Carvalho, Vera

Castanheira, Pedro

Domingues, Lucília

Faria, Tiago Q.

Gama, F. M.

Vilanova, Manuel

Universidade do Minho: RepositoriUM

 BIOLOGICAL ACTIVITY OF HETEROLOGOUS MURINE INTERLEUKIN-10 AND PRELIMINARY STUDIES ON THE USE OF A DEXTRIN NANOGEL AS A DELIVERY SYSTEM V. Carvalho1, P. Castanheira2, Tiago Q. Faria3, L. Domingues1, M. Vilanova4, F.M. Gama1 1IBB, Centro de Engenharia Biológica, Universidade do Minho, 2Biocant, Cantanhede, 3Faculdade de Ciências e Tecnolgia, Universidade de Coimbra, 4ICBAS, Universidade do Porto. Email: veracarvalho@deb.uminho.pt  KEYWORDS Protein delivery system; anti-inflammatory cytokine; interleukin-10; endotoxin-activated macrophages; dextrin nanogel. INTRODUCTION Interleukin-10 (IL-10) is an anti-inflammatory cytokine, which active form is a non-covalent homodimer with two intramolecular disulphide bonds that are essential to its biological activity, which includes reduction of tumor necrosis factor α (TNF-α) synthesis and down-regulation of class II major histocompatibility complex (MCH-II) molecules on monocytes/macrophages (de Waal Malefyt, 1991). Due to IL-10 potential applications in various medical fields, it is essential to develop systems that can effectively deliver the protein. A promising system is protein encapsulation by polymeric nanopartices (NPs), which minimize denaturation, and enables slow-release, while maintaining an effective concentration for the necessary period of time. In previous work, we have developed and characterized NPs obtained by self-assembling of hydrophobized dextrin (Gonçalves, 2007) whose properties makes them promising for IL-10 delivery.  METHODS The recombinant mutated (C149Y) murine IL-10 (rIL-10) was expressed, in form of inclusion bodies, in E. coli BL21 star. rIL-10 was recovered by a process of solubilization in 6M guanidine, renaturation and re-oxidation of disulphides. The dimeric form of rIL-10 was collected by gel filtration and further purified by ion-exchange chromatography. SDS-PAGE analysis was used to confirm molecular weight and purity. The amount of rIL-10 obtained was quantified by ELISA. rIL-10 bioactivity was assayed by its ability to inhibit the production of TNF-α, quantified by ELISA, and the surface expression of MHC-II molecules, monitorized by FACS analysis, in lipopolysacharide (LPS) and IFN-γ activated bone marrow derived macrophages (BMDM). To form the self-assembled NPs, lyophilized dextrin-VMA-SC16 was resuspended in culture medium, at room temperature. NPs formation was confirmed by dynamic light scattering. The complex NPs/rIL-10 was formed by dissolving rIL-10 in culture medium and then by mixing lyophilized dextrin-VMA-SC16. rIL-10 incorporation into NPs was verified quantifying the amount of rIL-10 free in solution by ELISA. The IL-10 stability was accessed by circular dichroism (CD). The release of rIL-10 from the complex NPs/rIL-10 was assessed in a BMDM culture, by measuring free rIL-10 by ELISA. Bioactivity of rIL-10 released from the NPs/rIL-10 complex was assayed as described previously. RESULTS  SDS-PAGE confirmed the expression and molecular weight (18 kDa) of the rIL-10 as well as their purity. Total recovery of dimeric rIL-10 was about 1-1.5 mg/L culture, quantified by ELISA. rIL-10 (in the range of 0.1 to 250 ng/mL) shown the ability to inhibit the TNF-α production and to reduce MHC-II expression on stimulated BMDM (figure 1A and B).  Figure 1: Biological activity of rIL-10 and cIL-10. (A) Percentage of inhibition of TNF-α production, by 0.1 ng/ml LPS and 1.0 ng/ml INF-γ stimulated BMDM. (B) Percentage of MHC-II expression induced by cIL-10 and rIL-10 treatment of stimulated BMDM. Data points are the means ± SD. (+) – positive control of macrophage activation; (-) – negative control of macrophage activation. rIL-10 incorporation by NPs was confirmed by quantifying, the amount of rIL-10 in free in solution,  Semana de Engenharia 2010 Guimarães, 11 a 15 de Outubro     Captions for figures and tables should be written in heading style. That means only important words are capitalized.  