Selective endocytic trafficking in live cells with fluorescent naphthoxazoles and their boron complexes

Article abstract of DOI:10.1039/c5cc02383a

Fluorescent naphthoxazoles and their boron derivatives have been synthesized and applied as superior and selective probes for endocytic pathway tracking in live cancer cells. The best fluorophores were compared with the commercially available acridine orange (co-staining experiments), showing far better selectivity.

Full text of DOI:10.1039/c5cc02383a

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DOI: 10.1039/C5CC02383A
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Selective Endocytic Trafficking in Live Cells with
Fluorescent Naphthoxazoles and their Boron
Complexes
Cite this: DOI: 10.1039/x0xx00000x
Gleiston G. Dias,a Bernardo L. Rodrigues,a Jarbas M. Resende,a Hállen D. R.
Calado,a Carlos A. de Simone,b Valter H. C. Silva,c Brenno A. D. Neto,d Marilia O. F.
Goulart,e Fabricia R. Ferreira,e Assuero S. Meira,f Claudia Pessoa,f,g José R. Correa*d
and Eufrânio N. da Silva Júnior*a
Received 00th January 2014,
Accepted 00th January 2014
DOI: 10.1039/x0xx00000x
www.rsc.org/
design, synthesis, characterization, single crystal X-ray,
photophysics, DFT calculations, bioimaging, cellular uptake and
dynamics in living cells (cancer cells) of new fluorescent
derivatives which have allowed for selectively visualizing the
whole endocytic pathway.
The structures of the synthesized fluorescent compounds are
visualized in Figure 1 and the synthetic details are found in the
supplementary material (Scheme S1).
Fluorescent naphthoxazoles and their boron derivatives have
been synthesized and applied as superior and selective probes
for endocytic pathway tracking in live cancer cells. The best
fluorophores were compared with the commercially available
acridine orange (co-staining experiments), showing far better
selectivity
.
Endocytosis, as the key regulator process for molecular
internalization in eukaryotic cells, has a fundamental role in cell
homeostasis.1
Signal transduction, neurotransmission,
intercellular communication and immune response are known to
be readily affected by endocytic pathway dysfunction.2 The
complexity of the cellular environment renders the
understanding of endocytosis as an outstanding challenge.
Cellular homeostasis, for instance, depends on the proper
communication of the cellular organelles and all other
components associated with correct signals throughout the
highly complex machinery. Deregulation in any of these
signalizing pathways could be the answer which triggers a
specific pathological process. All homeostasis unbalances may
also be associated with endocytosis dysfunctions. Fluorescence
techniques, in this sense, rose up, shedding light onto many
doubts over cellular endocytic mechanisms and pathways.3
Despite the great advance of using fluorescent bioprobes,4-7
many unanswered questions and challenges still remain to be
tackled. For some, the capacity of a bioprobe to transpose the cell
membrane is one of the most desirable features.8 Very recently,
some have shown the great potential of fluorogenic probes for
studying dynamic cellular uptake in live cells.9
Figure 1. Structures of the fluorescent compounds evaluated in this work. Inset:
fluorescence images under UV light (365 nm) in CH2Cl2 (50 µM).
All compounds afforded single crystals suitable for X-ray
characterization (Figure 2 and Tables S5 and S6) with different
packing depending on the size of the side ring.
With the knowledge that many quinonoid derivatives
sustaining the basic carbon scaffold are capable of transposing
the cell-membrane and can present luminescence, as we10-12 and
others13-15 have already demonstrated, we envisaged the
synthesis of fluorescent bioprobes from an affordable naturally
occurring naphthoquinone. In this sense, we aimed to use the cell
membrane permeability feature of such compounds to synthesize
fluorescent derivatives for dynamics and uptake in live cells
using the endocytic pathway. Herein, we disclose the rational
Figure 2. Molecular structure of
single crystal X-ray analysis.
