Antony D'Emanuele

The influence of surface modification on the cytotoxicity of PAMAM dendrimers

The influence of surface modification on the cytotoxicity of PAMAM dendrimers was examined using Caco-2 cells. Dendrimers were modified by conjugating either lauroyl chains or polyethylene glycol (PEG) 2000 onto the surface of cationic PAMAM dendrimers (G2, G3, G4). The cytotoxicity of unmodified dendrimers towards Caco-2 cells was appreciably higher for cationic (whole generation) compared with anionic (half generation) dendrimers and for both types increased with increasing size (generation) and concentration. A marked decrease in the cytotoxicity of cationic PAMAM dendrimers was noted when the surface was modified, with the addition of six lauroyl or four PEG chains being particularly effective in decreasing cytotoxicity. This decrease in cytotoxicity is thought to be due to a reduction/shielding of the positive charge on the dendrimer surface by the attached chains. The cytotoxicity of dendrimer-based delivery systems is likely to be very different from the parent dendrimer.

Engineering of Dendrimer Surfaces to Enhance Transepithelial Transport and Reduce Cytotoxicity

AbstractPurpose. To evaluate the cytotoxicity, permeation, and transport mechanisms of PAMAM dendrimers and surface-modified cationic PAMAM dendrimers using monolayers of the human colon adenocarcinoma cell line, Caco-2. Methods. Cytotoxicity was determined using the MTT assay. The effect of dendrimers on monolayer integrity was determined from measurements of transepithelial electrical resistance (TEER) and [14C]mannitol apparent permeability coefficient (Papp). The Papp of dendrimers through monolayers was measured in both the apical (A)-to-basolateral (B) and B→A directions at 4°C and 37°C and also in the presence and absence of ethylenediamine tetraacetic acid (EDTA) and colchicine. Results. The cytotoxicity and permeation of dendrimers increased with both concentration and generation. The cytotoxicity of cationic dendrimers (G2, G3, G4) was greater than that of anionic dendrimers (G2.5, G3.5) but was reduced by conjugation with lauroyl chloride; the least cytotoxic conjugates were those with six attached lauroyl chains. At 37°C the Papp of cationic dendrimers was higher than that of anionic dendrimers and, in general, increased with the number of attached lipid chains. Cationic dendrimers decreased TEER and significantly increased the Papp of mannitol. Modified dendrimers also reduced TEER and caused a more marked increase in the Papp of mannitol. The Papp values of dendrimers and modified dendrimers were higher in the presence of EDTA, lower in the presence of colchicine, and lower at 4°C than at 37°C. Conclusions. The properties of dendrimers may be significantly modified by surface engineering. Conjugation of cationic PAMAM dendrimers with lauroyl chloride decreased their cytotoxicity and increased their permeation through Caco-2 cell monolayers. Both PAMAM dendrimers and lauroyl-PAMAM dendrimer conjugates can cross epithelial monolayers by paracellular and transcellular pathways.

Polyamidoamine Starburst dendrimers as solubility enhancers.

The solubility of the hydrophobic drug ibuprofen has been compared in an aqueous solution of polyamidoamine (PAMAM) G4 dendrimer and sodium dodecyl sulphate (SDS). The PAMAM G4 dendrimer solution significantly enhanced the solubility of ibuprofen compared to 2% SDS solution. It was found that the solubility of ibuprofen in dendrimer solution was directly proportional to dendrimer concentration and inversely proportional to temperature. The influence of dendrimer solution pH on the solubility enhancement of ibuprofen suggests that it involves an electrostatic interaction between the carboxyl group of the ibuprofen molecule and the amine groups of the dendrimer molecule.

The use of thermoresponsive hydrogels for on-off release of molecules

Two responsive delivery devices are described from which the release of molecules is dependent on the temperature of the milieu. The devices utilise thermoresponsive hydrogels which possess a lower critical solution temperature (LCST). A novel thermoresponsive device based on the use of a thermoresponsive valve is described from which significant release of molecules only occurs at temperatures above the LCST of the hydrogel. Thermoresponsive hydrogel discs were also prepared from which significant release of molecules only occurs at temperatures below the LCST of the hydrogel. It was found that the release of molecules from both devices, the thermoresponsive valve-based device and the discs, could be controlled by changing the environmental temperature.

Surface modification of PAMAM dendrimers modulates the mechanism of cellular internalization.

