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Human Serum Albumin-Alginate Esters Microparticles Prepared by Transacylation: Effect of Ester Group
Imane Hadef, Mehdi Omri, Christophe Bliard, Barbara Rogé, Florence Edwards-Lévy
To cite this version:
Imane Hadef, Mehdi Omri, Christophe Bliard, Barbara Rogé, Florence Edwards-Lévy. Human Serum Albumin-Alginate Esters Microparticles Prepared by Transacylation: Effect of Ester Group. 1st European Conference on Pharmaceutics, Apr 2015, Reims, France. �hal-02894581�
3. Methods
3.1. Alginate ester synthesis
(3)3.3. Microparticle preparation
3.2. Alginate ester characterization
• The degree of esterification (DE) by
1H-NMR and titrimetry;
• Viscosity measurement of 2% ester aqueous solution (w/w);
• Critical aggregation concentration(CAC) via Coomassie blue test
(4).
3.4. Microparticle characterization
• Size distribution by laser diffraction granulometry;
• Morphology by optical microscopy;
• Inner structure after microparticle cross-section.
Human Serum Albumin-Alginate Esters Microparticles Prepared by Transacylation: Effect of Ester Group
Imane HADEF (a, b) ; Mehdi OMRI (a, b) ; Christophe BLIARD (b) ; Barbara ROGE (b) ; Florence EDWARDS-LEVY (a)
Institut de Chimie Moléculaire de Reims, CNRS UMR 7312
(a) U.F.R. Pharmacie, 51, rue Cognacq-Jay, 51096 Reims
(b) U.F.R. Sciences Exactes et Naturelles, Campus du Moulin de la Housse 51687 Reims florence.edwards@univ-reims.fr
1. Introduction
Microencapsulation using the transacylation reaction in a W/O emulsion is based on the creation of amide bonds between free amine functions of a protein and polysaccharide ester groups in the aqueous phase after alkalization. Several proteins have been implicated in this process, whereas commercial propylene glycol alginate (PGA) has been the only modified polysaccharide used up to now
(1,2). The intrinsic properties of PGA such as viscosity, molecular mass, ester type and substitution degree limit its use in microparticle preparation hence the need to study other potential polysaccharide esters.
2. Purpose
Study the effect of the alginate polysaccharide ester structure and properties on the transacylation reaction and on microparticle characteristics .
L-Guluronic
,D-Mannuronic
D- Mannuronic
L-Guluronic
O H
O O
OH
OH O O O
O
OH OH O
O O
O
O OH
O H
O O
OHO O
H O
O O
O H
OH O
H
H N H
PGA
+ Alkalization
+ Alginate
Protein
N
O H
Protein
O O OH
O H O
H
Transacylation reaction between protein and PGA Propylene Glycol Alginate (PGA)
4. Results
4.1. Alginate derivative DE
5. Conclusion
The replacement of commercial PGA by a variety of other synthetic alginate esters in the process of microparticle formation using the transacylation procedure was studied.
The best candidates for the reaction with HSA were alginate esters with the highest critical aggregation concentrations observed with the hydroxyalkylated esters.
Alkyl alginates or low DE alginate esters didn’t lead to microparticle formation under the given conditions of transacylation.
The use of low molecular weight alginates, displaying low aqueous solution viscosity, led to the formation of smaller microparticles.
6. References
1. Levy, M.-C. & Edwards-Levy, F. Coating alginate beads with cross-linked biopolymers: A novel method based on a transacylation reaction. J.
Microencapsul. 13, 169–183 (1996).
2. McDowell, R. New reactions of propylene glycol alginate. J. Soc. Cosmet.
Chem. 21, 441–457 (1970).
3. Della Valle, F., & Romeo, A. Polysaccharide esters and their salts. US Patent, No.4, 965, 353 (1990).
4. Rastello De Boisseson, M. et al. Physical alginate hydrogels based on hydrophobic or dual hydrophobic/ionic interactions: Bead formation, structure, and stability. J. Colloid Interface Sci. 273, 131–139 (2004).
y = 0.97x R² = 0.984 0
20 40 60 80 100
0 20 40 60 80 100
DE NMR (%)
DE titrimetry (%)
10% HSA-2% alginate ester (w/w) microparticles prepared by transacylation
4.2. Alginate ester microparticles
Alkyl alginate (propyl and ethyl) no microparticle.
