Labeling of Hyaluronic Acids with a Rhenium-tricarbonyl Tag and Percutaneous Penetration Studied by Multimodal Imaging

Article abstract of DOI:10.1021/acs.bioconjchem.7b00825

Hyaluronic acids were labeled with a rhenium-tricarbonyl used as single core multimodal probe for imaging and their penetration into human skin biopsies was studied using IR microscopy and fluorescence imaging (labeled SCoMPI). The penetration was shown to be dependent on the molecular weight of the molecule and limited to the upper layer of the skin.

Full text of DOI:10.1021/acs.bioconjchem.7b00825

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Communication
Labeling of hyaluronic acids with a Re-tricarbonyl tag and
percutaneous penetration studied by multimodal imaging
Lucas Henry, Nicolas Delsuc, Cécile Laugel, François Lambert, Christophe Sandt, Sarah Hostachy, Anne-
Sophie Bernard, Hélène Charlotte Bertrand, Laurence Grimaud, Arlette Baillet-Guffroy, and Clotilde Policar
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00825 • Publication Date (Web): 23 Jan 2018
Downloaded from http://pubs.acs.org on January 30, 2018
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Labeling of hyaluronic acids with a Re-tricarbonyl tag and
percutaneous penetration studied by multimodal imaging
a,b
a,b
c
a,b
d
Lucas Henry,
Nicolas Delsuc, Cécile Laugel, François Lambert, Christophe Sandt,
a,b
a,b
a,b
e
Sarah Hostachy, Anne-Sophie Bernard,
Arlette Baillet-Guffroy, and Clotilde Policar*
Hélène C. Bertrand, Laurence Grimaud,
c
a,b
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a
Laboratoire des Biomolécules, Département de chimie, École normale supérieure, UPMC
Univ. Paris 06, CNRS, PSL Research University, 24 rue Lhomond, 75005 Paris, France
Sorbonne Universités, UPMC Univ. Paris 06, École normale supérieure, CNRS, Laboratoire
b
des Biomolécules (LBM), 75005 Paris, France. Email : clotilde.policar@ens.fr
c
Laboratory of Analytical Chemistry, Lip(Sys)2, (EA 7357), Faculty of Pharmacy, Paris-Sud,
University of Paris-Saclay, 5 Rue Jean-Baptiste Clément, 92296 Chatenay-Malabry, France
d
SMIS beamline, Synchrotron SOLEIL Saint-Aubin, 91192 Gif-sur-Yvette Cedex, France
PASTEUR, Département de chimie, École normale supérieure, PSL Research University,
e
Sorbonne Universités, UPMC Univ. Paris 06, CNRS, 75005 Paris, France
Hyaluronic acids were labeled with a Re-tricarbonyl used as Single Core Multimodal Probe
for Imaging and their penetration into human skin biopsies was studied using IR
microscopy and fluorescence imaging. The penetration was shown to be dependent on the
molecular weight of the molecule and limited to the upper layer of the skin
Hyaluronic acid (HA) is a polymer of disaccharides made of D-glucuronic acid and D-N-
1
acetylglucosamine moieties, linked by alternating ß-1,4 and ß-1,3-glycosidic bonds, with
7
molecular weights (MW) up to 10 Da. HA is found in tissues and body fluids of almost all
2
living organisms. This highly hydrophilic molecule binds a thousand times its volume of
3
water, and is commonly used as a medicine, for instance injected into joints to treat
4
arthritis or in dermis to fill facial wrinkles. Low MW HAs are only used to maintain hydration
of epidermis after topical application whereas high MW HAs are also used as filing agent
3
after cutaneous injection.
In order to investigate their penetration into skin, HAs of two MW have been labeled with a
multimodal Re-tricarbonyl probe. Several Re(CO)
3
tags bearing a fluorescent and an IR
modality on the same molecular moiety have been recently developed. These SCoMPIs —for
5
-7
Single Core Multimodal Probes for Imaging— show two characteristic IR-absorption bands
-
in the transparency windows of biological tissues in the mid IR (between 1800 and 2200 cm
1
7-9
)
1
, namely E and A for the antisymmetric and symmetric CO-stretching vibrations. They
5
, 10-12
have already been characterized and used as organelle trackers
or for the labeling and
6
, 7, 13-16
11, 15-20
imaging of molecules of biological interest,
such as amino-acids and peptides,
21
22
23
24
peptide-nucleic acids, vitamins, hormones, or medicines. Interestingly, the A
1
-band,
IR-imaging is
a challenging technique. Its lateral resolution is limited to a few µm (λ/2, so about 2.5 µm at
9, 25-28
that is a singly degenerated band, can be used for quantification purpose.
