Biomedical subjects

Alexander T Florence

Publications and source records attributed to Alexander T Florence.

At least 19 recordsLinked to original sources

PubMed2006

Protein transduction by lipidic peptide dendrimers.

We investigated the potential of a new family of lipidic peptide dendrimers in protein transduction into cultured cells. Dendrimer-protein interaction was determined by gel retardation assays using purified recombinant protein. To assess intracellular protein delivery, two marker proteins were used: recombinant firefly luciferase and a Cy3-labeled monoclonal antibody to the c-myc proto-oncogene. Protein delivery was determined by luciferase assays and fluorescence microscopy, respectively. While there was minimal delivery of luciferase or antibody in the absence of the dendrimers, the latter increased protein delivery substantially. Luciferase delivery was concentration and cell type-dependent; the efficiency of delivery also varied with the number of terminal amino groups on the dendrimers. In previous reports, we showed that these dendrimers could be used for gene and drug delivery; the data we report herein suggest that they may also be capable of intracellular protein delivery. This finding has important implications for the use of these dendrimers in protein therapeutics and vaccinology.

Antibodies, Monoclonal↗

PubMed2006

An intrinsically fluorescent dendrimer as a nanoprobe of cell transport.

Dendrimers, spherical or quasi-spherical synthetic polymers in the nano-size range, have found useful applications as prospective carriers in drug and gene delivery. The investigation of dendrimer uptake by cells has been previously achieved by the incorporation of a fluorescent dye to the dendrimer either by chemical conjugation or by physical interaction. Here we describe the synthesis of two intrinsically fluorescent lysine based cationic dendrimers which lack a fluorophore, but which has sufficient fluorescence intensity to be detected at low concentrations. The nomenclature used to describe our compounds results in, for example the 6th generation dendrimer being notated as Gly-Lys(63) (NH2)(64); Gly denotes that the compound has a glycine in the core coupled to 63 lysine branching units (Lys(63)) and that the surface has 64 free amino groups (NH2)(64). The use of these dendrimers in probing transport avoids the need for fluorescent tagging with its attendant problems. The uptake of Gly-Lys(63) (NH2)(64) into Caco-2 cells was followed using confocal microscopy. Being cationic, it first adsorbs to the cell surface, enters the cytoplasm and reaches the nucleus within 35-45 min. Estimates of the diffusion coefficient of the dendrimer within the cell cytoplasm leads to a value of 6.27 ( +/- 0.49) x 10(-11) cm(2) s(-1), which is up to 1000 times lower than the diffusion coefficient of the dendrimer in water. Intrinsically fluorescent dendrimers of different size and charge are useful probes of transport in cells.

Biological Transport↗

PubMed2005

Supramolecular structures from dendrons and dendrimers.

This paper reviews aspects of the association of dendrons and dendrimers into a variety of supramolecular structures. There is such a wide range of primary dendron and dendrimer chemistries that it is still difficult to predict behaviour in aqueous media, and there are few studies in non-aqueous media. The aggregation of the primary units into larger and more complex forms leads to a wider range of potential carrier systems for drugs, genes and vaccines. This review deals principally with the association structures which can be formed. These include liquid crystalline structures and dendron block copolymer aggregates, surface monolayer formation, dendrimer derived nanoparticles, micellar structures and dendrisome (vesicle) formation. Of particular interest are DNA-dendrimer complexes and dendrimer-polyanion interactions. The in vivo behaviour of dendrons and dendrimers is of course crucial and is addressed. Dendrimer vesicle solubilisation by surfactants and emulsion stabilisation by dendrimers completes the survey of secondary structures. The challenge is to understand better the processes involved and to concentrate further on the design of the synthesis of dendrons and dendrimers which will associate into specific complex structures to increase the scope of dendrimer science.

DNA↗

PubMed2005

The diffusion of latex nanospheres and the effective (microscopic) viscosity of HPMC gels.

