Nanoparticle mediated delivery of 2'-O-methyl-RNA leads to efficient telomerase inhibition and telomere shortening in human lung cancer cells.

A promising approach for treatment of non-small cell lung cancer (NSCLC) is based on the inhibition of telomerase in cancer cells. The antisense oligonucleotide 2'-O-methyl-RNA binding to the RNA component of telomerase acts as a selective telomerase inhibitor. We developed chitosan-coated polylactide-coglycolide (PLGA) nanoparticles to mediate efficient delivery of 2'-O-methyl-RNA into human lung cancer cells. Cellular uptake of the inhibitor mediated by chitosan-coated PLGA nanoparticles was greatly enhanced compared to the uptake of antisense oligonucleotide alone as shown by flow cytometry analysis. Confocal laser scanning microscopy clearly demonstrated internalization of 2'-O-methyl-RNA. 2'-O-methyl-RNA-nanoparticle complexes exhibited nearly no acute cytotoxicity in human lung cancer cells and did not influence the viability of primary tumor lung fibroblasts. Human NSCLC A549 cells treated with 2'-O-methyl-RNA-nanoparticle complexes showed 87% viability compared to untreated control cells. 2'-O-methyl-RNA delivered by nanoparticle complexes inhibited telomerase activity in a sequence-specific manner. During long-term treatment (15 weeks) telomerase activity was continuously reduced by approximately 80%. Furthermore, nanoparticle mediated delivery of 2'-O-methyl-RNA resulted in significant telomere shortening from 5.9kb to 4kb (p=0.008) in A549 cells. In summary, our data demonstrate that nanoparticle mediated delivery of 2'-O-methyl-RNA induces effective telomerase inhibition and telomere shortening in human lung cancer cells and therefore represents a novel and promising strategy for the treatment of lung cancer.

Hyaluronic acid-modified DOTAP/DOPE liposomes for the targeted delivery of anti-telomerase siRNA to CD44-expressing lung cancer cells.

Cationic hyaluronic acid (HA)-modified DOTAP/DOPE liposomes were designed for the targeted delivery of anti-telomerase siRNA to CD44 receptor-expressing lung cancer cells. DOTAP/DOPE liposomes modified with 1%-20% (w/w) HA-DOPE conjugate were obtained by the ethanol injection method. Their size was below 170 nm and they exhibited zeta potentials higher than +50 mV. Lipoplexes prepared at different +/-ratios with siRNA were in the range of 200 nm and below and their zeta potentials were strongly dependent on the degree of modification and the +/-charge ratio. The presence of HA did not compromise binding, protection of siRNA from degradation, and complex stabilities in serum but rather resulted in an improvement of these properties. Liposome cytotoxicity, investigated by the MTT assay and LDH release after treatment of CD44(+) A549 cells and CD44(-) Calu-3, was demonstrated only at high concentrations. However, the addition of siRNA to HA-modified liposomes prevented cytotoxic effects compared to all other formulations. As shown by flow cytometry, transfection of siRNA into A549 cells was markedly improved with HA-modified liposomes, but not into Calu-3 cells. Using a qPCR-TRAP assay to test telomerase activity, no difference was demonstrated in the efficiency between HA-modified and nonmodified preparations. Moreover, some reduction in telomerase activity was observed with liposomes alone, lipoplexes prepared with nonsense siRNA and lipofectamine, indicative for some direct inhibitory effect of the lipids and siRNA on the expression of this enzyme. HA-modified DOTAP/DOPE liposomes represent a suitable carrier system for siRNA since properties like binding or protection of siRNA are not altered. They display an improved stability in cell culture medium and a reduced cytotoxicity. Furthermore, these novel lipoplexes could successfully be targeted to CD44-expressing A549 cells opening interesting perspectives for the treatment of lung cancer.

Efficient telomerase inhibition in human non-small cell lung cancer cells by liposomal delivery of 2'-O-methyl-RNA.

