Protein-based nanocarriers for photodynamic therapy and ...400 500 600 700 0.00 0.02 0.04 0.06 600...
Transcript of Protein-based nanocarriers for photodynamic therapy and ...400 500 600 700 0.00 0.02 0.04 0.06 600...
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Stefania Abbruzzetti
Dipartimento di Scienze Matematiche,
Fisiche e Informatiche
Università di Parma, Parma, Italy
e-mail: [email protected]
Protein-based nanocarriers for photodynamic
therapy and subdiffraction localization of the
theranostic agent
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Cristiano Viappiani
Pietro Delcanale
Chiara Montali
Stefano Bruno
Massimiliano Tognolini
Marco Cozzolino
Paolo Bianchini
Alberto Diaspro
Santi Nonell
Beatriz Rodriguez-Amigo
Paolo Foggi
F. Javier Luque
Axel Bidon-Chanal
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Develop of nanoscaled
chemical constructs that
have simultaneously
therapeutical and
diagnostical functions
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This theranostic approach is particularly interesting for PDT
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Principi PDT
Photodynamic effect
Nature Reviews Cancer 2003, 3, 380-387
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photosensitizer molecular oxygen
High extinction in the red/NIR
High
No dark toxicity
High bioavailability
The ideal
photosensitizer
= T S
P. Ogilby, Chem. Soc. Rev.,
2010, 39, 3181–3209
158 kJ/mole
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Photodynamic therapy of tumors
clinically approved, minimally invasive therapeutic procedure that
can exert a selective cytotoxic activity toward malignant cells.
3 s d 100 nm
P. Agostinis et al. CA Cancer J Clin., 2011, 61 (4), 250, 281.
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Protein based carriers for photodynamic therapy
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1O2
1O2
Stable and easily obtained human protein (myoglobin)
Well known natural photosensitizer (hypericin)
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Hyp
Fe-protoporfirin IX
Hypericin is very similar
to heme in size, shape,
symmetry and
hydrophobicity.
This suggests that the
apo-protein could form a
stable complex with Hyp.
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600 700 8000.0
0.5
1.0
1.5
Flu
ore
sce
nce
(a.u
.)
Wavelength (nm)
600 700 8000.0
0.5
1.0
1.5
Flu
ore
scen
ce(a
.u.)
600 700 8000.0
0.5
1.0
1.5
Flu
ore
scen
ce(a
.u.)
Hyp spontaneously binds to
apoMb in a 1:1 complex with
moderately high affinity.
Upon binding to apoMb
fluorescence emission is
recovered, similar to ethanol
F=0.14
J. Comas-Barceló et al.
RSC Advances, 2013, 3, 17874-17879.
Hyp-ApoMb complex
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Ka=(2.4±0.5)105 M-1
Kd =(4.2±0.8) M
Hyp+ApoMb kb
k-b
Hyp-ApoMb
600 7000.0
0.5
1.0
1.5
A
Flu
ore
sce
nce
(a.u
.)
Wavelength (nm)
0 10 20 300
2
4
6
F(a
.u.)
[apoMb]( M)0 10 20 30
0
2
4
6
F(a
.u.)
[apoMb]( M)
Hyp to apoMb binding
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Excitation fluorescence anisotropy
em = 620 nm
400 500 600-0.1
0.0
0.1
0.2
0.3
An
iso
tro
py
wavelength (nm)
400 500 600-0.1
0.0
0.1
0.2
0.3
An
iso
tro
py
wavelength (nm)
Hyp in ethanol
Hyp-apoMb in PBS
S0→ S2
S0→ S1
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Triplet-triplet transient absorbance
Hyp-ApoMb in air saturated PBS buffer
Hyp in air saturated DMSO
1.5 s
0.1 1 10 100
0.00
0.01
A(5
20
nm
)
Time( s)
0.1 1 10 100
0.00
0.01
11.5 s
Reactive states formation
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APBT
Antibacterial photosensitization-based treatment
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Excellent photoinactivation was observed with other Gram positive
bacteria like Listeria monocytogenes
Bacterial photoinactivation
B. subtilis
Hyp
Hyp-apoMb
S. aureus
B. subtilis
E. coli
Hyp = 10 M, ApoMb = 30 M
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300 400 500 600 700 8000.00
0.05
0.10
0.15A
bs o
r F
luo
wavelength (nm)
Excitation
566 nm
STED
715 nm
Emission
=
Excitation STED
pattern
Effective
PSF
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P. Delcanale, et al. Sci. Rep., 2015, 5, 15564.
STED imaging of Hyp-apoMb
521 nm
excited state absorption
647 nm
stimulated emission
592 nm
ground state bleaching
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confocal STED
B. subtilis
STED beam = 715 nm
Excitation = 566 nm
Detection = 605/70 nm
Improvement in resolution
2.5 m 2.5 m
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B. subtilis
E. coli
S. aureus
Gram-
Gram+
Gram+
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Changing the scaffold
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-Lactoglobulin
(2 LG)
KD = 8.6 µM-1
B. Rodríguez-Amigo et al. J. Dairy Sci., 2015, 98, 89–94.
Proteins may be endowed with multiple binding sites
Typical case is the dimeric protein -lactoglobulin
Internal binding site (e.g. retinoic acid) and clefts at dimer
interface
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Hyp in DMSO
Hyp in PBS
Hyp + 2 LG
Hyp binding to 2 LG
Kd = 0.71±0.03 µM.
