Properties of polysaccharides in sol and gel states.

M.Rinaudo Biomaterials Applications

Grenoble (France)

Guadalajara, Mexico, 2-4 May 2017

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3- Ionic gelation

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Test proposed to determine the ability to gelation of alginate or pectins.

Ionic selectivity

Structure of a pectin

Gelation of pectin.

Ionic Selectivity:

Ba>Sr>Ca

In dilute solution:

dimer formation

with Ba, Sr, Ca.

No specific interaction with Mg,

no dimer formation & no

gelation

Gel point of Pectin. Role of the

distribution of –COO- sites (DE=30%).

PE PH

PE=enzymic hydrolysis, blockwise distribution

PH=NaOH hydrolysis, random distribution

Theory and

oligomannuronic acid

Oligo-guluronic

&galacturonic

Alginate.

Influence of polymer

concentration on the

osmotic coefficient of

Ca counterion.

Dimer

Multimer

Alginates & pectins.

Influence of DP on activity

coefficient of Ca counterion

for different polyuronates.

R.Kohn (Slovakia)

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ManA ManAGulAGulAGulA ManAOH

OOH

HO

HOOCO

OH

O

OH

HOOC

O OH

OH

HOOC

O

O

HOOC

HO

OOH

O

OH

HOOC

O

OOH

O

HOOC

HO

O

O

OOH

HO

HOOCO

OH

O

OH

HOOC

O OH

OH

HOOC

O

O

HOOC

HO

OOH

O

OH

HOOC

O

OOH

O

HOOC

HO

O

O

MGGGGGGMMMMMMMMGMGMGMGMMMMMM

G-block M-block MG-block M-block

OH

OH

HO

HOOCOHO

OH

OH

HO

HOOCOHO

OH

HO

OH

OHHOOC O

OH

HO

OH

OHHOOC O

GulAManA polyanion

Alginate structure: fibers, beads in presence of calcium

Ionic gelation in alginate/pectins

Elastic modulus of alginate.

Role of M/G ratio.

With X4 M/G=0.28 ; X2 M/G=0.56 ; X5 M/G= 1.98. G units favour gelation.

Ionic selectivity for alginate in presence of

divalent salts (q= [COO-]/[Me]

Ionic selectivity Ba+2> Sr+2> Ca+2 -with Mg+2,no dimer

formation and no gelation.

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Role of DP on cooperative ionic interaction on oligo-uronic acid based on dimer formation Existence of a critical DP depending on sugar configuration

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Mechanism of gelation

First step of dimerisation Blockwise gelation

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0.1000 1.000 10.00frequency (Hz)

1000

10000

1.000E5

1.000E6G'

(Pa)

1000

10000

1.000E5

1.000E6

G'' (Pa)

AG acidic form

AG calcium form

Acidic and calcium alginate gel formed at 20g/L and 25°C

-Cooperative junction based on specific Ca fixation for the Ca salt form

-Cooperative H-bonds junction based on –COOH/-OH interaction

Mechanism of Ionotropic Gelation of Poly(HEMAm-g-GulA20) (side chain mechanism)

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Biohybrid glycopolymers.

- Obtention of purified guluronic and mannuronic oligomers with well defined DP (by controlled hydrolysis of alginates and SEC)

- -Preparation of oligoalginate-derived monomers (AlgiMERs) in two

steps without the need for protective group chemistry: glycosamine or oligoglycuronan-derived 1-amino-1-deoxy alditols react with 2-isocayanatoethyl methacrylate macromonomers (ManA16MAm and GulA20MAm) or ManA10Am (acrylamide derivative) (all reactions in water at exclusion of RAFT)

- Copolymerization studies of AlgiMERs (acrylamide,

methacrylamide) with N-(2-hydroxyethyl) methacrylamide (HEMAm) (radical polymerization or RAFT) give high molecular weight polymers

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Biohybrid glycopolymers. Strategy

alginate

=

=

β-D-mannuronic acid

α-L-guluronic acid

OOH

OH-OOCO

O

HO

OH-OOC

O

functionalization

vinyl glycomonomers

O O

(AlgiMERs)

depolymerization separation

R

Controlled polymerization

A schematic representation for the transformation of alginate into a biohybrid polymer with defined and tailored physico-chemical properties.

