Our second webinar in the MDC Connects Series 2021 | A Guide to Complex Medicines.
This slide deck takes a closer look at cellular internalisation and trafficking of complex medicines.
Dr Jamie Szczerkowski, Medicines Discovery Catapult
Multimodality Molecular Imaging โ An Overview With Special Focus on PET/CT
ย
Similar to MDC Connects Series 2021 | A Guide to Complex Medicines: Cellular Internalisation and Trafficking of Complex Medicines - Dr Jamie Szczerkowski (MDC)
Similar to MDC Connects Series 2021 | A Guide to Complex Medicines: Cellular Internalisation and Trafficking of Complex Medicines - Dr Jamie Szczerkowski (MDC) (20)
2. ยฉ 2021 Medicines Discovery Catapult. All rights reserved.
Why the interest in Complex Medicine
- Opens up the possibility for previously
undruggable targets
- Opportunity for cell / tissue specific targeting,
greater therapeutic index.
But has its own unique challenges:
Accumulation
Penetration
Cargo Delivery tech
Circulation
3. ยฉ 2021 Medicines Discovery Catapult. All rights reserved.
Why the interest in Complex Medicine
- Opens up the possibility for previously
undruggable targets
- Opportunity for cell / tissue specific targeting,
greater therapeutic index.
But has its own unique challenges:
Accumulation
Penetration
Cell specific binding
Internalisation
Efficacy
Circulation
Cargo delivery
Toxicity
X
Know your question
Plan your analysis
Choose your tool
4. ยฉ 2021 Medicines Discovery Catapult. All rights reserved.
Zeiss ELYRA 7
Zeiss LSM880
Zeiss AxioObserver
Zeiss AxioScan
Brightfield & widefield
Confocal, Multiphoton & Airyscan
EPI/TIRF/HILO & Super Resolution (SMLM)
Small
molecule
Proteins
Virus/
Vesicles
Bacteria/
mitochondria
Animal
Cell
Organoid
Mouse
brain
Mouse
1nm
10nm
100nm
1๐m
10๐m
100๐m
1mm
1cm
10cm
Fluorescence microscopy Super Resolution
Electron microscopy
Choose the right approach
Resolution
โข High Content Screening
โข Whole slide Imaging
โข Access to Opera Phenix
HCS (CRUK)
โข Widefield
โข Apotome
โข Confocal
โข Multiphoton
โข Airyscan super resolution
โข STORM super resolution
โข TIRF AND HILO
Speed / Scale
So what are your goals?
5. ยฉ 2021 Medicines Discovery Catapult. All rights reserved.
Zeiss ELYRA 7
Zeiss LSM880
Zeiss AxioObserver
Zeiss AxioScan
Resolution
โข High Content Screening
โข Whole slide Imaging
โข Access to Opera Phenix
HCS (CRUK)
โข Widefield
โข Apotome
โข Confocal
โข Multiphoton
โข Airyscan super resolution
โข STORM super resolution
โข TIRF AND HILO
Speed / Scale
So what are your goals?
Cell binding
Goals Equipment capabilities
Internalization
Method / Analysis
Hit-miss assay
Tissue or Spheroid
Real time tracking
Plate or slide scanning
Trafficking Higher resolution
Co-localisation
Incubation chamber
Depth penetration
3D reconstruction Z-stack capable
Higher speed acquisition
Sectioning or higher working distance
6. ยฉ 2021 Medicines Discovery Catapult. All rights reserved.
Fluorescence
>
+
Ctrl
Cell Mask
-
Ctrl
Cell ROI
Fluorescence
Overlap
Threshold Determined on
Negative control
205
250
235
175
โฆ
Mean:
225
Fluorescence Overlap = Fluorescence over
Threshold within Cell Region
Threshold =
Threshold
Intensity:
Fluorescence
Extract
Data
Percentage
area >
threshold
Intensity
values
7. ยฉ 2021 Medicines Discovery Catapult. All rights reserved.
5min 30min 60min 24h
anti CD33
Membrane
Nuclei
anti CD33
CD33 expressing
SHI-1 leukemia cells
Her-2
Nuclei
Membrane
Her-2
HER-2 expressing
Sk_Br3 breast
cancer cells
8. ยฉ 2021 Medicines Discovery Catapult. All rights reserved.
Initial mask (1) Whole cell (2) Membrane mask (3)
Cytoplasm region (4) Accepted Spots (5)
0 50 100 150
0.0
0.2
0.4
0.6
time (min)
Signal
in
cytoplasm
T1/2=19.13 min
9. ยฉ 2021 Medicines Discovery Catapult. All rights reserved.
Receptor binding
Internalisation
Endosome
Late Endosome
Recycling
Endosome
Lysosome
Active drug
LAMP1/anti CD33
RAB11/anti CD33
RAB5/anti CD33
RAB11
Recycling Endosome
RAB5
Early endosome
LAMP1
Lysosome
Intracellular Trafficking of Antibodies
10. ยฉ 2021 Medicines Discovery Catapult. All rights reserved.
Aim:
To evaluate the cellular internalisation of
silica nanoparticles and correlate
internalisation with efficacy.
Approach:
โข (+) charged Silica nanoparticles labelled
Alexa568
โข Plasmid DNA encoding GFP was loaded
on to the particles. Electrostatically
attracted (-) charged RNA / DNA
โข Cells were incubated with
nanoparticles
โข Efficiency of nanoparticle uptake and
subsequent GFP expression was
measured by microscopy
DNA (- charge)
Silicon (+ charged)
Image with confocal
Silicon particles
Labelled Red
11. ยฉ 2021 Medicines Discovery Catapult. All rights reserved.
Cell mask (Green)
Reconstruct
3D cell
Reconstruct
particle
In or out?
Silicon particles
(Red)
How many particles are internalised?
When and how are these particles internalised?
Cargo successfully released and protein expressed?
Link internalised particles to
GFP expression
Visualise internalisation in 3D
2D cell
Looks internalised
12. ยฉ 2021 Medicines Discovery Catapult. All rights reserved.
24Hr 48Hr 72Hr
Not the
same
images
The same images
Comparisons of individual images
13. ยฉ 2021 Medicines Discovery Catapult. All rights reserved.
Cell Mask ๏ 3D Reconstruction
Particles ๏ 3D Reconstruction
Particles 72 Hr
Cell border
Outside
On the cell
Inside
Distance
inside
14. ยฉ 2021 Medicines Discovery Catapult. All rights reserved.
โข Microscopy is a powerful tool for the
characterisation of complex medicines
โข Applicable to a wide variety of delivery
technology and cargos
โข Enable deep and adaptable evaluation over
varied scales and resolution in different
sample formats
โข To answer huge array of questions
Cells 2D/3D Mono- / Co-culture
Tissues Organ on a chip Live / fixed
Cell binding Internalization Trafficking
Co-localisation Subcellular distribution
Editor's Notes
ย therapeutic index quantitative measurement of the relative safety of a drug
Delivery technology โ can be cell targeted
In normal cells,ย CD33ย is involved in cell signaling, cell adhesion, and cell growth. Cancer cells that make too muchย CD33ย may grow more quickly. ... Cancers that may beย CD33 positiveย include acute myeloid leukemia and some types of lymphoma.
HER2-positive breast cancerย is aย breast cancerย that testsย positiveย for a protein called human epidermal growth factor receptor 2 (HER2). This protein promotes the growth ofย cancerย cells.