1. Affibody-functionalized bacterial cellulose tubes for biofiltration applications
Hannes 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 Chemical
and Biomolecular Engineering, Raleigh, USA
c Universidad Pontificia Bolivariana, School of Engineering, Medellenin, Colombia /

2. Objective: Develop a nanocellulose-based biofiltration system for affinity separation of HSA
Residue
Filtrate
Filtrate
Filtrate
Filtrate
Human serum albumin (HSA): the most abundant protein in human blood plasma
Feed
 66.5 kDa
The goal of this study was to prepare a semi permeable tube for affinity filter hsa from aqueous solutions. For that purpose we selected bacterial cellulose.

3. 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 ( 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
Why bacterial cellulose instead of wood based nanocellulose. Bacterial cellulose has rather interesting characteristics as an ability to tubular shapes that are difficult to achieve with wood base nfc. Some bacteria have a property to produe extracellularly cellulose fibrils. From those gluconobacteria xylinums is the most used strain.
P. Gatenholm, D. Klemm, MRS Bull. 2010, 35, 208

4. Applications of bacterial cellulose
Pure bacterial cellulose
Artificial blood vessels
Food
Kowalska-Ludwicka, Karolina et al. Archives of Medical Science : AMS 9.3 (2013): 527.
Nata de Coco
Scaffolds for tissue engineering
03/05/15
Bacterial cellulose has some potential applications already like artificial blood vessels, scaffolds for tissue engineering, skin hydratation in skin therapy, nanopaper and paper additives, and food.
Skin therapy
Nanopaper &
paper additives
de Olyveira et al. Bacterial Nanocellulose for Medicine Regenerative J. Nanotechnol. Eng. Med. 2, (2012) v
W. Czaja, A. Krystynowicz, S. Bielecki, R. J. Brown, 2006 Biomaterials, 27 2

5. Proteins Materials Tubes Bacterial cellulose tubes
Gluconoacetobacter medellinensis
In-situ modification
Carboxymethyl cellulose DS = 0.7, DP = g/mol
CMC concentration of 0-5 g/l
Ex-situ modification
TEMPO-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
In this study we grown bacterial cellulose tubes that were modified with carboxymethyl celullose for immobilizin anti-hsa affibodies onto bc fibrils. Affibodies are engineered proteins that mimic native antibodies.

6. 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
Carboxymethyl cellulose (CMC) O2
Air
Glucose
9 days
Silicone tube
HS media
Bacterial cellulose (BC) tube
Cellulose fibril
Closed vessel
CMC
Incubation
Here is a illustration of the preparation route of bc tubes with cmc modification. We used a closed vessel with a semi permeable silicone tube that artificially supplied oxugen for bacteria. Therefore bacterial grow only onto the tube and then the grown tube can be collected. CMC Was added in the growing medium that allows to in-situ modify tubes.
Carbon source + phosphorus + air + additives
Bacteria
Bacterial cellulose

7. with in-situ CMC modification
Conjugation of affibodies to CMC-modified BC tubes (via EDC/NHS chemistry)
Anti-HSA affibody
EDC/NHS
pH 5 buffer
Hollow BC tube
with in-situ CMC modification
BC fibrils -
Purification
Biofiltration
HSA
Filtrate
CMC
Affibodies were immobilized onto bc fibrils with ed/nhs chemistry that produces covalent linkages from cmc to affibody. Then tubes were washed and utilized in hsa separation.
Conjugations of 0.1 mg/ml anti-HSA to CMC with 0.1 M EDC M NHS in 10 mM NaOAc buffer at pH 5.

8. BC Characterization methods
Dry methods
Tubes were lyophilized via liquid nitrogen
SEM Imaging
Surface Topography of BC
Lyophilized via liquid nitrogen
Surface and cross-section
Surface chemistry with XPS
Surface chemical composition
Fluorescence imaging with Confocal Laser
Scanning Microscopy (CLSM)
Dansylated HAS
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
Functionalized bc tubes were characterized with different methods. Charge of BC tubes due to CMC modification was measured with conductometric titration. Water retention value reflected how the swelling properties of bc are changes when cmc is present. Chemistry and topography of bc tubes were also characterized. These analyses were carried out from tubes that were dried with lyophilisation via liquid nitrogen.

9. 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:
𝑑= 𝑙 𝑑 2 ∆ 𝑆𝑃𝑅 𝑎𝑛𝑔𝑙𝑒 𝑚( 𝑛 𝑎 − 𝑛 0 )
Surface coverage:
Γ=𝑑∗𝜌
We also investigated the immobilization of anti-hsa to cmc modified bc and detection of hsa by surface plasmon resonance and cellulose thin films. SPR is an optial method that is extremely sensitive to adsorption of molecules on sensor surface.
Matthew A. Cooper, Nature Reviews Drug Discovery 1, (2002)
SPR model from Jung et al. Langmuir 1998, 14,

10. Results Preparation of BC-tubes
Effect of added CMC on topography and physical characteristics of BC-tubes
Conjugation of anti-HSA to CMC-modified BC by SPR analysis
Filtering of HSA with anti-HSA-BC tubes
Then we go to results.

