Capsulight: In vitrophoto-killing of Helicobacter pyloriby using an innovative LED-based device. Paola Morici

Transcription

1 Capsulight: In vitrophoto-killing of Helicobacter pyloriby using an innovative LED-based device Paola Morici

2 Helicobacter pylori In 1893 Dr Robin Warren and Professor Marshall characterised H. pylori A spiral- or curved-shaped Gram negative bacterium (2,5-4 µm length, 0,5-1 µm width) Motile(2-6 polar flagella) Microaerophilic H. pylori colonizes the stomach of half the human population, causing severe mucosal inflammation and gastric diseases In 2005 Nobel Prize in Medicine for their discovery of "the bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease

3 Worldwide prevalence of H. pylori infection Prevalence varies greatly among countries and population groups It is highly dependent on the socioeconomic status of the country >80% prevalence in middle age adults in developing countries 20 50% prevalence in middle age adults in developed countries Michael D. Burkitt et al. Dis. Model. Mech. 2017;10: Published by The Company of Biologists Ltd

4 H. pylori infection Acquisition typically occurs in childhood Oral-oral or fecal-oral transmission Asymptomatic Syntoms Nausea Vomiting Bloating Heartburn Loss of appetite Weight loss Indigestion (dyspesia) Blood in stool etc... In adults, chronic gastritis can evolve towards severe gastric diseases Clinical course depends on bacterial and host factors Suerbaum S,MDandPMichetti,M.D.NEnglJMed2002; 347: In 1994 the World Health Organization has classified H. pylori as a class I carcinogen for gastric cancer

5 H. pylori virulence factors caga+/vaca+ H. pylori strains Increasedriskfor severe diseases and gastric cancer

6 H. pylori and biofilm Bacterial biofilm is a complex microbial ecosystem where bacterial cells are embedded in an extracellular polysaccharide matrix It plays a crucial role in promoting the survival and the spread of H.pylori inthehost Gastric biopsy (Cammarota G. et al, 2012) Chronic infections are most often associated with the biofilm formation

7 Clinical treatment Triple therapy(recommended as first line treatment): two antibiotics (Clarithromycin/Amoxicillin or Metronidazole; Levofloxacin/Amoxicillin) a proton-pump inhibitor PPI(i.e. esomeprazole and pantoprazole) The efficacy of conventional therapy is decreasing worldwide (<70 80%) mainly due to increased ANTIBIOTIC RESISTANCE rates

8 Antimicrobial PhotoDynamic Therapy (APDT) CELL DEATH

9 APDT and H. pylori Advantages Broad spectrum of action Multiple cellular targets Rapid killing No resistance induction Disadvantages Only for localised infections Use of an exogenousphotosensitizer: Photosensitization Insufficient drug uptake Gram-negative resistance Undesired uptake by healthy tissue. H. pylori naturally synthesizes and accumulates photoactive porphyrins that can be exploited to induce photokilling

10 Design of an ingestible robotic pill based on LED sources for the treatment of gastrointestinal disorders supported by Regione Toscana(Italy) Bando FAS Salute 2014 Elettronic boards LED Transparent enclosure Final capsule Batteries Characterization of the photophysical properties and study of the composition H. pylori endogenous porphyrins In vitro photokilling of H. pylori strains

11 Extraction of porphyrins from H. pylori Washing (Tris/EDTA) Washing (H 2 O) SONICATION in ethyl acetate/acetic acid (3:1, v/v) EXTRACTION H. pylori cultures (ATCC and a virulent strain, ATCC ) in Brucella Broth (BB)/10% FBS incubated at 37 for 2 days in a microaerophilic atmosphere. Raw extract in HCl (Protocol adapted from Mancini and Imlay, 2015)