-1
2000 cm or 5 µm) when the detection is optical but resolution down to 20-50 nm can be
29, 30
obtained using near-field techniques, such as AFM-IR.
IR-imaging can be recorded on
dedicated synchrotron beamlines (synchrotron-based FTIR spectromicroscopy or SR-FTIR-
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SM) but also on IR-microscopes that are commercially available with focal-plane array
detectors enabling fast imaging and this will certainly popularize FTIR-based
spectromicroscopy (FTIR-SM) in biology. One interest of the SCoMPIs as IR-probes is their
reliability for quantification in biological environments in which polarities have a wide range
of diversity. Generally speaking, luminescence cannot be directly implemented for absolute
6, 31, 32
quantification, since the quantum yield is highly dependent on the environment.
contrast, the epsilon of the A -band of the Re(CO) moiety is not strongly dependent on the
Therefore, this band can be used for absolute titration, as previously
In
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, 7, 28
solvent.
performed.
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3
These SCoMPIs Re(CO) probes are thus of interest, both for IR-mapping
and quantification purposes, and their grafting onto biomolecules worthy to explore.
To graft the probe onto the HA, a Ugi multicomponent condensation involving the carboxylic
33
34, 35
acid functions of HA was selected for its high efficiency in water,
the most appropriate
35
solvent for HA. A graftable SCoMPI bearing an amine moiety was synthesized in five steps
and was reacted with HA, as described in Scheme 1 and SI.
HAs in two ranges of MW were used (10 kDa and 400-1000 kDa), with a low or high probe to
1
HA (probe/HA) ratio (Table 1). In order to easily monitor the reaction completion using H-
NMR, the Ugi reaction was performed in deuterated solvent (D
growing in the aromatic range (7.5-9.5 ppm) were assigned to the probe grafted on HA
Figure S3, SI). When no more evolution could be seen, the tagged HAs were purified by
2 3
O/CD OD 1/1). New signals
(
filtration over a membrane with a 3000 MW cut-off, in order to remove low-molecular
weight reactants, and the fraction containing HA was then lyophilized.
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a
b
c
Br
Br
N3
NH3+Br-
NHBoc
NHBoc
N
N
N
N
NHBoc
1
2
3
NH3 Cl-
+
1
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NHBoc
N
N
e-f
N
d
N
Cl
OC
Cl
N
N
N
N
N
N
N
Re
Re
N
NHBoc
CO
OC
CO
CO
CO
3
4
5
C-
N+
OH
NH3+Cl-
+
O
HOH2C
HO
O
N
Re
CO
O
N
N
H
X
O
O
Cl
OC
O
N
OH
+
+
HO
NH
OH
CO
n
O
5
O
HOH C
2
H
O
HO
O
OC
CO
O
H O/MeOH
2
OH
O
HO
HN
OC
Re Cl
N
1
/1
NH
OH
or
OH
X =
O
N N
N
n
O
N
6-10
Scheme 1. Synthesis of the graftable SCoMPI 5 and labeling of HA using a Ugi condensation. (a) Boc
THF, 1h, r.t., quantitative. (b) NaN , NaI, acetone/H O, 40h, 35°C, 66%. (c) 2-ethynylpyridine, CuSO
ascorbate, acetone/H O, 3h, r.t, 62%. (d) Re(CO) Cl, toluene, 1h30, 80°C, 88%. (e) CH Cl , TFA, 1h, 25°C. (f)
CH OH, HCl, 10 min, 25°C, 95%.
