FORMULASI DAN SITOTOKSISITAS NANOPARTIKEL RIBOSOME-INACTIVATING PROTEIN Mirabilis jalapa L. DENGAN ALGINAT-KITOSAN VISKOSITAS RENDAH TERKONJUGASI ANTIBODI ANTI-EpCAM TERHADAP SEL T47D

PSYCHA ANINDYA W., Prof. Dr. Sismindari, SU., Apt.

2015 | Tesis | S2 Bioteknologi

Kanker payudara merupakan penyebab kematian tertinggi pada wanita. Terapi kanker payudara masih terkendala tingginya toksisitas dan kurangnya selektivitas. Ribosome-Inactivating Protein (RIP) dari daun Mirabilis jalapa L. telah terbukti sitotoksik terhadap sel kanker payudara. Meskipun demikian, RIP yang diberikan secara langsung mudah terdegradasi di dalam tubuh, sehingga RIP diformulasikan menjadi nanopartikel. Penelitian ini bertujuan untuk memformulasikan ekstrak protein daun M. jalapa L. (RIP-MJ) menjadi nanopartikel dengan polimer alginat-kitosan dan mengkonjugasikan nanopartikel tersebut dengan antibodi anti-EpCAM untuk meningkatkan efektivitas terapi. RIP-MJ diekstrak dari daun tanaman M. jalapa L. bunga merah. Nanopartikel diformulasi menggunakan metode kompleks polielektrolit dengan alginat dan kitosan viskositas rendah. Selanjutnya nanopartikel dikonjugasikan dengan antibodi anti-EpCAM menggunakan EDAC dengan reaksi karbodiimida. Karakterisasi nanopartikel meliputi ukuran partikel, nilai indeks polidispersitas, potensial zeta, morfologi partikel, dan efisiensi enkapsulasi. Nanopartikel selanjutnya diuji sitotoksisitasnya terhadap sel T47D dan sel Vero dengan metode MTT assay. Konsentrasi optimal yang dapat membentuk suspensi nanopartikel adalah RIP-MJ 0,05%; alginat 0,4%; dan kitosan 1% (b/v). Nanopartikel RIP-MJ memiliki nilai efisiensi enkapsulasi 98,57% berbentuk heksagonal dengan rata-rata ukuran partikel sebesar 130,73 nm, indeks polidispersitas 0,380 dan potensial zeta +26,33 mV. Nilai IC50 untuk sel T47D pada perlakuan nanopartikel RIP-MJ terkonjugasi dan tidak terkonjugasi anti-EpCAM (13,269-14,868 μg/mL) lebih kecil daripada nilai IC50 untuk sel Vero pada kedua perlakuan tersebut (27,841-33,619 μg/mL). Nilai IC50 kedua sel pada perlakuan nanopartikel baik terkonjugasi dan tidak terkonjugasi anti-EpCAM lebih rendah daripada nilai IC50 perlakuan RIP-MJ tanpa formulasi (>500 μg/mL). Kata kunci: ribosome-inactivating protein, Mirabilis jalapa L. nanopartikel, kitosan, alginat, anti-EpCAM

Breast cancer is the leading cause of women death over the world. The effectivity of breast cancer therapy is limited due to high toxicity and lack of selectivity. Ribosome-Inactivating Protein (RIP) from Mirabilis jalapa L. leaves has cytotoxic effect on breast cancer cell lines but less toxic on normal cells. However, proteins including RIP can easily be degraded after administration. Therefore, RIP needs to be formulated into nanoparticles to increase its resistance against enzymatic degradation. The objectives of this study were to develop the protein extract of M. jalapa L. leaves (RIP-MJ) into nanoparticles, to conjugate the nanoparticle with Anti-EpCAM antibody, and to determine its cytotoxicity and selectivity on breast cancer cell line. RIP-MJ was extracted from red-flowered M. jalapa L. leaves. The nanoparti-cles were formulated based on polyelectrolyte complexation using low viscosity chitosan and alginate. The nanoparticles were then chemically conjugated with anti-EpCAM antibody using EDAC based on carbodiimide reaction. RIP-MJ nanoparticles were characterised for the particle size, polydispersity index, zeta potential, particle morphology, and entrapment efficiency. The cytotoxicity of RIP-MJ nanoparticles on T47D and Vero cells was then determined with MTT assay. The optimal concentration of RIP-MJ, low viscosity chitosan and alginate to form an ophalescent particle in solution were 0.05%, 1%, and 0.4% (m/v) respectively. RIP-MJ nanoparticles are hexagonal with high entrapment efficiency of 98.57%, average size of 130.73 nm, polydispersity index of 0.380 and zeta potential +26.33 mV. The IC50 value of both anti-EpCAM-conjugated and non-conjugated RIP-MJ nanoparticles for T47D cells (13.269-14.868 μg/mL) were lower than the IC50 value of that for Vero cells (27.841-33.619 μg/mL). The IC50 values for both cells after conjugated and non-conjugated RIP-MJ administration were much lower than IC50 values obtained after non-formulated RIP-MJ administration (>500 μg/mL). Keywords: ribosome-inactivating protein, Mirabilis jalapa L. nanoparticle, chitosan, alginate, anti-EpCAM.

Kata Kunci : ribosome-inactivating protein, Mirabilis jalapa L. nanopartikel, kitosan, alginat, anti-EpCAM; ribosome-inactivating protein, Mirabilis jalapa L. nanoparticle, chitosan, alginate, anti-EpCAM