Water-soluble peptide-coated nanoparticles: Control of the helix structure and enhanced differential binding to immune cells

Article abstract of DOI:10.1021/ja107588q

The stabilizing action of C^α-tetrasubstituted α-amino acids inserted into a sequence of short peptides allowed for the first time the preparation of water-soluble nanoparticles of different materials coated with a helix-structured undecapeptide. This peptide coating strongly favors nanoparticle uptake by human immune system cells.

Full text of DOI:10.1021/ja107588q

Published on Web 12/10/2010
Water-Soluble Peptide-Coated Nanoparticles: Control of the Helix Structure
and Enhanced Differential Binding to Immune Cells
Iria M. Rio-Echevarria,†,‡ Regina Tavano,§,| Valerio Causin,† Emanuele Papini,§,| Fabrizio Mancin,†,*
and Alessandro Moretto†,*
Dipartimento di Scienze Chimiche, UniVersita` di PadoVa, Via Marzolo 1, 35131 PadoVa, Italy, Dipartimento di
Biologia, UniVersita` di PadoVa, Via U. Bassi 58/B, 35131 PadoVa, Italy, Dipartimento di Scienze Biomediche
Sperimentali, UniVersita` di PadoVa, Via U. Bassi 58/B, 35131 PadoVa, Italy, and Centro di Ricerca
Interdipartimentale per le Biotecnologie InnoVatiVe (CRIBI), UniVersita` di PadoVa, Via U. Bassi 58/B,
35131 PadoVa, Italy
Received August 22, 2010; E-mail: fabrizio.mancin@unipd.it; alessandro.moretto.1@unipd.it
Abstract: The stabilizing action of CR-tetrasubstituted R-amino
acids inserted into a sequence of short peptides allowed for the
first time the preparation of water-soluble nanoparticles of different
materials coated with a helix-structured undecapeptide. This
peptide coating strongly favors nanoparticle uptake by human
immune system cells.
Self-organization of simple building blocks to generate large
structures is an intriguing strategy for the formation of complex
systems.1 In particular, monolayer-protected nanoparticles rank
among the top representatives of such an approach.2 In their
synthesis, a mixture of molecular precursors is converted by the
addition of appropriate reagents into a collection of monodisperse
and well-organized objects wherein an inorganic core is coated with
a tightly packed three-dimensional (3D) monolayer of organic
species.3 It is not surprising that in the past decade such organization
has been exploited for several important applications, ranging from
catalysis4 and sensing5 to energy conversion,6 development of new
materials,7 and biomedicine.8
A target that has been extensively addressed over the years is
the preparation of water-soluble nanoparticles coated with peptides
that have a defined helix secondary structure. Viewed from the
Figure 1. (top) Chemical structures of the nanoparticle-protecting peptides
outside, such nanoparticles closely resemble artificial globular
proteins, where the biocompatibility of the coating, the chiral space
organization, and the functional group crowding could pave the
way for the realization of complex functional systems.9 To date,
such a goal has been frustrated by the complex interligand
interactions that can be established between spatially close peptide
chains.10 These interactions strongly affect the monolayer structure
as well as the nanoparticle solubility. As a result, the few examples
of similar systems reported to date are soluble in nonaqueous
solvents only.11
To address this problem, we turned our attention to peptides con-
taining R-aminoisobutyric acid (Aib). Ca-tetrasubstituted R-amino
acids are known to stabilize helix structures, even in short
peptides,12 and we reasoned that they could help to maintain this
structure even after nanoparticle grafting11 and exposure to aqueous
solutions. For this study, we selected the water-soluble undecapep-
tide H-Ala3-(Aib-Ala)4-OMe (OMe ) methoxy), which is known
to form a helix in solution13 and is characterized by a free
1 (upper structure) and 2 (lower structure). (bottom) Representative TEM
micrographs (left) and CD spectra (right) of 1 and undecapeptide-coated
Au, Ag, Pt, and ormosil (Si) nanoparticles (see the SI for the CD spectrum
of 2).