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O

OH

OH

HOOC

OH

O

OH

OH

HOOC

O

OH

O

OH

OH

HOOC

OH

OH

OH

OH

HOOC

O

NH

O

CH3

X-1 X-1

(1-4)--L-guluronan

i) NH4OAc, NaBH

3CN

ii) Methacryloyl chloride

GulAXMAm X = 10, 20

NH

O

OH

NH

O

CH3

GulAx

+

Azo-initiator

D2O, 60°C

GulAXMAm

HEMAm

n

co

ONH

GulAx

ONH

OH

m

Poly(HEMAm-co-GulA

X)

Shematic representation of the synthesis of poly(HEMAm-g-GulAx) via conventional radical copolymerization (random grafting)

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They were then copolymerized with 2-hydroxyethylmethacrylamide in aqueous solution to yield high molar mass biohybrid glycopolymers containing between 25 and 52% by mass of oligosaccharide grafted chains.

In this study, it was demonstrated that alginate-extracted oligosaccharides and aqueous radical polymerization can be combined for the flexible design of biohybrid glycopolymers capable of ionotropic gelation under very mild conditions.

(a) Formation of gel beads when an aqueous solution of poly(HEMAm-g- GulA20MAm) (c= 71 g. L-1) was dripped into 0.5 mol /L CaCl2. Note the progressive gelation from the outside to the inside of the polymer (b) Dynamic rheological characterization of the gel in oscillatory mode.

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poly(HEMAm-g-ManA16) poly(HEMAm-g-GulA20)

Hydrogels of poly(HEMAm-g-ManA16) (left, laying on the dialysis membrane) and poly(HEMAm-g-GulA20) (right) obtained by dialysis against with CaCl2. …..ManA16 with 4% grafting ….GulA 20 with 2.7% grafting

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At C= 1.5 C*

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Synergistic mixtures: Xanthan/glucomannan or Xanthan/galactomannan

galactomannan

xanthan

Gelation involving two polysaccharides

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Cooling and heating curves for G’ on 1 g/L deacetylated Xanthan + 4.2 g/L galactomannan nin 5mM NaCl; DSC exotherm on the same solution.

Role of the conformation of xanthan on the gel formation: in acidic conditions, xanthan is helical but disordered after neutralisation.

Complex forms with disordered xanthan conformation

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« Complex » formed better when deacetylated xanthan and galactomannan are mixed.

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Conclusions

- Physical gels are often obtained with stereoregular or blockwise structured polysaccharides involving different mechanisms but based on junction zones

- The gelation is directly related to the nature of counterions, and thermodynamics conditions (temperature, ionic concentration, pH)

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Sixth part

Specific applications of some polysaccharides: -HA -chitosan -alginates,pectins - methylcelluloses

General applications as thickeners, gelling agents, stabilisers for emulsion or solid particles suspension… but also as good film and fiber forming systems

Hyaluronan, viscoelastic fluid used for visco-supplementation (arthrosis)

Walk corresponds to low frequency deformation and viscous character. 27

Hyaluronan, viscoelastic fluid.

Running corresponds to larger frequency & elastic character.

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Bacterial HA is used for viscosupplementation

(in physiological conditions)

Synovial fluid f0=0.22 cycle/s (or Hz).

Walking frequency 0.5 Hz

Running frequency 2.5 Hz

Osteoarthritis with C and MW of HA decrease

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0

10

20

30

40

50

1 2 3 4 5 6 7pH

Test of the influence of pH on the viscosity of HA at 10g/L. Gel-like at pH~2.5 based on H-bond network

and/or –NH3+/-COO- interaction

-This gelation allows to produce

good films after drying

(medical application, with slow release).

