Protein Stabilization and Delivery: A Case ... - gea.ku.edu
Transcript of Protein Stabilization and Delivery: A Case ... - gea.ku.edu
![Page 1: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/1.jpg)
Protein Stabilization and Delivery: A Case Study with Invasion Plasmid Antigen D
Nicole Montoya
Institute of Sustainable Engineering
Department of Chemical and Petroleum Engineering
University of Kansas, Lawrence
![Page 2: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/2.jpg)
Vaccine Cold Chain
• Transporting and storing vaccines at 2-8ºC from
manufacturing to delivery site
• 50% vaccine wastage in the past 15 years
• Main problems:
• Inadequate cold chain capacity
• Lack of functioning cold chain equipment
• Poor temperature monitoring and
maintenance systems
WHO/UNICEF Achieving immunization targets with the comprehensive effective vaccine management (EVM) framework.
![Page 3: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/3.jpg)
Ultimate Goal
Desorbing
Agent
Mem
bra
ne
Mem
bra
ne
Silica Immobilized Vaccine
Mem
bra
ne
Mem
bra
ne
Silica + Desorbing Agent
Desorbing agent is pushed
through membrane
Free Vaccine
Vaccine diffuses through
membrane Vaccine is now ready for
administration
To develop and manufacture a syringe-like device that transports and
stores silica immobilized vaccine
![Page 4: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/4.jpg)
Background
Shigellosis & IpaD
• Shigellosis is a gastrointestinal disease (1 million deaths annually)
• Invasion Plasmid Antigen D (IpaD): antigen protein → target protein for stabilization
Mesoporous Silica & IpaD
![Page 5: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/5.jpg)
Physicochemical Characterization (BET)
Silica GelAverage Pore Diameter
(nm)
Accessible Pore Volume
(cm3/g)
Accessible Surface Area
(m2/g)
Sample 1 (S1) 3.9 ± 1.5 0.07 ± 0.01 67.4 ± 3.3
Sample 2 (S2) 8.1 ± 1.5 0.30 ± 0.01 125.6 ± 3.3
Sample 3 (S3) 15.0 ± 1.5 1.28 ± 0.01 333.2 ± 3.3
Sample 4 (S4) 17.7 ± 1.5 1.41 ± 0.01 319.3 ± 3.3
Sample 5 (S5) 24.2 ± 1.5 2.18 ± 0.01 370.7 ± 3.3
Sample 6 (S6) 30.2 ± 1.5 3.78 ± 0.01 300.0 ± 3.3
Sample 7 (S7) 36.3 ± 1.5 2.87 ± 0.01 286.1 ± 3.3
Langmuir 2020, XXXX, XXX, XXX-XXX Publication Date:October 23, 2020
https://doi.org/10.1021/acs.langmuir.0c02400
![Page 6: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/6.jpg)
Materials and Methods
Adsorption Washing Heating DesorptionCircularDichroism
• 30 mg silica gel + 0.7 mL IpaD at 1.5 mg/mL
• Mix for 20 hours
• Measure supernatant protein concentration
• Wash silica to remove unbound proteins
• Heat silica-IpaD complex at 95C for 2 hours
• Remove proteins from silica with 0.7 mL of 10% LDAO
• CD analysis on IpaD to evaluate secondary structure
LDAO Structure:
![Page 7: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/7.jpg)
Effect of silica pore diameter on IpaD adsorption
Key Results:
• Percent IpaD adsorption increases
as pore volume increases
• Silicas with pore diameter > 15 nm
adsorb more than 90% IpaD
Pore Diameter (nm)
0 10 20 30 40
Perc
ent
Ipa
D A
dso
rbed
0
20
40
60
80
100
S4
S1
S2
S3
S6 S7S5
Langmuir 2020, 36, 14276-14287 Publication Date:October 23, 2020
https://doi.org/10.1021/acs.langmuir.0c02400
![Page 8: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/8.jpg)
Adsorption Isotherm Silica Gel S3
Langmuir Model Equation:
𝐶𝐼𝑝𝑎𝐷,𝑎𝑑𝑠 =𝐾𝐶𝑚𝐶𝐼𝑝𝑎𝐷,𝑓𝑟𝑒𝑒
1+𝐾𝐶𝐼𝑝𝑎𝐷,𝑓𝑟𝑒𝑒
Linearized Form:𝐶𝐼𝑝𝑎𝐷,𝑎𝑑𝑠
𝐶𝐼𝑝𝑎𝐷,𝑓𝑟𝑒𝑒=
1
𝐾𝐶𝑚+
𝐶𝐼𝑝𝑎𝐷,𝑓𝑟𝑒𝑒
𝐶𝑚
• Cm: maximum monolayer coverage
• K= kads/kdes
Free IpaD Concentration (mg/mL)
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Adso
rbed I
pa
D (
mg
/g)
0
50
100
150
200
Langmuir 2020, 36, 14276-14287 Publication Date:October 23, 2020
https://doi.org/10.1021/acs.langmuir.0c02400
![Page 9: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/9.jpg)
Adsorption Isotherm Silica Gel S3
Cm=181 mg/g
K=21
Free IpaD Concentration (mg/mL)
0.0 0.2 0.4 0.6 0.8 1.0
Fre
e/A
dso
rbed I
pa
D C
once
ntr
ati
on (
g/m
L)
0.000
0.002
0.004
0.006
0.008
Langmuir 2020, 36, 14276-14287 Publication Date:October 23, 2020
https://doi.org/10.1021/acs.langmuir.0c02400
IpaD Unit Cell Dimensions 10.07 nm by 11.2 nm
Unit Surface Area 112.8 nm2/protein
