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CHEM-E2155 Biopolymers

Alginates

Content- Alginates in general

• Overview

• Structure

• Properties

• Gel forming

• Applications

- Different alginates

• Algal alginate

• Bacterial alginate

• Future of alginate production

- Production of alginates with Pseudomonas aeruginosa

• Cultivation and purification

• Material development – Electrospinning

• Cross-linking

• Hydrogels

• Bioengineered alginates

- Conclusion

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www.themarinedetective.com

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Alginates in general

Overview- Term alginates can refer to alginic acid, its salts, or any of its derivatives.

- Alginates can be refined from brown seaweed or synthetized with Pseudomonas or Azotobactergenera.

- Alginates form hydrogels, scaffolds, elastomers and nanocomposites when combined with polyurethanes.

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http://www.irishseaweeds.com/kelp-laminaria-digitata/Laminaria digitata Pseudomonas aeruginosa

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Structure- Alginate is a natural polysaccharide, linear, unbranched,

non-repeating copolymer.

- β-D-mannuronic acid (M) and its C5-epimer α-L-guluronic acid (G) linked via β-1,4-glycosidic bonds.

• The block distribution is important for the alginate properties

G-blocks are responsible of gelling. M-residues do not take part

in gel networking but make a capsule and trigger the

immunogenic response.

MG-blocks form flexible chains and are more soluble in lower pH than the mono-block structures.

High amount of G-blocks provide heat-stable and strong (rigid) gels with high porosity, low shrinkage, and low swelling after drying.

High amount of M-blocks grant weaker elastic gels that are less heat- stable but are more durable to freeze-thaw processing. The pore size is smaller and the structure softer.

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Szekalska, M., Pucilowska, A., Szymanska, E., Ciosek, P. & Winnicka, K.

2016, "Alginate: Current Use and Future Perspectives in Pharmaceutical

and Biomedical Applications", International Journal of Polymer Science,

vol. 2016.

Properties- Alginates are abundant biocompatible,

biodegradable and low cost polysaccharides with excellent attributes.

• Retain water

• Stabilize

• Temperature-independent gelling

- Solubility to H2O

• pH of the solvent (regulates the electrostatic charges)

• Total ionic strength of the solute

• Gelling ions in the solvent (e.g. Ca2+).

- Viscosity

• Increases when pH decreases while the carboxylate groups of alginate form hydrogen bonds.

• Increases as the molecular weight increases

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https://dir.indiamart.com/delhi/sodium-alginate.html

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Gel forming (1/2)

- Alginates form thermostable gels in mild conditions.

- Aligned G-blocks form diamond shaped holes in which the bivalent counter ions bind forming a structure called egg-box.

- Regarding the amount of calcium ions present, these interactions can be either permanent or temporary, leading to viscous solutions.

- The high amount of G-blocks provide the gels with high porosity, low shrinkage, and low swelling after drying. A high amount of M-blocks grant the gels smaller pore size and softer structure.

- Gelling can be achieved with ionic, cell or covalent cross-linking, or with thermal gelation.

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Gel forming (2/2)

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http://www.kimica-alginate.com/alginate/chemical_structure.htmlhttps://www.researchgate.net/publication/264637062_Alginate_Particles_as_Platform_for_Drug_Delivery_by_the_Oral_Route_State-of-the-

Art/figures?lo=1&utm_source=google&utm_medium=organic

Egg-box model of alginate gelling behaviour

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http://www.openbiomedical.org/the-starting-

material-for-organs-of-the-future/

https://www.dentalcompare.com/News/1468

71-New-Dental-Product-IMAGE-Dust-Free-

Alginate-Dental-Impression-Material-from-

DUX-Dental/

https://food-hacks.wonderhowto.com/how-to/make-water-bottles-you-can-eat-0154909/

http://culinaryphysics.blogspot.fi/2014/09/alginates-in-food-sodium-alginate-uses-in-food.html

