Stefan Romeijn

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.

Cyclodextrins in nasal drug delivery.

Nasal drug delivery is an attractive approach for the systemic delivery of high potency drugs with a low oral bioavailability due to extensive gastrointestinal breakdown and high hepatic first-pass effect. For lipophilic drugs nasal delivery is possible if they can be dissolved in the dosage form. Peptide and protein drugs often have a low nasal bioavailability because of their large size and hydrophilicity, resulting in poor transport properties across the nasal mucosa. Cyclodextrins are used to improve the nasal absorption of these drugs by increasing their aqueous solubility and/or by enhancing their nasal absorption. With several cyclodextrins very efficient nasal drug absorption has been reported, but also large interspecies differences have been found. Studies concerning the safety of cyclodextrins in nasal drug formulations demonstrate the non-toxicity of the cyclodextrins and also clinical data show no adverse effects. Therefore, some cyclodextrins can be expected to become effective and safe excipients in nasal drug delivery.

ABSORPTION ENHANCING EFFECT OF CYCLODEXTRINS ON INTRANASALLY ADMINISTERED INSULIN IN RATS

The absorption enhancing effect of α-, β-, and γ-cyclodextrin (CD), dimethyl-β-cyclodextrin (DMβCD), and hydroxypropyl-β-cyclodextrin (HPβCD) on intranasally administered insulin was investigated in rats. Coadministration of 5% (w/v) DMβCD to the insulin solution resulted in a high bioavailability, 108.9 ± 36.4% (mean ± SD, n = 6), compared to i.v. administration, and a strong decrease in blood glucose levels, to 25% of their initial values. Coadministration of 5% α-CD gave rise to an insulin bioavailability of 27.7 ± 11.5% (mean ± SD, n = 6) and a decrease in blood glucose to 50% of its initial value. The rate of insulin absorption and the concomitant hypoglycemic response were delayed for the α-CD-containing solution as compared to the DMβCD preparation. The other CDs, HPβCD (5%), β-CD (1.8%), and γ-CD (5%), did not have significant effects on nasal insulin absorption. DMβCD at a concentration of 5% (w/v) induces ciliostasis as measured on chicken embryo tracheal tissue in vitro, but this effect is reversible. In conclusion, DMβCD is a potent enhancer of nasal insulin absorption in rats.

Absorption enhancers in nasal drug delivery: efficacy and safety

Abstract Efficacy and safety of nasal absorption enhancers depend on many different parameters, such as their influence on nasal epithelial membrane barriers, the enzymatic activities in the nasal cavity, and the mucociliary clearance. Large interspecies differences appear to exist in the nasal absorption of drugs. Nasal absorption enhancers, although very effective in some cases, differ substantially in efficacy and safety. For a number of enhancers like surfactants, bile salts and fusidate derivatives a good relationship (similarity in rank order) can be established between the morphological damage and the influence on the ciliary beat frequency (CBF) of nasal epithelial tissue, making CBF measurements a valuable tool in the search for safe nasal absorption enhancers.

Effects of N-trimethyl chitosan chloride, a novel absorption enhancer, on caco-2 intestinal epithelia and the ciliary beat frequency of chicken embryo trachea.

N-trimethyl chitosan (TMC) polymers are quaternized chitosans in different degrees of trimethylation. These polymers enhance the absorption of macromolecules through mucosal epithelia by triggering the reversible opening of tight junctions and only allow for paracellular transport. To investigate the safety of these novel absorption enhancers cytotoxicity and ciliotoxicity studies have been performed. Intestinal Caco-2 cell monolayers were chosen to study possible membrane damaging effects of these polymers, using confocal laser scanning microscopy visualization of nuclear staining by a membrane impermeable fluorescent probe during transport of the paracellular marker Texas red dextran (MW 10 000). Ciliated chicken embryo trachea tissue was used to study the effect of the polymers on the ciliary beat frequency (CBF) in vitro. In both studies the TMC polymers of different degrees of substitution (20, 40 and 60%) were tested at a concentration of 1.0% (w/v). No substantial cell membrane damage could be detected on the Caco-2 cells treated with TMCs, while the effect on the CBF in vitro was found to be marginal. TMC60 and TMC40 enhance paracellular transport of Texas red dextran in Caco-2 cell monolayers, whereas TMC20 is ineffective. In conclusion, TMCs of high degrees of substitution may be effective and safe absorption enhancers for peptide and protein drug delivery.

