Spektroskopia EPR w badaniu nanoleków

1. 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,

2. Electron Paramagnetic Resonance (EPR) Spectroscopy Warunek rezonansowy hν=gμBB

3. EPR – the nuclear hyperfine interaction Znacznik spinowy • Oddziaływania nadsubtelne: oddziaływanie spinowego momentu magnetycznego elektronu ze spinowym momentem magnetycznym jądra. • Wpływ na parametry widma mają oddziaływania dipolowe i wymienne

4. EPR – correlation time and the spectrum of the radical NO. 10-12s 10-11 s 10-10 s 10-9 s 10-8 s 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

5. EPR X-band L-band

6. Materiały 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 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 pannustissue • free radicals/reactive oxygen species (ROS) play an important role in RA inflammation.

7. Materiały ROS Scavenger Antioxidant for excess generation of ROS TEMPO( 2,2,6,6-tetramethylpiperidine-1-oxyl) MATERIAL Low molecularROS scavengers are easily eliminated from the system resulting in poor therapeutic effect. Polyion Complex (PIC) Redox Flower Micelle hydrophilic PEG shell (prevents aggregation, biocompatible) Injctable PIC hydrogel TEMPO (ROS scavanger) PEG: poly(ethylene glycol) PMNT: poly[p-4-(2,2,6,6-tetramethyl-1-piperidine-1-oxyl)aminomethylstyrene 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 protonated pH + + PMNT-PEG-PMNT triblock copolymer + + + + + + N N N N N N N N N N N N N N N N unprotonated pH PIC hydrogel flower micelle solution injectable fluid which exhibis in situ gelformation + _ 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 Advantages: N N N N N N N

8. 280K 150K chloroform DMF dry polymer TEMPO crystal H2O micelles

9. Lineartransformationmethod 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)

12. Fig. PMNT-PEG-PMNT in H2O, DMF and chloroform (5mg/mL) peak-to-peak line width (ΔHpp) versus temperature [K]

13. Materiały

14. Materiały Scheme 1. Schematic representation of the magnetic nanocarrier studied

15. Materiały Schemat syntezy nanocząstek magnetytu pokrytych chitozanem i znakowanych TEMPO z dołaczonym antybiotykiem

16. Materiały Mikrofotografie TEM A,20nm, B50nm, C10nm magnetycznych nanocząstek

17. Widma EPR znakowanych TEMPO nanocząstek magnetytu z dopaminą

18. Fig. 4. Changes ofresonance field (Hr) versustemperatureforinvestigatedsample with (FC)andwithout field cooling(ZFC). Fig. 6. Changes ofg-factorvalueversustemperatureforinvestigatedsamplewith (FC) andwithout field cooling(ZFC). Zmiany parametrów widm EPR w funkcji temperatury i tzw. FC i ZFC

19. 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.

20. Zależności temperatorowe nanocząstek magnetytu parametrów EPR:szerokości linii, intensywności i wsp. g *linia szeroka od rdzenia

21. Fig. 5. The temperature dependences of fast rotation correlation times of nitroxide spin probe for the orientations 0and 90. Czasy korelacji

22. Tomografia EPR

28. Wolne rodniki

29. Wolne rodniki

30. Wolne rodniki

31. Wolne rodniki Dyfuzja w ciągu 3 godzin

32. Współczynniki dyfuzji nanocząstek w hydrożelu

33. Dziękuję za uwagę

37. Nie mam żadnych wykresów Hałupki

38. Materiały Dexamethasone - DEX, a member of the GC class of steroidinflammatory and immunosuppressingdrug 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-linedrugs to preventjointsdestruction: DMARDs first-linedrugs to reduceinflammation and pain: NSAIDs GC LONG TERM TREATMENT CAUSING MANY SIDE EFFECTS

39. . . AIBN toluene HS-PEG-SH ・S-PEG-S・ PCMS-PEG-PCMS PCMS-PEG-PCMS (4-amino-TEMPO) PMNT-PEG-PMNTTriblock 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

40. Fig. 7. Changes ofcorrelationtime(τ) versustemperatureforinvestigatedsample with (FC)andwithout field cooling(ZFC).