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Light dose effects on S. aureus photoinactivation
by the complex Hyp- 2 LG for Hyp (8 µM) and 2 LG (40 µM).
Bacterial photoinactivation
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Absorption and fluorescence emission
suggest non perfect solvation
Multiple binding at each cleft
Solvent exposure of Hyp bound at the large cleft
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Preferential distribution of DMSO molecules around the protein
surface of dimeric LG.
DMSO density of 0.3 g cm3.
Amino acids with hydrophobic side chains.
Tuning local solvent composition at the
protein surface
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Delcanale, P. et al. Journal of Materials Chemistry B, 2017, 5 (8), 1633-1641.
Preferential distribution of DMSO
molecules around the protein
surface of dimeric LG bound to Hyp
molecules as monomers in the
narrow cleft, and either (A) monomer
or (B) dimer in the wide cleft.
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400 500 600 7000.00
0.02
0.04
0.06
600 650 7000
20
40
60
80
100
Ab
so
rba
nce
Em
issio
n (
a.u
.)
400 500 600 7000.00
0.02
0.04
0.06
600 650 7000
20
40
60
80
100
Ab
so
rba
nce
Em
issio
n (
a.u
.)
400 500 600 7000.00
0.02
0.04
0.06
600 650 7000
20
40
60
80
100
Ab
so
rba
nce
Em
issio
n (
a.u
.)
400 500 600 7000.00
0.02
0.04
0.06
600 650 7000
20
40
60
80
100
Ab
so
rba
nce
Em
issio
n (
a.u
.)
400 500 600 7000.00
0.02
0.04
0.06
600 650 7000
20
40
60
80
100
Ab
so
rba
nce
Em
issio
n (
a.u
.)
Hyp in DMSO
Hyp in PBS
Hyp in 20% PBS–DMSO
Hyp2 LG in PBS
Hyp2 LG in 20% PBS–
DMSO
Absorption and Fluorescence emission indicate better
solvation of Hyp when bound to 2 LG.
Further improvement is observed when DMSO is
added
Spectral improvements for mixed solvents?
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400 450 500 550 600-0.2
0.0
0.2
Anis
otr
opy
400 450 500 550 600-0.2
0.0
0.2
Anis
otr
opy
400 450 500 550 600-0.2
0.0
0.2
Anis
otr
opy
Hyp in DMSO
Hyp2bLG in PBS
Hyp2bLG in 20% PBS–
DMSO
The interaction with the protein is fundamental
for the observed spectral improvement
No anisotropy observed in the absence of the protein
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10-4
10-2
100
102
0.0
0.5
1.0
1.5
2.0
G
10-2
100
102
0.0
0.1
0.2
0.3
(ms)
D = 70 μm2/s
Hyp bound to
2βLG
D = 40 μm2/s
Hyp bound to 2βLG
higher viscosity of the mixture
different hydrodynamic radius of the DMSO-
coated protein.
FCS demonstrates formation of the complex
Hyp-2 LG
PBS PBS-DMSO
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sample solvent τF (ns) ФF τT (μs) ФT τΔ (μs) ФΔ
Hyp DMSO 5.5±0.1 (100%)
0.35±0.02 12 1.4±0.1* 0.35 13 5.5±0.1 0.28±0.05 29
Hyp PBS + S. aureus 0.2 (48%) 4.2 (52%)
Hyp PBS-DMSO 20%
3.5 (100%)
+ S. aureus 0.5 (48%) 3.6 (52%)
Hyp2βLG PBS 3.9 (35%) 6.7 (65%)
0.03±0.01 10±2* 8.6 & 9.6 £
0.050±0.002 2.3±0.1 0.065±0.010
+ S. aureus 4.6 (45%) 7.0 (55%)
8±1 *
Hyp2βLG PBS-DMSO 20%
0.2 (20%) 5.6 (80%)
0.06±0.01 8.2±0.4* 8.4 & 7.5 £
0.170±0.002 2.5±0.1 0.123±0.05
+ S. aureus 5.5 (100%) 9±1 *
*: Laser Flash Photolysis; &:Time-Resolved NIR phosphorescence detection; £:Fluorescence Correlation Spectroscopy
Photophysical parameters of Hyp and Hyp2βLG in
PBS or PBS-DMSO 20% incubated with S. aureus
cells
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0 10 20 3010
-8
10-6
10-4
10-2
100
CF
U/m
L
Light Dose (J/cm2)
A
0 10 20 30 40
B
Unexpectedly, bioavailability is decreased
Hyp
Hyp-2 LG
PBS PBS-DMSO 20%
S. aureus
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Funding