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Synthesis of chitosan-graft-poly(sodium-l-glutamate) for preparation of protein nanoparticles

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Abstract

In this manuscript we have designed a synthetic approach for the preparation of a series of chitosan-graft-poly(l-glutamate) copolymers with different lengths of poly(l-glutamate) grafts. First, organosulfonic chitosan salt, soluble in DMSO, was prepared in order to effectively initiate ring-opening polymerization of γ-benzyl-l-glutamate N-carboxyanhydride. The chitosan-graft-poly(γ-benzyl-l-glutamate) copolymers were fully deprotected by applying tetrabutylammonium hydroxide. The molar mass characteristics and chemical composition of graft copolymers with various lengths of polypeptide grafts were determined by SEC-MALS, FT-IR and various NMR spectroscopic techniques. The synthesized chitosan-graft-poly(sodium-l-glutamate) copolymers were used in combination with trimethyl chitosan for the preparation of nanoparticles (NPs) of a recombinant granulocyte colony-stimulating factor (GCSF). The suspensions of NPs with typical average diameter of 200–300 nm were obtained with polydispersity index values below 0.26. The achieved loading efficiency was up to 95 % and the final loading of GCSF protein in NPs was up to 45 %. The time, temperature and pH stability of NPs was also studied.

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Abbreviations

1D:

One-dimensional

2D:

Two-dimensional

AcOH:

Acetic acid

AE:

Association efficiency

BGlu:

γ-Benzyl-l-glutamate

Chi:

Chitosan

Chi-g-PBGlu:

Chitosan-graft-poly(γ-benzyl-l-glutamate)

Chi-g-PGlu:

Chitosan-graft-poly(l-glutamate)

COSY:

Correlation spectroscopy

CSA:

(±)Camphor-10-sulfonic acid

DCA:

Dichloroacetic acid

DLS:

Dynamic light-scattering

DMAc:

N,N-Dimethyl acetamide

DMF:

N,N-Dimethyl formamide

DMSO:

Dimethyl sulfoxide

d H :

Average hydrodynamic diameter

dn/dc :

Refractive-index increment

Đ M :

Dispersity

FL:

Final loading

FT-IR:

Fourier transform infrared spectroscopy

GCSF:

Granulocyte colony-stimulating factor

GE:

Grafting efficiency

gHSQCad:

Gradient Heteronuclear Single Quantum Coherence adiabatic version

GlcN:

Glucosamine

Glu:

Glutamate repeating unit

IL:

Initial loading

MALS:

Multi-angle laser light-scattering

M n :

Number-average molar mass

MSA:

Methanesulfonic acid

M w :

Weight-average molar mass

NaAc:

Sodium acetate

NCA:

N-Carboxyanhydride

NMR:

Nuclear magnetic resonance spectroscopy

NP:

Nanoparticle

PBGlu:

Poly(γ-benzyl-l-glutamate)

PdI:

Polydispersity index

PEG:

Poly(ethylene glycol)

PGlu:

Poly(l-glutamate)

pI:

Isoelectric point

RI:

Refractive index

ROP:

Ring-opening polymerization

SEC:

Size-exclusion chromatography

TBAH:

Tetrabutylammonium hydroxide

TFA:

Trifluoroacetic acid

THF:

Tetrahydrofuran

TMC:

Trimethyl chitosan

TMS:

Tetramethylsilane

TMSI:

Trimethylsilyl iodide

TMSPA:

3-Trimethylsilyl-2,2′,3,3′-d 4-propanoic acid sodium salt

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Acknowledgments

The authors gratefully acknowledge the financial support of the Ministry of Higher Education, Science and Technology of the Republic of Slovenia through the Slovenian Research Agency (Program P2-0145 and project L2-4166).

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Authors and Affiliations

  1. Laboratory for Polymer Chemistry and Technology, National Institute of Chemistry, Ljubljana, Hajdrihova 19, 1001, Ljubljana, Slovenia

    Peter Perdih, David Pahovnik & Ema Žagar

  2. Lek Pharmaceuticals d.d., Sandoz Development Center Slovenia, Verovškova 57, 1526, Ljubljana, Slovenia

    Mateja Cegnar & Janez Kerč

  3. Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000, Ljubljana, Slovenia

    Ana Miklavžin & Janez Kerč

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  1. Peter Perdih
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  2. David Pahovnik
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  4. Ana Miklavžin
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  6. Ema Žagar
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Correspondence to Ema Žagar.

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Perdih, P., Pahovnik, D., Cegnar, M. et al. Synthesis of chitosan-graft-poly(sodium-l-glutamate) for preparation of protein nanoparticles. Cellulose 21, 3469–3485 (2014). https://doi.org/10.1007/s10570-014-0362-8

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