Chitosans for delivery of nucleic acids.

Alternatives to efficient viral vectors in gene therapy are desired because of their poor safety profiles. Chitosan is a promising non-viral nucleotide delivery vector because of its biocompatibility, biodegradability, low immunogenicity and ease of manufacturing. Since the transfection efficiency of chitosan polyplexes is relatively low compared to viral counterparts, there is an impetus to gain a better understanding of the structure-performance relationship. Recent progress in preparation and characterisation has enabled coupling analysis of chitosans structural parameters that has led to increased TE by tailoring of chitosan's structure. In this review, we summarize the recent advances that have lead to a more rational design of chitosan polyplexes. We present an integrated review of all major areas of chitosan-based transfection, including preparation, chitosan and polyplexes physicochemical characterisation, in vitro and in vivo assessment. In each, we present the obstacles to efficient transfection and the strategies adopted over time to surmount these impediments.

Immobilized carboxymethylated dextran coatings for enhanced ELISA.

We here report the development of a new generation of enzyme-linked immunosorbent assay (ELISA) that takes advantage of a low-fouling carboxymethylated dextran (CMD) layer chemically grafted on ELISA wells. In our approach, the overnight capture antibody adsorption step found in classical ELISA was replaced by a covalent attachment step to the CMD layer completed in 15 min. As a model, the potential of our approach was highlighted using commercially available anti-human epidermal growth factor (EGF) antibodies to quantify EGF present in various samples. Of interest, the grafted CMD layer was found to be as efficient as the commonly used bovine serum albumine (BSA) to reduce non-specific adsorption, thus eliminating the need of a time-consuming BSA blocking step normally required in classical ELISA. Our results demonstrated similar specificity, affinity, and intra- and inter-assay variations regardless of the diluent used in the assay (BSA-based diluent or protein-free buffer solution) when compared to standard ELISA. Finally, accuracy and precision of the CMD-based ELISA were verified by a spike and recovery test. Dilutions of recombinant human EGF in serum from healthy human volunteers showed almost-perfect linearity and mean recovery rates ranging between 90 and 110%.

Adva-27a, a novel podophyllotoxin derivative found to be effective against multidrug resistant human cancer cells.

BACKGROUND/AIM: Multidrug resistance poses a serious challenge in cancer therapy. To address this problem, we designed and synthesized Adva-27a, a novel non-ester GEM-difluorinated C-glycoside derivative of podophyllotoxin. MATERIALS AND METHODS: Adva-27a activity was evaluated in a variety of assays including inhibition of topoisomerase IIα, cytotoxic activity in drug-sensitive and drug-resistant cancer cell lines, metabolic stability in human liver microsomes and pharmacokinetic properties in rats. RESULTS: Adva-27a exhibited dose-dependent human topoisomerase IIα inhibitory activity and dose-dependent growth inhibitory activity in several drug-sensitive and two multidrug-resistant cancer cell lines. In the multidrug-resistant cell lines, MCF-7/MDR (breast cancer) and H69AR (small-cell lung cancer), Adva-27a was significantly more potent than etoposide. The metabolic stability of Adva-27a in human liver microsomes and its pharmacokinetic properties in rats were better than those of etoposide. CONCLUSION: Our studies have identified Adva-27a as a novel topoisomerase II inhibitor with superior cytotoxic activity against multidrug-resistant human cancer cells and more desirable pharmacokinetic properties than etoposide.

Low molecular weight chitosan nanoparticulate system at low N:P ratio for nontoxic polynucleotide delivery.

Chitosan, a natural polymer, is a promising system for the therapeutic delivery of both plasmid DNA and synthetic small interfering RNA. Reports attempting to identify the optimal parameters of chitosan for synthetic small interfering RNA delivery were inconclusive with high molecular weight at high amine-to-phosphate (N:P) ratios apparently required for efficient transfection. Here we show, for the first time, that low molecular weight chitosan (LMW-CS) formulations at low N:P ratios are suitable for the in vitro delivery of small interfering RNA. LMW-CS nanoparticles at low N:P ratios were positively charged (ζ-potential ~20 mV) with an average size below 100 nm as demonstrated by dynamic light scattering and environmental scanning electron microscopy, respectively. Nanoparticles were spherical, a shape promoting decreased cytotoxicity and enhanced cellular uptake. Nanoparticle stability was effective for at least 20 hours at N:P ratios above two in a slightly acidic pH of 6.5. At a higher basic pH of 8, these nanoparticles were unravelled due to chitosan neutralization, exposing their polynucleotide cargo. Cellular uptake ranged from 50% to 95% in six different cell lines as measured by cytometry. Increasing chitosan molecular weight improved nanoparticle stability as well as the ability of nanoparticles to protect the oligonucleotide cargo from nucleases at supraphysiological concentrations. The highest knockdown efficiency was obtained with the specific formulation 92-10-5 that combines sufficient nuclease protection with effective intracellular release. This system attained >70% knockdown of the messenger RNA, similar to commercially available lipoplexes, without apparent cytotoxicity. Contrary to previous reports, our data demonstrate that LMW-CS at low N:P ratios are efficient and nontoxic polynucleotide delivery systems capable of transfecting a plethora of cell lines.