https://www.dreamstime.com/stock-

photo-green-alginate-face-mask-

cosmetology-clinic-customer-skin-

therapy-green-alginate-face-mask-

image98817263

http://www.silverson.com/us/resource-

library/application-reports/dispersion-of-beer-

foam-head-retaining-agents/

https://www.healthproductsforyou.com/p-

lohmann-rauscher-suprasorb-a-plus-ag-

calcium-alginate-dressing.html

http://www.acatris.com/applications/ice-

cream/single-ingredients-ice-cream/

http://rsta.royalsocietypublishing.org/co

ntent/368/1917/1981

https://ninithi.wordpress.com/2015/07/24/patching-up-weak-hearts-with-gold-nanowires/

http://tasmouldingandcasting.com.au/products/alginates/

https://www.recipetineats.com/french-vinaigrette/

http://www.jiejing-alginate.com/sale-8513432-

food-grade-sodium-alginate-600-800cps.html

http://www.acatris.com/applications/ice-cream/single-

ingredients-ice-cream/

http://www.mdpi.com/1422-0067/17/12/1976/htm

https://www.artmolds.com/alginate-uses http://www.hidetanning.net/Taxi

dermyEyes2.html

http://carolinaeyeprosthetics.com/making-

a-prosthetic-eye/

https://www.stanwinstonschool.c

om/blog/dental-casting-for-

making-fake-teeth

https://www.amrita.edu/research/project/controlled-drug-delivery-studies-biological-

macromolecules-sodium-alginate-and-lignosulphonic-acid-filmshttp://www.wound-treatment.jp/english/index_e.htm

https://oecotextiles.wordpress.com/category/printing/http://www.jiejing-alginate.com/sale-8513477-sodium-

alginate-for-welding-rods.html

https://www.snpinc.com/markets/paper-coating/https://www.artmolds.com/alginate-uses

http://www.scielo.br/sci

elo.php?pid=S1413-

78522009000400011&

script=sci_arttext&tlng=

en

https://www.researchgate.net/publication/25911

4828_Towards_ready-to-use_3-

D_scaffolds_for_regenerative_medicine_Adhesi

on-

based_cryopreservation_of_human_mesenchy

mal_stem_cells_attached_and_spread_within_

alginate-gelatin_cryogel_scaffolds/figures?lo=1

https://www.nature.com/articles/boneres201714

https://www.micr

ofluidicfuture.co

m/blog/microfluidi

c-cartilage-

scaffold

https://www.artmolds.com/alginate-uses

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187776/

Applications- Food (thickening/gelling/binder/softening agent, emulsifier, stabilizer, texture-improver, filling

material)

• e.g. beer, processed meat, ice cream, yoghurt, bread, sauces, pet food, fish feed

- Medical (wound/burn dressing, dental/prosthetic impression material, tablet binder, disintegrate, controllable drug release, immobilization, chelator, scaffold)

- Pharmaceutical (health food/weight loss supplement, treatment of reflux and heart burn)

- Tissue engineering (scaffolds)

- Paper manufacturing, printing (paint and dye thickener, 3D-printing), fertilizer

- Cosmetics (thickener, moisture retainer)

- Beauty (spa treatments, face masks, body casts)

- Textile printing (substrate of color paste)

- Welding rods

- Direct fabrication of artificial living tissue

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Example I: 3D Bioprinting- Direct fabrication of artificial living tissue, integrate living cells in three-

dimensional biomaterials

- Multicellular building blocks (bioinks) are dispensed layer by layer.

- Requirements: printability, biocompatibility, biomimicry and necessary structural/mechanical properties

- Alginates-based bioinks (vascular, cartilage and bone tissue printing)

+ Good printability and excellent biocompatibility.

+ Molecules can be trapped in an alginate matrix

+ Fast gelling when mixed with multivalent cations

+ Tunable structural and mechanical properties (viscosity, shear-thinning, pore size)

o Incorporation of other biomaterials (nanocellulose, gelatin, hydroxyapatite, biosilica)

o Different hydrogel-fabrication methods

+ Tunable degradation kinetics

o Oxidation

o Modifying the molecular weight distribution

- minimal cellular adhesion and slow degradation properties.

