Gerrit Borchard

Chitosans for gene delivery

Efficient non-viral gene delivery based on cationic polymers as DNA-condensing agents is dependent on a variety of factors, e.g. complex size, complex stability, toxicity, immunogenicity, protection against DNase degradation, and intracellular trafficking and processing of the DNA. This review examines the advances in the application of chitosan and chitosan derivatives to non-viral gene delivery, and gives an overview of transfection studies which have been performed recently using chitosans as transfection agents.

Chitosan and its derivatives in mucosal drug and vaccine delivery.

Numerous studies have demonstrated that chitosan and their derivatives (N-trimethyl chitosan, mono-N-carboxymethyl chitosan) are effective and safe absorption enhancers to improve mucosal (nasal, peroral) delivery of hydrophylic macromolecules such as peptide and protein drugs and heparins. This absorption enhancing effect of chitosans is caused by opening of the intercellular tight junctions, thereby favouring the paracellular transport of macromolecular drugs. Chitosan nano- and microparticles are also suitable for controlled drug release. Association of vaccines to some of these particulate systems has shown to enhance the antigen uptake by mucosal lymphoid tissues, thereby inducing strong systemtic and mucosal immune responses against the antigens. The aspecific adjuvant activity of chitosans seems to be dependent on the degree of deacetylation and the type of formulation. From the studies reviewed it is concluded that chitosan and chitosan derivatives are promising polymeric excipients for mucosal drug and vaccine delivery.

The potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption. III: Effects of chitosan-glutamate and carbomer on epithelial tight junctions in vitro

Two mucoadhesive polymers, chitosan-glutamate and carbomer, were studied in an in vitro model (Caco-2 cell monolayers) with respect to their ability to enhance intestinal peptide drug delivery. Preparations of the polymers at concentrations of 0.5, 1.0, and 1.5% w/v (chitosan), and of 0.5 and 1.0% w/v (carbomer) were applied to the apical side of Caco-2 cell monolayers. The effects on transepithelial electrical resistance (TEER), paracellular transport of a FITC-dextran of a molecular weight of 4400 (FD-4) and [14C]mannitol were measured. Paracellular transport of FD-4 was visualized by means of confocal laser scanning microscopy (CLSM). Furthermore, the impact of lowering the pH of the polymer solutions to pH 4 on the integrity of the cell layer was determined. The results show that both polymers were able to decrease TEER of Caco-2 cell layers significantly. In the case of carbomer, CLSM revealed a partial opening of epithelial tight junctions. Lowering of the pH in the control and polymer solutions to pH 4 resulted in every case in the irreversible damage of a large percentage of the cells, as shown by CLSM. Transport studies with [14C]mannitol and FD-4 showed only during co-application of carbomer significantly increased fluxes, whereas no difference from the control solution could be detected for chitosan-glutamate. A threshold value of about 50% of TEER reduction has been identified, which allows for transport of hydrophilic compounds across the cell monolayers of the Caco-2 cell model.

Chitosan for mucosal vaccination

The striking advantage of mucosal vaccination is the production of local antibodies at the sites where pathogens enter the body. Because vaccines alone are not sufficiently taken up after mucosal administration, they need to be co-administered with penetration enhancers, adjuvants or encapsulated in particles. Chitosan easily forms microparticles and nanoparticles which encapsulate large amounts of antigens such as ovalbumin, diphtheria toxoid or tetanus toxoid. It has been shown that ovalbumin loaded chitosan microparticles are taken up by the Peyers patches of the gut associated lymphoid tissue (GALT). This unique uptake demonstrates that chitosan particulate drug carrier systems are promising candidates for oral vaccination. Additionally, after co-administering chitosan with antigens in nasal vaccination studies, a strong enhancement of both mucosal and systemic immune responses is observed. This makes chitosan very suitable for nasal vaccine delivery. In conclusion, chitosan particles, powders and solutions are promising candidates for mucosal vaccine delivery. Mucosal vaccination not only reduces costs and increases patient compliance, but also complicates the invasion of pathogens through mucosal sites.

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 microparticles for oral vaccination : preparation, characterization and preliminary in vivo uptake studies in murine Peyer's patches

Although oral vaccination has numerous advantages over parenteral injection, degradation of the vaccine in the gut and low uptake in the lymphoid tissue of the gastrointestinal tract still complicate the development of oral vaccines. In this study chitosan microparticles were prepared and characterized with respect to size, zeta potential, morphology and ovalbumin-loading and -release. Furthermore, the in vivo uptake of chitosan microparticles by murine Peyers patches was studied using confocal laser scanning microscopy (CLSM). Chitosan microparticles were made according to a precipitation/coacervation method, which was found to be reproducible for different batches of chitosan. The chitosan microparticles were 4.3+/-0.7 microm in size and positively charged (20+/-1 mV). Since only microparticles smaller than 10 microm can be taken up by M-cells of Peyers patches, these microparticles are suitable to serve as vaccination systems. CLSM visualization studies showed that the model antigen ovalbumin was entrapped within the chitosan microparticles and not only associated to their outer surface. These results were verified using field emission scanning electron microscopy, which demonstrated the porous structure of the chitosan microparticles, thus facilitating the entrapment of ovalbumin in the microparticles. Loading studies of the chitosan microparticles with the model compound ovalbumin resulted in loading capacities of about 40%. Subsequent release studies showed only a very low release of ovalbumin within 4 h and most of the ovalbumin (about 90%) remained entrapped in the microparticles. Because the prepared chitosan microparticles are biodegradable, this entrapped ovalbumin will be released after intracellular digestion in the Peyers patches. Initial in vivo studies demonstrated that fluorescently labeled chitosan microparticles can be taken up by the epithelium of the murine Peyers patches. Since uptake by Peyers patches is an essential step in oral vaccination, these results show that the presently developed porous chitosan microparticles are a very promising vaccine delivery system.