Hydroxyalkyl alginate with ↗DE microparticles with lacework inner structure.
1-Bromo-2-propanol was less reactive during esterification
2-Hydroxypropyl alginate with ↘ DE No microparticle.
↘ alginate molecular weight ↘ aqueous solution viscosity
↘microparticle size.
4.3. Critical aggregation concentrations(CAC)
0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80
-5 -4 -3 -2 -1 0 1 2
Relative O.D at 618nm
Log [C] alginate ester (g/l)
2OH-Propyl alginate (DE15%)
2OH-Propyl alginate (DE30%) Commercial PGA (DE84%)
For alkyl esters, hydrophobic microdomains were formed due to the association of alkyl chains at lower polymer concentrations compared to hydroxyalkyl esters .
Increasing the esterification degree or the length of the ester chain led to the formation of
hydrophobic microdomains at lower polymer concentrations, except for propylene glycol alginates (2OH-propyl alginate) for which increasing the esterification degree reduced significantly their hydrophobicity.
The formation of hydrophobic microdomains seemed unfavorable to the reaction with HSA.
0 0.5 1 1.5 2 2.5
-5 -4 -3 -2 -1 0 1 2
Relative O.D at 618nm
Log [C ] alginate ester (g/l)
3OH-Propyl alginate (DE76%)
3OH-Propyl alginate (DE52%)
The spectral change of Coomassie blue was studied in the presence of alginate derivatives in water at increasing concentrations.
0 0.5 1 1.5 2 2.5 3 3.5 4
-5 -4 -3 -2 -1 0 1 2
Relative O.D at 618nm
Log [C] alginate ester (g/l)
Ethyl alginate (DE82%)
OH-Ethyl alginate (DE82%)
CAC
Absorbance change of Coomassie blue as a function of alginate ester concentration: effect of ester type and substitution degree Alginate microparticle size in relation with aqueous solution
viscosities
Propyl Alginate (DE= 80%) 1H-NMR spectrum (D2O, 318K, 600MHz)
Esterification degree by 1H-NMR vs titration
Good agreement between
1H-NMR and titrimetric results.
The DE could be controlled by adjusting the quantity of bromide reagent.
R DE (%) Particles Particle size
(µm)
Cross- sections
2-Hydroxypropyl
O O
OH Commercial
PGA (84%)
908
Reference MM~1000KD
Ethyl
O O
43% No microparticle
Synthetic alginate esters MM~ 10 KD 82%
2-Hydroxyethyl
O O O
H
58% 343
82% 243
Propyl
O O
25%
No microparticle 60%
90%
80%
2-Hydroxypropyl
O O
OH 19%
No microparticle 31%
3-Hydroxypropyl
O O
H
O
48% 297
76% 364
-CH3 propyl
-CH2- propyl -CH2-O propyl
Alginate backbone
Microparticles are stable, biocompatible and biodegradable
0 20 40 60 80 100 120
0 100 200 300 400 500 600 700 800 900 1000
Viscosity (mPa.s)
particle size (µm)
microparticle size 2% ester solution viscosity
COOH
COOH
Neutralization by TBAOH Freeze drying
COOTBA
COOTBA
COOTBA
COOTBA
DMSO RBr, 50°C, 24h
COOR
COOTBA COOR
COOTBA
Dialysis Stirring in NaCl
2,5N, 24h
Dialysis Freeze drying
COOR
COONa
Alginic Acid
R = CH3CH2-, OHCH2CH2-, CH3CH2CH2-, CH3CHOHCH2- or OHCH2CH2CH2-
NaOH/EtOH
2 - Alkalization
pH 7 Buffer solution
3 - Neutralization 1 - Emulsification
2% Alginate ester + 10% HSA (Human
serum albumin)
Isopropyl myristate + 5%
surfactant
Microparticles
15min 5min
R Equivalent
RBr DE (%)
Ethyl
O O
0.5eq 43%
1eq 82%
2-Hydroxyethyl
O O O
H
0.5eq 58%
1eq 82%
Propyl
O O
0.3eq 25%
1eq 90%
2eq 80%
2-Hydroxypropyl
O O
OH 0.5eq 19%
1eq 31%
3-Hydroxypropyl
O O
H
O
0.5eq 48%
1eq 76%