Chitosan-based therapeutic nanoparticles for combination gene therapy and gene silencing of in vitro cell lines relevant to type 2 diabetes.

Glucagon like peptide 1 (GLP-1), a blood glucose homeostasis modulating incretin, has been proposed for the treatment of type 2 diabetes mellitus (T2DM). However, native GLP-1 pharmacokinetics reveals low bioavailability due to degradation by the ubiquitous dipeptydil peptidase IV (DPP-IV) endoprotease. In this study, the glucosamine-based polymer chitosan was used as a cationic polymer-based in vitro delivery system for GLP-1, DPP-IV resistant GLP-1 analogues and siRNA targeting DPP-IV mRNA. We found chitosans to form spherical nanocomplexes with these nucleic acids, generating two distinct non-overlapping size ranges of 141-283 nm and 68-129 nm for plasmid and siRNA, respectively. The low molecular weight high DDA chitosan 92-10-5 (degree of deacetylation, molecular weight and N:P ratio (DDA-Mn-N:P)) showed the highest plasmid DNA transfection efficiency in HepG2 and Caco-2 cell lines when compared to 80-10-10 and 80-80-5 chitosans. Recombinant native GLP-1 protein levels in media of transfected cells reached 23 ng/L while our DPP-IV resistant analogues resulted in a fivefold increase of GLP-1 protein levels (115 ng/L) relative to native GLP-1, and equivalent to the Lipofectamine positive control. We also found that all chitosan-DPP-IV siRNA nanocomplexes were capable of DPP-IV silencing, with 92-10-5 being significantly more effective in abrogating enzymatic activity of DPP-IV in media of silenced cells, and with no apparent cytotoxicity. These results indicate that specific chitosan formulations may be effectively used for the delivery of plasmid DNA and siRNA in a combination therapy of type 2 diabetes.

Excess polycation mediates efficient chitosan-based gene transfer by promoting lysosomal release of the polyplexes.

The optimal ratio of the polycation's amine to DNA phosphate group (N:P) for efficient polymer-based transfection always employs excess polycation versus DNA. Most of the excess polycation remains free in solution, unassociated with the polyplexes, but is essential for efficient transfection. The mechanism by which excess polycation increases transfection efficiency is not identified. We hypothesised that excess chitosan facilitates intracellular lysosomal escape of the polyplexes. We highlight here the essential role of excess chitosan by rescuing poorly transfecting low N:P ratio polyplexes, by adding free chitosan before or after polyplex addition to cells. We examined polyplex uptake, the kinetics of rescue, intracellular trafficking, and the effects of lysosomotropic agents. We found the facilitating role of excess chitosan to be downstream of cellular uptake. Live-cell confocal quantification of intracellular trafficking revealed prolonged colocalisation of low N:P polyplexes within lysosomes, compared to shorter residence times for both rescued or N:P 5 samples, followed by observation of free pDNA in the cytosol. These data demonstrate that excess polycation mediates enhanced transfection efficiency by promoting the release of polyplexes from the endo-lysosomal vesicles, revealing a critical intracellular barrier overcome by excess polycation and suggesting possible avenues for further optimisation of polymer-based gene delivery.

Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing.

Chitosan, a well known natural cationic polysaccharide, has been successfully implemented in vitro and in vivo as a nonviral delivery system for both plasmid DNA and siRNA. While using chitosan/siRNA polyplexes to knock down specific targets, we have underestimated the effect of nucleic acids binding to chitosan when extracting RNA for subsequent quantitative PCR evaluation of silencing. In vitro transfection using chitosan/siRNA-based polyplexes reveals a very poor recovery of total RNA especially when using low cell numbers in 96 well plates. Here, we describe a method that dramatically enhances RNA extraction from chitosan/siRNA-treated cells by using an enzymatic treatment with a type III chitosanase. We show that chitosanase treatment prior to RNA extraction greatly enhances the yield and the integrity of extracted RNA. This method will therefore eliminate the bias associated with lower RNA yield and integrity when quantifying gene silencing of chitosan-based systems using quantitative real time PCR.

New ELISA approach based on coiled-coil interactions.