N-terminal amino group that allows easy conjugation of the peptide
with nanoparticle anchoring moieties such as trityl (Trt)-protected
mercaptopropionic acid (for metal nanoparticles; peptide 1 in Figure
1) or 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane (for silica
nanoparticles; peptide 2 in Figure 1) via standard solution chemistries.
Significantly, these conjugates are water-soluble, like the parent
peptide itself, and the helix structure is maintained even in this
solvent. Moreover, the peptide conjugates work as universal nano-
particle capping agents, allowing for the straightforward prepara-
tion of different peptide-protected nanoparticles with gold, silver,
platinum, and organic-modified silica (ormosil) cores using standard
protocols [Figure 1 and Figure S1 in the Supporting Information
(SI)]. Metal nanoparticles (1-2 nm diameter) were prepared by
chemical reduction (with NaBH4) of the corresponding metal salts
in the presence of peptide conjugate 1, in a methanol/water mixture.
In addition to TEM analysis, formation of the nanoparticles was
confirmed by UV-vis absorption spectra, where weak (because of
the small nanoparticle size) metal-dependent plasmonic bands were
† Dipartimento di Scienze Chimiche.
‡ Dipartimento di Biologia.
§ Dipartimento di Scienze Biomediche Sperimentali.
| CRIBI.
9
8
J. AM. CHEM. SOC. 2011, 133, 8–11
10.1021/ja107588q  2011 American Chemical Society

C O M M U N I C A T I O N S
observed (Figure S4). Ormosil nanoparticles (10-20 nm diameter)
were prepared by ammonia-catalyzed polymerization of surfactant-
stabilized microemulsions of vinyltriethoxysilane (VTES) in water
and in the presence of peptide conjugate 2.14
All of the nanoparticles prepared are fully soluble not only in
water but also in alcoholic (methanol, ethanol) solvents. The peptide
coating is so stable that the nanoparticles can be purified by
reversed-phase HPLC, even using trifluoroacetic acid-containing
eluents (Figures S5-S7).
Most importantly, circular dicroism (CD) analysis confirmed that
the peptide chains retain a helix structure after nanoparticle
grafting.15 In particular, two negative bands at 225-227 nm (peptide
n f π* transition) and 206-208 nm (peptide π f π* transition)
that are typical of helix structures16 were detected in the spectra of
both the free peptide in water and the peptide-coated nanoparticles
(Figure 1). Interestingly, the ratio of the intensities of the n f π*
and π f π* bands (R) for the free peptide in water was 0.56 (Figure
1 and Figure S10), and the π f π* band was centered at 206 nm;
these values have been attributed to a significant population of
concomitant 310- and R-helix conformations.16 When the peptide
was grafted onto the surface of Ag and Pt nanoclusters, the R values
increased to 0.67 and 0.75, respectively, and a red shift in the π f
π* band was observed, indicating an increase in the amount of the
R-helix structure. Finally, R values close to 1 (with the position of
the π f π* band reaching 208 nm) were detected in the case of
Au and ormosil particles, as expected for a full R-helix structure
of the peptide. Thermogravimetric analysis (TGA) (Figures S2 and
S3) indicated that this trend is related to the packing of the peptides
on the particle surfaces. In fact, in the case of Ag and Pt
nanoparticles, the calculated peptide footprints were 0.11 and 0.12
nm2, respectively, which are similar to the values reported for 2
nm gold nanoparticles coated with short peptides in the 310-helix
conformation.11a In the case of Au and ormosil nanoparticles, the
peptide footprints increased to 0.65 and 0.69 nm2, respectively.17
Apparently, when they are grafted onto the nanoparticles, the
peptides switch their conformation from the 310-helix to the bulkier
R-helix, likely to achieve optimal packing on the nanoparticle
surface. Interestingly, in the case of small (1-2 nm) gold nano-
particles coated with small 310-helix-forming peptides, it has been
reported that the peptide density on the particle surface is not
controlled by the peptide-to-metal ratio used in the synthesis.11a
The results reported here may indicate that, on the contrary, the
nature of the metal core could be crucial in determining the degree
of peptide surface coverage and consequently the kind of helix
structure assumed.