Dynamic light scattering (DLS) has been used to measure the diffusion coefficients of 108+/-7 (+/-S.D.) and 495+/-23 nm positively charged amino latex nanospheres (ALNs) and negatively charged carboxyl latex nanospheres (CLNs) (48+/-7, 91+/-9.8 and 483+/-10 nm) in three different media, water, glycerol aqueous solutions and hydroxypropyl methylcellulose (HPMC) gels. The translational diffusion coefficients (D) of these latex spheres in water were found to be 13.00 (+/-0.12), 5.11 (+/-0.06), 0.89 (+/-0.01) microm2/s for 48, 91 and 483 nm CLN, and 3.26 (+/-0.01) and 0.88 (+/-0.03) microm2/s for 108 and 495 nm ALN, respectively. In Newtonian glycerol aqueous solutions as anticipated the diffusion could be predicted by the Stokes-Einstein relationship over a range of system viscosities. In HPMC gels the results show the deviation of the diffusion coefficient from the Stokes-Einstein equation when the viscosity of the medium is increased. In addition, there was an increase in the polydispersity index (PI) from 0.217 to 0.928 with 108 nm ALN on increasing HPMC concentrations from 0.2% to 0.8% (w/v), which implied an interaction between the positively charged nanospheres and the gel. From the D values, the "effective" or "microscopic" viscosities of the HPMC medium were calculated, and ranged from 0.899 to 0.925 mPa s.

Diffusion↗

PubMed2005

Hydrophobic dendrimer-derived nanoparticles.

Lipidic polylysine dendrimers, synthesized using Fmoc solid phase peptide techniques, have been formulated as nanoparticles by precipitation from solution in dichloromethane. The effect of concentration on the diameter and stability of nanoparticles formed from two short homologous series of dendrimers--one fifth generation and one sixth generation series and with surface C4, C10 or C12 groups--was investigated using photon correlation spectroscopy. The increase in generation from fifth to sixth resulted in increased diameter for each chain length. An increase in the surface lipidic chain length from C4 to C12 had no effect on the particle diameter of aggregates derived from fifth generation dendrimers, and a small and variable effect on the sixth generation derived nanoparticles. Using pyrene (excitation 340 nm) as a hydrophobic fluorescent probe, a decrease in intensity peak I1 (374 nm)/I3 (385 nm) in the emission spectra (340-600 nm) was observed in the two dendrimers studied, fifth generation dendrimers with C10 or C12 surface lipidic chains, as the dendrimer concentration increased, reaching a plateau at higher concentrations, indicating that a more compact form of the aggregates with a more hydrophobic interior was obtained. Apart from the hydrophobicity of the dendrimers and dendrimer concentration, the flexibility of the dendrimers might have a significant effect in determining nanoparticle size. The aggregates derived from the fifth generation dendrimers with C10 or C12 surface lipidic chains are stable in purified intestinal fluid but not in purified stomach fluid, in which further aggregation of the nanoparticulate dendrimer aggregates occurs as an effect of pH, salts, proteins and enzymes in these fluids. This study demonstrates, inter alia, the importance of testing nanoparticulate delivery systems in relevant physiologically based fluids prior to their use in vivo.

Chemical Phenomena↗

PubMed2005

Novel anhydrous emulsions: formulation as controlled release vehicles.

Novel anhydrous emulsions, which may offer some advantages as depot or reservoir vehicles for lipophilic drugs in controlled delivery systems, were formulated using castor oil as the disperse phase and dimethicone or cyclopentasiloxane as the continuous phase. Among the emulsifiers studied only silicone surfactants (cyclomethicone/dimethicone copolyols) which were miscible in silicone oil stabilized the emulsions. Cyclomethicone/PEG/PPG-18/18 Dimethicone and Cyclopentasiloxane/PEG/PPG-18/18 Dimethicone were more effective in lowering the interfacial tension between castor oil and both dimethicone and cyclopentasiloxane. Emulsions formulated using either of these two surfactants were found to be stable against phase separation and exhibited least globule growth over 168 h. The average particle size was found to be 2-6 microm in these systems formed by probe sonication. Slow release patterns of 3H-dehydroepiandrosterone (DHEA) and 3H-dexamethasone solubilized in the disperse castor oil phase into an aqueous dialyzing medium were observed over 48 h.

Castor Oil↗

PubMed2005

Release of DNA from dendriplexes encapsulated in PLGA nanoparticles.

Biodegradable PLGA particles of less than 1 microm can encapsulate DNA and DNA-dendron complexes (dendriplexes) providing sustained DNA release for transfecting cells in gene delivery. Two polylysine-based dendrons prepared by solid state peptide synthesis were used to condense pRedN-1 DNA (7.5 kbp), a fluorescent protein vector. The dendrons had 16 free surface amino groups attached to seven lysine groups, bound to a lipid core, one containing three C18 chains and the other a single C10 chain. Increased lipophilicity and molar charge ratios are key factors in producing compact and reproducible dendriplexes, shown by the hydrodynamic diameter which is of the order of 800 nm (p.d.>0.5) at a 2:1 molar charge ratio, a value which decreases to around 200 nm at a 5:1 charge ratio. At lower charge ratios the dendriplexes are negative and have a zeta potential in order of -18 mV. As the ratio increases (5:1, 10:1) the complexes bear a positive potential (13+/-2 mV). This suggests that at the 2:1 ratio the DNA is not fully condensed. The DNA was radiolabelled with 35S dCTP (deoxycytidinetriphosphate) with the removal of the un-incorporated radiolabelled nucleotides. The encapsulation efficiency of dendriplexes in PLGA particles is higher than that for uncomplexed DNA. When the results are normalised for DNA content and particle surface area, complexation of the DNA was found to decrease release rate.

