Targeted therapy against VEGF and mTOR pathways has been established as the standard-of-care for metastatic clear cell renal cell carcinoma (ccRCC); however, these treatments frequently fail and most ...patients become refractory requiring subsequent alternative therapeutic options. Therefore, development of innovative and effective treatments is imperative. About 80%-90% of ccRCC tumors express an inactive mutant form of the von Hippel-Lindau protein (pVHL), an E3 ubiquitin ligase that promotes target protein degradation. Strong genetic and experimental evidence supports the correlate that pVHL functional loss leads to the accumulation of the transcription factor hypoxia-inducible factor 2α (HIF2α) and that an overabundance of HIF2α functions as a tumorigenic driver of ccRCC. In this report, we describe an RNAi therapeutic for HIF2α that utilizes a targeting ligand that selectively binds to integrins αvβ3 and αvβ5 frequently overexpressed in ccRCC. We demonstrate that functional delivery of a HIF2α-specific RNAi trigger resulted in HIF2α gene silencing and subsequent tumor growth inhibition and degeneration in an established orthotopic ccRCC xenograft model.
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Effective in vivo delivery of small interfering (siRNA) has been a major obstacle in the development of RNA interference therapeutics. One of the first attempts to overcome this obstacle utilized ...intravenous injection of cholesterol-conjugated siRNA (chol-siRNA). Although studies in mice revealed target gene knockdown in the liver, delivery was relatively inefficient, requiring 3 daily injections of 50 mg/kg of chol-siRNA to obtain measurable reduction in gene expression. Here we present a new delivery approach that increases the efficacy of the chol-siRNA over 500-fold and allows over 90% reduction in target gene expression in mice and, for the first time, high levels of gene knockdown in non-human primates. This improved efficacy is achieved by the co-injection of a hepatocyte-targeted and reversibly masked endosomolytic polymer. We show that knockdown is absolutely dependent on the presence of hepatocyte-targeting ligand on the polymer, the cognate hepatocyte receptor, and the cholesterol moiety of the siRNA. Importantly, we provide evidence that this increase in efficacy is not dependent on interactions between the chol-siRNA with the polymer prior to injection or in the bloodstream. The simplicity of the formulation and efficacy of this mode of siRNA delivery should prove beneficial in the use of siRNA as a therapeutic.
The protective effect of poly(ethylene glycol) and some other polymers on nanoparticulate carriers including liposomes is considered in terms of statistical behavior of macromolecules in solution, ...when polymer flexibility plays a key role. According to the mechanism proposed, surface-grafted chains of flexible and hydrophilic polymers form dense “conformational clouds” preventing other macromolecules from the interaction with the surface even at low concentration of protecting polymer. Using liposomes as an example, experimental evidence is presented of the importance of protecting polymer flexibility in liposome steric protection. Further possible applications of the suggested model are discussed. The possibility of using protecting polymers other than poly(ethylene glycol) is analyzed, and examples of such polymers are given based on polymer-coated liposome biodistribution data. General requirements for protecting polymers are formulated, and differences in steric protection of liposomes and particles are discussed. The scale of protective effect is interpreted as the balance between the energy of hydrophobic anchor interaction with the liposome membrane core or with the particle surface and the energy of polymer chain free motion in solution.
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The RNA interference (RNAi)-based therapeutic ARC-520 for chronic hepatitis B virus (HBV) infection consists of a melittin-derived peptide conjugated to N-acetylgalactosamine for hepatocyte targeting ...and endosomal escape, and cholesterol-conjugated RNAi triggers, which together result in HBV gene silencing. To characterize the kinetics of RNAi trigger delivery and 5΄-phosphorylation of guide strands correlating with gene knockdown, we employed a peptide-nucleic acid (PNA) hybridization assay. A fluorescent sense strand PNA probe binding to RNAi duplex guide strands was coupled with anion exchange high performance liquid chromatography to quantitate guide strands and metabolites. Compared to PCR- or ELISA-based methods, this assay enables separate quantitation of non-phosphorylated full-length guide strands from 5΄-phosphorylated forms that may associate with RNA-induced silencing complexes (RISC). Biodistribution studies in mice indicated that ARC-520 guide strands predominantly accumulated in liver. 5΄-phosphorylation of guide strands was observed within 5 min after ARC-520 injection, and was detected for at least 4 weeks corresponding to the duration of HBV mRNA silencing. Guide strands detected in RISC by AGO2 immuno-isolation represented 16% of total 5΄-phosphorylated guide strands in liver, correlating with a 2.7 log10 reduction of HBsAg. The PNA method enables pharmacokinetic analysis of RNAi triggers, elucidates potential metabolic processing events and defines pharmacokinetic-pharmacodynamic relationships.
Protease-triggered siRNA delivery vehicles Rozema, David B; Blokhin, Andrei V; Wakefield, Darren H ...