The aim of this study was to investigate the influence of dendrimer surface properties on cellular internalization and intracellular trafficking in the human colon adenocarcinoma HT-29 cell line. Third-generation (G3) polyamidoamine (PAMAM) dendrimers were modified to contain either two lauroyl chains (G3L2), two propranolol molecules (G3P2), or two lauroyl and two propranolol molecules (G3L2P2) at the dendrimer surface. Surface-modified and unmodified dendrimers were labeled with fluorescein isothiocyanate (FITC) at an average molar ratio of 1:1. The mechanisms of cellular internalization and intracellular trafficking of dendrimers were analyzed by confocal laser scanning microscopy and flow cytometry. The internalization of G3 and G3P2 dendrimers involved both caveolae-dependent endocytosis and macropinocytosis pathways; internalization of G3L2P2 dendrimer appeared to involve caveolae-dependent, and possibly clathrin-dependent, endocytosis pathways; and internalization of G3L2 dendrimer occurred via caveolae-dependent, clathrin-dependent, and macropinocytosis pathways. Subcellular colocalization data indicated that unmodified and all surface-modified G3 PAMAM dendrimers were internalized and trafficked to endosomes and lysosomes. It is therefore apparent that the initial mode of dendrimer internalization into HT-29 cells is influenced by the surface properties of G3 PAMAM dendrimer.

The Use of Sonication for the Efficient Delivery of Plasmid DNA into Cells

AbstractPurpose. Ultrasonic methods have considerable potential for the introduction of macromolecules into cells. In this paper we demonstrate that, under controlled conditions, application of 20kHz ultrasound to a suspension of yeast cells facilitates the delivery of plasmid DNA into these cells. Methods. Aliquots of growing yeast cells (Saccharomyces cerevisae, strain AH22) were suspended in buffer and exposed to 20kHz ultrasound from a laboratory (probe-type) sonicator in the presence of microgram quantities of plasmid DNA. Efficiency of DNA delivery was scored as the number of cells transformed. Results. Cell transformation was optimal at 30 seconds sonication using an output of 2.0 watts and resulted in a 20 fold enhancement over control values. At extended sonication times, fewer cells showed evidence of transformation because of reduced cell viability. The increased DNA uptake and the decreased cell viability were both attributable to acoustic cavitation events during sonication. The extent of acoustic cavitation was measured and it was found that there was an increase in cavitation events with increased sonication time. Cell viability was shown to be directly related to the number of cavitation events. The effects of sonication on plasmid DNA were investigated and indicated that the structural integrity of plasmid DNA was unaffected by the sonication conditions employed. Conclusions. Under controlled conditions, ultrasound is an effective means of delivering plasmid DNA into cells. The subsequent expression of DNA molecules in cells depends upon a balance between transient cell damage and cell death.

On-off transport through a thermoresponsive hydrogel composite membrane.

A novel thermoresponsive composite membrane is described through which the permeation of molecules is dependent on the temperature of the milieu. The thermoresponsive composite membranes utilise a thermosensitive crosslinked polymeric hydrogel, which possesses a volume phase transition temperature (VPTT). The hydrogel was polymerised in situ within the pores of a sintered glass filter, through which significant permeation of molecules only occurred at temperatures above the VPTT of the hydrogel. It was found that the permeation of molecules through the thermoresponsive composite membranes could be modulated by changing the environmental temperature.

Preparation, characterisation, and drug release from thermoresponsive microspheres

Abstract An emulsion polymerisation method for the preparation of thermoresponsive hydrogel microspheres based on N-isopropylacrylamide is described in this paper. It was found that microsphere size distribution could be altered by varying the rate of stirring during the polymerisation process and also by modifying the concentration of lecithin. Microspheres were imaged in the fully hydrated state using environmental scanning electron microscopy (ESEM). Thermoresponsive release of a model drug from the microspheres was found to be a function of microsphere size, with improved control as microsphere size increased.

Effect of geometry on the erosion characteristics of polyanhydride matrices

The aim of this study was to investigate the effect of device geometry on the erosion characteristics of poly[1,3-bis(p-carboxyphenoxy) propane:sebacic acid], p(CPP:SA), matrices in phosphate buffer to obtain an insight into the mechanism of polyanhydride erosion. Matrices with different dimensions were prepared using a melt moulding technique and erosion was followed by determining changes in polymer molecular weight, water absorption, mass loss and release of monomers with time of immersion in phosphate buffer pH 7.4 at 37°C. In all matrices the erosion process was characterised by an initial lag phase during which there was a rapid decrease of polymer molecular weight but no significant water absorption, mass loss or release of monomers. The rates of absorption of water and loss of mass after the lag period showed characteristics of both surface and bulk eroding systems.

Responsive polymeric drug delivery systems : Meeting the patient's needs

SummaryCurrent approaches to the development of drug delivery systems are based typically on the premise that the relationship between the plasma concentration and therapeutic effect of a drug is invariant with time. Release kinetics from these systems are, therefore, usually engineered to be either zero-order or a simple function of time. It has been recognised for some time, however, that this approach may not be appropriate for certain drugs and it has been suggested that therapeutic efficacy may be improved by the utilisation of triggered, pulsed and programmed delivery systems. This article considers an approach to the development of responsive drug delivery system based on the utilisation of functional polymers, which aim to improve therapeutic efficacy by varying drug release in accordance with a patients’s varying requirements.