Chemical Phenomena↗

PubMed2005

The relative flow of the walls of phospholipid tether bilayers.

Lipid nanotubes or "tethers" can be formed from liposomes or niosomes, pulled from the parent vesicles by micromanipulation. The tethers are cylindrical multibilayer tubes. Here, we describe the movement of these multilamellar walls, initiated by creating a surface tension gradient along the tether. The movement of lipid can give rise to a visible moving boundary. In the case of bilayer membranes, a tangential gradient in surface tension produces membrane bulk flow toward regions of higher surface tension. The flow of the bilayers comprising the tether nanotubes seems to be restricted to the inner bilayers, creating a velocity gradient in the bilayers. In this study, we discuss the implementation of tension-driven flows as a transport method in a tether-vesicle network. Interactions between fluid within the tether channels and the lipid layers are important, leading to anomalies in the transport of fluids and particles compared to bulk systems.

Cholesterol↗

PubMed2005

Dendrisomes: vesicular structures derived from a cationic lipidic dendron.

The behavior of a novel synthetic lipidic cationic lysine-based dendron (partial dendrimer) in aqueous media and its ability, with and without cholesterol, to self-assemble into higher order structures was studied to gain an understanding of these structures as potential drug carriers. The dendron was prepared by solid-phase peptide synthesis. A reverse-phase evaporation (REV) technique was used to prepare cationic vesicular aggregates of the dendron with different molar ratios of cholesterol. The size and zeta potential of these supramolecular aggregates or "dendrisomes" was determined by photon correlation spectroscopy (PCS). Dendrisome morphology and thermotropic properties were studied by transmission electron microscopy (TEM) and differential scanning calorimetry (DSC). Radiolabeled penicillin G was used as a model of a negatively charged water-soluble compound to investigate the encapsulation efficiency of the dendrisomes. In vitro release of the drug was determined using as a comparator a REV liposome formulation. Dendrisomes of all compositions have higher encapsulation efficiencies and slower release rates compared to the comparator. Cholesterol was found both to increase the size of the aggregates from around 310 to 560 nm and to increase shape irregularities, but did not change the positive zeta potential, in the order of +50 mV, of the dendrisomes. Cholesterol decreases penicillin G entrapment efficiency but increases solute leakage at 25 degrees C.

Chemical Phenomena↗

PubMed2005

Versatile peptide dendrimers for nucleic acid delivery.

Dendrimers are nonviral vectors that have attracted interest on account of a number of features. They are structurally versatile because their size, shape, and surface charge can be selectively altered. Here we examine the functions of a new family of composite dendrimers that were synthesized with lipidic amino acid cores. These dendrimers are bifunctional because they are characterized by positively charged (lysine) modules for interaction with nucleic acids and neutral lipidic moieties for membrane lipid-bilayer transit. We assessed their structure-function correlations by a combination of molecular and biophysical techniques. Our assessment revealed an unexpected pleitropy of functions subserved by these vectors that included plasmid and oligonucleotide delivery. We also generated a firefly luciferase cell line in which we could modulate luciferase activity by RNA interference. We found that these vectors could also mediate RNA suppression of luciferase expression by delivering double-stranded luciferase transcripts generated in vitro. The structural uniqueness of these lipidic peptide dendrimers coupled with their ease and specificity of assembly and the versatility in their choice of cargo, puts them in a new category of macromolecule carriers. These vectors, therefore, have potential applications as epigenetic modifiers of gene function.

Animals↗

PubMed2005

Investigation of the association and flexibility of cationic lipidic peptide dendrons by NMR spectroscopy.