FOOTNOTES AND ENDNOTES Do not use footnotes or endnotes, instead incorporate such material into the text directly or parenthetically. If you want to acknowledge a grant or support, include an extra acknowledgements section at the end of the paper.  REFERENCES In text, references should be cited by the last name of the author and the year of publication, all in parentheses. The Reference List should be organized alphabetically by the name of the author, followed by the author's initials, year of publication, and other complete information about the published work. It may not be numbered. Only references that may be readily obtained should be cited in the list. Others may be referred to as "personal communication" in the text. In the reference list, multiple entries with the same author are arranged chronologically. Italicize the name of the publication in which the article is found, or the title itself if a separate publication. For Ph.D. and M.S. theses, the institutions granting  the degree should be given. References to proceedings must include the full name of the proceedings, how to obtain it, year of publication, and page numbers of article cited. A reference to part of a book must include the range of pages in which the material is cited. Names of periodicals must be written out in full, and the range of pages cited. Citing websites as references should not be done.  Examples of References In text: (Smith 1978) -- one author (Jones and Miller 1983) -- two authors (Arthur et al. 1985) -- more than two authors (Andrews 1982a) -- a trailing lowercase letter should distinguish multiple papers by the same author(s) published during a single year. (Dijkstra 1972; Hoare et al. 1980; Smith and Kim 1984b) -- more references  In reference List (a 9 point font is standard for the reference list): The examples below show what a reference should look like in a journal (Balci a d Sargent 1981), for a book (Felker et al. 1980), for a book chapter (Balci and Sargent 1983), in a conference proceedings (Gass 1978), for a special publication (Nat onal Bureau of Standards 1976), for a technic l report (Balci 1985), and for an internal report (Inglehart and Shedler 1983).  REFERENCES Balci, O. and R.G. Sargent. 1981. “A Methodology for Cost-Risk Analysis in the Statistical Validation of Simulation Models.” Communications of the ACM 24, No.4 (Apr), 190-197. Balci O. and R.G. Sargent. 1983. “Validation of Multivariate Response Trace-Driven Simulation Models”. In Performance 1983, A.K. Agrawalla and S.K. Tripathi (Eds.). North-Holland, Amsterdam, 309-323. Balci O. 1985. “Guidelines for Successful Simulation Studies”.  Technical report TR-85-2. Department of Computer Science, Virginia Tech, Blacksburg, Va. (Nov). Felker, D.B.; F. Pickering; V.R. Holland; and W.L. Harper. 1980. Data Processing Document tion St ndards, Procedures and Applications. P entice-Hall, Englewood Cliffs, N.J. Gass, S.L. 1978. “Computer Model Documentation”. In Proceedings of the 1978  Winter Simulation Conference (Miami Beach, FL, Dec.4-6). IEEE, Picataway, N.J., 281-287. National Bureau of Standards. 1976. Guidelines for Documentation of Computer Programs and Automated Data Systems. Federal Information Processing Standards Publication 38. Government Printing Office, Washington, D.C. (Feb). Inglehart, D.L. and G.S. Shedler. 1983. "Simulation Output Analysis for Local Areas Computer Networks." Research Report RJ 4020 (45068), Research Division, IBM, San Jose, CA (Sep). AUTHOR BIOGRAPHIES Please include a brief biography (no more than 300 words) of all authors at the end of the manuscript. The section heading is AUTHOR BIOGRAPHIES. This allows the viewing and reading audience to become familiar with the background of the authors.  Start the paragraph devoted to each author's name, without indentation. In boldface FULL CAPITALS. If there are multiple authors, separate each paragraph with a blank line. Authors are encouraged to include email and webaddress in the last line of the biography.  If space permits, a small picture of 2.25 x 3 cm should be included as shown below.  