11. CMC-modified BC tubes Length 20 cm Diameter 1 cm Wall thickness (wet):
Here you can see a photo image of grown cmc-modified bc tubes. Those were hollow and uniform without visible cracks and voids.
Length 20 cm
Diameter 1 cm
Wall thickness (wet):
1.8 ± 0.2 mm

12. Effect of CMC on BC tube morphology
Inner surface
Cross-section
Pure BC
SEM images ravelled changes on the topography when cmc was added in the growing medium. The inner surface looks little bit more porous but the cross sections were similar. Also bc fibrils in surface of cmc bc look more flexible.
BC grown with 2 g/l CMC

13. Water binding capacity and irreversible changes upon drying
Small effect of CMC on WRV of never dried BC
Drying: Significant effect on water binding
Highest charge obtained with CMC additions above 2 g/l
Never dried
Dried
Here you can see the effect of cmc addition on water binding capacity of never dried bc. CMC increases the vrw capacity. It has been reported that bc goes through irreversible structural changes when bc is dried. Therefore we thought that cmc could help with this due to increased charge in bc wall. And CMC really increased the water binding capacity of bc when dried. On right side we can see the increase on the carboxyl groups as a function of added cmc.

14. Effect of CMC hydrogel on permanent BC structural changes upon drying
TEMPO
CMC
Dried samples!
CMC
TEMPO
We investigated also the effect of hydrogel like nature of cmc on structural changes within drying. As an alternative carboxylation route tempo-mediated oxidation was utilized here. With this condition we can get similar charge. When we compare water binding capasitied we can see a gap between curves that tells that not only carboxyl groups explains the increased swelling ability.

15. Conjugation of anti-HSA affibodies onto cellulose verified by SPR and cellulose thin films
EDC/NHS
+CMC
+Anti-HSA affibody
+ HSA
Ethanolamine
81 ng/cm2
Specific
Here you can see spr sensogram on immobilization of affibodies on cmc modified cellulose. Without edc activation no any affibody adsorption was not observed. The immobilized affibodies boosted the hsa adsorption eight fold.
141 ng/cm2
84.4 ng/cm2
Ref (no EDC/NHS)
10 ng/cm2
Non-specific
Ref (no EDC/NHS)
= Rinsing
= Rinsing

16. Effect of CMC on the adsorption of HSA on cellulose
= Rinsing
Anti-HSA biointerface
HSA adsorption
Biointerface 80 ng/cm2
Unmodified cellulose 57 ng/cm2
CMC-modified cellulose 10 ng/cm2
Cellulose
CMC modification lowers the non-specific adsorption of HSA on cellulose
Hydrogel-like structure
Anionic charge of CMC
CMC modified cellulose
The hydrogel like structure and anionic charge of cmc layer has also an effect on adsorption of cmc. Here you can see that hsa adsorbs more on pure cellulose than on cmc modified cellulose.

17. Filtration properties of BC
Molecular cut-off
Never dried membrane up to 66 kDA (BSA)
Sokolnicki et al J. Membr. Sci
Dried membrane 20 kDa (PEG)
Shibazaki et al J. Appl. Pol. Sci
Pressure resistance
Up to 880 mmHg (1.17 bar)
Bodin et al Biotechnol. Bioeng. 97(2)
Chemistry
No electrostatic interactions with proteins
Highly stable due to high crystallinity degree
vanderhart et al Macromolecules
BC pellicle
Finally we tested the affinity filtration of hsa with anti-hsa immobilized bc-tubes. BC has reported filtration properties as molecular cut of up to 66 kda by bsa and 20 kDa by peg. The pressure resistance up tp 1.17 bar.

18. Biofiltration of fluorescence stained HAS with affibody-functionalized BC tubes
CMC-BC with anti-HSA
Dansylated HSA on…
CMC-BC without anti-HSA
Anti-HSA-CMC-BC
Dansylated -HSA
Anti-HSA-TEMPO-BC
Her you can see fluorescence images of dansylated hsa adsorbed on cmc-bc with and without immobilized anti-hsa affibodies. The results were identical with spr results and with affibody the fluroesence signal was higher illustrating higher adsorption. We also compared the cmc modification to tempo-oxidation on affibody immobilization. CMC modification strategy gave better results.
Background CMC-BC
High fluorescence when anti-HSA was conjugated to CMC-BC
CMC modification decreases the background noise compared to TEMPO-oxidation.

19. 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
We can conclude that
Orelma et al., Affibody conjugation onto bacterial cellulose tubes and bioseparation of human serum albumin, RSC Advances, 4, (2014).

20. 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 ( )
Dr Joseph M. Campbell for performing XPS; Anu Anttila, Ritva Kivela, and Marja Karkkainen for technical assistance.
Finaly I wanna thank lignocel project and the academy finland for supporting this study. These persons are thanked for technical assistance.