Figure 2. Lengths and footprints of ormosil nanoparticle-grafted peptides
2 (R-helix) and 3 (unordered with minimum and maximum possible lengths
corresponding to the lengths of the 310-helix and the extended chain,
respectively).
trialkoxysilane derivative of the cyanine dye CHROMIS 678-Z (4)
(see the SI) was used to ensure covalent grafting of the dye to the
silica network, thus preventing it from leaking out of the nanopar-
ticles. Biological tests were simultaneously performed on dye-doped
nanoparticles that were either uncoated or coated with either
peptide-silane derivative 2 (Figure 1) or 3 (Figure 2). Derivative
3, which is based on the pentameric sequence H-Aib-Ala4-OMe,
was selected as an unordered reference analogue of 2 on the basis
of the following considerations: (i) the Aib-Ala4 sequence grants
water solubility, the maximum possible conservation of the residues
present in 2, and the inability to form helix structures (which is
different from sequences with more Aib residues or with the Aib
residue in a more central position); (ii) the length of the nanopar-
ticle-grafted unordered 3 spans the range between those of the 310-
helix and the extended (ꢀ-sheet like) conformation20 and hence
should be very similar to the length of 2 in the R-helix conformation
(Figure 2). As expected, the CD spectrum of the 3-coated ormosil
nanoparticles (Figure S11) demonstrated that in these nanoparticles
the grafted peptides are in an unordered conformation. Moreover,
TGA analysis indicated that the footprint of 3 on the ormosil
particles is 0.43 nm2. This value is smaller than that of 2 on the
same nanoparticles (0.69 nm2; see above) and reflects well the lower
bulkiness of the unordered conformation in comparison with the
R-helix.
In addition to their ability to form 310-helices even in short
sequences, Aib-rich peptides show several other intriguing proper-
ties. They are produced as secondary metabolites by several fungi
and are characterized by antibacterial, antiviral, and antifungal
activities, all of which are due to their ability to bind cell membranes
and increase their permeability.18 Moreover, the presence of CR-
tetrasubstituted R-amino acids makes these peptides highly resistant
to proteolysis, which is crucial for the development of effective
peptide-based drugs.19 On this basis, nanoparticles coated by such
peptides may present interesting biological properties.
Dye-doped ormosil nanoparticles were incubated with the
different cell lines for 6 h at 37 °C in culture medium containing
fetal calf serum (FCS). Their uptake and acute toxicity effects were
investigated by cytofluorimetry and confocal microscopy experi-
ments. Neither the coated nor uncoated nanoparticles showed any
acute toxicity to the cell lines investigated under the experimental
conditions employed.
An initial investigation of the interaction of these nanoparticles
and biological systems was then performed by measuring their
uptake by different cell lines, including both epithelial cancer cells
(HeLa and A431) and cells from the immune system (human
macrophages and primary blood monocytes). For these studies, we
used 15 nm ormosil nanoparticles since they can be easily doped
with fluorescent dyes without affecting either the photophysical
The results of the flow cytofluorimetric uptake analysis (Figure
3) are remarkable. Epithelial cells (HeLa, A431) associated to the
different nanoparticles with a dose-dependent behavior related to
the intrinsic uptake ability of the different cell lines but substantially
independent of the features of the nanoparticle surface. On the
contrary, association to human macrophages and monocytes was
strongly dependent on the presence of the peptide coating on the
nanoparticles. It is worth nothing that (i) these cells efficiently
properties of the latter or the surface of the nanoparticles.14
A
9
J. AM. CHEM. SOC. VOL. 133, NO. 1, 2011
9

C O M M U N I C A T I O N S
Figure 3. (top) Dose-dependent nanoparticle uptake by HeLa cells, A431
cells, macrophages, and monocytes treated with ormosil nanoparticles: (red)
uncoated; (green) coated with the unordered pentapeptide; (blue) coated
with the helix undecapeptide. Uptake is expressed as mean fluorescence
intensity (MFI) as determined by cytofluorimetry. A logarithmic scale is
used to allow a better appreciation of the trends for the single cell lines.
