Maya Thanou

Targeting nanoparticles to cancer.

Nanotechnology applications in medicine, termed as nanomedicine, have introduced a number of nanoparticles of variable chemistry and architecture for cancer imaging and treatment. Nanotechnology involves engineering multifunctional devices with dimensions at the nanoscale, similar dimensions as those of large biological vesicles or molecules in our body. These devices typically have features just tens to hundred nanometers across and they can carry one or two detection signals and/or therapeutic cargo(s). One unique class of nanoparticles is designed to do both, providing this way the theragnostic nanoparticles (therapy and diagnosis). Being inspired by physiologically existing nanomachines, nanoparticles are designed to safely reach their target and specifically release their cargo at the site of the disease, this way increasing the drugs tissue bioavailability. Nanoparticles have the advantage of targeting cancer by simply being accumulated and entrapped in tumours (passive targeting). The phenomenon is called the enhanced permeation and retention effect, caused by leaky angiogenetic vessels and poor lymphatic drainage and has been used to explain why macromolecules and nanoparticles are found at higher ratios in tumours compared to normal tissues. Although accumulation in tumours is observed cell uptake and intracellular drug release have been questioned. Polyethyleneglycol (PEG) is used to protect the nanoparticles from the Reticulo-Endothelial System (RES), however, it prevents cell uptake and the required intracellular drug release. Grafting biorecognition molecules (ligands) onto the nanoparticles refers to active targeting and aims to increase specific cell uptake. Nanoparticles bearing these ligands are recognised by cell surface receptors and this leads to receptor-mediated endocytosis. Several materials are suggested for the design of nanoparticles for cancer. Polymers, linear and dendrimers, are associated with the drug in a covalent or non-covalent way and have been used with or without a targeting ligand. Stealth liposomes are suggested to carry the drug in the aqueous core, and they are usually decorated by recognition molecules, being widely studied and applied. Inorganic nanoparticles such as gold and iron oxide are usually coupled to the drug, PEG and the targeting ligand. It appears that the PEG coating and ligand decoration are common constituents in most types of nanoparticles for cancer. There are several examples of successful cancer diagnostic and therapeutic nanoparticles and many of them have rapidly moved to clinical trials. Nevertheless there is still a room for optimisation in the area of the nanoparticle kinetics such as improving their plasma circulation and tumour bioavailability and understanding the effect of targeting ligands on their efficiency to treat cancer. The need to develop novel and efficient ligands has never been greater, and the use of proper conjugation chemistry is mandatory.

Oral drug absorption enhancement by chitosan and its derivatives.

Chitosan is a non-toxic, biocompatible polymer that has found a number of applications in drug delivery including that of absorption enhancer of hydrophilic macromolecular drugs. Chitosan, when protonated (pH<6.5), is able to increase the paracellular permeability of peptide drugs across mucosal epithelia. Chitosan derivatives have been evaluated to overcome chitosans limited solubility and effectiveness as absorption enhancer at neutral pH values such as those found in the intestinal tract. Trimethyl chitosan chloride (TMC) has been synthesized at different degrees of quaternization. This quaternized polymer forms complexes with anionic macromolecules and gels or solutions with cationic or neutral compounds in aqueous environments and neutral pH values. TMC has been shown to considerably increase the permeation and/or absorption of neutral and cationic peptide analogs across intestinal epithelia. The mechanism by which TMC enhances intestinal permeability is similar to that of protonated chitosan. It reversibly interacts with components of the tight junctions, leading to widening of the paracellular routes. Mono-carboxymethylated chitosan (MCC) is a polyampholytic polymer, able to form visco-elastic gels in aqueous environments or with anionic macromolecules at neutral pH values. MCC appears to be less potent compared to the quaternized derivative. Nevertheless, MCC was found to increase the permeation and absorption of low molecular weight heparin (LMWH; an anionic polysaccharide) across intestinal epithelia. Neither chitosan derivative provokes damage of the cell membrane, and therefore they do not alter the viability of intestinal epithelial cells.

Preparation and NMR characterization of highly substitutedN-trimethyl chitosan chloride

N,N,N-Trimethyl chitosan chloride (TMC) is a chemically modified chitosan with improved aqueous solubility, compared with the native chitosan. It is essential to follow a synthesis procedure in which the degree of substitution of the final product can be controlled by means of the number of reaction steps, the duration of each reaction step and the amount of methyl iodide as reagent. A two-step reaction yields products with high degrees of substitution (40–80%). Comparison of the NMR spectra of the product TMC, after a two-step reaction, indicates that there is a peak assigned to the substituted amino group that shifts from 2.5 to 3.1 ppm upon acidification. This peak must be assigned to N(CH3)2 and not to N(CH3)+3. A three-step reaction procedure yields products with a degree of substitution > 80%, but with substantially decreased water-solubility.

