Spektroskopia EPR
w badaniu nanoleków
Ryszard Krzyminiewski1,2, Bernadeta Dobosz1,2, Joanna
Kurczewska3, Grzegorz Schroeder3, Magdalena Hałupka-Bryl2,
Magdalena Bednarowicz2, Tomasz Kubiak1
1Zakład
Fizyki Medycznej, Wydział Fizyki UAM, Umultowska 85, 61-614 Poznań,
2Centrum NanoBioMedyczne, Umultowska 85, 61-614 Poznań,
3Zakład Chemii Supramolekularnej, Wydział Chemii UAM, Umultowska 89B, 61-614 Poznań,
Electron Paramagnetic Resonance
(EPR) Spectroscopy
Warunek rezonansowy
hν=gμBB
EPR – the nuclear hyperfine
interaction
- Oddziaływania nadsubtelne: oddziaływanie spinowego
momentu magnetycznego elektronu ze spinowym
momentem magnetycznym jądra.
- Wpływ na parametry widma mają oddziaływania dipolowe i
wymienne
Znacznik spinowy
EPR – correlation time and the
spectrum of the radical NO.
10-12 s
Czas korelacji τ informuje o dynamice
molekuł
w danym środowisku, ich
10-11 s
rotacji itd
10-10 s
ΔH(0) szerokość linii centralnej a I(+1), I(0) i
I(-1)
to
amplitudy
linii
niskopolowej,
10-9 s
centralnej i wysokopolowej
10-8 s
EPR
X-band
L-band
Materiały
Reactive Oxygen Species
Reactive Oxygen Species (ROS): chemically reactive molecules
containing oxygen causing oxidative demage.
oxidative stress
Rheumatoid arthritis (RA):
- chronic, systemic
inflammatory disorder,
affecting the joints,
causing pain and
stiffness, leading to
destruction of the
articular cartilage.
- characterized by
development of a
pannus tissue
- free radicals/reactive
oxygen species (ROS)
play an important role
in RA inflammation.
higher risk of systemic
diseases e.g. Alzheimer,
cancer, atherosclerosis
and…
…they play important role
in chronic inflammation
pathogenesis
ROS scavenging activity
plays an importnant role
in suppresing
inflammation caused by
oxidative stress
CHRONIC
INFLAMMATION
Arthriti
s
Materiały
ROS Scavenger
Antioxidant for excess generation of ROS
TEMPO( 2,2,6,6-tetramethylpiperidine-1-oxyl)
MATERIAL
Low molecular ROS scavengers are easily eliminated from the system
resulting in poor therapeutic effect.
Polyion Complex (PIC) Redox Flower Micelle
hydrophilic PEG shell
(prevents aggregation, biocompatible)
TEMPO
PEG: poly(ethylene glycol)
(ROS scavanger)
PMNT: poly[p-4-(2,2,6,6polyion core
tetramethyl-1-piperidine-1(nanoreservoir
oxyl)aminomethylstyrene
for drugs)
Injctable PIC hydrogel
the novel redox
flower micelle
developed by utilizing
the pH-responsive
character of PMNTPEG-PMNT triblock
copolymer
protonated
pH
unprotonated
pH
protonated
PMNT-PEG-PMNT triblock copolymer
N
N N N
N
N N N
14
+ N+N+N +
pH
N
+ N+ +N+
N
N
unprotonated
pH
12
micelle 1 day
10
8
micelle over 30 days
electrostatic
interactions
6
hydrophobic
4
hydrophilic
hydrophobic
PIC hydrogel
flower micelle solution
injectable
fluid which
exhibis in situ
gel formation
COO-
COO-
COO-
selfassembley
+
disassembley
and gelation in
37 ℃ and 150mM
ionic strenght
COO-
2
N N N
N
0
0
50
100
150
200
N
_
+
_
+
_+
_
+
+
+
+ +
_
_
+
+
+
+
_
easy to operate
N N N
Advantages:
biocompatibil
prolonging duration of ROS scavengers
local controlling of drug release
+
150K
chloroform
DMF
dry polymer
TEMPO crystal
H2O
micelles
280K
Linear transformation method
The shape of the signal can be described by:

