Investigation of Nuvec Particles for Effective Gene Delivery N. Ponnappan¹, J. Szczerkowski¹, M. Bachman¹, M. Main¹, D. Templeton², R. Harris², M. Janssen², N. Theobald², D. Yilmaz¹ 1. Medicines Discovery Catapult, Alderley Park, Cheshire, SK10 4ZF, UK. 2. N4 Pharma, Weston house Bradgate Park View, Chellaston, Derbyshire, DE73 5UJ
md.catapult.org.uk
Abstract
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30 ng plasmid/ug Nuvec 50 ng plasmid/ug Nuvec 100 ng plasmid/ug Nuvec
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10 15 20 25 30 60 35 40 45 50
Time (h)
Figure 1. Plasmid DNA (pmaxGFP) loading on Nuvec particles at various mass ratios (ng of plasmid per ug of Nuvec). A: Size distribution analysis of pmaxGFP:Nuvec particles by DLS. B: Assessment of zeta potential of particles by zeta nanosizer. C: Mass spectrometry analysis of binding of plasmid DNA to Nuvec particles.
100 100ngngplasmid/ug plasmid/ugNuvec Nuvec
No treatment
Concentration (uM)
Concentration (uM)
3030ngngplasmid/ug plasmid/ugNuvec Nuvec
0. 5
Transfection Efficiency % Cell viability % Transfection Efficiency %
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1515ngngplasmid/ug plasmid/ugNuvec Nuvec
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Transfection Efficiency %
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ro l 0. 05
190 ± 22 ng DNA / µg particle (95% CI, r2 = 0.987)
5 5ngngplasmid/ug plasmid/ugNuvec Nuvec
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CPX NoNotreatment treatment
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30 ng plasmid/ug 50 ng plasmid/ug 100 ng plasmid/ug 150 Nuvec Nuvec Nuvec
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Figure 2. Representative microscopy images (A) & graph showing the transfection efficiency 20 (B) of HEK293T cells transfected with plasmid:Nuvec at different mass ratios over a period of 48 h. 0 U
nt gf re p at m ed R N A o N uv nly ec on ly fr e ly op sh hi liz ed
150 ng/ug Nuvec
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Size (nm)
15 ng plasmid/ug Nuvec
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-20
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on % Transfection Efficiency t
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DNA binding capacity
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150ng/ug Nuvec 150ng/ug Nuvec
DNA binding capacity
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Genistein
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Cell border 0
24Hr GFP
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U n m tre R at N e N A d uv on ec ly on ly fr es -2 h 0º C 4º C R T
Figure 4. Effect of endocytic inhibitors on the transfection efficiency of plasmid DNA loaded Nuvec (A) & viability of cells (B). Control are the HEK293T cells with no endocytic inhibitor.
p gf
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% GFP Cells
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Figure 5. Transfection efficiency of freshly 40 lyophilized GFP mRNA:Nuvec at day 0 and that20stored at different conditions for 6 days after0lyophilization
Conclusions GFP - Cells
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Day 6
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GFP + Cells
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U p nt m re R at N e N A d uv on ec ly on ly fr es -2 h 0º C 4º C R T
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nt re p at m ed R N A o N uv nly ec on ly fr e ly op sh hi liz ed
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Transfection Efficiency %
Transfection Efficiency %
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on tr ol 0. 05
Percentage GFP
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Concentration (uM)
Figure 3. High resolution microscopy images of HEK293T cells treated with Alexa-568 labelled Nuvec particles. 3D reconstruction of images were carried out and percentage of cells expressing GFP and number of Nuvec particles internalised were assessed
100 ng plasmid/ug Nuvec 20
Concentration (uM)
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on tr ol 0. 93
Non-GFP
48Hr
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50 ng plasmid/ug Nuvec 40
Concentration (uM)
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30 ng plasmid/ug Nuvec 60
Transfection Efficiency %
Cell viability %
Distance inside
% Particles Internalised
70
0
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Day 0
15 ng plasmid/ug Nuvec 80
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150
Inside
72Hr
Genistein
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Reconstruct
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5 ng plasmid/ug Nuvec 100
on tr ol 0. 05
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Concentration (uM)
No treatment
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On the cell
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Cell viability %
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Percentage GFP
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CPX
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93
GFP Expression Over time
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150
93
40
Percentage GFP
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% GFP Cells
GFP - Cells
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60
GFP + Cells
24Hr
Genistein
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Transfection Efficiency % 9. 3
70
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A A
Reconstruction
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Cells
Particle
GFP Expression Over time
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24Hr
48Hr
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• Nuvec particle efficiently binds to nucleic acids – plasmid DNA & mRNA, and mediates their intracellular delivery mainly through macropinocytic pathway 2D
Reconstruct
Outside
?