Quaternized chitosan oligomers as novel gene delivery vectors in epithelial cell lines

Quaternized modifications of chitosan present characteristics that might be useful in DNA condensing and efficient gene delivery. Trimethylated chitosan (TMO) was synthesized from oligomeric chitosan (<20 monomer units). TMOs spontaneously formed complexes (chitoplexes) with RSV-alpha3 luciferase plasmid DNA. These complexes were characterized by photon correlation spectroscopy and were investigated for their ability to transfect COS-1 and Caco-2 cell lines in the presence and absence of fetal calf serum and compared with DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium sulphate) lipoplexes. Additionally, their effect on the viability of the respective cell cultures was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results showed that quaternized chitosan oligomers were able to condense DNA and form complexes with a size ranging from 200 to 500 nm. Chitoplexes proved to transfect COS-1 cells, however, to a lesser extent than DOTAP-DNA lipoplexes. The quaternized oligomer derivatives appeared to be superior to oligomeric chitosan. The presence of fetal calf serum (FCS) did not affect the transfection efficiency of the chitoplexes, whereas the transfection efficiency of DOTAP DNA complexes was decreased. Cells remained 100% viable in the presence of chitosan oligomers whereas viability of DOTAP treated cells decreased to approximately 50% in both cell lines. Both DOTAP-DNA lipoplexes and chitoplexes resulted in less transfection efficiency in Caco-2 cell cultures than in COS-1 cells; however quaternized chitosan oligomers proved to be superior to DOTAP. Effects on the viability of Caco-2 cells were similar to the effects observed in COS-1 cells. We conclude that trimethylated chitosan-DNA complexes present suitable characteristics and the potential to be used as gene delivery vectors.

Chitosan and its derivatives as intestinal absorption enhancers

Chitosan is a non-toxic, biocompatible polymer that has found a number of applications in drug delivery including that of absorption enhancer of hydrophilic macromolecular drugs. Chitosan, when protonated (pH<6.5), is able to increase the paracellular permeability of peptide drugs across mucosal epithelia. Chitosan derivatives have been evaluated to overcome chitosans limited solubility and effectiveness as absorption enhancer at neutral pH values such as those found in the intestinal tract. Trimethyl chitosan chloride (TMC) has been synthesized at different degrees of quaternization. This quaternized polymer forms complexes with anionic macromolecules and gels or solutions with cationic or neutral compounds in aqueous environments and neutral pH values. TMC has been shown to considerably increase the permeation of neutral and cationic peptide analogs across Caco-2 intestinal epithelia. The mechanism by which TMC is enhancing the intestinal permeability is similar to that of protonated chitosan. It reversibly interacts with components of the tight junctions, leading to widening of the paracellular routes. This chitosan derivative does not provoke damage of the cell membrane, and does not alter the viability of intestinal epithelial cells. Co-administrations of TMC with peptide drugs were found to substantially increase the bioavailability of the peptide in both rats and juvenile pigs compared with administrations without the polymer.

Effect of degree of quaternization of N-trimethyl chitosan chloride for enhanced transport of hydrophilic compounds across intestinal caco-2 cell monolayers.

N-Trimethyl chitosan chloride (TMC) is a permanently quaternized chitosan derivative with improved aqueous solubility compared to native chitosan. TMC is able to open the tight junctions of intestinal epithelia at physiological pH values, where chitosan is insoluble and therefore ineffective. TMCs with degrees of substitution of 40 and 60% were synthesized according to a novel synthesis procedure and their effect on the permeability of the tight junctions of the intestinal Caco-2 monolayers was studied, measuring the transepithelial electrical resistance and the transport of a mainly paracellularly transported compound, [14C]-mannitol. Toxicity studies using nucleic stains were done to establish the transport as a cause of opening of the tight junctions and not of possible cytotoxicity. TMC60 showed higher transport enhancement ratios than TMC40 in all concentrations tested (0.05-1. 0%, w/v). Both derivatives did not affect the viability of the Caco-2 cell monolayers. These results suggest that high charge density is necessary for TMC to substantially improve the paracellular permeability of intestinal epithelia. It is expected that TMC40 and TMC60 will enhance the intestinal permeation of hydrophilic macromolecular drugs such as peptides and proteins.

Enhanced fluid flow through nanoscale carbon pipes.

Recent experimental and theoretical studies demonstrate that pressure driven flow of fluids through nanoscale ( d < 10 nm) carbon pores occurs 4 to 5 orders of magnitude faster than predicted by extrapolation from conventional theory. Here, we report experimental results for flow of water, ethanol, and decane through carbon nanopipes with larger inner diameters (43 +/- 3 nm) than previously investigated. We find enhanced transport up to 45 times theoretical predictions. In contrast to previous work, in our systems, decane flows faster than water. These nanopipes were composed of amorphous carbon deposited from ethylene vapor in alumina templates using a single step fabrication process.