2
O, DIPEA,
3
2
4
, sodium
2
5
2
2
3
IR-spectroscopy was used to characterize the tagging of the HAs. The number of bands of
CO-stretching of M(CO)s observed in the 2000 cm wavenumber range depends on the local
symmetry, with the doubly degenerated band labeled E possibly split in the case of a low
symmetry. Their energy position vary with the local environment, depending on the
hydrophobicity of the environment for instance, and splittings can be observed upon
aggregation or in the solid state when there is a composite environment. The IR-spectra of
4 and 5 have been recorded in several solvents (Figure S4 and table S2, SI). They show the
two bands in the 2000 cm energy range. As previously published in the case of pyridyl-
triazolyle based Re(CO)
shown in figure S4 and table S2, the positions of the A
depending on the solvent nature, with shifts of about 10 to 20 cm . Similar shifts have
already been reported, with frequencies higher in hydrophobic than in hydrophilic
environment. The spectra of the tagged HAs show several sets of A
exemplified with 7 in figure 1. The presence of several sets of A and E bands is indicative of
the different local environments of the Re(CO) moiety which can be found in a hydrophobic
or hydrophilic environment depending on the fact that the SCoMPI is exposed to the solvent
or buried within the polymer, where its environment may be composite and solid-state like.
-1
8
8
, 9
-1
5
-7, 28
3
, the E-band is split into a doublet due to a low symmetry.
As
1
band and E bands are shifted
-1
3
6
1
and E bands, as
1
3
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Figure 1. IR spectra obtained using ATR mode between 1820 and 2075 cm of 5 in acetone and 7 in water (2
-1
mg·mL ). See also Figure S4 and Table S2 for the spectra of 5 in a range of solvents.
The number of grafted probes per monomeric unit (one D-glucuronic acid and one D-N-
acetylglucosamine moiety) was evaluated by H-NMR when possible (compounds soluble
enough: 6 and 7) and FTIR using a calibration curve with a mixture of HA and 5 (SI). The
results obtained by FTIR for the lightest compounds 6 and 7 were in good agreement with
those obtained by H-NMR. This validates the FTIR protocol that was then applied for the
heavier compounds (8, 9, 10) for which NMR quantification could not be used (see Table 1).
To investigate their ability to permeate through skin, 200 µL of a 2mg·mL aqueous solution
of labeled HAs was applied to the surface of human skin biopsies mounted in a Franz cell
chamber. After a 7- or 24-hour exposure, skin samples were washed, frozen and cut in 10-
1
1
-1
2
µm thick sections that were mounted on CaF windows.
The distribution of the labeled HAs in the skin was investigated using synchrotron radiation
FTIR spectromicroscopy (SR-FTIR-SM, see Figure 2) or FTIR spectromicroscopy (FTIR-SM, see
SI, figures S8-S10). IR spectra were recorded in the 800-4000 cm range, and maps were
generated by integrating specific bands (see SI): A band (2055-2005 cm ) and CH bands in
the 2868-2838 cm range. Areas with high concentration of CH
-1
-1
1
2
-
1
2
indicate a lipid-rich layer,
18, 37
corresponding to the stratum corneum (SC).
Luminescence imaging was also performed
after Hoechst staining to reveal cells nuclei of the viable epidermis (VE).
3
8
18
Table 1. Distribution of HAs in the skin
Molecule
MW of HA
Labeling %
Labeling %
Soluble in
water
Yes
Distribution in the skin
a
b
(kDa)
(IR)
(NMR)
7h
24h
6
10
~5%
~3-4%
Not observed
Hotspots mapped by IR
and fluorescence in the SC
(Figure S8)
7
10
~20%
~20%
Yes
Hardly detectable by IR,
hotspots mapped by
fluorescence in the SC
Homogeneously
distributed in the SC and
very weak amount in the
VE (Figure 2, IR and
fluorescence)
(
Figure S9)
8
9
10
~40%
~20%
~5%
-
-
-
No
No
Yes
/
/
/
/
400-1000
400-1000
1
0
Not observed
Inhomogeneous, hotspots
in the SC (Figure S10)
a
b
%
of functionalized carboxylic acid functions determined by IR. % of functionalized carboxylic acid functions
1
determined by H NMR.
After a 24-hour exposure, 6 was hardly detectable by infrared imaging but was detected by
fluorescence microscopy at some spots where it accumulated (Figure S8, SI). The labeling
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ratio of 6 was probably too low for an IR-detection. Compound 7, with a labeling rate c.a.
four times higher, was thus investigated: its distribution after a 7-hour exposure was non-
homogenous in the skin section, with spots of high accumulation (or hotspots) and large
areas showing no product (Figure S9, SI). After 24 hours, an intense and homogenous signal
was observed both by IR and fluorescence and it was mainly observed in the SC. Only some
weak signals were seen at few areas in the VE (Figure 2). The high MW analog with 5%
tagging (10) was investigated and showed an inhomogeneous distribution at 24 h.