Journal of controlled release,
07/2015, Volume:
209
Journal Article
Peer reviewed
Open access
The safe and efficacious delivery of membrane impermeable therapeutics requires cytoplasmic access without the toxicity of nonspecific cytoplasmic membrane lysis. We have developed a mechanism for ...control of cytoplasmic release which utilizes endogenous proteases as a trigger and results in functional delivery of small interfering RNA (siRNA). The delivery approach is based on reversible inhibition of membrane disruptive polymers with protease-sensitive substrates. Proteolytic hydrolysis upon endocytosis restores the membrane destabilizing activity of the polymers thereby allowing cytoplasmic access of the co-delivered siRNA. Protease-sensitive polymer masking reagents derived from polyethylene glycol (PEG), which inhibit membrane interactions, and N-acetylgalactosamine, which targets asialoglycoprotein receptors on hepatocytes, were synthesized and used to formulate masked polymer-siRNA delivery vehicles. The size, charge and stability of the vehicles enable functional delivery of siRNA after subcutaneous administration and, with modification of the targeting ligand, have the potential for extrahepatic targeting.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
This review deals with diagnostic applications of polymeric micelles composed of amphiphilic block-copolymers. In aqueous solutions these polymers spontaneously form particles with diameter 20–100 ...nm. A variety of diagnostic moieties can be incorporated covalently or non-covalently into the particulates with high loads. Resulting particles can be used as particulate agents for diagnostic imaging using three major imaging modalities: gamma-scintigraphy, magnetic resonance imaging and computed tomography. The use of polyethyleneoxide–diacyllipid micelles loaded with chelated
111In/Gd
3+ as well as iodine-containing amphiphilic copolymer in percutaneous lymphography and blood pool/liver imaging are discussed as specific examples.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The hypothetical model is built explaining the molecular mechanism of protective action of poly(ethylene glycol) on liposomes in vivo. The protective layer of the polymer on the liposome surface is ...considered as a statistical ‘cloud’ of polymer possible conformations in solution. Computer simulation was used to demonstrate that relatively a small number of liposome-grafted molecules of hydrophilic and flexible polymer can create a dense protective conformational cloud over the liposome surface preventing opsonizing protein molecules from contacting liposome. A more rigid polymer fails to form this dense protective cloud, even when hydrophilic. Computer simulation was also used to reveal possible heterogeneity of reactive sites on a polymer-coated liposome surface, and to estimate the optimal polymer-to-lipid ratio for efficient liposome protection. Experiments have been performed with the quenching of liposome-associated fluorescent label (nitrobenzoxadiazole or fluorescein) with protein (rhodamine-ovalbumin or anti-fluorescein antibody) from solution. It was shown that poly(ethylene glycol) grafting to liposomes hinders protein interaction with the liposome surface, whereas liposome-grafted dextran (more rigid polymer) in similar quantities does not affect protein-liposome interaction. Highly-reactive and low-reactive populations of chemically identical reactive sites have been found on polymer-coated liposomes. Experimental data satisfactory confirm the suggested mechanism for the longevity of polymer-modified liposome.
DNA can be condensed with an excess of polycations in aqueous solutions forming stable particles of submicron size with positive surface charge. This charge surplus can be used to deposit alternating ...layers of polyanions and polycations on the surface surrounding the core of condensed DNA. Using poly-L-lysine (PLL) and succinylated PLL (SPLL) as polycation and polyanion, respectively, we demonstrated layer-by-layer architecture of the particles. Polyanions with a shorter carboxyl/backbone distance tend to disassemble binary DNA/PLL complexes by displacing DNA while polyanions with a longer carboxyl/ backbone distance effectively formed a tertiary complex. The ζ potential of such complexes became negative, indicating effective surface recharging. The charge stoichiometry of the DNA/PLL/SPLL complex was found to be close to 1:1:1, resembling polyelectrolyte complexes layered on macrosurfaces. Recharged particles containing condensed plasmid DNA may find applications as non-viral gene delivery vectors.
Positron emission tomography/computed tomography (PET/CT) hybrid imaging can be used to gain insights into a synthetic siRNA delivery system targeted to the liver. Either siRNA or the delivery ...vehicle was labeled with 64Cu via 1, 4, 7, 10- tetraazacyclododecane- 1, 4, 7, 10- tetraacetic acid (DOTA) chelation. This study confirmed that the siRNA delivery system was successfully targeted to the liver. Incorporation of the siRNA into the delivery system protected the siRNA from renal filtration long enough so that the siRNA could be delivered to the liver. PET/CT imaging was important for confirming biodistribution and for determining differences in the distribution of labeled siRNA, siRNA incorporated into the delivery system, and the delivery system without siRNA.
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IJS, KILJ, NUK, PNG, UL, UM
The utilization of micelle forming block-copolymers as long-circulating drug carriers for sparingly soluble or amphiphilic drugs or diagnostic agents is reviewed with the emphasis on the use of ...phosphatidylethanolamine-polyethyleneoxide (PE-PEO) conjugates as structural amphiphilic polymers for formulation of polymeric micelles. PE-PEO conjugates spontaneously form particles with average diameter 10–30 nm depending on the molecular weight of PEO block and which are stable upon dilution at ambient temperature. Topoisomerase II inhibitor ellipticine was shown to be successfully incorporated into PE-PEO (5 kDa) conjugate micelles in vitro. Percutaneous lymphatic delivery of amphiphilic diagnostic agents incorporated into PE-PEO micelles was demonstrated using magnetic resonance imaging and gamma-scintigraphy. For water-soluble low molecular weight drugs one can expect hydrophobic prodrug approach to be most suitable for use with this drug delivery system.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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