Transfection efficiency and toxicity of polyethylenimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures.

In the present study, we evaluated polyethylenimine (PEI) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting nondifferentiated COS-1 (green monkey fibroblasts) and well-differentiated human submucosal airway epithelial cells (Calu-3). Studying the effect of particle size, zeta potential, presence of serum proteins or chloroquine, it appeared that transfection efficiency depends on the experimental conditions and not on the MW of the PEI used. Comparing transfection efficiencies in both cell lines, we found that PEI was 3 orders of magnitude more effective in COS-1 than in Calu-3 cells, because Calu-3 cells are differentiated and secrete mucins, which impose an additional barrier to gene delivery. Transfection efficiency was strongly correlated to PEI cytotoxicity. Also, some evidence for PEI-induced apoptosis in both cell lines was found. In conclusion, our results indicate that PEI is a useful vector for nonviral transfection in undifferentiated cell lines. However, results from studies in differentiated bronchial epithelial cells suggest that PEI has yet to be optimized for successful gene therapy of cystic fibrosis (CF).

PLGA–PEI nanoparticles for gene delivery to pulmonary epithelium

Abstract Pulmonary gene delivery is thought to play an important role in treating genetically related diseases and may induce immunity towards pathogens entering the body via the airways. In this study we prepared poly (d,l-lactide-co-glycolide) (PLGA) nanoparticles bearing polyethyleneimine (PEI) on their surface and characterized them for their potential in serving as non-viral gene carriers to the pulmonary epithelium. Particles that were synthesized at different PLGA–PEI ratios and loaded with DNA in several PEI–DNA ratios, exhibited narrow size distribution in all formulations, with mean particle sizes ranging between 207 and 231 nm. Zeta potential was strongly positive (above 30 mV) for all the PEI–DNA ratios examined and the loading efficiency exceeded 99% for all formulations. Internalization of the DNA-loaded PLGA–PEI nanoparticles was studied in the human airway submucosal epithelial cell line, Calu-3, and DNA was detected in the endo-lysosomal compartment 6 h after particles were applied. Cytotoxicity of these nanoparticles was dependent on the PEI–DNA ratio and best cell viability was achieved by PEI–DNA ratios 1:1 and 0.5:1. These findings demonstrate that PLGA–PEI nanoparticles are a potential new delivery system to carry genes to the lung epithelium.

Mucoadhesive Polymers in Peroral Peptide Drug Delivery. II. Carbomer and Polycarbophil Are Potent Inhibitors of the Intestinal Proteolytic Enzyme Trypsin

AbstractPurpose. The evaluation of the inhibitory action of two mucoadhesive poly(acrylates), polycarbophil and carbomer, registered by the Food and Drug Administration (FDA), on the intestinal proteolytic enzyme trypsin. Methods. The effect of the polymers on trypsin activity by measuring the degradation of a trypsin specific substrate. Binding of Ca2+ ions and proteins (125I-BSA) to the poly(acrylates). The influence of the polymers on the secondary trypsin structure by circular dichroism. Results. Trypsin inhibition was found to be time-dependent upon addition of Ca2+ in the degradation experiment. Only when Ca2+ was added within 10 min after trypsin incubation, recovery of the enzyme could be observed. Both polymers showed a strong Ca2+ binding ability. Carbomer, which had a higher inhibitory effect on trypsin activity, also revealed a higher Ca2+ binding affinity than polycarbophil. The amount of Ca2+ depleted out of the trypsin structure and the reduction of enzyme activity were comparable. Immobilization of trypsin by binding to the polymers could not be observed at pH 6.7. Circular dichroism studies suggested that, under depletion of Ca2+ from trypsin, the secondary structure changed its conformation, followed by an increased autodegradation of the enzyme. Conclusions. The poly(acrylates) investigated may have potential to protect peptides from tryptic degradation and may be used to master the peroral delivery of peptide drugs.

Drug transport and metabolism characteristics of the human airway epithelial cell line Calu-3

Pulmonary drug delivery serves two purposes, namely the application of locally active compounds for treatment of diseases afflicting the lung, and the utilization of the pulmonary epithelia as absorption sites for macromolecular drugs. To elucidate the mechanisms involved in the pulmonary absorption and metabolism of compounds on a cellular level, cell culture models have shown to be, though limited, rather useful in predicting in vivo conditions. The Calu-3 cell line has been employed recently as a model for the airway epithelium in a number of drug transport and metabolism studies. The results of these studies, as well as an evaluation of the predictive potency of the model, are presented here.