Introduction Cytotoxic T cells (CTL) attack cancer cells by recognizing cancer-specific antigens presented on the cellular surface. However, some cancer cells suppress the expression of MHC-class I to escape from such an immune system. It is known that double-stranded RNA (dsRNA) recognized by Toll-like receptor 3 (TLR3), inducing interferon response. This results in the induction of MHC-class I expression. Therefore it is expected to express MHC-class I molecule again by delivering dsRNA to cancer cells specifically. However, nucleic acids are easily degraded by enzymes and rapidly excluded via kidney after injection. We have studied hyaluronic acid (HA) as delivery carrier to cancer cells. HA is a linear polymer consisting of glucuronic acid and N-acetylglucosamine. Because cancer cells express CD44, a one of major HA receptor, on cellular surface, we think that HA-modified dsRNA can be efficiently delivered to cancer cell through CD44. Method As a model of the conjugation between HA and dsRNA, we prepared single chain oligo nucleotides modified with an amino group at the 5’-terminal to make a conjugate with carboxyl group of HA. We used 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as crosslinking agents. EDC and NHS, DMSO solution were added to HA ( 1.0 in 10 mM MES solution and incubated for 2 hours. After that, oligo nucleotide solution added and incubated overnight. The molar ratio in the reaction mixture was HA : oligo nucleotide = 1 : 25 (HA/ON 25) and 1: 50 (HA/ON 50). Results The conjugation was evaluated with gel permeation chromatography. When we determined the reaction yields from the peak areas between free oligo nucleotide and reaction mixture by UV measurement (260 nm), HA/ON25 and HA/ON50 were 20% and 6%, respectively. This difference in the reaction efficiency is considered to be attributed to the water content in the reaction mixture because the activated ester of HA-NHS is soon hydrolyzed in water.

Development of tumor-specific double-stranded RNA delivery system using hyaluronic acid

256th National Meeting (2018)

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