The de novo designed heterodimeric E/K coiled-coil system has been previously demonstrated to be an excellent capture/dimerization system applicable to various needs in both biotechnology and pharmaceutical fields. Those include controlled protein dimerization, capture, purification and Western-blot detection. We here report the development of a new generation of ELISA test based on coiled-coil interactions for the direct quantitation of coil-tagged epidermal growth factor (EGF). The new approach was evaluated for its specificity, plate storability and reusability as well as for convenience when compared to commercially available systems. Our results show a similar affinity/sensitivity to standard capturing antibody-based ELISA systems and an improved affinity/sensitivity when compared to the commercially available Ni-NTA capture system. The E/K coiled-coil ELISA system was validated with respect to recovery, intra- and inter-assay variations. The practical working range was estimated to be between 5.2 and 34,000 pM. Furthermore, the storability and reusability of the plates was greater than the two aforementioned systems, suggesting that the E/K coiled-coil system is a good alternative to traditional tags such as poly-histidine for the development of ELISA tests aiming at quantitating coil-tagged proteins.

Identification and characterization of a putative baculoviral transcriptional factor IE-1 from Choristoneura fumiferana granulovirus.

A gene that encodes a protein homologue to baculoviral IE-1 was identified and sequenced in the genome of the Choristoneura fumiferana granulovirus (ChfuGV). The gene has an 1278 nucleotide (nt) open-reading frame (ORF) that encodes 426 amino acids with an estimated molecular weight of 50.33 kDa. At the nucleotide level, several cis-acting regulatory elements were detected within the promoter region of the ie-1 gene of ChfuGV along with other studied granuloviruses (GVs). Two putative CCAAT elements were detected within the noncoding leader region of this gene; one was located on the opposite strand at -92 and the other at -420 nt from the putative start triplet. Two baculoviral late promoter motifs (TAAG) were also detected within the promoter region of the ie-1 gene of ChfuGV. A single polyadenylation signal, AATAAA, was located 18nt downstream of the putative translational stop codon of ie-1 from ChfuGV. At the protein level, the amino acid sequence data that was derived from the nucleotide sequence in ChfuGV IE-1 was compared to those of the Cydia pomonella granulovirus (CpGV), Xestia c-nigrum granulovirus (XcGV) and Plutella xylostella granulovirus (PxGV). The C-terminal regions of the granuloviral IE-1 sequences appeared to be more conserved when compared to the N-terminal regions. A domain, similar to the basic helix-loop-helix like (bHLH-like) domain in NPVs, was detected at the C-terminal region of IE-1 from ChfuGV (residues 387 to 414). A phylogenetic tree for baculoviral IE-1 was constructed using a maximum parsimony analysis. A phylogenetic estimation demonstrates that ChfuGV IE-1 is most closely related to that of CpGV.

Molecular characterization of three new avian infectious bronchitis virus (IBV) strains isolated in Quebec.

Three unrecognized field isolates of Infectious Bronchitis Virus (IBV) were recovered from commercial broiler chickens vaccinated with live Mass viral strain (H120). These isolates were identified by immunofluorescence using monoclonal antibodies produced against reference serotypes: Mass, Conn, and Ark. RT-PCRs were performed on viral RNAs to amplify S1 gene using a specific set of primers S1OLIGO3' and S1OLIGO5'. Restriction polymorphism (RFLP) of PCR products was determined by the use of HaeIII restriction enzyme. As expected, patterns of PCR products were different from common pattern of strains assigned to Mass serotype M41, Beaudette, H120, and Florida. Molecular analysis showed a nucleotide insertion in hypervariable region one (HVR-1) of S1 gene of only Quebec isolates (Qu16, Qu_mv and Q_37zm). However, New Brunswick IBV isolate (NB_cp) did not display these insertions. Major amino acid changes involved insertion of two stretches (aa118-119: Arg-Ser and aa141-145: Sys-Ser-Asn-Ala-Ser-Cys) located at N-terrminal and C-terminal regions of HVR-2. It is speculated that cysteine residue located upstream and downstream of Cys-Ser-Asn-Ala-Ser-Cys segment might be involved in the formation of loop structure and disulfide bond that could trigger important epitope changes. Insertion of new NXT and NXS (X not equal to P) glycosylation motifs scattered along S1 region and insertion of cysteine residues in HVR are contributing to the antigenic shifting of Quebec isolates. Fragment insertions were thought to be induced by inter-serotype recombination between vaccine strain (H120) that belongs to Mass serotype and another strain belonging to Ark serotype. Phylogenetic tree based on amino acid sequences showed that Quebec isolates formed a new phylogenetic cluster.