Alexandre A. Vetcher

Intratracheal Versus Intravenous Liposomal Delivery of siRNA, Antisense Oligonucleotides and Anticancer Drug

PurposeTo compare systemic intravenous and local intratracheal delivery of doxorubicin (DOX), antisense oligonucleotides (ASO) and small interfering RNA (siRNA).Methods“Neutral” and cationic liposomes were used to deliver DOX, ASO, and siRNA. Liposomes were characterized by dynamic light scattering, zeta-potential, and atomic force microscopy. Cellular internalization of DOX, ASO and siRNA was studied by confocal microscopy on human lung carcinoma cells. In vivo experiments were carried out on nude mice with an orthotopic model of human lung cancer.ResultsLiposomes provided for an efficient intracellular delivery of DOX, ASO, and siRNA in vitro. Intratracheal delivery of both types of liposomes in vivo led to higher peak concentrations and much longer retention of liposomes, DOX, ASO and siRNA in the lungs when compared with systemic administration. It was found that local intratracheal treatment of lung cancer with liposomal DOX was more efficient when compared with free and liposomal DOX delivered intravenously.ConclusionsThe present study outlined the clear advantages of local intratracheal delivery of liposomal drugs for the treatment of lung cancer when compared with systemic administration of the same drug.

Nonviral Nanoscale-Based Delivery of Antisense Oligonucleotides Targeted to Hypoxia-Inducible Factor 1α Enhances the Efficacy of Chemotherapy in Drug-Resistant Tumor

Purpose: To enhance the efficacy of cancer treatment, we propose a complex approach: simultaneous delivery to the tumor of a chemotherapeutic agent and a suppressor of hypoxia-inducible factor 1α (HIF1A). Experimental Design: The novel complex liposomal drug delivery system was developed and evaluated in vitro and in vivo on nude mice bearing xenografts of multidrug-resistant human ovarian carcinoma. The proposed novel complex drug delivery system consists of liposomes as a nanocarrier, a traditional anticancer drug (doxorubicin) as a cell death inducer, and antisense oligonucleotides targeted to HIF1A mRNA as a suppressor of cellular resistance and angiogenesis. Results: The system effectively delivers active ingredients into tumor cells, multiplies the cell death signal initiated by doxorubicin, and inhibits cellular defensive mechanisms and angiogenesis by down-regulating BCL2, HSP90, and vascular endothelial growth factor proteins. This, in turn, activates caspases, promotes apoptosis, necrosis, and tumor shrinkage. The proposed novel complex multipronged approach enhances the efficiency of chemotherapy. Conclusions: The proposed combination therapy prevents the development of resistance in cancer cells, and thus, increases the efficacy of chemotherapy to an extent that cannot be achieved by individual components applied separately. It could form the foundation for a novel type of cancer therapy based on simultaneous delivery of an anticancer drug and a suppressor of HIF1A.

Fractionation of SWNT/nucleic acid complexes by agarose gel electrophoresis

We show that aqueous dispersions of single-walled carbon nanotubes (SWNTs), prepared with the aid of nucleic acids (NAs) such as RNA or DNA, can be separated into fractions using agarose gel electrophoresis. In a DC electric field, SWNT/NA complexes migrate in the gel in the direction of positive potential to form well-defined bands. Raman spectroscopy as a function of band position shows that nanotubes having different spectroscopic properties possess different electrophoretic mobilities. The migration patterns for SWNT/RNA and SWNT/DNA complexes differ. Parallel elution of the SWNT/NA complexes from the gel during electrophoresis and subsequent characterization by AFM reveals differences in nanotube diameter, length and curvature. The results suggest that fractionation of nanotubes can be achieved by this procedure. We discuss factors affecting the mobility of the nanotube complexes and propose analytical applications of this technique.

Gel mobilities of linking-number topoisomers and their dependence on DNA helical repeat and elasticity

Agarose-gel electrophoresis has been used for more than thirty years to characterize the linking-number (Lk) distribution of closed-circular DNA molecules. Although the physical basis of this technique remains poorly understood, the gel-electrophoretic behavior of covalently closed DNAs has been used to determine the local unwinding of DNA by proteins and small-molecule ligands, characterize supercoiling-dependent conformational transitions in duplex DNA, and to measure helical-repeat changes due to shifts in temperature and ionic strength. Those results have been analyzed by assuming that the absolute mobility of a particular topoisomer is mainly a function of the integral number of superhelical turns, and thus a slowly varying function of plasmid molecular weight. In examining the mobilities of Lk topoisomers for a series of plasmids that differ incrementally in size over more than one helical turn, we found that the size-dependent agarose-gel mobility of individual topoisomers with identical values of Lk (but different values of the excess linking number, DeltaLk) vary dramatically over a duplex turn. Our results suggest that a simple semi-empirical relationship holds between the electrophoretic mobility of linking-number topoisomers and their average writhe in solution.