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187776/

Example II: Controlled drug delivery –Encapsulation to calcium alginate hydrogels

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- Goal: Maintains prolonged therapeutic effect at a reduced dosing frequency

- Encapsulation within a calcium alginate hydrogel results in sustained release of the incorporated drug.

- Alginate

• non-toxic, biocompatibility, biodegradability, mucoadhesiveness, mild gelation conditions

• pH sensitiveness customized release profiles

• Forms two types of gels depending on the pH

o At low pH hydration of alginic acid leads to the formation of

a high-viscosity “acid gel” due to intermolecular binding.

After gelation the water molecules are physically entrapped

inside the alginate matrix, but are still free to migrate.

o Alginate gels easily in the presence of a divalent cations.

Dried sodium alginate beads re-swell, creating a diffusion

barrier decreasing the migration of small molecules (e.g.,

drugs). http://pubs.rsc.org/en/content/articlepdf/2015/nr/c5nr02196k

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Example III: Injectable hydrogels for cartilage and bone tissue engineering

- Goal: Repairing damaged cartilage and bone tissue

- Alginate

+ High water content, similarity to the natural extracellular matrix (ECM), porous framework for cell transplantation and proliferation, minimal invasive properties, and ability to match irregular defects.

- Not strong enough to maintain the structural shape of the regenerated tissue.

Hybrids (e.g. calcium phosphate–alginate cement hydrogel system)

- Lack of cell adhesion ability

Blends (e.g. oxidized alginate/hyaluronic acid hydrogel, alginate and O-

carboxymethyl chitosan with the addition of fibrin nanoparticles

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https://www.nature.com/articles/boneres201714

Different alginates

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Algal alginates (SA)

- Algal alginates are found in all species of brown seaweed in intercellular matrix

- Alginate is a structure-forming component giving the algae mechanical strength and flexibility in the same way as cellulose in plants.

- Alginates occur as a sodium-magnesium-calcium-strontium gel in algae.

- Different brown algae species have adapted to varying habitat conditions of periodic drying regarding the tide and waves with different constitution of MM-, GG- and MG-blocks, leading to disparity in properties like elasticity, stiffness, and water-binding capacity.

- Moreover, the growth conditions and seasonal variations, and even the different tissues of the same algae have an effect on the sequential structure

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http://www.kimica-alginate.com/alginate/botanical_source.html

Production

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https://www.artmolds.com/brown-seaweeds-alginate

Harvesting Processing

(Szekalska et al. 2016)

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Bacterial alginates (BA)

- BA is an exopolymeric polysaccharide having a role on maintaining the hydration of the cells, pathogeny, virulence factors, and in maturation and formation of biofilms under desiccate conditions.

- P. aeruginosa produces alginates in order to survive in water-limited conditions. Alginates are part of the thick biofilms that protect the bacteria from antimicrobial agents, common bactericides, host defense mechanisms and opsonic killings. Moreover, alginates’ ability to rinse reactive oxygen helps it to prevent macrophages, neutrophils, and plant attacks.

- A. vinelandii needs alginates for the formation of a dormant desiccation-resistant cyst.

- Bacterial alginate composition and molecular mass differs depending on the species, stage of the life-cycle and surroundings.

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http://agrobio.hu/bakteriumok/galeria/bakteriumtorzsek-a-bactofil-

termekcsaladban/

https://fineartamerica.com/featured/3-pseudomonas-aeruginosa-

bacteria-sem-steve-gschmeissner.html

Pseudomonas aeruginosa

Azotobacteri vinelandii

Biosynthesis pathway of alginates in Pseudomonas aeruginosa

- The production mechanism of alginate in P. aeruginosa and A. vinelandii is very similar

- Precursor synthesis

- Polymerization

- Modification

• Modification at polymer level

o Transacetylases (AlgI, AlgJ,

AlgF)

o C5-epimerases (AlgG)

o Lyases (AlgL)

- Secretion (AlgE)

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BA vs SA

- The biggest difference between algal and bacterial alginates is that the bacterial M-residues are O-acetylated to various degrees at the O2 and/or O3 position. Acetylation changes the morphology of the biofilm to make it more resistant to antimicrobial agents.

