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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: hannes.orelma@aalto.fi / orlando.rojas@aalto.fi

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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.