Other polysaccharides give gel under

-COOH form (alginate, pectin…)

HA in acidic medium H-bond network gelation (thermoreversible gelation)

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Hyaluronan: biocompatible, hydrating polymer.

Application for cosmetics.

SEM microphotograph: (a) nonwoven chitin* fabric; (b) sponge chitin sheet.

*Very low solubility in usual solvents (DMAc/LiCl is often used)

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Chitin transformation & applications

Applications of chitosan

Agriculture Defensive mechanism in plants Stimulation of growth Seed coating, Frost protection Time release of fertilizers, nutrients …into the soil

Water & waste treatment Floculant to clarify water (drinking water ,pools) Removal of metal ions Ecological polymer (eliminate synthetic polymers) Reduce odors

Food &beverages Not digestible by human (dietary fiber) Bind lipids (reduce cholesterol) preservative Thickener & stabilizer for sauces Protective, fungi static, antibacterial coating for fruits

Cosmetics & toiletries Maintain skin moisture Treat acne Improve suppleness of hair Reduce static electricity in hair Tone skin Oral care (toothpaste, chewing gum)

Biopharmaceutics Immunologic, Antitumoral Hemostatic and anticoagulant Cicatrisant, Bacteriostatic 33

Main properties of chitin and chitosan* in biomedical applications**.

Biodegradability Film forming

Biocompatibility Hydrating agent

Bioadhesivity Renewable

Polycationic substance

Absorption promoters

Antifungal Non toxicity

Antibacterial Non allergenic

Immunoadjuvant Anticholesteremic agent

Antithrombogenic

*chitosan is obtained by deacetylation of chitin

** they are processed under different forms: bead, microsphere,fiber,film,sponge,solution, gel, tablet, capsule

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How Electrospinning Work?

(http://nano.mtu.edu) 2005. Michigan Technological University. Used with permission

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Chit 5% MW~100,000 + PEO powder MW=106

70/30 (w/w)%

Chitosan/PEO 60/40 (w/w)%

Chitosan/PEO

Morphologies of the nanostructures

Perspectives: Biomedical applications

Tissue engineering Wound healing …..

SEM image; Nanofibrous structure (left) promoted the attachment of human osteoblasts and chondrocytes and maintained characteristic cell morphology.

Nanofibers Casted film

Bhattarai et al. (2005) Electrospun chitosan-based nanofibers and their cellular compatibility

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Production of gel beads

(Na-alginate drops into CaCl2)

Use of these particles for bacteria or fungi encapsulation

Alginate application

Encapsulation of microorganisms in gels for metabolite production (alginate, agar, carrageenan)

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Pectins is a gelling polysaccharide

The mechanism of gelation depends on the degree of esterification (DE)

-high DE, gelification in acidic medium in the presence of sucrose

-DE< 50%, gelification in the presence of calcium (same model as for alginate)

Application of methylcellulose which forms a gel when temperature increases.

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DNA-CHITOSAN ELECTROSTATIC

COMPLEX FORMATION:

STOICHIOMETRY AND CONFORMATION

DNA-CHITOSAN ELECTROSTATIC COMPLEX

FORMATION:

STOICHIOMETRY AND CONFORMATION

Conclusions

-Due to their original chemical structure (nature of repeat unit, functionnality allowing easy chemical modification) -To their macromolecular characteristics ( stereoregular conformation, blockwise or random repetition of sugar units) - To their biological properties (antimicrobial and biocompatible properties) Polysaccharides have a large potential of developments and new applications But in carefull conditions of preparation & purification

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* Polysaccharides are hydrophilic polymers with many applications in:

- Food, cosmetics, pharmaceutical and biomedical domains - Some of them are biocompatible and biodegradable (mainly chitosan, HA) * They are easy to process under film, powder, bead, fiber, capsule…

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Thank you for your attention

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