Measurements of DNA-loop formation via Cre-mediated recombination

The Cre-recombination system has become an important tool for genetic manipulation of higher organisms and a model for site-specific DNA-recombination mechanisms employed by the λ-Int superfamily of recombinases. We report a novel quantitative approach for characterizing the probability of DNA-loop formation in solution using time-dependent ensemble Förster resonance energy transfer measurements of intra- and inter-molecular Cre-recombination kinetics. Our method uses an innovative technique for incorporating multiple covalent modifications at specific sites in covalently closed DNA. Because the mechanism of Cre recombinase does not conform to a simple kinetic scheme, we employ numerical methods to extract rate constants for fundamental steps that pertain to Cre-mediated loop closure. Cre recombination does not require accessory proteins, DNA supercoiling or particular metal-ion cofactors and is thus a highly flexible system for quantitatively analyzing DNA-loop formation in vitro and in vivo.

Multifunctional Nanotherapeutics for Cancer

Nanotechnology, as a field of applied science, focuses on the development, production, characterization and application of materials, and devices at the level of molecules and atoms with a typical size between 10 �9 nm and 10 �6 µm. Nanotherapeutics, a rapidly expanding area of medicine, uses nanotechnology products for highly specific medical interventions at the molecular scale for curing diseases or repairing damaged tissues. Although some nanotechnology products can be applied alone as therapeutic or imaging agents, they are being most often used as pharmaceutical nanocarriers for delivering drugs or imaging agents to the site of the action in desired quantities and releasing therapeutic loads with a specific time profile. Linear and branched polymers, dendrimers, quantum dots, nanoparticles, nanospheres, nanotubes, nanocrystals, nanogels, liposomes, micelles, as well as other types of nanocarriers are being employed in different fields of medicine for diagnostics, imaging, treatment, and prophylaxis of many pathological conditions (Fig. 1) In contrast to the earlier developed nanotherapeutics, which had a relatively simple two-component drug–carrier composition, modern nanocarriers often include other active ingredients that perform different specific functions for enhancing cellular uptake and efficiency of the main drug, preventing adverse side effects, providing drug release with a predetermined profile in the certain compartment of an organ, tissue, or cell, and preventing the development and/or suppression of the existent drug resistance, etc. The increase in complexity and performed functions of nanocarriers actually converts them into multifunctional nanotherapeutical products. This chapter is mainly focused on reviewing modern multifunctional approaches in nanotherapeutics designed for effective cancer treatment.

ELECTROPHORETIC FRACTIONATION OF CARBON NANOTUBE DISPERSIONS ON AGAROSE GELS

We show that various aqueous suspensions of single-walled and multi-walled carbon nanotubes, dispersed using either common surfactants or DNA, can be separated into components using agarose gel electrophoresis. In a DC electric field, carbon nanotubes and nanotube–DNA complexes migrate in the gel in the direction of positive potential to form well-defined bands. Raman spectroscopy as a function of band position shows that nanotubes having different spectroscopic properties have distinct electrophoretic mobilities. The results suggest that the separation of nanotubes according to structure can be achieved by this procedure. We discuss the factors affecting the mobility of the nanotube complexes and propose analytical applications of this technique.

PCR-based synthesis of repetitive single-stranded DNA for applications to nanobiotechnology

Recent data show that assembly of repetitive-sequence, single-stranded DNA molecules (ssDNA) and carbon nanotubes (CNTs) depend on the specific sequence repeat. Therefore, it is of practical interest to assess various methods for generating single-stranded DNA molecules that contain repetitive sequences. Existing automated synthesis procedures for generating long (> 100 nt) ssDNA molecules generate ssDNA products of variable purity and yield. An alternative to automated synthesis is the polymerase chain reaction (PCR), which provides a powerful tool for the amplification of minute amounts of specific DNA sequences. Here we show that a modified asymmetric PCR method allows synthesis of long ssDNAs comprised of tandem repeats of the repetitive vertebrate telomeric sequence (TTAGGG)n, and is also applicable to arbitrary (repetitive or nonrepetitive) DNA. Long, repetitive deoxynucleotides produced by automated synthesis are surprisingly heterogeneous with respect to both length and sequence. Benefits of the method described here are that long, repetitive ssDNA sequences are generated with high sequence fidelity and yield.