(bottom) Comparison (linear scale) of the uptake of the different cell lines
treated with the different nanoparticles (50 µg/mL) for 6 h at 37 °C.
Figure 4. Confocal microscopy images showing the intracellular distribu-
tion of nanoparticles in human macrophages treated with the different
nanoparticles (50 µg/mL) for 6 h at 37 °C. The left column shows the
Lysotracker probe localization, the central column the localization of the
nanoparticles, and the right column the superposition of the first two columns
to highlight the colocalization. Arrows in the bottom-row panels, which
show magnified details of the upper panels, point to selected examples of
acidic endo/phagosomes loaded with helix peptide-coated nanoparticles.
internalize uncoated ormosil nanoparticles and (ii) surface func-
tionalization with neutral hydrophilic molecules greatly decreases
the uptake.14b,21 Here, in the case of macrophages, the amount of
peptide-coated nanoparticles internalized was up to 45-fold greater
than that of the uncoated nanoparticles, irrespective of the peptide
derivative present in the coating (Figure 3).
The results obtained with the monocytes are even more interest-
ing. In this case, not only was the uptake of both of the peptide-
coated nanoparticles still much more efficient than that of the
uncoated ones, but also this cell line even appeared to be capable
of recognizing the organization of the particle surface. Indeed, the
particles coated with the helical undecapeptide 2 were taken up
2-3-fold more efficiently than those protected with the unordered
pentapeptide 3.
Confocal microscopy experiments (Figure 4) confirmed that the
peptide-coated nanoparticles associate to macrophages much more
effectively than do uncoated nanoparticles. Moreover, such an
analysis demonstrated that virtually all of the cell-associated
nanoparticles were engulfed and transported into the cell cytoplasm
with a compartmentalized signal that tended to be distributed around
the perinuclear region. High colocalization of the nanoparticle
signals with the Lysotracker lysosome probe suggested that they
were endocytosed and accumulated in acidic endolysosomal/
phagosomal membrane compartments, as expected for this kind of
cell. In contrast, but in full agreement with flow cytofluorimetry
data, uncoated nanoparticles gave a very low fluorescence signal
under these conditions, indicating relatively less efficient cell
internalization.
we have found that these systems, which form a precise
helical-spherical domain, have intriguing biological properties.
Indeed, we observed that peptide-coated and uncoated nanoparticles
associate to two cancer cell lines with the same efficacy, while the
presence of the peptide induces a marked gain of uptake by
monocytes and antigen-presenting macrophages. Such ability is
strongly related to the biomimetic peptide coating and could be
strengthened, in the case of monocytes, by the presence of an
organized helix structure. Several factors, such as different surface
features, hydrophobicity, and even specific recognition by cell-
dependent receptors, may be the basis of this behavior, which will
require further investigation. Possible applications of these proper-
ties range from the control of inflammatory phenomena and
immune-system-related disorders to the development of immuniza-
tion strategies. Such developments, as well as the behavior of
peptide-coated nanoparticles based on Ag, Au, and Pt cores, are
currently under investigation in our laboratories.
Acknowledgment. Financial support for this research was partly
provided by the European Union (FP7, Grant 201031 NANO-
PHOTO). The authors thank Prof. C. Toniolo and Prof. P. Scrimin
for encouragement and helpful discussions, F. Selvestrel for the
synthesis of 4, and E. Longo for help with chemical characterization.