The antisense oligonucleotide 2'-O-methyl-RNA is a selective telomerase inhibitor targeting the telomerase RNA component and represents a potential candidate for anticancer therapy. The poor cellular uptake of 2'-O-methyl-RNA is a limiting factor that may contribute to the lack of functional efficacy. To improve delivery of 2'-O-methyl-RNA and consequently antitumoral efficiency in human lung cancer cells, we have investigated several transfection reagents. The transfection reagents DOTAP, MegaFectin 60, SuperFect, FuGENE 6 and MATra-A were tested for intracellular delivery. A FAM-labeled 2'-O-methyl-RNA was used to assess the intracellular distribution by confocal laser scanning microscopy in A549 human non-small cell lung cancer cells. Telomerase activity was measured using the telomeric repeat amplification protocol. Cell viability after transfection was quantified by the MTT assay. All transfection reagents enhanced 2'-O-methyl-RNA uptake in A549 cells but the cationic lipid reagents DOTAP and MegaFectin 60 were most efficient in the delivery of 2'-O-methyl-RNA resulting in telomerase inhibition. Among both DOTAP exhibited the lowest cytotoxicity. Our experiments show that DOTAP is the most suitable transfection reagent for the delivery of 2'-O-methyl-RNA in human lung cancer cells according to its relatively low cytotoxicity and its ability to promote efficient uptake leading to the inhibition of telomerase.

Chitosan-coated PLGA nanoparticles for DNA/RNA delivery: effect of the formulation parameters on complexation and transfection of antisense oligonucleotides.

Cationically modified poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles have recently been introduced as novel carriers for DNA/RNA delivery. The colloidal characteristics of the nanoparticles--particle size and surface charge--are considered the most significant determinants in the cellular uptake and trafficking of the nanoparticles. Therefore, our aim was to introduce chitosan-coated PLGA nanoparticles, whose size and charge are tunable to adapt for a specific task. The results showed that biodegradable nanoparticles as small as 130 nm and adjustable surface charge can be tailored controlling the process parameters. As a proof of concept, the overall potential of these particulate carriers to bind the antisense oligonucleotides, 2'-O-methyl-RNA, and improve their cellular uptake was demonstrated. The study proved the efficacy of chitosan-coated PLGA nanoparticles as a flexible and efficient delivery system for antisense oligonucleotides to lung cancer cells.

Coupling of biotin-(poly(ethylene glycol))amine to poly(D,L-lactide-co-glycolide) nanoparticles for versatile surface modification.

Generally, polymeric nanoparticles (NP) for drug targeting are designed to entrap the drug moiety in the core and to present the targeting moiety on the surface. However, in most cases, common preparation techniques of polymeric NP need to be specifically arranged for each compound to be entrapped or attached. In the present work, we introduce a method for versatile conjugation of targeting moieties to the surface of preformed, polymeric NP. Moreover, due to taking advantage of biotin-avidin interactions, our regime opens the additional possibility of a rapid fluorescence labeling of NP. Poly(D,L-lactide-co-glycolide) (PLGA) NP in the size of 210 nm were prepared by the classic oil-in-water method. Such NP were functionalized with biotin-(poly(ethylene glycol))amine (BPEG) by means of cyanuric chloride chemistry. The amount of surface-associated biotin was 850 pmol per milligram of polymer, corresponding to roughly 2650 molecules of biotin per NP. When drawn to scale, such surface coating appeared to be well-suited for subsequent binding of avidin or avidin-linked ligands. By resonant mirror measurements, we could prove specific binding of biotinylated NP to a NeutrAvidin (NAv)-coated surface. Furthermore, after coupling of NAv-linked fluorescence dyes to BPEG-functionalized NP, differences in binding and uptake could be demonstrated using two epithelial cell lines (Caco-2, A549).

Optimization of the TRAP assay to evaluate specificity of telomerase inhibitors.

Telomerase inhibition represents a promising approach to anticancer treatment. In order to clarify the therapeutic potential of telomerase inhibitors we examined different substances (small molecule compounds BIBR1532 and BRACO19, as well as hTR antisense oligonucleotides 2'-O-methyl RNA and PNA) in A-549, MCF-7, and Calu-3 cell lines in a cell-free TRAP assay. We demonstrated that each of the tested agents inhibited telomerase in all used cell lines and that the antisense oligonucleotides represent the most potent inhibitors. Interestingly, upon evaluating the specificity of telomerase inhibitors we found out that not all agents acted specifically against telomerase. We observed that BRACO19 and PNA had an inhibitory effect also on PCR amplification of the TSR8 oligonucleotide which is provided in the TRAP(EZE) kit as a PCR control. By modifying the experimental protocol and using a different reverse primer we were able to enhance PNA selectivity, although the PCR inhibition of the TSR8 control template by BRACO19 could not be prevented. We propose an explanation for the lack of target specificity and suggest caution when testing putative telomerase inhibitors, as it appears that some of those substances may not affect specifically telomerase or telomeric G-rich sequences and thus can lead to the misinterpretation of experimental results.