11/15/2017

 Biological Roles of O2 and principles ofquenched-phosphorescence O2 sensing

 Demonstration in in vivo studies
 Demonstration in ex-vivo models
 3D tissue models and physiological studies inDmitri B. Papkovsky

vitro

School of Biochemistry and Cell Biology,
University College Cork, Ireland

Oxygenation and Physiological Relevance

Oxygenation and (Tumour) Biology
Tumour Metabolism

Cell Models & 3D culture

Therapeutic Efficacy

Glycolytic Flux ([H+] /h)

1.E-07
8.E-08Cell Culture~18.6%

6.E-08Oxygen4.E-08

Anoxia0‘Hypoxia’
5O2%1015‘Normoxia’
20Inspired air

2.E-08~1% O2~7% O2~21% O2Kidney
Liver

0.E+00iO2 = 17%Muscle

iO2 = 0.5%

McKeown SR. Defining normoxia, physoxia and hypoxia
in tumours—implications for treatment response.
Br J Radiol 2014;87:20130676•••

Therapeutic Efficacy
Delineating hypoxia signalling (HIF stabilisation)
Ischemia Reperfusion Modelling‘Hypoxia’ ‘Normoxia’‘Physoxia’

HyperoxicQ) Is culturing cells at 18.6% O2 physiologically relevant ? A) No. It is Hyperoxic w.r.t. to most tissues.
ROS / Metabolism / Signalling?

111/15/2017

In situ [O2] determines cell physiology !!

Zhdanov et al - Integr. Biol. 2010, 2:443

S*
T*

Analytical ApproachFeatures

In vivo imaging with intravascularO2 probes

Systemic administration in live
animals (IV):• High doses, rapid clearance(~3h), once-off• Poorly suitable for in vitro use• Bulk tissue is dark
• Complex synthesis, costs

Intracellular O2 probes & microimaging

Local administration in tissue:• low doses, controlled location• long retention time (many days)• Low toxicity• Micro & macro imaging,

Miscellaneous: O2 probes
Imaging systems,

Various technologies, often 2D orpoint measurements, semiquantitative

phosphorescencehnfluorescencensO2ms•Non-chemical , reversible•

Quantitative, real-time, stable•

Optical, minimally invasive

Relationship : [O2] = (to/t -1)/ K q2

11/15/2017• Small molecules• Bioconjugates, e.g. peptides• Polymeric NPs (by inclusion)• Polymeric NPs (by conjugation)• Multi-functional NP composites

Intensity, a.u.

600
500
400300Probe name, composition

lexclemto (ms)K SVRu(bpy)2(pic)2+ - CPP conjugate
PtCP - CPP conjugates
Ir-BTP coumarin C343 conjugate

458 nm390 nm405 nm

775 ns50-70 ms
5.6 msND
0.006 mM -1
0.064 mmHg -1

IrOEP – CPP complexes
PtTFPP Pt-Glc conjugate

386
395

610 nm
650 nm620, 680 nm
480 nm (ref)654
650

58-69 ms
57 ms

0.074 mM -1
0.03 mM -1

PdTCBP-HiLyte680 dendrimer in PAAG NPs modified
with TAT peptide (30-50 nm)

442, 632 nm
678 nm (ref)790 nm (O2)699 nm (ref)Not reported(250 ms for G2)

0.034 mM -1[Ru(dpp(SO3Na)2)3]Cl2 in PAA NPs (45 nm)

454 nm

608 nm

3.88-4.06 ms

ND

PtTFPP in RL100 polymer (35 nm NPs)

395 nm

650 nm69.1 ms

0.04 mM -1

PtTFPP-naphtalimide dye in PS NPs (410-430 nm)

395 nm

650 nm
490 nm (ref)ND

ND

PtTFPP and PFO in RL100 NPs (70 nm)

405 nm - 1P
760 nm – 2P

650 nm
430 nm (ref)66 ms

0.041 mM -1[Ru(dpp)3](TMSPS)2 in amino modified PS NPs (121nm)

488 – 1P
830 nm – 2P

630 nm

ND~0.8?