Compounds 8 and 9 were not soluble enough for an epidermal application. The results are
summarized in Table 1.
1
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Figure 2. Map of a 10 µm-thick skin cryosection after a 24-hour exposure with 7, mounted on a CaF window
2
(
2
scale bar = 10 µm) (a-b) SR-FTIR-SM images based on the integration of absorption bands: (a) A
1
-band (2055-
-
1
-1
005 cm ), (b) CH (2868-2838 cm ), using a false color scale from blue (low) to red (high intensity), (c, d)
2
Bright field image merged with (c) the luminescence signal of 7 (ex 350/50x, em 560/80m) and (d) nuclei
-1
staining (Hoechst, blue, ex 350/50x em 460/50m); 128 scans, 8 cm spectral width. D=dermis, VE=viable
epidermis, SC=stratum corneum.
FTIR-SM was also used to investigate modifications of lipids and proteins of the skin in
-
1
hotspots of HA after permeation of 7. The CH
lipid acyl chains is specific of the organization of lipids,
2
IR band in the 2850 cm range assigned to
3
9, 40
and the amide I band can be
analyzed to probe a modification of the proteins structure. Spectra were recorded at the
4
1
surface of the skin exposed to 7 for 24 hours, at some points with various intensities of the
A -band and at the surface of the skin not exposed (Figures S11 and S12, SI). No variation in
1
-1
peak position was seen at 2850 and 1500-1700 cm , which suggests that 7 does not induce
any modification of the organization of lipids or proteins.
3
In conclusion, we were able to efficiently label HAs of different MW with a Re(CO) SCoMPI
at different rates using a Ugi reaction. Their penetration into human skin biopsies was
monitored using IR and fluorescence microscopies. The label percentage was an important
parameter to ensure detection without modifying to a too large extent the physico-chemical
properties of the biopolymer: if too low, the compound (6, 10 kDa, 3-4%) was not seen, but
when too high (8, 10 kDa, 40% and 9, 400-1000 kDa, 20%), solubility was impaired and hence
penetration as well. After a 7-hour exposure, 7 (10 kDa, 20%) was localized at some spots in
the SC (Figure S9), whereas 10 (400-1000 kDa, 5%) was not detected (not shown). After a 24-
hour exposure, 7 was homogeneously distributed in the SC and slightly in the VE (Figure 2),
but was not detected in the deeper layers of the skin whereas 10 was only located at some
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spots in the SC (Figure S9). The present data showed that the penetration of a cosmetic
relevant compound in the skin can be efficiently probed using a SCoMPI. As expected, the
penetration is highly dependent on the size of HAs, with no modification of the skin
structure.
Funding sources
The authors declare no competing financial interest. École Normale Supérieure (ENS), CNRS
(UMR7203, 8640), and Université Pierre et Marie Curie Paris 6 are acknowledged for their
financial supports. The authors wish to thank ENS for LH’s PhD fellowship, PSL-Inocellchem
in the frame of the contract ANR-10-IDEX-0001-02-PSL, maturation project C1532 - DM1503
MeCOX (for the development of SCoMPI probes) and Fédération pour la recherche
biomédicale FRM DEI20151234413 (funding for an IR-microscope, namely a Cary 620
infrared microscope equipped with a 128×128 pixels Stingray MCT detector) for financial
supports.
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Acknowledgements
We thank Zoher Gueroui for access and help with the fluorescence microscope, the SOLEIL
committee for beamtime and members from SMIS beamline (project 20150269).
Supporting Information
The following file is available free of charge: supporting information about synthesis,
methods and additional data (PDF).
Abbreviations
HA, hyaluronic acid; SC, stratum corneum; VE, viable epidermis; MW, molecular weight; (SR)-
FTIR-SM, (synchrotron radiation) Fourier transform infrared spectromicroscopy; ROI, region
of interest; SCoMPI, Single core multimodal probe for imaging; DIPEA,
diisopropylethylamine; TFA, trifluoroacetic acid
Notes and references
(
1)
Weissmann, B., and Meyer, K. (1954) The Structure of Hyalobiuronic Acid and of
Hyaluronic Acid from Umbilical Cord1,2. J. Am. Chem. Soc. 76, 1753-1757.