Mono‐N‐carboxymethyl chitosan (MCC), a polyampholytic chitosan derivative, enhances the intestinal absorption of low molecular weight heparin across intestinal epithelia in vitro and in vivo

The synthesis and evaluation of mono-N-carboxymethyl chitosan (MCC) as an intestinal permeation enhancer for macromolecular therapeutics is presented. MCCs were synthesized from two different viscosity grade chitosans to yield both high and low viscosity grade products. These MCCs were tested on Caco-2 cells for their efficiency to decrease the transepithelial electrical resistance (TEER) and to increase the paracellular permeability of the anionic macromolecular anticoagulant low molecular weight heparin (LMWH). For in vivo studies, LMWH was administered intraduodenally with or without MCC to rats. Both types of experiments were performed at pH 7.4. Results show that both viscosity grade MCCs managed to significantly decrease the TEER of Caco-2 cell monolayers when they were applied apically at concentrations of 3-5% (w/v). Transport studies with Caco-2 cells revealed substantial increases of LMWH permeation in the presence of both viscosity grade MCCs compared with controls. In rats, 3% (w/v) low viscosity MCC significantly increased the intestinal absorption of LMWH, reaching the therapeutic anticoagulant blood levels of LMWH. Both in vitro and in vivo results indicate that the polyampholytic chitosan modification MCC is a suitable and functional polymer for the delivery and intestinal absorption of anionic macromolecular therapeutics like LMWH.

N-Trimethylated Chitosan Chloride (TMC) Improves the Intestinal Permeation of the Peptide Drug Buserelin In Vitro (Caco-2 Cells) and In Vivo (Rats)

AbstractPurpose. To evaluate N-trimethyl chitosan chloride (TMC) of highdegrees of substitution as intestinal permeation enhancers for thepeptide drug buserelin in vitro using Caco-2 cell monolayers, and toinvestigate TMCs as enhancers of the intestinal absorption of buserelinin vivo, in rats. Methods. TMCs were tested on Caco-2 cells for their efficiency toincrease the paracellular permeability of the peptide buserelin. For thein vivo studies male Wistar rats were used and buserelin wasadministered with or without the polymers intraduodenally. Both types ofexperiments were performed at pH 7.2. Results. Transport studies with Caco-2 cell monolayers confirmed thatthe increase in buserelin permeation is dependent on the degree oftrimethylation of TMC. In agreement with the in vitro results, in vivodata revealed highly increased bioavailability of buserelin followingintraduodenal co-administration with 1.0% (w/v) TMCs.Intraduodenally applied buserelin resulted in 0.8% absolute bioavailability,whereas co-administrations with TMCs resulted in mean bioavailabilityvalues between 6 and 13 %. Chitosan HCl (1.0% pH = 7.2) did notsignificantly increase the intestinal absorption of buserelin. Conclusions. Both the in vitro and in vivo results indicate that TMCsare potent mucosal permeation enhancers of the peptide drug buserelinat neutral pH values.

Mono-N-carboxymethyl chitosan (MCC) and N-trimethyl chitosan (TMC) nanoparticles for non-invasive vaccine delivery.

Mucosal application of a vaccine can effectively induce both systemic and mucosal immune responses. In general, mucosal applications of antigens result in poor immune responses. Therefore, adjuvant/delivery systems are required to enhance the immune response. Chitosan is a cationic biopolymer which exerts advantages as a vaccine carrier due to its immune stimulating activity and bioadhesive properties that enhance cellular uptake and permeation as well as antigen protection. Similar effects are also shown by chitosan derivatives. In this study, the nanoparticulate systems were prepared by using differently charged chitosan derivatives, N-trimethyl chitosan (TMC, polycationic), and mono-N-carboxymethyl chitosan (MCC, polyampholytic) for mucosal immunisation. The derivatives were synthesised and characterised in-house. The aqueous dispersions of the derivatives were also prepared for comparison. The cytotoxicity studies (MTT assay) on Chinese hamster ovary (CHO-K1) cell lines showed that cell viability was in the order of MCC, chitosan and TMC. Nanoparticles were prepared using ionic gelation method and loaded with tetanus toxoid (TT). Nanoparticles with high loading efficacy (>90% m/m), particle size within the range of 40-400nm, with a negative surface charge for MCC and positive surface charge for TMC and chitosan were obtained. The structural integrity of the TT in the formulations was confirmed by SDS-PAGE electrophoresis analysis. The effective uptake of the FITC-BSA loaded nanoparticles into the cells was demonstrated by cellular uptake studies using J774A.1 cells. Immune responses induced by the formulations loaded with tetanus toxoid were studied in vivo in Balb/c mice. Enhanced immune responses were obtained with intranasal (i.n.) application of nanoparticle formulations. Chitosan and TMC nanoparticles which have positively charged surfaces induced higher serum IgG titres when compared to those prepared with MCC which are negatively charged and smaller in size. Nanoparticle formulations developed in this study can be used as promising adjuvant/delivery systems for mucosal immunisation.