Research Report 1 : SAINT:DOPE Based Liposome Polycation Particles for siRNA Delivery siRNA induced Knockdown by SAINT:DOPE based LPD and LP Particles in Endothelial Cells

Bijlsma, K.T. (2013) Research Report 1 : SAINT:DOPE Based Liposome Polycation Particles for siRNA Delivery siRNA induced Knockdown by SAINT:DOPE based LPD and LP Particles in Endothelial Cells. Master's Thesis / Essay, Biology.

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Abstract

Since the discovery of RNA interference as post-translational gene silencing pathway via small RNA sequences, it gave rise to the idea to use this mechanism for pharmacological intervention. Short interfering RNA (siRNA) is one class of small RNAs that binds to target messenger RNA (mRNA) which leads to gene silencing by cleavage of this mRNA. Systemic administration of “naked” siRNA will lead to rapid excretion and degradation by serum RNases. siRNA is unable to cross cell membranes and therefore has to be delivered into the target cells. This can be achieved by formulating siRNA into a drug carrier. Drug delivery systems that are currently used for siRNA delivery in vitro and in vivo are lipid based drug delivery systems, like liposome polycation DNA particles (LPD). These nanoparticles developed by Huang et al. have a size of ±200 nm, contain an inner solid core consisting of siRNA, carrier DNA and protamine. This negatively charged core is wrapped by a supported bilayer, containing cationic lipids for membrane interaction and particle stability. LPD particles can be shielded with high concentrations of poly ethylene glycol (PEG) molecules to avoid clearance by the reticuloendothelial system. Endothelial cells play a major role in many inflammatory (vascular) diseases, what makes them an interesting target for pharmacological intervention. By coupling anti-E-selectin to the PEG molecules of LPD, disease specific endothelial cells can be targeted. Investigating the potency of LPD particles to deliver siRNA to endothelial cells (HUVECs), we formulated LPD with cationic SAINT:DOPE liposomes. These liposomes are known to effectively deliver siRNA and proteins into cells. Besides LPD, we came up with a new particle: liposome protamine particle (LP). We studied these particles for size, shielding by PEG, targeting potential with anti-E-selectin to TNF-α challenged HUVECs and targeting specific knockdown of model gene VE-cadherin in HUVECs. The results show that LPD and LP particles can be shielded by PEGylation. SAINT:DOPE LP particles can be targeted to activated HUVECs with anti-E-selectin antibodies. Our data demonstrated that SAINT:DOPE LPD and LP particles deliver sufficient amounts of siRNA to HUVECs, causing knockdowns of more than 90% of VE-cadherin mRNA at siRNA concentrations of 600 and 300 pmol/ml, respectively. Unfortunately, this knock down was not anti E-selectin specific.

Item Type:	Thesis (Master's Thesis / Essay)
Degree programme:	Biology
Thesis type:	Master's Thesis / Essay
Language:	English
Date Deposited:	15 Feb 2018 07:52
Last Modified:	15 Feb 2018 07:52
URI:	https://fse.studenttheses.ub.rug.nl/id/eprint/10895

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