PtTBP in RL100 NPs

440, 614 nm

760 nm57 ms~0.02 mM -1

PtTFPP in PS NPs (50 nm)
PtTFPP and PFO in acrylic polymer NPs (95 nm).

395
405 – 1P
760 – 2P

61 ms
68 ms

ND
0.086 mM -1

WPF-Ir4 and WPF-Ir8 NPs (19 nm).

405 nm

650650 nm
430 nm (ref)630 nm
450 nm (ref)0.6 ms

0.006 mmHg ?

[Ru(dpp)3]2+Cl2 and NaYF4:Yb/Tm@NaYF4 inmesoporous silica NP (50 nm)

980 nm – UC

613 nm477 nm (ref)nsND

PtTFPP in PS-silane hybrid NPs (77 nm)

395605 nm

MsND

NanO24002002001001000

Prof. Sergey Borisov, Graz, Austria••••MM2300300 350 400 450 500 550 600 650 700
Wavelength, nm

0300 350 400 450 500 550 600 650 700
Wavelength, nm

Biocompatible polymer
Average size 35-50 nm
Z potential +45mV
Stable, bright, low toxicity

Detection Modes:

Fercher A. et al – ACS Nano, 2011

Dmitriev RI et al . – Adv . Funct . Mater 2012311/15/2017

Conjugated Polymer NPs
Luminescence
Intensity, a.u.

1.2x106
1.0x106

pO2, kPa

(a)

00.98
1.96
3.92
7.82
11.74
19.56

8.0x105
6.0x105
4.0x105
2.0x105• Hydrophilic, water-soluble, neutral• Efficient cell and tissue penetration

0.0400

500

600

700

4x105

2x10

• Stable calibration

pO2, kPa

(b)

00.98
1.96
3.92
7.82
11.74
19.56

3x105
51x1050500

600• High photostability, low toxicity• Moderate brightness

700

800

Wavelength, nm
4R0/R

Luminescence
Intensity, a.u.

Wavelength, nm(c)Advantages:

SII-0.1+
SI-0.15+/0.05-••32•105101520

Enhanced brightness - up to 10-fold, 1P, 2P
Tunable spectra, surface charge, cellspecificity
Improved stability, tissue staining andpenetration

Pt-Glc structure

pO2, kPa

Dmitriev RI et al . – Biomater. Sci., 2014

Dmitriev RI et al . – ACS Nano, 2015

Procedure:
◦◦
◦
◦
◦

Anaesthesia, surgery
Probe/Sensor application
Mounting cranial window
Commercial Intensity based imager
Sacrificing animal4

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VSD - Cell depolarisation◦ fast, localised response - ~50 ms

O2 Probe - tissue oxygenation, metabolism, hemodynamics◦ Delayed, bi-phasic response◦ Affects larger area
◦ Resembles BOLD-MRI, fNIRS signals

Tsytsarev et al – J. Neurosci Meth, 2013,

Ex-Vivo Tissue: Experimental procedure
Euthanasia
Tissue excision& mounting

 Distal colon
 Urinary bladder Carotid arterysupercontinuum ps laser
Fianium, 400-650 nm, 4W

Axio Examiner microscope (Zeiss)
DCS-120 Confocal FLIM (B&H)

Pt-GlcPins

Pins

1-3 h at 37oC
Pins

Pt-GlcStimulations
Pins

Pins

Pins

511/15/2017

Occluded Carotid Artery Oxygenation
Control carotid artery

6 weeks after ligation180

iO2 [mM]

0• Bright staining of the tunica intima layer• Dramatic effect of ligation on tissue oxygenation
Zhdanov AV et al . – CMLS , 2016

KOWT

Control
DSS

010 20 30 40 50 60

HKOI
105

1.4
1.3

Normalised OCR [%]

80
7060
50
4030
20
100

OCR [nM/min × mg protein]GO2 levels in the samples [mM]

WT

1.2
1.1
10.9
0.8

0.7
Control

DSS

100

95

S1p = 0.116

S20135iO2 [mM]

Prominent differences in ROS generation, but
marginal differences in tissue O 2 levels !