Nasal Insulin Delivery with Dimethyl-β-Cyclodextrin as an Absorption Enhancer in Rabbits: Powder More Effective than Liquid Formulations

The nasal absorption of insulin using dimethyl-β-cyclodextrin (DMβCD) as an absorption enhancer in rabbits was studied. The nasal administration of insulin/DMβCD liquid formulations did not result in significant changes in serum insulin and blood glucose concentrations. In contrast, previous experiments in rats showed that the addition of DMβCD to the liquid nasal formulation resulted in an almost-complete insulin absorption, with a concomitant strong hy-poglycaemic response. Apparently, the effect of the cyclodextrin derivative on insulin absorption differs between animal species following nasal delivery of insulin/DMβCD solutions. On the other hand, nasal administration of the lyophilized insulin/DMβCD powder dosage form in rabbits resulted in increased serum insulin concentrations, and a maximum decrease in blood glucose of about 50%. The absolute bioavailability of the nasally administered insulin/DMβCD powder was 13 ± 4%, compared to 1 ± 1% for both an insulin/ DMβCD liquid and an insulin/lactose powder formulation. It is concluded that insulin powder formulations with DMβCD as an absorption enhancer are much more effective than liquid formulations.

Effects of absorption enhancers on rat nasal epithelium in vivo: release of marker compounds in the nasal cavity.

AbstractPurpose. The assessment of the effects of nasal absorption enhancers on the rat nasal epithelium and membrane permeability in vivo after a single nasal dose of the enhancers. Methods. The release of marker compounds (protein, cholesterol and acid phosphatase) from the nasal epithelium was measured using a lavage technique. The nasal membrane permeability was determined after intravenous administration of a systemic tracer (FITC-albumin). Results. The effects of the absorption enhancers could be classified into four categories. The first consisted of HPβCD (5%), DMβCD (2%) and RAMEB (2%) and was not different from the control (physiological saline). For the second category, DMβCD (5%), effects were significantly higher than for the control. The third category, SGC (1%), was more active than DMβCD (5%) but less active than the last group. The fourth, most membrane damaging, category consisted of STDHF (1%), laureth-9 (1%) and LPC (1%). Administration of these three enhancers also resulted in release of acid phosphatase, indicating that severe membrane damage occurred. The release of cholesterol from nasal epithelium was largely dependent on the cholesterol solubilisation of the absorption enhancers. The amount of cholesterol released by laureth-9 and LPC was the largest. Conclusions. The results of this in vivo study are in agreement (i.e. similarity in rank order) with morphological and ciliotoxicity studies of nasal absorption enhancers, demonstrating that this invivo model is a valuable tool to classify nasal absorption enhancers according to their effects on the rat nasal epithelium.

Serial cerebrospinal fluid sampling in a rat model to study drug uptake from the nasal cavity