Shiedlin, A., Bigelow, R., Christopher, W., Arbabi, S., Yang, L., Maier, R. V., Wainwright,
N., Childs, A., and Miller, R. J. (2004) Evaluation of Hyaluronan from Different
Sources:ꢀ Streptococcus zooepidemicus, Rooster Comb, Bovine Vitreous, and Human
Umbilical Cord. Biomacromolecules 5, 2122-2127.
(
2)
(
3)
Pavicic, T., Gauglitz, G. G., Lersch, P., Schwach-Abdellaoui, K., Malle, B., Korting, H. C.,
and Farwick, M. (2011) Efficacy of Cream-Based Novel Formulations of Hyaluronic
Acid of Different Molecular Weights in Anti-Wrinkle Treatment. J. Drugs Dermatol. 10,
9
90-1000.
(4)
Kogan, G., Šoltés, L., Stern, R., and Gemeiner, P. (2007) Hyaluronic acid: a natural
biopolymer with a broad range of biomedical and industrial applications. Biotechnol.
Lett 29, 17-25.
(
5)
Clède, S., Lambert, F., Sandt, C., Gueroui, Z., Refregiers, M., Plamont, M. A., Dumas,
P., Vessieres, A., and Policar, C. (2012) A rhenium tris-carbonyl derivative as a single
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core multimodal probe for imaging (SCoMPI) combining infrared and luminescent
properties. Chem. Commun. 48, 7729-31.
(
(
(
6)
7)
8)
Clède, S., and Policar, C. (2015) Metal-carbonyl units for vibrational and
luminescence imaging: towards multimodality. Chem. Eur. J. 21, 942-58.
Hostachy, S., Policar, C., and Delsuc, N. (2017) Re(I) carbonyl complexes: Multimodal
platforms for inorganic chemical biology. Coord. Chem. Rev. 351, 172-188.
Stephenson, G. R. (2006) Organometallic bioprobes, Bioorganometallics—
Biomolecules, labeling, medecine (Jaouen, G., Ed.) pp 215-262, Wiley-VCH, Weinheim.
Salmain, M., Vessières, A., Jaouen, G., and Butler, I. S. (1991) Fourier transform
spectroscopic method for the quantitative trace analysis detection of transition-
metal carbonyl-labeled bioligands. Anal. Chem. 63, 2323-2329.
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10) Amoroso, A. J., Arthur, R. J., Coogan, M. P., Court, J. B., Fernández-Moreira, V., Hayes,
A. J., Lloyd, D., Millet, C., and Pope, S. J. A. (2008) 3-Chloromethylpyridyl bipyridine
fac-tricarbonyl rhenium: a thiol-reactive luminophore for fluorescence microscopy
accumulates in mitochondria. New J. Chem. 32, 1097.
(11) Raszeja, L. J., Siegmund, D., Cordes, A. L., Guldenhaupt, J., Gerwert, K., Hahn, S., and
Metzler-Nolte, N. (2017) Asymmetric rhenium tricarbonyl complexes show superior
luminescence properties in live cell imaging. Chem. Commun. 53, 905-908.
12) Agorastos, N., Borsig, L., Renard, A., Antoni, P., Viola, G., Spingler, B., Kurz, P., and
Alberto, R. (2007) Cell-specific and nuclear targeting with [M(CO)(3)](+) (M=(99m)Tc,
Re)-based complexes conjugated to acridine orange and bombesin. Chem. Eur. J. 13,
3842-52.
13) Fernandez-Moreira, V., Thorp-Greenwood, F. L., and Coogan, M. P. (2010)
Application of d6 transition metal complexes in fluorescence cell imaging. Chem.
Commun. 46, 186-202.
(
(
(
(
14) Coogan, M. P., and Fernandez-Moreira, V. (2014) Progress with, and prospects for,
metal complexes in cell imaging. Chem. Commun. 50, 384-399.
15) Coogan, M. P., Doyle, R. P., Valliant, J. F., Babich, J. W., and Zubieta, J. (2014) Single
amino acid chelate complexes of the M(CO)3+ core for correlating fluorescence and
radioimaging studies (Mꢀ=ꢀ99mTc or Re). J. Label. Compd. Radiopharm. 57, 255-261.