90O243.5
32.5
21.5
10.50050
TOPMIDDLE

100
BOTTOM611/15/2017

Detailed Analysis - Group comparison
Title
Non-parametric Chart
Mann-Whitney
U-test
125Line 1Line 2

5040iO2 [microM]

75

Pt-Glc intensity

60
100

2530

20
aver

100Cell border

400iO2

40060

20050

200100 200 300
Distance [micrometres]Line 2

averm +/- SEM values for 25%, 50% and 75% quartiles show thedifference in actual O 2 levels

Line 1O2 [microM]

65

Pircalabioru G et al . – Cell Host Microbe. 2016,19:651.

5Calculated HIF prolyl hydroxylase 2 activities

RestingiO2 [mM] v [%Vmax]
5-10
2-3.8
10-15
3.8-5.7
15-20
5.7-7.4
20-30
7.4-10.7
30-40
10.7-13.8
40-50
13.8-16.7

AntA (80 min)

FCCP / EGTA

85

O2 [mM]

Putative
5 PHD2 activity, v [%Vmax]

12-15 mM O2
4.6-5.7 %Vmax
03040
35
LT [ms]4550160120
8040040 mM O2
13.8 %Vmax

160

Resting
FCCP / EGTA
AntA

Calculated local PHD2 activity :Km (O2) = 250 mM;
v = Vmax[S]/(Km+[S])120

iO2 [mM]**256 mM
9 mM

Average iO2 [mM]

O2 [mM]

30min

987654321080
min
50
min

Relative LT frequency [ *105]100

10-13 mM O2
3.8-4.9 %Vmax

80

35 mM O2
12.3 %Vmax

40

0 20 40 60 80
AntA treatment [min]

0100
02550
75
Cell cross section [mm]Zhdanov AV – Am . J. Cell Physiol.. 2015

O2 [mM]

5Zhdanov A et al, AJP-CP, 20157

11/15/2017•Multicellular spheroids•

Cell co-cultures•Organoids•

Engineered tissue scaffolds•Vascularised tissue

Environmental control and standardization remain bottlenecks
FLIM platforms can address these8

11/15/2017• Neurospheres cultured
at 21% and 4% O2 withNanO2 probe.
• Imaged at 21% O2.

Jenkins J. et al . – Biochem . J. 2016

Intestinal Organoid (SIO) Models:◦◦◦◦Establish cultures
Characterise O2 and respiration activity
Standardize, reduce heterogeneity
Conduct physiological studies

500μm

O2 gradient between basal (blue) andapical (violet) membranes (n=10):9

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



Biophysics Lab (University College Cork):◦ Dr. Alex Zhdanov, Dr. Ruslan Dmitriev◦ Dr. Irina Okkelman◦ Dr. James Jenkins◦ All former lab members

Profs. Sergei Borisov (Graz University of Technology, Austria)
Dr. H. Dussmann (Royal College of Surgeons Ireland)
Dr. V.P. Baklaushev (Pirogov Medical University, Russia)
Dr. M. Tangney, (CCRC, University College Cork)

T – probe (NanO2 analog) –Anal Chem, 2016, 88: 10566
pH probe - J. Mater. Chem. B, 2014, 2: 6792
Cell Cycle assay (Hoechst 2334 and dBrU) –PLOS One, 2016, 11: e0167385
K+-probe - Adv Funct Mater (in press)





More in development

Prof John Cryan, Dr Anna Golubeva
Dr Silvia Melgar, Dr Niall Hyland
Prof Ulla Knaus, Dr Gabriella AvielloFunding:
Science Foundation Ireland, Enterprise Ireland

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