Affibody-functionalized bacterial cellulose tubes for biofiltration applications Hannes Orelma a,...
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Transcript of Affibody-functionalized bacterial cellulose tubes for biofiltration applications Hannes Orelma a,...
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Affibody-functionalized bacterial cellulose tubes for biofiltration applicationsHannes Orelmaa, Luis O. Moralesa, Leena-Sisko Johanssona, Ingrid C. Hoegerb, Ilari Filpponena, Cristina Castroc, Janne Lainea, Orlando J. Rojasa
a Aalto University, Biobased Colloids and Materials group (BiCMat), Finland. b North Carolina State University, Departments of Forest Biomaterials and Chemicaland Biomolecular Engineering, Raleigh, USAc Universidad Pontificia Bolivariana, School of Engineering, Medellenin, Colombia
E-mail: [email protected] / [email protected]
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Objective: Develop a nanocellulose-based biofiltration system for
affinity separation of HSA
Feed
Filtrate
Filtrate
Filtr
ate
Filtrate
Residue
66.5 kDa
Human serum albumin (HSA): the most abundant protein in human blood plasma
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Microbial cellulose / bacterial cellulose
• Acetobacter, Agrobacterium, Alcaligenes, Pseudomonas, Rhizobium or Sarcina are able to produce cellulose extra-cellularly– Acetobacter xylinum/Gluconacetobacter xylinus is considered to be
the most efficient strain– A continuous source of air and carbon is required – Cellulose yield of 35-40% in relation to glucose in culture medium
• Acetobacter microfibrils usually have large crystal structures and thickness in the range of 6-10 nm
• Bacterial cellulose has a high degree of polymerization (4000-10000 anhydroglucose units) and it forms a pellicle
– It can contain more than 99 % water– Strong in wet state!
Klemm et al. 2011, Angewandte Chemie International Edition,50, 5438
P. Gatenholm, D. Klemm, MRS Bull. 2010, 35, 208
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Applications of bacterial cellulose
Skin therapy
Artificial blood vessels
Scaffolds for tissue engineering
Nanopaper & paper additives
Food
W. Czaja, A. Krystynowicz, S. Bielecki, R. J. Brown, 2006 Biomaterials, 27 2
Kowalska-Ludwicka, Karolina et al. Archives of Medical Science :
AMS 9.3 (2013): 527.
Pure bacterial cellulose
Nata de Coco
de Olyveira et al. Bacterial Nanocellulose for Medicine Regenerative J. Nanotechnol.
Eng. Med. 2, 034001 (2012) v
http://healthcarenutritionlifestyle.blogspot.fi/
2012/11/be-slim-with-nata-de-coco.html 03/05/15
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MaterialsTubesBacterial cellulose tubes
Gluconoacetobacter medellinensis
In-situ modificationCarboxymethyl cellulose DS = 0.7, DP = 250 000 g/mol
CMC concentration of 0-5 g/l
Ex-situ modificationTEMPO-mediated oxidation of BC with TEMPO, NaBr, and NaOCl system at
pH of 10 TEMPO-oxidation time: 1-30 min
Proteins Human Serum Albumin (HSA) Dansylated HSA for fluorescence imaging Anti-HSA affibodies H2N
Preparation of BC tubes Strain and incubation conditions
Hestrin-Schramm (HS) medium of pH of 4.5
Gluconoacetobacter medellinensis
Closed vessel with permeable silicone tubes
Static incubation at 28 C for 9 days
O2 O2O2
O2
O2 O2
O2O2
O2O2
O2Air
Glucose
Glucose
9 days
Silicone tube HS media Bacterial cellulose (BC) tube
O2
Glucose
Cellulose fibrilClosed vessel CMC
CMC
Incubation
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Carboxymethyl cellulose (CMC)
BacteriaCarbon source + phosphorus + air + additives
Bacterial cellulose
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Conjugation of affibodies to CMC-modified BC tubes (via EDC/NHS chemistry)
O- Na+
O
O
O
N
O
O
NH
O
Anti-HSA affibody
EDC/NHS
pH 5 buffer
Hollow BC tubewith in-situ CMC
modification
BC fibrils
- - --
--- - -
---
--
---
Purification
Biofiltration
HSA
Filtrate
- -
--
-
-
-
-
CMC
Conjugations of 0.1 mg/ml anti-HSA to CMC with 0.1 M EDC + 0.4 M NHS in 10 mM NaOAc buffer at pH 5.