16) Albada, B., and Metzler-Nolte, N. (2016) Organometallic–Peptide Bioconjugates:
Synthetic Strategies and Medicinal Applications. Chem. Rev. 116, 11797-11839.
17) Henry, L., Schneider, C., Mutzel, B., Simpson, P. V., Nagel, C., Fucke, K., and
Schatzschneider, U. (2014) Amino acid bioconjugation via iClick reaction of an
oxanorbornadiene-masked alkyne with a Mn(I)(bpy)(CO)3-coordinated azide. Chem.
Commun. 50, 15692-5.
18) Clède, S., Delsuc, N., Laugel, C., Lambert, F., Sandt, C., Baillet-Guffroy, A., and Policar,
C. (2015) An easy-to-detect nona-arginine peptide for epidermal targeting. Chem.
Commun. 51, 2687-9.
19) Raszeja, L., Maghnouj, A., Hahn, S., and Metzler-Nolte, N. (2011) A novel
organometallic ReI complex with favourable properties for bioimaging and
applicability in solid-phase peptide synthesis. ChemBioChem 12, 371-6.
(
(
(
(
(20) Bartholoma, M., Valliant, J., Maresca, K. P., Babich, J., and Zubieta, J. (2009) Single
amino acid chelates (SAAC): a strategy for the design of technetium and rhenium
radiopharmaceuticals. Chem. Commun., 493-512.
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(21) Gasser, G., Pinto, A., Neumann, S., Sosniak, A. M., Seitz, M., Merz, K., Heumann, R.,
and Metzler-Nolte, N. (2012) Synthesis, characterisation and bioimaging of a
fluorescent rhenium-containing PNA bioconjugate. Dalton Trans. 41, 2304-2313.
(
22) Santoro, G., Zlateva, T., Ruggi, A., Quaroni, L., and Zobi, F. (2015) Synthesis,
characterization and cellular location of cytotoxic constitutional organometallic
isomers of rhenium delivered on a cyanocobalmin scaffold. Dalton Trans. 44, 6999-
7
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(23) Clède, S., Lambert, F., Sandt, C., Kascakova, S., Unger, M., Harte, E., Plamont, M. A.,
Saint-Fort, R., Deniset-Besseau, A., Gueroui, Z., et al. (2013) Detection of an estrogen
derivative in two breast cancer cell lines using a single core multimodal probe for
imaging (SCoMPI) imaged by a panel of luminescent and vibrational techniques.
Analyst 138, 5627-38.
(
24) Imstepf, S., Pierroz, V., Rubbiani, R., Felber, M., Fox, T., Gasser, G., and Alberto, R.
2016) Organometallic Rhenium Complexes Divert Doxorubicin to the Mitochondria.
Angew. Chem. Int. Ed. 55, 2792-2795.
(
(25) Salmain, M., and Vessières, A. (2006) Organometallic complexes as tracers in non-
isotopic immunoassay, Bioorganometallics—Biomolecules, labeling, medecine
(
Jaouen, G., Ed.) pp 263-302, Wiley-VCH, Weinheim.
(
26) Policar, C., Waern, J. B., Plamont, M. A., Clède, S., Mayet, C., Prazeres, R., Ortega, J.-
M., Vessières, A., and Dazzi, A. (2011) Subcellular Imaging in the Mid-IR of a Metal-
Carbonyl Moiety Using Photothermal Induced Resonance. Angew. Chem. Int. Ed. 50,
860-864.
(
(
27) Hostachy, S., Swiecicki, J. M., Sandt, C., Delsuc, N., and Policar, C. (2016)
Photophysical properties of single core multimodal probe for imaging (SCoMPI) in a
membrane model and in cells. Dalton Trans. 45, 2791-2795.
28) Clède, S., Lambert, F., Saint-Fort, R., Plamont, M. A., Bertrand, H., Vessieres, A., and
Policar, C. (2014) Influence of the side-chain length on the cellular uptake and the
cytotoxicity of rhenium triscarbonyl derivatives: a bimodal infrared and luminescence
quantitative study. Chem. Eur. J. 20, 8714-22.