1-
4
(top) and packing (bottom) depicted from
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Molecular structures of
for the side pyrane ring. Compound
1
and
2
are basically planar except
4 is in turn perfectly planar
LUMO (basically of π-type) and electron density difference were
also calculated and are in accordance with the good stability
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DOI: 10.1039/C5CC02383A
with methyl groups and fluorine atoms out of the plane. Distinct
crystal packing for all compounds are also noted when
comparing the four structures (Figure 2).
The photophysical properties of these molecules were
investigated and pointed to the stability of these compounds in
the excited state, as shown in Table 1.
observed in the excited state for compounds 1-4 (Figure 3).
Electrochemical (cyclic voltammetry, CV) analyses were
also performed (Figures S45-S46). All the analysed compounds
were reduced in two reduction waves (Figure S45 and Table S9).
The first electron uptake was associated with a relatively stable
radical anion whereas the second one is, presumably, a dihydro-
derivative, in accordance with similar compounds.16 Differences
in current were noticed comparing the boron-free and boron-
containing derivatives. As shown in Figures S45-46, comparison
of the synthesized compounds allowed us to suggest that
complexation with boron slightly facilitates the reduction
process (Table S9), with Epc values less negative than those for
the boron-free compounds.
Table 1. UV-Vis and fluorescence emission data (in different solvents) for 1,
2, 3 and 4 (10 µM of concentration).
Compound
Solvent
Stokes shift
(nm)
λ
max (abs)
(nm)
348
λ
max (em)
(nm)
396
Hexane
CH3Ph
CH2Cl2
EtOAc
MeOH
MeCN
DMSO
Hexane
CH3Ph
CH2Cl2
EtOAc
MeOH
MeCN
DMSO
Hexane
CH3Ph
CH2Cl2
EtOAc
MeOH
MeCN
DMSO
Hexane
CH3Ph
CH2Cl2
EtOAc
MeOH
MeCN
DMSO
48
353
351
350
348
348
353
348
353
371
350
348
348
352
354
358
357
355
354
354
359
373
378
377
355
355
355
359
402
407
403
470
412
414
421
433
445
477
469
461
412
398
409
414
408
410
416
419
427
445
442
411
412
422
417
49
56
53
122
64
61
73
80
74
127
121
113
60
44
51
57
53
56
62
60
54
67
65
56
57
67
58
1
2
3
Figure 3. Optimized geometries, HOMO and LUMO plots, and electron density
difference map. Diagram energy levels calculated with DMSO as the solvent.
Calculations at PBE1PBE/6-311+G(2d,p)//CAM-B3LYP/6-31+g(d) level of theory.
Structures calculated with implicit DMSO as the solvent showed similar orbitals
localizations.
4
The quantum yields measured in triplicate with the respective standard
deviations are 0.80 0.07, 0.58 0.05, 0.61 0.05 and 0.42 0.03.
Finally, live cell-imaging experiments (PC3 cell lineages -
human prostate cancer cells) were conducted to evaluate the
In a general way, the boron complexes showed larger Stokes
shift values, most likely due to the higher planarity and more
potential of the fluorescent compounds as bioprobes. Dye
1
showed no fluorescent signal when incubated with live cells
(Figure S41) whereas compounds 2-4 displayed a bright blue
emission signal. These three compounds proved to be capable of
staining the canonical endocytic pathway, meaning they are
capable of selectively staining early endosome, late endosome
and lysosome (Figures 4-6) during the cellular uptake process.
Panels (A) and (C) in Figures 4-5 show all membranous
compartments strongly stained; there are no fluorescent signals
inside of the nucleus. Acridine orange (AO) is widely used in
order to stain endocytic pathways, however, AO also stains
nucleic acids as can be observed in Figures 4-6. Under the same
experimental conditions, 2-4 did not stain nucleic acids and the
efficient
observed when structures
correspondent boron complexes, showing that the planarity
introduced with the formation of the complex favors more
electronic conjugation in the structure. Beyond the red shift,
and displayed a shoulder at 356 and 360 nm, respectively,
indicating an slight effect over its conformation as a consequence
of the solvatochromic effect. The Stokes shifts for the BODIPY-
like compound are larger than those observed for the other
π
-conjugation. Bathochromic displacements are
1
and were compared with their
3
3
4
2
structures (Table 1).