IpaD Molecular Weight 39 kDa
181 𝑚𝑔 𝐼𝑝𝑎𝐷
𝑔 𝑠𝑖𝑙𝑖𝑐𝑎𝑥𝑚𝑜𝑙 𝐼𝑝𝑎𝐷
3.9𝑥107𝑚𝑔𝑥6.02𝑥1023 𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠
1 𝑚𝑜𝑙𝑥112.8 𝑛𝑚2
𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠𝑥
(1 𝑚)2
(1𝑥109 𝑛𝑚)2=316 𝑚2 𝐼𝑝𝑎𝐷
𝑔 𝑠𝑖𝑙𝑖𝑐𝑎
![Page 10: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/10.jpg)
Adsorption Isotherm Silica Gel S4 and S5
Silica Gel N2 Surface Area (m2/g) IpaD Surface Area (m2/g)
S3 333 ± 3.3 316 ± 8.9
S4 319 ± 3.3 317 ± 11.4
S5 371 ± 3.3 372 ± 20.2
Free IpaD Concentration (mg/mL)
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Fre
e Ip
aD
/Ad
sorb
ed I
paD
Con
cen
trati
on
(g/m
L)
0.000
0.002
0.004
0.006
0.008
0.010
Free IpaD Concentration (mg/mL)
0.0 0.2 0.4 0.6
Fre
e/A
dso
rbed
Ip
aD
Con
cen
trati
on
(g/m
L)
0.000
0.001
0.002
0.003
0.004
Silica Gel S4 Silica Gel S5
![Page 11: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/11.jpg)
Silicas with Smaller Pore Size (<15 nm)
Free IpaD Concentration (mg/mL)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Ad
sorb
ed I
pa
D C
on
cen
tra
tio
n (
mg
/g)
0
50
100
150
200
Free IpaD Concentration (mg/mL)
1.30 1.32 1.34 1.36 1.38 1.40 1.42 1.44
Adso
rbed I
paD
Conce
ntr
ati
on (
mg/g
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Silica Sample Pore Diameter
S1 (black) 3.9 nm
S3 (red) 15 nm
• IpaD cannot fit inside the pores
and adsorption is negligible
Langmuir 2020, 36, 14276-14287 Publication Date:October 23, 2020
https://doi.org/10.1021/acs.langmuir.0c02400
![Page 12: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/12.jpg)
Silicas with Larger Pore Size (>25 nm)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
Free IpaD Concentration (mg/mL)
Fre
e/A
dso
rbed I
paD
Conce
ntr
ati
on (
g/m
L)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
0.000
0.002
0.004
0.006
0.008
Free IpaD Concentration (mg/mL)
Fre
e/A
dso
rbed I
paD
Conce
ntr
ati
on (
g/m
L)
294𝑚𝑔 𝐼𝑝𝑎𝐷
𝑔 𝑠𝑖𝑙𝑖𝑐𝑎𝑥
𝑚𝑜𝑙 𝐼𝑝𝑎𝐷
3.9𝑥107𝑚𝑔𝑥6.02𝑥1023 𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠
1𝑚𝑜𝑙𝑥630.0 𝑛𝑚3
𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠𝑥
(1 𝑐𝑚)3
(1𝑥107 𝑛𝑚)3=
2.87 𝑐𝑚3 𝐼𝑝𝑎𝐷
𝑔 𝑠𝑖𝑙𝑖𝑐𝑎
Silica
Sample
N2 Accessible Surface
Area (m2/g)
IpaD Surface Area
m2/g
N2 Volume
(cm3/g)
IpaD Volume
(cm3/g)
SA6 300.0 ± 3.3 603.4 ± 43.5 3.78 ± 0.01 3.49 ± 0.23
SA7 286.1 ± 3.3 514.2 ± 46.8 2.87 ± 0.01 2.87 ± 0.26
• Hypothesis: Multilayer adsorption instead of monolayer adsorption
Langmuir 2020, 36, 14276-14287 Publication Date:October 23, 2020
https://doi.org/10.1021/acs.langmuir.0c02400
![Page 13: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/13.jpg)
Types of Pore Interaction with IpaD
A. Pore size less than 15 nm: IpaD does not
fit into the pore
B. Pore size between 15-25 nm: monolayer
coverage, hydrogen bonds depicted by red
line
C. Pore size larger than 25 nm: multilayer
coverage, van der Waals forces
Langmuir 2020, 36, 14276-14287 Publication Date:October 23, 2020
https://doi.org/10.1021/acs.langmuir.0c02400
![Page 14: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/14.jpg)
Circular Dichroism: Secondary Structure
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
190 200 210 220 230 240 250 260
Mo
lar
elli
pti
city
(
)
Wavelength (nm)
IpaD heated on silica
Native IpaD
Denatured IpaD
Key Results:
• Adsorbed IpaD after heat treatment
(and desorption) displays similar “W”
shaped CD signal as the native
unheated IpaD
• IpaD was heated to 95C for 150
minutes while attached to S4
![Page 15: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/15.jpg)
Langmuir Publication
LANGMUIRpubs.acs.org/Langmuir
The ACS journal of fundamental interface science
December 1, 2020 Volume 36 Issue 47
![Page 16: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/16.jpg)
Acknowledgements
• Dr. David Corbin
• Dr. Philip Gao
• Prof. Shiflett and Prof. Allgeier Research
Groups
• Dr. Ana Rita Morais
• Simon Velasquez Morales
• Kaylee Barr
• Rhianna Roth
• Eric Hartman
Visit our website: www.shiflettresearch.com
![Page 17: Protein Stabilization and Delivery: A Case ... - gea.ku.edu](https://reader031.fdocuments.us/reader031/viewer/2022012514/618d8dd464babc52e278cc71/html5/thumbnails/17.jpg)
Thank you for you attention! Questions?