In summary, we have de novo designed, synthesized, and
characterized a new set of water-soluble nanoparticles coated with
peptides in helix conformations. Apparently, the helix structure
switches from the 310- to the R-helix conformation with decreasing
peptide density on the nanoparticles. This is controlled either by
the nature of the nanocrystal core (for metal nanoparticles) or the
synthetic conditions (for ormosil nanoparticles). More importantly,
Supporting Information Available: Experimental details and
synthesis and characterization of the peptide ligands and the nanopar-
ticles. This material is available free of charge via the Internet at http://
pubs.acs.org.
9
10 J. AM. CHEM. SOC. VOL. 133, NO. 1, 2011

C O M M U N I C A T I O N S
Ceccato, M.; Donkers, R. L.; Fabris, L.; Pace, G.; Maran, F. Langmuir
2006, 22, 10584–10589. (c) Higashi, N.; Kawahara, J.; Niwa, M. J. Colloid
Interface Sci. 2005, 288, 83–87. (d) Higuchi, M.; Nagata, K.; Abiko, S.;
Tanaka, M.; Kinoshita, T. Langmuir 2008, 24, 13359–13363. (e) Schade,
M.; Moretto, A.; Donaldson, P. M.; Toniolo, C.; Hamm, P. Nano Lett.
2010, 10, 3057–3061.
References
(1) For example, see: Balzani, V.; Credi, A.; Venturi, M. Chem.sEur. J. 2002,
8, 5524–5532.
(2) (a) Shenhar, R.; Rotello, V. M. Acc. Chem. Res. 2003, 36, 549–561. (b)
Goesmann, H.; Feldmann, C. Angew. Chem., Int. Ed. 2010, 49, 1362–1395.
(3) (a) Templeton, A. C.; Wuelfing, M. P.; Murray, R. W. Acc. Chem. Res.
2000, 33, 27–36. (b) Daniel, M. C.; Astruc, D. Chem. ReV. 2004, 104,
293–346. (c) Gentilini, C.; Pasquato, L. J. Mater. Chem. 2010, 20, 1403–
1412.
(12) (a) Karle, I. L.; Balaram, P. Biochemistry 1990, 29, 6747–6756. (b) Toniolo,
C.; Crisma, M.; Formaggio, F.; Peggion, C. Biopolymers 2001, 60, 396–
419.
(13) (a) Pavone, A.; Benedetti, E.; Di Blasio, B.; Pedone, C.; Santini, A.; Bavoso,
A.; Toniolo, C.; Crisma, M.; Sartore, L. J. Biomol. Struct. Dyn. 1990, 7,
1321–1331. (b) Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Santini,
A.; Bavoso, A.; Toniolo, C.; Crisma, M.; Sartore, L. J. Chem. Soc., Perkin
Trans. 2 1990, 1824–1837.
(14) (a) Roy, I.; Ohulchanskyy, T. Y.; Pudavar, H. E.; Bergey, E. J.; Oseroff,
A. R.; Morgan, J.; Dougherty, T. J.; Prasad, P. N. J. Am. Chem. Soc. 2003,
125, 7860–7865. (b) Rio-Echevarria, I. M.; Selvestrel, F.; Segat, D.;
Guarino, G.; Tavano, R.; Causin, V.; Reddi, E.; Papini, E.; Mancin, F. J.
Mater. Chem. 2010, 20, 2780–2787.
(15) The helix structure of the peptides grafted to the nanoparticles was further
confirmed by 1H NMR experiments performed on Au nanoparticles (Figures
S8 and S9).
(16) (a) Formaggio, F.; Crisma, M.; Rossi, P.; Scrimin, P.; Kaptein, B.;
Broxterman, Q. B.; Kamphuis, J.; Toniolo, C. Chem.sEur. J. 2000, 6,
4498–4504. (b) Toniolo, C.; Polese, A.; Formaggio, F.; Crisma, M.;
Kamphuis, J. J. Am. Chem. Soc. 1996, 118, 2744–2745.