F ( x) 
 K ( x  x ) ( x )dx 

K ( x  x )
 ( x )
- a function describing the shape and
width of particular spectral lines, e.g.
Gauss
- a function describing positions and
intensities of particular components of
the signal
The Fourier Transform of F(x)
~
1 / 2
F ( y )  2 

 F ( x) exp  iyx dx

Determination of the function (x)
 ( x)  (2 )
1/ 2
~( y )
F
 K~( y) exp(ixy)dy

Fig. PMNT-PEG-PMNT in H2O,
DMF and chloroform (5mg/mL)
peak-to-peak line width (ΔHpp)
versus temperature [K]
DMF
water
chloroform
g – Factor
Hp [mT]
2,0054
2,0055
2,0058
333,9
332,7
333,0
Materiały
Materiały
Scheme 1. Schematic representation of the magnetic nanocarrier studied
Materiały
Schemat syntezy
nanocząstek
magnetytu
pokrytych
chitozanem i
znakowanych
TEMPO z
dołaczonym
antybiotykiem
Materiały
Mikrofotografie TEM A,20nm, B50nm, C10nm magnetycznych nanocząstek
Widma EPR znakowanych TEMPO nanocząstek
magnetytu z dopaminą
Fig. 4. Changes of resonance field (Hr) versus temperature for investigated sample
with (FC) and without field cooling ( ZFC).
Fig. 6. Changes of g-factor value versus temperature for investigated sample
with (FC) and without field cooling (ZFC).
Zmiany parametrów widm EPR
w funkcji temperatury i tzw. FC i
ZFC
Widma EPR magnetytu
pokrytego chitozanem i
znakowanego TEMPO ,
temperatura 120K
rejestracja ZFC i FC ,
badania orientacji w polu
magnetycznym
Centralny fragment
widma EPR 254K i
132K.
Zależności temperatorowe
nanocząstek magnetytu
parametrów EPR:szerokości linii,
intensywności i wsp. g *linia
szeroka od rdzenia
Fig. 5. The temperature dependences of fast rotation
correlation times of nitroxide
spin probe for the orientations 0 and 90.
Czasy korelacji
Tomografia EPR
Wolne rodniki
Wolne rodniki
Wolne rodniki
Wolne rodniki
Dyfuzja w ciągu 3 godzin
Współczynniki dyfuzji nanocząstek w hydrożelu
Dziękuję za uwagę
Nie mam żadnych
wykresów Hałupki
Materiały
AVAILABLE
TREATMENTS
second-line
drugs to prevent
joints
destruction:
first-line drugs to
DMARDs
reduce inflammation
and pain:
NSAIDs
LONG TERM
GC
TREATMENT CAUSING
MANY SIDE EFFECTS
Current strategies are to design
nanosystem for delivery of
therapeutic agents specifically to the
site of inflammation, therefore
avoiding potential systemic and offtarget unwanted effects.
Dexamethasone
- DEX, a member of the GC class
of steroid inflammatory and
immunosuppressing drug
Prednisolone
- PRD, a synthetic glucocorticoid,
a derivative of cortisol, which is
used to treat a variety of
inflammatory diseases
- active metabolite of the drug
prednisone
AIBN
toluene
HS-PEG-SH
.
・S-PEG-S・
.
PCMS-PEG-PCMS
PCMS-PEG-PCMS
(4-amino-TEMPO)
PMNT-PEG-PMNT Triblock Copolymer
-radical telomerization of CMS using SH-PEG-SH as telogen – 60 ℃,
24h in oil bath
-purification by reprecipitation by 30-fold amount of diethyl ether
-freeze-drying with benzene
-amination of PCMS-PEG-PCMS with 4-amino TEMPO in DMSO
-dialysis against methanol (48h)
-evaporation
-freeze-drying
PMNT-PEG-PMNT
synthesis
Fig. 7. Changes of correlation time (τ) versus temperature for investigated sample with (FC) and
without field cooling (ZFC).