Out or in
• The cells transfected with GFP plasmid loaded Nuvec particles exhibit up to 70% transfection efficiency Out
3D
On the cell
In
Reconstruct
• Our data for transfection efficiency of lyophilized GFP mRNA loaded Nuvec stored at -20°C indicates the ability of Nuvec particles to protect the nucleic acids from degradation. The freeze-dried formulations of Nuvec-based nucleic acid therapeutics would be convenient and cost-effective Day 0 Inside
50
Cell border 0
24Hr Non-GFP
48Hr GFP
Non-GFP
72Hr GFP
Non-GFP
GFP
Transfection Efficiency %
% Particles Internalised
70
Distance inside
100 80 60
gf
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nt re p at m ed R N A o N uv nly ec on ly fr e ly op sh hi liz ed
Medicines Discovery Catapult (MDC) 40 Funded by Innovate UK, MDC is national centre set up to help UK SMEs, biotechs, academics and innovators with access to cutting edge technology, 20 expertise, data and networks they need to progress their drug discovery programs. 0 Through collaborative programmes of R&D we are tackling the most challenging issues in drug discovery, addressing systemic problems and bottlenecks and using innovative technologies to enable “fast-to-patient” medicines discovery. 13201 - A0 MDC Posters Full Set v3.indd 3
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ol
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Microscopy and flow 50 cytometry analysis
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on tr
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Concentration (uM)
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on tr ol 0. 05 C 150
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Percentage GFP
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on tr ol 0. 93
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on tr ol 0. 05 C
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50*** **
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Percentage GFP
Percentage GFP
150
on tr ol 0. 93
100ng/ug Nuvec 100ng/ug Nuvec C
Size (nm)
200
C
50ng/ug Nuvec 50ng/ug Nuvec
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50 ng/ug Nuvec
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DNA bound (ng)
25 ng/ug Nuvec 25 ng/ug Nuvec
10 0
No DNA
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25 ng/ug Nuvec
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No DNA
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Zeta Potential-pDNA Zeta Potential-pDNA
No DNA
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Incubation at 37°C, 5% C02 for different time-periods
Cell viability %
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DLS-size-pDNA
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B
Genistein
Concentration (uM)
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CPX
B
Zeta Potential (mV)
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Mass spectrometry analysis to assess the binding 100 100Nuvec particles of DNA/mRNA molecule to A Cyt D CPX
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DLS-size-pDNA DLS-size-pDNA
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Concentration (uM)
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Transfection of HEK293T cells seeded in 96-well plate with nucleic acid: Nuvec complex
Concentration (uM)
Genistein
Cell viability %
Incubation at room temperature for 20 min
Results 40
0. 93
ol on tr C
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Lyophilisation of complex
A
0
Concentration (uM)
Loading of Nuvec with different amounts of plasmid DNA/mRNA
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Size distribution and zeta potential analysis using DLS & zetasizer
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Method
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Percentage GFP
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Percentage GFP
Genistein
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CPX
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Our data demonstrates that Nuvec particles provide protection to RNA/DNA, efficiently deliver DNA/RNA into the cells and result in protein expression. Furthermore, our data provides insights into the mechanisms involved in the cellular uptake of Nuvec particles. Altogether our findings provide guidance for the rational design of Nuvec for efficient gene delivery applications.
0. 5
Here we carried out biological characterisation of Nuvec particles using a range of methods. We determined the size, zeta potential and DNA/ RNA loading capacity of different size Nuvec particles using DLS, zeta sizer and mass spectrometry respectively. Next, we determined the transfection efficiency of Nuvec at different Nuvec:nucleic acid ratios using both live cell imaging and flow cytometry methods. Furthermore, we investigated the stability of mRNA upon lyophilisation of
0. 5
Nuvec:mRNA complex and demonstrated that Nuvec particles protect the RNA from degradation even after lyophilisation. Additionally, we studied the cellular internalisation of Nuvec using high resolution microscopy. We analysed 3D reconstructed microscopy images and quantified the number of Nuvec particles inside the cells. Finally, we studied the mechanism of cellular internalisation in HEK293T cells upon inhibition of different endocytosis pathways.
5
Effective delivery of nucleic acids into the cells holds great potential for vaccine development and cancer treatments. N4 Pharma has been developing Nuvec® – a unique non-viral adjuvant delivery system for the effective delivery of nucleic acids into the cells. Nuvec silica nanoparticles have a unique irregular (spiky) surface structure, coupled with polyethyleneimine (PEI), that simply and effectively traps and protects nucleic acids (such as mRNA / pDNA) as it travels into the cells.
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