(
(
29) Dazzi, A., and Policar, C. (2011) AFM-IR: photothermal infrared nanospectroscopy:
Application to cellular imaging, Biointerface Characterization by Advanced IR
Spectroscopy (Pradier C.M. and Chabal, Y. J., Ed.) pp 245-278, Elsevier, Amsterdam.
30) Dazzi, A., and Prater, C. B. (2017) AFM-IR: Technology and Applications in Nanoscale
Infrared Spectroscopy and Chemical Imaging. Chem. Rev. 117, 5146-5173.
(31) Joshi, T., Pierroz, V., Mari, C., Gemperle, L., Ferrari, S., and Gasser, G. (2014) A
Bis(dipyridophenazine)(2-(2-pyridyl)pyrimidine-4-carboxylic acid)ruthenium(II)
Complex with Anticancer Action upon Photodeprotection. Angew. Chem. Int. Ed. 53,
960-2963.
2
(
32) Kitanovic, I., Can, S., Alborzinia, H., Kitanovic, A., Pierroz, V., Leonidova, A., Pinto, A.,
Spingler, B., Ferrari, S., Molteni, R., et al. (2014) A Deadly Organometallic
Luminescent Probe: Anticancer Activity of a ReI Bisquinoline Complex. Chem. Eur. J.
20, 2496-2507.
(33) Fudala, R., Mummert, M. E., Gryczynski, Z., and Gryczynski, I. (2011) Fluorescence
detection of hyaluronidase. J. Photochem. Photobiol., B 104, 473-477.
(
34) El Kaim, L., and Grimaud, L. (2009) Beyond the Ugi reaction: less conventional
interactions between isocyanides and iminium species. Tetrahedron 65, 2153-2171.
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(35) Cheron, N., Ramozzi, R., El Kaim, L., Grimaud, L., and Fleurat-Lessard, P. (2012)
Challenging 50 years of established views on Ugi reaction: a theoretical approach. J.
Org. Chem. 77, 1361-6.
(
36) Creaser, C. S., Fey, M. A., and Stephenson, G. R. (1994) Environment sensitivity of IR-
active metal carbonyl probe groups. Spectrochim. Acta, Part A 50, 1295-1299.
37) Fernández, E., Rodríguez, G., Hostachy, S., Clède, S., Cócera, M., Sandt, C., Lambert, F.,
de la Maza, A., Policar, C., and López, O. (2015) A rhenium tris-carbonyl derivative as
a model molecule for incorporation into phospholipid assemblies for skin
applications. Colloids Surf., B 131, 102-107.
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38) Clède, S., Policar, C., and Sandt, C. (2014) Fourier transform infrared (FT-IR)
spectromicroscopy to identify cell organelles: correlation with fluorescence staining
in MCF-7 breast cancer cells. Appl. Spectrosc. 68, 113-7.
39) Merle, C., and Baillet-Guffroy, A. (2009) Physical and chemical perturbations of the
supramolecular organization of the stratum corneum lipids: In vitro to ex vivo study.
Biochim. Biophys. Acta 1788, 1092-1098.
(40) Cotte, M., Dumas, P., Besnard, M., Tchoreloff, P., and Walter, P. (2004) Synchrotron
FT-IR microscopic study of chemical enhancers in transdermal drug delivery: example
of fatty acids. J. Controlled Release 97, 269-281.
(
41) Mendelsohn, R., Flach, C. R., and Moore, D. J. (2006) Determination of molecular
conformation and permeation in skin via IR spectroscopy, microscopy, and imaging.
Biochim. Biophys. Acta 1758, 923-933.
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Figure 1. IR spectra obtained using ATR mode between 1820 and 2075 cm-1 of 5 in ethanol and 7 in water
(2 mg·mL-1)
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Figure 2. Map of a 10 µmꢀthick skin cryosection after a 24ꢀhour exposure with 7, mounted on a CaF2
window (scale bar = 10 µm) (aꢀb) SRꢀFTIRꢀSM images based on the integration of absorption bands: (a)
A1ꢀband (2055ꢀ2005 cmꢀ1), (b) CH2 (2868ꢀ2838 cmꢀ1), using a false color scale from blue (low) to red
(
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60/80m) and (d) nuclei staining (Hoechst, blue, ex 350/50x em 460/50m); 128 scans, 8 cmꢀ1 spectral
width. D=dermis, VE=viable epidermis, SC=stratum corneum.
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