JOHN J. SMITH was born in Antwerp, Belgium and went to the Free University of Brussels, where he studied chemical technology and obtained his degree in 1973. He worked for a couple of years for the Brussels Chemical Company Actin before moving in 1986 to the University of Anytown where he is now leading a large research group in the field of simulation for X-Y-Z Analysis. His e-mail address is : JJSmith@anytown.com and his Web-page can be found at http://www.anytown.com/~smith.  2.25 x 3 cm that was negligible. The secondary structure of free soluble rIL-10 is mainly helical (Zdanov, 1995), and the complexation in the nanogel did not induce any conformational change (figure 2). Further CD studies showed that rIL-10 stability is significantly increased when it is complexed with the dextrin nanogel.  Figure 2: CD spectra of free rIL-10 (0.25 mg/ml) and complex nanogel/rIL-10 (1 mg/ml nanogel and 0.25 mg/ml rIL-10) at 37ºC in PBS. In the presence of 20% serum, rIL-10 is being released over time in a BMDM culture. After two hours of incubation with the NPs/rIL-10 complex, the rIL-10 reaches a maximum concentration; a stable value of about 35 ng/mL rIL-10 being estimated after twenty-four hours.   Figure 3:Biological activity of rIL-10 released from nanogel/rIL-10 complex. (A) TNF-α concentration (pg/ml), produced by 0.1 ng/ml LPS and 1.0 ng/ml INF-γ stimulated BMDM, treated with rIL-10 and nanogel/rIL-10. (B) MHC-II induced, in stimulated BMDM, by 50 ng/ml rIL-10 and nanogelrIL-10. Data points are the means±SD. (+) – positive control of macrophage activation; (-) – negative control of macrophage activation. The rIL-10 released from the NPs/rIL-10 complex was able to inhibit TNF-α production and MHC-II expression at the same level as the soluble rIL-10 (figure 3A and B). DISCUSSION & CONCLUSIONS A mutated form of murine IL-10 was successfully expressed in E. coli. This recombinant protein was recovered and purified from inclusion bodies and demonstrated biological activity similar to a commercially available IL-10. Dextrin self-assembled nanogel was able to efficiently encapsulate and protect rIL-10 from denaturation at 37ºC, and also enables rIL-10 to be released in biologically significant amounts over time. The biological activity of the released rIL-10 was confirmed by the evaluation of the production of TNF-α and expression of MHC-II on endotoxin-stimulated BMDM. The simplicity of the preparation of the nanogel/rIL-10 complex, associated to the enhancement of protein stability and controlled release, makes this a very promising system. IL-10 has potential application in various medical fields, such as in acute inflammatory diseases, namely rheumatoid arthritis, and these results point to dextrin nanogel as a promising carrier of IL-10, which enables the protein sustained release.  REFERENCES de Waal Malefyt, R., Abrams, J., Bennett, B., Figdor, C. G. and de Vries, J. E. 1991. Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med 174: 1209-1220. Goncalves, C., Martins, J. A. and Gama, F. M. 2007. “Self-assembled nanoparticles of dextrin substituted with hexadecanethiol”. Biomacromolecules 8: 392-398. Zdanov, A., Schalk-Hihi, C., Gustchina, A., Tsang, M., Weatherbee, J. and Wlodawer, A. 1995. “Crystal structure of interleukin-10 reveals the functional dimer with an unexpected topological similarity to interferon gamma”. Structure 3: 591-601. ACKNOWLEDGEMENTS  Vera Carvalho was supppoted 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. AUTHOR BIOGRAPHIES VERA CARVALHO was born in V. N. Famalicão, Portugal. In 2002 got her degree in Biochemistry in Universidade do Porto. In 2006 concluded her master degree in Biomedical Engineering also in Universidade do Porto. Since 2002 she had several grants in the Biological Engineering Department of Universidade do Minho where she researches in the biomedical and biotechnology areas.  Email:veracarvalho@deb.uminho.pt -20 -10 0 10 20 30 190 210 230 250 Ellipticity x 10-3  (deg cm2  dmol-1) Wavelength (nm) rIL-10 Nanogel/rIL-10 

Biological activity of heterologous murine interleukin-10 and preliminary studies on the use of a dextrin nanogel as a delivery system

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