(4) (a) Prins, L. J.; Mancin, F.; Scrimin, P. Curr. Org. Chem. 2009, 13, 1050–
1064. (b) Bonomi, R.; Selvestrel, F.; Lombardo, V.; Sissi, C.; Polizzi, S.;
Mancin, F.; Tonellato, U.; Scrimin, P. J. Am. Chem. Soc. 2008, 130, 15744–
15745. (c) Schatz, A.; Reiser, O.; Stark, W. J. Chem.sEur. J. 2010, 16,
8950–8967.
(5) Bunz, U. H. F.; Rotello, V. M. Angew. Chem., Int. Ed. 2010, 49, 3268–
3279.
(6) Imahori, H.; Fukuzumi, S. AdV. Mater. 2001, 13, 1197–1198.
(7) Nie, Z. H.; Petukhova, A.; Kumacheva, E. Nat. Nanotechnol. 2010, 5, 15–
25.
(8) (a) Giljohann, D. A.; Seferos, D. S.; Daniel, W. L.; Massich, M. D.; Patel,
P. C.; Mirkin, C. A. Angew. Chem., Int. Ed. 2010, 49, 3280–3294. (b)
Boisselier, E.; Astruc, D. Chem. Soc. ReV. 2009, 38, 1759–1782.
(9) In a different but related application, peptide-coated water-soluble nano-
particles wherein the peptides are designed to fold in a helix structure under
controlled conditions have been described. See: Aili, D.; Mager, M.; Roche,
D.; Stevens, M. M. Nano Lett. [Online early access]. DOI: 10.1021/
nl1024062. Published Online: Aug 26, 2010. Aili, D.; Mager, M.; Roche,
D.; Stevens, M. M. Chem. Soc. ReV. 2010, 39, 3358–3370. Aili, D.; Enander,
K.; Baltzer, L.; Liedberg, B. Nano Lett. 2008, 8, 2473–2478. Stevens,
M. M.; Flynn, N. T.; Wang, C.; Tirrell, D. A.; Langer, R. AdV. Mater.
2004, 16, 915–918.
(10) (a) Pengo, P.; Baltzer, L.; Pasquato, L.; Scrimin, P. Angew. Chem., Int.
Ed. 2007, 46, 400–404. (b) Levy, R.; Thanh, N. T. K.; Doty, R. C.; Hussain,
I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Fernig, D. G. J. Am. Chem.
Soc. 2004, 126, 10076–10084. (c) Pengo, P.; Broxterman, Q. B.; Kaptein,
B.; Pasquato, L.; Scrimin, P. Langmuir 2003, 19, 2521–2524.
(11) (a) Fabris, L.; Antonello, S.; Armelao, L.; Donkers, R. L.; Polo, F.; Toniolo,
C.; Maran, F. J. Am. Chem. Soc. 2006, 128, 326–336. (b) Holm, A. H.;
(17) Peptide footprints for the ormosil nanoparticles were calculated by TGA
analysis assuming a 1.5 g/cm3 density of the ormosil core (see the SI).
(18) For example, see: Taira, J.; Shibue, M.; Osada, S.; Kodama, H. Int. J. Pept.
Res. Ther. 2010, 16, 277–282, and references therein.
(19) De Zotti, M.; Biondi, B.; Formaggio, F.; Toniolo, C.; Stella, L.; Park, Y.;
Hahm, K. S. J. Pept. Sci. 2009, 15, 615–619.
(20) Garcia, A. E. Polymer 2004, 45, 669–676.
(21) Tavano, R.; Segat, D.; Reddi, E.; Kos, J.; Rojnik, M.; Kocbek, P.; Iratni,
S.; Scheglmann, D.; Colucci, M.; Rio-Echevarria, I. M.; Selvestrel, F.;
Mancin, F.; Papini, E. Nanomedicine 2010, 5, 881–896.
JA107588Q
9
J. AM. CHEM. SOC. VOL. 133, NO. 1, 2011 11