- Bacterial alginates form flexible gels whereas seaweed alginate produces rigid ones. The selective ion binding in BA is higher with calcium whereas in SA it is higher with sodium.

- The molecular weight of alginates produced by A. vinelandii varies with respect to the dissolved oxygen levels. A vinelandii is unique, since it appears to be capable of modifying the ratio of M- and G-blocks, and thus the material properties of the alginate. Pseudomonas genus does not contain GG-blocks.

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Problems with alginate production- Algal alginates are not ideal sources for commercial demands.

- Marine pollution and climate conditions (e.g. El Niño) affect the seaweed alginate supply, production has to be located on shore areas. Some environmental concerns are associated with the seaweed harvesting and processing.

- The use of algae as a food product in Asia limits its refinery to alginate.

- The low price of the traditional algal alginates acts as a deterrent for the establishment of commercially feasible bacterial production processes. A. vinelandii is the most used host for bacterial alginate production, but the yields are rather low (4 g/l).

- One additional trouble with the production of bacterial alginate is that the production organisms are possibly pathogenic; biocompatibility and purity have to been taken into consideration, especially when the desired applications related to medical or pharmaceutical fields.

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https://www.healthline.com/health/why-

is-brown-seaweed-good-for-youhttp://www.pseudomonas-aeruginosa.org/

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Future of alginate production

- The industrial production of algal alginates in 2005 was 30 000 metric tons, which was barely 10 % of the macro algae biomaterial growing annually. Since there are huge reserves of naturally existing biomaterial, macro algae can be cultivated, and because of the possibility to make alginates by fermentation, the source potential can be considered as unlimited.

- It is believed that the growth in the market will be qualitative instead of quantitative, from commodity applications to high-tech, tailored refinery products.

- New host organisms and techniques have to be applied with the latest knowledge of bacterial alginate biosynthesis in order to make homogenous alginates economically . When the parameters, such as alginates molecular weight, acetylation level and monomer composition, and sequence pattern, defining the material properties of alginates can be precisely controlled. This whole new field opens for both bioengineered alginates and alginate-biopolymer compounds.

- By genetically engineering the alginate producing organisms, it is possible to produce novel alginates with unique or improved properties for medical and industrial applications.

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Bioengineered alginates

- To capitalize modern-day techniques and to develop solutions for demanding issues, e.g., in tissue engineering and wound healing, it is important to create homogenous structures and to control the molecular weight and the composition of the polymeric alginate

- The monomer composition, and the degree of polymerization and acetylation govern the alginate properties. Different applications require different attributes.

- The polymerization could be controlled with either genetic tools or fermentation conditions. The monomer composition could be manipulated with knock-out or knock-in mutants. A third method is that of tailor-made polymers produced in vivo by expressing recombinant alginate-epimerases in an alginate-producing host

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Production of alginates with Pseudomonas aeruginosa

Pseudomonas aeruginosa- P. aeruginosa is a biosafety Level 2 adaptable ubiquitous opportunistic pathogen

- It is a motile, aerobic, Gram-negative, rod-shape bacterium, with a flexible metabolism. Even though its metabolism is aerobic it can live in anaerobic conditions by using nitrogen as an electron acceptor. It has minimal nutritional requirements, can use several different carbon sources, and thrives in temperature up to 42 °C.

- Alginate overproduction, also called mucoidy, helps P. aeruginosa invade and adapt to a newhost environment by formation of the extracellular matrix defending it from the host defensesystem both physically and by making bacteria cells less susceptible to body’s defense. Mucoidlayer helps P. aeruginosa colonize by acting as an additional or alternative cell surfaceadhesin and by anchoring cells to the surface. Mucoid alginate biofilm has a high resistance to antibiotics

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Cultivation and Purification

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Scraping all the material to 1x PBS

Stirring with magnetic stirrer (1 h)

to separate the extracellular

material from the bacteria

Centrifugation (4h, 17700xg, 4 ◦C)

