied in vitro cell viability using an Alamar blue
assay with HeLa cell lines and found the cells to be ~80% viable
even at 250 µg/ mL of polymer solution (Figure S8).
9
10
11
12
13
14
15
16
17
In summary, we have designed and synthesized a polypeptide,
the amphiphilic nature of which provides
a
nanoscale
supramolecular assembly that can stably encapsulate hydrophobic
guest molecules in aqueous media. The polypeptide is engineered
to present a protein-specific ligand in its hydrophilic face. We
show that the binding interaction between the ligand moiety and
the complementary protein causes the assembly to fall apart. This
binding-induced disassembly has been shown to be specific to
bCAII and to cause release of guest molecules. The extent of
guest release in response to protein binding was found to be
substantial (~85%). This feature, along with the simplicity of the
synthetic route, highlights the utility of peptide-based assemblies
Figure 3. DLS of P1 aggregates in presence of a) lysozyme. b) BSA
(Concentration of P1 = 50 µM). c) Chemical structure of P2. d) Time
dependent DLS of P2 in presence of bCA-II protein.
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
for protein-induced supramolecular disassembly.
Although
fact that the structurally identical polymer P2, except for the
ligand presence, did not disassemble in the presence of bCA-II
rules out this alternate hydrolysis based disassembly pathway
(Figure 3d).
responsive molecular assemblies have been consistently targeted
for applications such as delivery and diagnostics,10 systems that
respond to protein activity are very limited. These are interesting,
because aberrant protein activity is the basis for all genetic
diseases. While there have been significant efforts on systems that
respond to enzymatic activity variations,11 assemblies that
respond to non-enzymatic proteins are very limited. The
polypeptides, outlined here, are poised to make a significant
impact in this area with their biocompatible, biodegradable, and
high fidelity responsive disassembly characteristics.
Since the protein binding causes a disassembly, it is possible
that we can utilize this process to cause the guest molecules to be
released. Since the hydrophobic guest is insoluble in water, it is
likely that it will simply precipitate out of solution. To test this
hypothesis, DiI-encapsulated P1 (50 µM) was treated with bCAII
(30 µM) and the possible guest release of the DiI was monitored
by absorption spectroscopy. Indeed, the absorbance of DiI
decreased overtime in presence of bCA-II, while there was no
such change in the absence of bCA-II (Figure 4a and 4b). These
observations indicate that the observed process is a protein-
specific molecular release (~85% release over 33h, Figure 4c). To
further test whether the process is specific to bCA-II, in a control
experiment, we exposed the same solution to non-complementary
proteins with different surface charges, viz. pepsin, BSA and
lysozyme. The guest release was found to be relatively
insignificant (Figure S6). Similarly, we also monitored the
possible guest release from P2 aggregate and it was <20%. More
Supporting Information
Detailed synthetic procedures and characterizations of the
polymers. This material is available free of charge via the Internet
ACKNOWLEDGMENT
We thank the NIGMS of the National Institutes of Health (GM-
065255) for support.
REFERENCES
(1) (a) Gao,Y.; Zhao, F.; Wang, Q.; Zhanga, Y.; Xu, B. Chem. Soc. Rev.
2010, 39, 3425–3433. (b) Breslow, R. Acc. Chem. Res. 1995, 28,
146-153. (c) Zhao, H.; Foss, F.W.; Breslow, R. Jr. J. Am. Chem. Soc.
2008, 130, 12590–12591. d) Bruns, C. J.; Stoddart, J. F. Acc. Chem.
Res. 2014, 47, 2186−2199. (e) Coskun, A.; Banaszak, M.; Astumian,
R. D.; Stoddart, J. F.; Grzybowski, B. A. Chem. Soc. Rev. 2012, 41,
19–30.
(2) (a) Schreiber, G.; Haran, G.; Zhou, H.-X. Chem. Rev. 2009, 109,
839-860. (b) Keskin, O.; Gursoy, A.; Ma, B.; Nussinov, R. Chem.
Rev. 2008, 108, 1225−1244. (c) Ito, T.; Tashiro, K.; Muta, S.;
Ozawa, R.; Chiba, T.; Nishizawa, M.; Yamamoto, K.; Kuhara, S.;
Sakaki, Y. Proc. Natl. Acad. 2000, 97, 1143-1147.
(3) (a) Ramírez-Tapia, L. E.; Martin, C. T. J. Biol. Chem. 2012, 287,
37352-37361. (b) Papantonis, A.; Cook, P. R. Chem. Rev. 2013, 113,
8683−8705. (c) Michaelis, J.; Treutlein, B. Chem. Rev. 2013, 113,
8377−8399.
(4) (a) Wong, P.; Hampton, B.; Szylobryt, E.; Gallagher, A. M.; Jaye,
M.; Burgess, W. H. J. Biol. Chem. 1995, 270, 25805–25811. (b)
Spivak-Kroizman, T.; Lemmon, M. A.; Dikic, I.; Ladbury, J. E.;
Pinchasi, D.; Huang, J.; Jaye, M.; Crumley, G.; Schlessinger, J.;
Lax, I. Cell 1994, 79, 1015-1024. (c) Powell, A. K.; Fernig, D. G.;
Turnbull, J. E. J.; Biol. Chem. 2002, 277, 28554-28563.
Figure 4. DiI release from P1 micelle a) in presence and b) in absence
of bCA-II protein (Concentration of P1 = 50 µM); c) Plot of % release
with time; d) Plot of % release of DiI from control polymer P2 in
presence and absence of bCA-II.
(5) (a) Zhou, M.; Smith, A. M.; Das, A.K.; Hodson, N.W.; Collins,
R.F.; Ulijn, R.V.; Gough, J. E. Biomaterials. 2009, 30, 2523–2530.
3
ACS Paragon Plus Environment