Abstract
Short interfering RNA (siRNA) targeted against anti-apoptotic Bcl-2 protein proved to knockdown its expression and trigger cancer cell death. We used degradable, pH-sensitive, comb-like [P(EAA-co-BMA)-b-PNASI-g-P(HMA-co-TMAEMA)] polymer to condense anti-Bcl-2 siRNA into “smart” particles, which proved to shuttle their cargo past the endosomal membrane and into the cytoplasm of HeLa and UM-SCC-17B cancer cells. HeLa and UM-SCC-17B cancer cells were treated with anti-Bcl-2 particles followed by quantifying Bcl-2 mRNA and protein levels using qRT-PCR and western blotting, respectively. “Smart” anti-Bcl-2 particles selectively suppress Bcl-2 mRNA and protein levels in HeLa cells by 50%–60% and 79%–81%, respectively. Similarly, “smart” anti-Bcl-2 particles inhibited Bcl-2 mRNA levels by 30%, 40%, and 20% upon incubation with UM-SCC-17B cancer cells for 48, 72, and 96 h, respectively. Bcl-2 protein expression in UM-SCC-17B cancer cells was inhibited by 30% after treatment for 72 h. Results show that pH-sensitive comb-like polymer complex anti-Bcl-2 siRNA forming “smart” nanoparticles that deliver their cargo into the cytoplasm of HeLa and UM-SCC-17B cancer cells causing Bcl-2 knockdown at the mRNA and protein levels.
Highlights
IntroductionB-cell lymphoma 2 (Bcl-2) is a family of proteins that includes more than 20 apoptotic regulators with opposing functions but share at least one conserved Bcl-2 homology (BH) domain [1,2,3]
B-cell lymphoma 2 (Bcl-2) is a family of proteins that includes more than 20 apoptotic regulators with opposing functions but share at least one conserved Bcl-2 homology (BH) domain [1,2,3].Anti-apoptotic proteins such as Bcl-2, Bcl-XL, and Bcl-w appear to inhibit apoptotic cell death through their binding to the pro-apoptotic proteins [1,2,3]
We report the ability of P(EAA-co-butyl methacrylate (BMA))b-PNASI-g-P(HMA-co-trimethyl aminoethyl methacrylate (TMAEMA)) polymer to condense anti-Bcl-2 Short interfering RNA (siRNA) into “smart” particles that deliver their cargo past the endosomal membrane and into the cytoplasm of HeLa cervical cancer and UM-SCC-17B head and neck cancer cells to knockdown the expression of anti-apoptotic Bcl-2 protein at the mRNA and protein levels
Summary
B-cell lymphoma 2 (Bcl-2) is a family of proteins that includes more than 20 apoptotic regulators with opposing functions but share at least one conserved Bcl-2 homology (BH) domain [1,2,3]. We reported the design and synthesis of a new degradable, comb-like, pH-sensitive, and membrane-destabilizing polymer namely P(EAA-co-BMA)-b-PNASI-g-P(HMA-co-TMAEMA), which incorporates two blocks in the backbone [11]. We report the ability of P(EAA-co-BMA)b-PNASI-g-P(HMA-co-TMAEMA) polymer to condense anti-Bcl-2 siRNA into “smart” particles that deliver their cargo past the endosomal membrane and into the cytoplasm of HeLa cervical cancer and UM-SCC-17B head and neck cancer cells to knockdown the expression of anti-apoptotic Bcl-2 protein at the mRNA and protein levels. Comb-like polymer (A) condenses siRNA molecules forming “smart” particles (B); after internalization into target cells through adsorptive endocytosis (C); the acid-labile hydrazone linkages get hydrolysed releasing the membrane-active fragments that rupture the endosomal membrane (D) and release the encapsulated cargo into the cytoplasm.
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