Considering the solvent effect is important for investigating
this sort of derivatives, especially in the presence of polar
solvents, therefore some theoretical calculations were performed
aiming to shed some light on their behaviour in the ground and
excited state. In this sense, DMSO was used (implicit treatment)
as the medium for the calculations at PBE1PBE/6-
311+G(2d,p)//CAM-B3LYP/6-31+g(d) level of theory. HOMO,
cell nuclei are visualized as black voids. Compounds 2, 3 and 4
could be found at different localizations inside the cytoplasm,
and therefore are found associated with different membranous
compartments. Vesicles found at the cell periphery close to the
plasmatic membrane is correlated with early endosomes (Figures
4 and 5). Vesicles found between the plasmatic membrane and
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the nucleus are correlated with late endosomes (Figures 5), tested compounds irrespective of any affinity with the caveolar
whereas vesicles found near to the nucleus are correlated with structure. Caveolar vesicles formation is a stable structure due to
lysosomes (Figure 6). The overlay images between the tested the lipids composition in these regions.17,18 This feature keeps
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compounds and acridine orange (panel (C) in the cited Figures) the vesicles open by a time period greateDr OthI:a1n0.t1h0a39t/oCb5sCeCr0v2e3d83inA
provided the evidence that these compounds are staining the membrane regions associated with clathrin-mediated vesicles
membranous compartments belonging to the endocytic pathway, formation.17,19 Most likely the tested compounds accumulate in
but in this case selectively. AO is widely used to stain acidic the open vesicles during their formation. As a consequence they
organelles in studies regarding the endocytic and autophagic are found trapped inside the vesicles during their bud off from
pathways applied to eukaryotic cells, however, as demonstrated, the cells membrane therefore targeting the endoplasmic
with high affinity to nucleic acid components, which produce a reticulum and matching with our finds related to vesicles staining
non-specific pattern in the cells nuclei. 2-4 were able to stain near to the nuclei. To be sure about this hypothesis, an assay
specifically the membranous compartments belonging to the using the commercially available anti-caveolin-1 antibody (red
endocytic pathway without nucleus cross staining.
emitter), which is the universal marker specific for caveolar
vesicle formation, has been performed (Figures 7-9).
Figure 4. Live PC3 cancer cells stained with
with cells stained with acridine orange (green emission). (B)
(C) Overlay of (A) and (B). was found accumulated inside of spherical
2
(blue emission). (A) Positive control
2
as the bioprobe and
2
membranous organelles associated with endosomes (white arrow heads). White
arrows show the nucleus stained with acridine orange (non-specific staining). N =
cell nucleus. Reference scale bar 20 µm.
Figure 7. Live PC3 cancer cells stained with
(universal caveolar marker). (A) Shows the cell nucleus stained with commercially
available DAPI (blue), and the cytoplasmic endocytic vesicles stained by each
2 and immunodetection of caveolin-1
2
(also in blue). (B) Shows the fluorescence distribution related to caveolin-1
immunodetection (red). (C) Shows the overlay of the two fluorescent signals (blue
and red). Arrows show vesicles belong to canonical endocytic pathways (absence
of caveolin-1 detection) and arrowheads show the vesicles belonging to caveolae-
mediated endocytosis (vesicle with double staining - blue and red). (D) Shows the
Figure 5. Live PC3 cancer cells stained with
with cells stained with acridine orange (green emission). (B)
(C) Overlay of (A) and (B). was found accumulated inside of spherical
3
(blue emission). (A) Positive control
3
as the bioprobe and
cells normal morphological aspects. Reference scale bar 25 µm.