12 Spectroscopic analysis Different behavior under different environmental conditions HCl MeOH ABSORPTION B band Q bands ABSORPTION: In MeOH, absorbance spectra show a B band (around 400 nm) and 4 smaller bands(q bands; nm) In HCl, the protonation induces the FLUORESCENCE coalescence of the 4 Q bands in two broad bands FLUORESCENCE: Fluorescence spectra show a marked displacement of the peaks Battisti A, Morici P, Signore G, Ghetti F, Sgarbossa A. Compositional analysis of endogenous porphyrins from Helicobacter pylori. Biophys Chem ,

13 Compositional analysis of the bacterial extracts (HPLC-MS) m/z[m+h + ] = 563 m/z[m+h + ] = 655 m/z[m+h + ] PP IX CP I CP III A = CP I 655 B = CP III 655 C = PP IX 563 D =? 710 Battisti A, Morici P, Signore G, Ghetti F, Sgarbossa A. Compositional analysis of endogenous porphyrins from Helicobacter pylori. Biophys Chem ,

14 Compositional analysis of the bacterial extracts (HPLC-MS) A B C D A B C D

15 Compositional analysis of the bacterial extracts (HPLC-MS) CP PP IX? A B C D CP PP IX? A B C D CP PP IX?

16 About peak D HPLC-MS 0 Long retention time

17 About peak D HPLC-MS Absorption Long retention time Porphyrin-like absorption spectrum

18 About peak D HPLC-MS Absorption Fluorescence Long retention time Porphyrin-like absorption spectrum CFS CFS Residues: FS extract CFS Residual fluorescence detected

19 H. pylori biofilm 1 Hp strains were cultured on Brucella agar plates supplemented with 7% laked horse blood and incubated at 37 for 3 days in microaerophilic conditions. 2 Bacteria were harvested and suspended in brain heart infusion (BHI) broth supplemented with 0.5% β-cyclodextrin and 0.4% yeast extract CFU/ml 4 Bacterial suspension was inoculated into each well of the 12-well culture plates containing sterilized round glass coverslips placed vertically 3 Incubation for 4 and 7 days. After incubation, coverslips were washed twice with PBS to remove non-adhered bacterial cells. 5 Confocal microscopy Fluorescence lifetime imaging (FLIM) Viability (PI/Syto9) Negative control: BHI broth without Hp cells

20 Fluorescence imaging of bacterial biofilms PI/Syto9 DIC Fluo λ ex = 640 nm λ em = nm

21 Fluorescence lifetime τ monomer > τ dimer > τ aggregate Extracts(λex= 405 nm) Bacterial biofilms

22 Design of an ingestible robotic pill based on LED sources for the treatment of gastrointestinal disorders supported by Regione Toscana(Italy) Bando FAS Salute 2014 Elettronic boards LED Transparent enclosure Final capsule Batteries Characterization of the photophysical properties and study of the composition H. pylori endogenous porphyrins In vitro photokilling of H. pylori strains

23 In vitro photo-killing assay: LED-based DEVICE Cover Petri Support Electronic boards Base with batteries Main features Completely integrated in a small case Up to 1 hour lifetime Programmableillumination(time, delivered power, omogeneity in terms of power) Possibilityto deliverallavailablepower in a short time LEDs configurations at: 460 nm 405 nm 500 nm 630 nm Final capsule Elettronic boards LED Transparent enclosure 27 mm Batteries

24 Determination of wavelength of the LEDs Typical absorption spectrum of the natural photosensitizers of H. pylori Different light absorption by human mucosa tissue 405nm 460nm 500nm 630nm 2 LED-based DEVICES Relative intennsity [a. u.] Light emission of LEDs nm nm 0,75 0,5 0, Wavelength [nm] 4LEDsources at405nm 4LEDsources at630nm

25 In vitro photo-killing assay H. pylori strains used: ATCC 43504, ATCC (J99), a virulent strain(caga+ and vaca+) H. pylori cells in exponential growth phase (in Brucella broth/10%fbs) diluted ( CFU/ml) in PBS(phosphate-buffered saline) buffer Irradiationinmicroaerophilicatmosphereat37 o C Finalcolonycount (CFU/ml)after5days OD600 5,00 4,50 4,00 3,50 3,00 2,50 2,00 1,50 1,00 0,50 0,00 Growth curve of ATCC strain Time (h)