Drug transport from the nasal cavity to the brain has gained much interest in the last decade. In the present study, a model was developed to determine the uptake of drugs into the cerebrospinal fluid (CSF) after nasal delivery in rats. CSF samples were taken using a cisternal puncture method. In this method, a needle is advanced through the skin and muscles overlying the atlanto-occipital membrane into the cisterna magna, while the rat is fixed in a stereotaxic frame. This method appears to be superior over cannulation of the atlanto-occipital membrane for CSF sampling. The major advantages of the puncture method is the ability of serial and simultaneous CSF and blood sampling for over 2 h in the same rat. To obtain maximal drug absorption from the nasal cavity and uptake into CSF, different positions of the rats head (upright-90 degrees, supine-90 degrees, supine-45 degrees and supine-70 degrees angles) were tested in nasal delivery studies using hydrocortisone (HC) as a model drug. Putting the rat in the supine-90 degrees angle position increased the absorption of HC into plasma and CSF 2-fold compared to the upright-90 degrees angle position. The supine-70 degrees angle position did not change the HC plasma and CSF levels compared to the supine-90 degrees angle position. However, the supine-70 degrees angle position showed the fastest CSF sampling rate, enabling more accurate CSF sampling and therefore preferred for further studies. In conclusion, the cisternal puncture method using the supine-70 degrees and 90 degrees angle position is a suitable method to study drug transport from the nasal cavity into the CSF, with the ability of multiple CSF sampling.

Immune Modulation by Adjuvants Combined with Diphtheria Toxoid Administered Topically in BALB/c Mice After Microneedle Array Pretreatment

PurposeIn this study, modulation of the immune response against diphtheria toxoid (DT) by various adjuvants in transcutaneous immunization (TCI) with microneedle array pretreatment was investigated.MethodsTCI was performed on BALB/c mice with or without microneedle array pretreatment using DT as a model antigen co-administrated with lipopolysaccharide (LPS), Quil A, CpG oligo deoxynucleotide (CpG) or cholera toxin (CT) as adjuvant. The immunogenicity was evaluated by measuring serum IgG subtype titers and neutralizing antibody titers.ResultsTCI with microneedle array pretreatment resulted in a 1,000-fold increase of DT-specific serum IgG levels as compared to TCI. The immune response was further improved by co-administration of adjuvants, showing a progressive increase in serum IgG titers when adjuvanted with LPS, Quil A, CpG and CT. IgG titers of the CT-adjuvanted group reached levels comparable to those obtained after DT-alum subcutaneous injection. The IgG1/IgG2a ratio of DT-specific antibodies decreased in the following sequence: plain DT, Quil A, CT and CpG, suggesting that the immune response was skewed towards the Th1 direction.ConclusionsThe potency and the quality of the immune response against DT administered by microneedle array mediated TCI can be modulated by co-administration of adjuvants.

Confocal laser scanning microscopic visualization of the transport of dextrans after nasal administration to rats: effects of absorption enhancers.

AbstractPurpose. To visualize the transport pathway(s) of high molecular weight model compounds across rat nasal epithelium in vivousing confocal laser scanning microscopy. Furthermore, the influence of nasal absorption enhancers (randomly methylated β-cyclodextrin and sodium taurodihydrofusidate) on this transport was studied. Methods. Fluorescein isothiocyanate (FITC)-labelled dextrans with a molecular weight of 3,000 or 10,000 Da were administered intranasally to rats. Fifteen minutes after administration the tissue was fixed with Bouin. The nasal septum was surgically removed and stained with Evans Blue protein stain or DiIC18(5) lipid stain prior to visualization with the confocal laser scanning microscope. Results. Transport of FITC-dextran 3,000 across nasal epithelium occurred via the paracellular pathway. Endocytosis of FITC-dextran 3,000 was also shown. In the presence of randomly methylated β-cyclodextrin 2% (w/v) similar transport pathways for FITC-dextran 3,000 were observed. With sodium taurodihydrofusidate 1% (w/v) the transport route was also paracellular with endocytosis, but cells were swollen and mucus was extruded into the nasal cavity. For FITC-dextran 10,000 hardly any transport was observed without enhancer, or after co-administration with randomly methylated β-cyclodextrin 2% (w/v). Co-administration with sodium taurodihydrofusidate 1% (w/v) resulted in paracellular transport of FITC-dextran 10,000, but morphological changes, i.e. swelling of cells and mucus extrusion, were observed. Conclusions. Confocal laser scanning microscopy is a suitable approach to visualize the transport pathways of high molecular weight hydrophilic compounds across nasal epithelium, and to study the effects of absorption enhancers on drug transport and cell morphology.