3
membranous organelle correlated with early endosomes (white arrow heads), late
endosomes (yellow arrow heads) and lysosomes (red arrows). White arrows show
the nucleus stained with acridine orange (non-specific staining). N = cell nucleus.
Reference scale bar 20 µm.
Figure 6. Live PC3 cancer cells stained with
with cells stained with acridine orange (green emission). (B)
(C) Overlay of (A) and (B). was localized inside of spherical membranous
4
(blue emission). (A) Positive control
4
as the bioprobe and
4
organelles belong to the endocytic pathway. Red arrows show the staining
membranous compartment correlated with lysosomes. White arrows show the
nucleus stained with acridine orange (non-specific staining). N = cell nucleus.
Figure 8. Live PC3 cancer cells stained with
(A) Shows the cell nucleus stained with DAPI (blue), and the cytoplasmic endocytic
vesicles stained by each (also in blue). (B) Shows the fluorescence distribution
3 and immunodetection of caveolin-1.
Reference scale bar 20 µm.
3
related to caveolin-1 immunodetection (red). (C) Shows the overlay of the two
fluorescent signals (blue and red). Arrows show vesicles belong to canonical
endocytic pathways (absence of caveolin-1 detection) and arrowheads show the
vesicles belonging to caveolae-mediated endocytosis (vesicle with double staining
The possibility of caveolae-mediated endocytosis was also
evaluated to confirm the selective endocytic trafficking. Cells
through caveolae-mediated endocytosis may also internalize the
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b
- blue and red). (D) Shows the cells normal morphological aspects. Reference scale
bar 25 m.
Institute of Physics, Department of Physics and Informatics, University
µ
of São Paulo, 13560-160, São Carlos, SP, Brazil.
c University Unit of Exact Sciences and Technology, State University from
View Article Online
DOI: 10.1039/C5CC02383A
Figure 7 shows few vesicles stained near to cytoplasmic
membrane correlated with caveolar vesicle bud off and near to
the nucleus of the cell. This feature indicates the vesicles target
Goiás, 75024-010, Anápolis, GO, Brazil.
d
Laboratory of Medicinal & Technological Chemistry, Institute of
endoplasmic reticulum which is the main docking point for Chemistry, University of Brasilia, 70904970, P.O.Box 4478, Brasilia, DF,
caveolar vesicles. In other words, the caveolar entering is one
possibility as well as the canonical endocytic pathway. The same
behaviour could be noted for all tested compounds indicating the
new bioprobes are capable of probing selectively the endocytic
trafficking in live cells irrespective of the preferred
internalization path. Similar results from that in Figure 7 have
been observed in Figures 8 and 9.
Brazil. E-mail: correa@unb.br
e Institute of Chemistry and Biotechnology, Federal University of Alagoas,
57072-970, Maceió, AL, Brazil.
f
Department of Physiology and Pharmacology, Federal University of
Ceará, 60430-270, Fortaleza, CE, Brazil.
g Fiocruz-CE, 60180-900, Fortaleza, CE, Brazil.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/c000000x/
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Figure 9. Live PC3 cancer cells stained with
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vesicles stained by each (also in blue). (B) Shows the fluorescence distribution
4 and immunodetection of caveolin-1.
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In summary, four new fluorescent derivatives have been
synthesized, characterized and successfully used as live cell-
imaging probes, capable of selectively staining the endocytic
pathway. The compounds also showed no cytotoxicity when
evaluated by MTT assay. Compounds 2-4 were able to permeate
intact live cells, thus being trapped inside of acidic
compartments, turning them into suitable molecules for
investigating the lysosome biogenesis, host pathogens
interaction and autophagic processes, as will be disclosed in due
course. The selective endocytic trafficking in live cells and its
dynamics have been probed using the synthesized compounds,
which in turn, were capable of selectively reveal the
internalization endocytic process in live cell through both the
caveolar vesicles or the canonical pathway. The new designed
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