H2N
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BC Characterization methods
Wet methods• Conductometric titration (SCAN-CM 65:02)
Charge density of BC after CMC addition
• Water retention value (SCAN-C 62:00)
Bound water and irreversible structural changes of BC
Dry methodsTubes were lyophilized via liquid nitrogenSEM Imaging
Surface Topography of BC Lyophilized via liquid nitrogenSurface and cross-section
Surface chemistry with XPSSurface chemical composition
Fluorescence imaging with Confocal LaserScanning Microscopy (CLSM)
Dansylated HAS
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Interaction analysis with Surface Plasmon Resonance (SPR)
SPR and cellulose model surfaces• Multimode SPR Navi 200, Oy Bionavis Ltd.• Angular scan mode• Langmuir-Schaefer deposited TMSC based
cellulose surfaces• Cellulose II content up to 70%
• Thickness:
• Surface coverage:
Matthew A. Cooper, Nature Reviews Drug Discovery 1, 515-528 (2002)SPR model from Jung et al. Langmuir 1998, 14, 5636-5648
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CMC-modified BC tubes
Length 20 cmDiameter 1 cm
Wall thickness (wet): 1.8 ± 0.2 mm
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Effect of CMC on BC tube morphology
Pure BC
BC grown with 2 g/l CMC
Inner surface Cross-section
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0 1 2 3 4 5
0100200300400500600700800900
100011001200
CMC added in the medium (g/l)
WR
V (
%)
0
200
400
600
Cha
rge
(eq
/g)
Water binding capacity and irreversible changes upon drying
Never dried
Dried
1. Small effect of CMC on WRV of never dried BC
2. Drying: Significant effect on water binding
3. Highest charge obtained with CMC additions above 2 g/l
0 1 2 3 4 5 60
100
200
300
400
500
600
TEMPO-oxidation (min)
Cha
rge
(eq
/g)
CMC added in the medium (g/l)
0 5 10 15 20 25 30
0 1 2 3 4 5 60
100
200
300
400
500
600
WR
V (
%)
CMC added in the medium (g/l)
0 5 10 15 20 25 30
TEMPO-oxidation (min)
Effect of CMC hydrogel on permanent BC structural changes upon drying
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CMC
TEMPOCMC
TEMPO Dried samples!
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Conjugation of anti-HSA affibodies onto cellulose verified by SPR and cellulose thin films
0 5 10 15 20 25 30 35 40 45 50 55 60 65 700.0
0.2
0.4
0.6
0.8
1.0
1.2
SP
R a
ngle
()
Time (min)
Ref (no EDC/NHS)
Cellulose
+CMC
O
O
N
O
O
NH
O
= Rinsing
EDC/NHS
+Anti-HSA affibody
Ethanolamine
0 5 10 15 20 25 30 35 40 45 50 55 600.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
SP
R a
ngle
()
Time (min) = Rinsing
NH
O
+ HSA
Ref (no EDC/NHS)
Specific
Non-specific
84.4 ng/cm2141 ng/cm2
81 ng/cm2
10 ng/cm2
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Effect of CMC on the adsorption of HSA on cellulose
CMC modification lowers the non-specific adsorption of HSA on cellulose
Hydrogel-like structure Anionic charge of CMC
HSA adsorptionBiointerface 80 ng/cm2Unmodified cellulose 57 ng/cm2CMC-modified cellulose 10 ng/cm2
Anti-HSA biointerface
Cellulose
CMC modified cellulose
= Rinsing
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Filtration properties of BC
BC pellicle
Molecular cut-off• Never dried membrane up to 66 kDA (BSA)
Sokolnicki et al. 2006. J. Membr. Sci. 272. 15-27.
• Dried membrane 20 kDa (PEG) Shibazaki et al. 1993. J. Appl. Pol. Sci. 50. 965-969.
Pressure resistance• Up to 880 mmHg (1.17 bar)
Bodin et al. 2006. Biotechnol. Bioeng. 97(2). 425-434.
Chemistry• No electrostatic interactions with proteins• Highly stable due to high crystallinity degree
vanderhart et al. 1984. Macromolecules. 17. 1465-1472.
Biofiltration of fluorescence stained HAS with affibody-functionalized BC tubes
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1E-3 0.01 0.10
5
10
15
20
25
30
35
Nor
mal
ized
fluo
resc
ence
inte
nsity
HSA concentration (mg/ml)
Anti-HSA-CMC-BC
Anti-HSA-TEMPO-BC
• High fluorescence when anti-HSA was conjugated to CMC-BC• CMC modification decreases the background noise compared
to TEMPO-oxidation.
Dansylated HSA on…
CMC-BC with anti-HSA
CMC-BC without anti-HSA
Background CMC-BC
Dan
syla
ted
-HSA
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Conclusions
• Bacterial tubes were incubated in the presence of CMC
• CMC lowers irreversible changes in BC upon drying
• Affibodies were covalently conjugated to BC tubes via EDC/NHS chemistry
• BC tubes were utilized for specific binding of HSA
• Orelma et al., Affibody conjugation onto bacterial cellulose tubes and bioseparation of human serum albumin, RSC Advances, 4, 51440-51450 (2014).
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Funding and acknowledgements
• Lignocell project of the Finnish Centre for Nanocellulosic Technologies financially supported by the Finnish Funding Agency for Technology and Innovation (TEKES) and UPM
• Academy of Finland’s Centres of Excellence Programme (2014-2019)
• Dr Joseph M. Campbell for performing XPS; Anu Anttila, Ritva Kivela, and Marja Karkkainen for technical assistance.