BIOLOGICAL ACTIVITY OF HETEROLOGOUS MURINE INTERLUKIN-10 AND

PRELIMINARY STUDIES ON THE USE OF A DEXTRIN NANOGEL AS A DELIVERY

SYSTEM
University of Minho Author* VERA CARVALHO
School of Engineering
Centre of Biological Engineering Supervisors: Miguel Gama, Co-Supervisors Lucília Domingues; Manuel Vilanova
* veracarvalho@deb.uminho.pt

INTRODUCTION RESULTS AND DISCUSSION

Interleukin-10 (IL-10) is an anti-inflammatory cytokine, which active rIL-10 expression and purification rIL-10 release from the complex nanogel/rIL-10
form is a non-covalent homodimer with two intramolecular
disulphide bonds that are essential to its biological activity, which The two fractions rIL-10 were recovered as In the presence of 20% serum, rIL-
includes reduction of tumor necrosis factor α (TNF-α ) synthesis non-covalent homodimers and the yield of 10 is being released over time in a
and down-regulation of class II major histocompatibility complex protein (about 1.0-1.5 mg/l culture) was BMDM culture. After two hours of
(MCH-II) molecules on monocytes/macrophage. Due to IL-10 high enough to the subsequent assays, incubation with the NPs/rIL-10
potential applications in various medical fields, it is essential to and stable between batches. complex, the rIL-10 reaches a
develop systems that can effectively deliver the protein. A promising maximum concentration; a stable
system is protein encapsulation by polymeric nanogels, which value of about 35 ng/mL rIL-10
minimize denaturation, and enables slow-release, while maintaining being estimated after twenty-four
an effective concentration for the necessary period of time. In hours.
previous work, our group have developed and characterized SDS-PAGE: MW- molecular weight marker (75 kDa, 50 kDa, 37 kDa, 25
kDa, 20 kDa, 15 kDa, 10 kDa); 1 – Insoluble fraction applied to Superdex
IL-10 release profile on a BMDM culture and on culture medium alone.

nanogels obtained by self-assembling of hydrophobized dextrin
whose properties makes them promising for IL-10 delivery.
200; 2 – Fractions eluted from Superdex 200 and applied to Mono S; 3, 4 –
first peak eluted from Mono S; 5, 6 – second peak eluted from Mono S.
Biological activities of the rIL-10 released from the nanogel
rIL-10 biological activities

STRATEGY
pET 28aIL-10
Site directed mutagenesis
Restriction analysis
Cloning in E. coli BL21 star
Expression in LB medium (37ºC,
0.5 mM ITPG, 3 h incubation)

Inclusion bodies
Renaturation and re-oxidation
(6M Guanidine)
Gel filtation
Ion-exchange chromatography
IL-10 C149Y mutant Circular dichroism (CD)
(rIL-10)
Evaluation of rIL-10
CD
and nanogel/rIL-10
Dextrin nanogels
stability
LPS and INF-γ activated
bone marrow derived
macrophage
Nanogel/rIL-10
Bone marrow derived
macrophage
Engenharia para a Qualidade de Vida: SAÚDE, LAZER e AMBIENTE – Semana da Escola de Engenharia -11 a 16 de Outubro de 2010
TNF-α concentration (pg/ml) produced by 0.1 ng/ml LPS and 1.0 ng/ml INF-γ stimulated MHC-II induced, in stimulaterd BMDM, by 50ng/ml rIL-10 (cIL-10) and NPs/rIL10 (represented
BMDM, treated with 50 ng/ml rIL-10 and NPs/rIL10 (represented by Nanogel/rIL10). (+) – by Nanogel/rIL10). (+) – positive control of macrophage activation; (-) – negative control of
positive control of macrophage activation; (-) – negative control of macrophage activation. macrophage activation.
Percentage of inhibition of TNF-α production by 0.1 ng/ml LPS and 1.0 ng/ml INF-γ stimulated
BMDM. cIL-10 – commercial available IL-10 (eBiocience); rIL-10 – recombinant IL-10.
Percentage of MHC-II expression induced by commercial IL-10 (cIL-10) and recombinant IL-10 (rIL-
10) treatment. (+) – positive control of macrophage activation; (-) – negative control of macrophage
The rIL-10 released from the nanogel/rIL-10 complex was able to inhibit
activation.
TNF- production and MHC-II expression at the same level as the
It is noticeable that rIL-10 ability to reduce TNF- production and MHC-II soluble rIL-10.
expression is similar to that of tested cIL-10, in the analyzed range of
concentrations. CONCLUSIONS
üA mutated form of murine IL-10 was successfully expressed in E. coli.
rIL-10 incorporation by the nanogel and stability assays üThe recombinant protein was recovered and purified from inclusion
bodies and demonstrated biological activity similar to a commercially
rIL-10 incorporation by the dextrin nanogel was confirmed by quantifying,
available IL-10.
by ELISA, the amount of free rIL-10 that was negligible.
Evaluation of rIL-10 üDextrin self-assembled nanogel was able to efficiently encapsulate and
biological activity: protect rIL-10 from denaturation at 37ºC, and also enables rIL-10 to be
The secondary structure of free released in biologically significant amounts over time.
•TNF-α production
(ELISA) soluble rIL-10 is mainly helical, üThe biological activity of the released rIL-10 was confirmed by the
•MHC-II surface and the complexation in the evaluation of the production of TNF- and expression of MHC-II on
expression (FACS nanogel did not induce any endotoxin-stimulated BMDM.
analysis) conformational change. Further The simplicity of the preparation of the nanogel/rIL-10 complex,
CD studies showed that rIL-10 associated to the enhancement of protein stability and controlled
stability is significantly increased release, makes this a promising delivery system.
Evaluation of rIL-10 when it is complexed with the
release: dextrin nanogel.
•IL-10 quantification
(ELISA)
CD spectra of free rIL-10 (0.25 mg/ml) and nanogel/rIL-10 (1 mg/ml nanogel, 0.25 mg/ml rIL-10)
at 37ºC in PBS ACKNOWLEDGEMENTS
Vera Carvalho was supported by the grant SFRH/BD/27359/2006 from
Fundação para a Ciência e Tecnologia (FCT), Portugal.This study was
financially supported by FCT through the project PTDC/BIO/67160/2006.