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. Czas korelacji τ informuje o dynamice molekuł w danym środowisku, ich rotacji itd ΔH(0) szerokość linii centralnej a I(+1), I(0) i I(-1) to amplitudy linii niskopolowej, centralnej i wysokopolowej 10-11 s 10-10 s 10-9 s 10-8 s

EPR X-band L-band

Reactive Oxygen Species Materiały 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. Reactive Oxygen Species Reactive Oxygen Species (ROS): chemically reactive molecules containing oxygen causing oxidative demage. higher risk of systemic diseases e.g. Alzheimer, cancer, atherosclerosis and… oxidative stress …they play important role in chronic inflammation pathogenesis ROS scavenging activity plays an importnant role in suppresing inflammation caused by oxidative stress CHRONIC INFLAMMATION Arthritis

Materiały MATERIAL Advantages: 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) PEG: poly(ethylene glycol) PMNT: poly[p-4-(2,2,6,6-tetramethyl-1-piperidine-1-oxyl)aminomethylstyrene TEMPO (ROS scavanger) Injctable PIC hydrogel the novel redox flower micelle developed by utilizing the pH-responsive character of PMNT-PEG-PMNT triblock copolymer polyion core (nanoreservoir for drugs) protonated pH unprotonated pH PMNT-PEG-PMNT triblock copolymer protonated pH N N N N + + N + + + N N N N N + N + N + N N N N unprotonated pH injectable fluid which exhibis in situ gel formation flower micelle solution PIC hydrogel self- assembley disassembley and gelation in 37 ℃ and 150mM ionic strenght + _ _ + + electrostatic interactions _ + + + _ _ _ + + + + + hydrophobic hydrophilic hydrophobic + + _ COO- COO- COO- COO- + + easy to operate biocompatibil prolonging duration of ROS scavengers local controlling of drug release N N N N N N N N Advantages:

150K 280K chloroform DMF dry polymer TEMPO crystal H2O micelles

Linear transformation method The shape of the signal can be described by: 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) Determination of the function (x)

Fig. PMNT-PEG-PMNT in H2O, DMF and chloroform (5mg/mL) peak-to-peak line width (ΔHpp) versus temperature [K] g – Factor Hp [mT] DMF 2,0054 333,9 water 2,0055 332,7 chloroform 2,0058 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

Zmiany parametrów widm EPR w funkcji temperatury i tzw. FC i ZFC 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

LONG TERM TREATMENT CAUSING MANY SIDE EFFECTS Materiały 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 Current strategies are to design nanosystem for delivery of therapeutic agents specifically to the site of inflammation, therefore avoiding potential systemic and off-target unwanted effects. AVAILABLE TREATMENTS second-line drugs to prevent joints destruction: DMARDs first-line drugs to reduce inflammation and pain: NSAIDs GC LONG TERM TREATMENT CAUSING MANY SIDE EFFECTS

PMNT-PEG-PMNT synthesis . . 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).