Enzyme digestion with Dnase and

RNase (4h, 37 ◦C)

Heat (80 C, 30 min),

centrifugation (4 ◦C, 20

min)

Presipitation with

EtOH (1h, 4 ◦C)

Drying with aeration

Agarose gel

electrophoresis

SDS-PAGE

MALDI-TOF

FT-IR

Material development - Electrospinning- Electrospinning is easy-to-use, simple, adaptable and

affordable technique to create fine nanofibers, micro- and nanoscale engineered scaffold topographies, and high porosity, high surface to volume ratio materials mimicking the natural extracellular matrix (ECM). These attributes improve the cell attachment, drug loading, and mass transfer properties.

- Syringe pump forces the polymer solution through the needle creating a drop at the tip of the needle. The needle is connected to the high voltage power supply, which injects the charge in the solution. If the electrostatic force overcomes the surface tension of the polymer solution, Taylor cone is formed, and fiber jet emitted. Chaotic whipping occurs, and the solvent evaporates while travelling to the collector, leading to the deposition of solid polymer fibers to the collector.

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(Sill, von Recum 2008)

(Tino Koponen)

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SA-PEO-DMSO-Triton X-100 SA-PEO-Triton X-100 BA-PEO-DMSO-Triton X-100 BA-PEO-Triton X-100

Cross-linking- Electrospun alginate nanofibers are promising candidates for tissue engineered applications. The

lack of structural stability in aqueous environment and limited cell attachment can be assessed with post-electospinning modifications, such as cross-linking.

- Cross-linking can be either chemical or ionic. With ionic cross-linking, the toxicity can be avoided. One example of ionic cross-linking is to use ethanol as a pre-cross-linking solution, which allows the non-ethanol soluble alginate not to dissolve. Cross-linking is then conducted with CaCl2. After the cross-linking, the nanofiber mats are soaked in water in order to get rid of the carrier polymers. By removing the carrier polymer, the fiber diameter is reduced. This procedure allows the creation of alginate-only nanofiber scaffolds, and the removal of inert carrier polymers.

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Alginate Hydrogels

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Alginate worms

Cross-linked calcium alginate hydrogel (http://people.clarkson.edu/~amelman/alginate_hydrogels.html)

Wet spinning

Block Fractioning

- Total yield

• SA 97 %

• BA 65 %

- BA contained minimal amount of GG blocks, and over 70 % MG blocks

- SA contained over 50 % GG blocksresponsible of strong gels

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0

10

20

30

40

50

60

70

80

90

Fraction 1 MG-block Fraction 2 MM-block Fraction 3 GG-block

[%]

BA [%] SA [%

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Single gene deletions of AlgG with 1-step PCR

- Bacterial alginates are synthetized as polymannuronate and modified at the polymeric level in periplasm

- Mannuronan C-5 epimerase converts the polymannuronate chain into guluronic acid residues. P. aeruginosa has only one C-5 epimerase, AlgG.

- Alginate derived from P. aeruginosa lacks consecutive α-L-guluronic acid residues.

- We hypothesize that by knocking-out the only mannuronan C-5 epimerase (AlgG), converting single mannuronan residues to guluronicacid residues, we would end up with mannuronan-only alginates which would have an effect on gel forming properties, chain formation, and immunogenic activity.

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(Rahme, 2008)

Conclusion- β-D-mannuronic acid (M) and α-L-guluronic acid (G) linked via β-1,4-glycosidic bonds form

blocks with either consecutive G-residues (GG-block) or M-residues (MM-block), or altering M and G (MG-blocks)

- Unique physicochemical properties and diverse biological activities.

• Capacity to retain water, stabilize, and form viscous gels

• Almost temperature-independent transition from soluble form to gel

• Tunable

- Exceptional application potential

• Long and successful history in applications, from the food industry to cosmetics and from textile-printing to pharmaceuticals.

• New possibilities e.g. in tissue engineering, bioinks and controlled drug delivery.

• Even more prospects in blends, hybrids, core-shell structures, and in bioengineered alginates.

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