26 In vitro photo-killing of H. pylori: LEDsat405 nm Microaerophilic conditions 4 LEDs 25 ma Irradiance: 8, mw/m 2 Illumination time: min LIGHT DOSE= Irradiance (W/cm 2 ) Time (sec) Survival fraction 1,00E+01 1,00E+00 1,00E-01 1,00E-02 1,00E-03 1,00E-04 1,00E-05 1,00E Light Doses: 5 min 2,6 J/cm 2 10 min 5,2 J/cm 2 15 min 7,8 J/cm 2 20 min 10,4 J/cm Time (min)

27 Photo-killing effect The photo-killing effect depends on: Photosensitizer Wavelengths Oxygen concentration LIGHTDOSE=Irradiance(W/cm 2 ) Time(sec)

28 In vitro photo-killing of H. pylori:ledsat405 nm Aerophilic conditions 4 LEDs 25 ma Irradiance: 8, mw/m 2 Illumination time: min 1,00E+01 1,00E Survival fraction 1,00E-01 1,00E-02 1,00E-03 1,00E-04 1,00E-05 1,00E Time (min)

29 In vitro photo-killing of H. pylori:ledsat405 nm Microaerophilic vs Aerophilic Survival fraction 1,00E+01 1,00E+00 1,00E-01 1,00E-02 1,00E-03 1,00E-04 1,00E-05 H. pylori Aerophilic Microaerophilic 1,00E Time (min) 1,00E+01 H. pylori Aerophilic 1,00E+00 Microaerophilic Survival fraction 1,00E-01 1,00E-02 1,00E-03 1,00E-04 1,00E-05 1,00E Time (min)

30 In vitro photo-killing of H. pylori:ledsat405 nm 1,00E+01 1,00E Microaerophilic condition Set up 10 min Illumination time: 5 min Intensity (ma) Irradiance (mw/m 2 ) Dose (J/cm 2 ) Survival fraction 1,00E-01 1,00E-02 1,00E-03 1,00E-04 1,00E , ,6 50 2, ,4 75 3, , , ,2 1,00E Dose (J/cm 2 ) 1,00E Irradiance 8, mw/m 2 Microaerophilic condition Set up 10 min Illumination time: min Survival fraction 1,00E+00 1,00E-01 1,00E-02 1,00E-03 1,00E-04 1,00E-05 1,00E Time (min) Dose (J/cm 2 ) 5 2,6 10 5,2 15 7, ,4 Dose (J/cm 2 )

31 In vitro photo-killing of H. pylori 4 LEDs at 630 nm 4 LEDs-75 ma Irradiance: 2, mw/m 2 Illumination: 13 minutes Dose 18,02 J/cm 2 1,00E+07 1,00E+06 Untr IRR Log CFU/ml 1,00E+05 1,00E+04 1,00E+03 1,00E+02 1,00E+01 Hamblin et al ,00E nm

32 Conclusions The spectroscopic features of Hp endogenous porphyrins were investigated in terms of absorption, steady-state and time-resolved fluorescence, both on bacterial extracts and intact cells CPI,CPIIIandPPIXwere identifiedasthemaincomponents of theraw bacterialextracts Smaller amounts of other porphyrinic compounds were found to contribute to the global red fluorescence of the bacterial extracts Redfluorescence of Hpbiofilmswasimagedfor thefirsttime LED-based devices at 405 nm determined a significantly reduction of viability of H. pylori Further steps of experimentation: to determine the action spectrum of H. pylori photokilling to evaluate the efficacy of the LED-based device also against the in vitro H. pylori biofilm

33 ACKNOWLEDGEMENTS Antonella Sgarbossa Francesco Ghetti Giovanni Checcucci Antonella Battisti Paola Morici Giovanni Signore Arianna Menciassi Giuseppe Tortora Franco Fusi Barbara Orsini Giovanni Romano Paola Faraoni Alessio Gnerucci Laboratori Victoria S. R. L.

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