The cationic peptide dendrons synthesized and studied are lower generation polylysine-based partial dendrimers with or without lipid chains in the core. The dendrons with lipidic chains can be utilized as protein and liposomal mimics because of their unique structural properties. The full assignments of three different dendrons (L)7(NH2)8, (C14)1(L)7(NH2)8 and (C14)3(L)7(NH2)8 were obtained in D2O and H2O/D2O using a 500 MHz NMR spectrometer. The hydrophobic lipidic core of branched polylysine dendrons was found to induce aggregation upon increasing concentration. Because non-lipidic dendrons do not self-assemble, the behaviour and internal structural features of two different dendrons with one and three C14 hydrocarbon chains were explored. The critical association concentration clearly depends on the number of core hydrophobic residues and the association starts at 0.025 mM for (C14)1(L)7(NH2)8 and 0.05 mM for (C14)3(L(7(NH2)8. Chemical shift analysis also revealed that the hydrophobic chains of the dendrons associate in the core, whereas the polar head groups (NH2) are mainly located at the surfaces of the aggregates. The T1 relaxation time measurements showed that the mobility of the hydrocarbon chain is greater with the monomeric form of dendron (C14)1(L)7(NH2)8) than that of monomer (C14)3(L)7(NH2)8. The inter-chain hydrophobic interactions restrict the flexibility of the dendron with three hydrocarbon chains. As expected, the flexibility of the monomeric form is higher than that of the aggregated state for both of the dendrons.

Lipoproteins↗

PubMed2004

Issues in oral nanoparticle drug carrier uptake and targeting.

Some of the broader issues relating to the exploration of the use of nanoparticulate drug carriers by the oral route to achieve absorption of molecules which are poorly absorbed from the gastrointestinal tract are considered briefly here. These relate to both the pharmaceutical and biological characteristics of the carrier and carrier-gut interactions, the dynamic nature of such interactions, the varied modes of uptake, and the difficulties in targeting to the gut epithelium to encourage more efficient uptake of nanoparticles. These have the unhelpful habit of aggregating and flocculating hence increasing their effective size, when small size aids uptake and translocation. Post-absorption events can be equally hazardous and need further research. The question is asked whether or not it is wise load the gut lumen with molecules such as insulin even when protected in a carrier, and the suggestion is made that targets need to be rethought. The epithelium of the gut and the lymphoid tissue itself offers much scope for therapeutic interventions through nanoparticle delivery.

Administration, Oral↗

PubMed2003

Microtubules formed by capillary extrusion and fusion of surfactant vesicles.

Polyhedral non-ionic surfactant vesicles formed from mixtures of polyoxyethylene-5-cetyl ether (C(16)EO(5)) or polyoxethylene-5-stearyl ether (C(18)EO(5)) with poly-24-oxyethylene cholesteryl ether (Solulan C24) and low amounts of cholesterol, when extruded from microcapillaries under pressure fuse to form multi-lamellar tubules up to about 80 microm in length. The diameter of the extruded tubules depends on the exit diameter of the capillaries used, in this paper generally around 1 microm. Under some circumstances, instead of linear tubules, the tubules form as concentric whorls which can be unraveled into their constituent tubules, indicating the strength of the systems. Vesicles can be formed within these tubular structures to act as a model for vesicular flow in elastic capillaries. Microparticles encapsulated with the primary polyhedral vesicles after extrusion are seen within the tubules, promising the possibility of a model for the study of microparticle flow within vessels. Preliminary studies on the use of the tubules as templates for polymerisation are also presented.

Microspheres↗

PubMed2003

A vesicular shuttle: transport of a vesicle within a flexible microtube.

Micromanipulation of the external bilayers of nonionic surfactant vesicles (niosomes) and liposomes allows the formation of tethers, which are fluid state lipid/surfactant lamellar nanotubes. The technique allows investigation of some of the factors affecting tether formation and vesicle-tether interactions. In this paper the movement of a vesicle along, or more precisely in, tethers derived from the vesicle, has been studied. When a vesicle is supported by bipolar tethers, stretching the tether on one side of the vesicle, initiates the movement of the vesicle in the opposite direction, at velocities ranging up to 2.5 microm s(-1) thus creating a 'vesicular shuttle'. Movement of the vesicle occurs inside the tether structures, a process akin to the movement of a sphere in a flexible tube with a diameter much less than that of the sphere. The factors involved in the movement of vesicles in the tethers include the radial stretching and subsequently contraction of the tethers and the minimisation of elastic energies stored in the tether membranes. Vesicle velocity is not constant: there is deceleration near the end of the trajectory. While the relevance to the design of novel delivery systems is as yet tenuous, the system allows vesicle-vesicle collisions to be observed when the vesicle is propelled towards a stationary vesicle, and directly observation of the flow properties of vesicles in flexible 'capillaries', a neglected topic.

Algorithms↗

PubMed2003

Some properties of extruded non-ionic surfactant micro-tubes.

Polyhedral non-ionic surfactant vesicles (niosomes) undergo complex shape transitions as a result of mechanical stress. When extruded under pressure from capillaries with exit diameters smaller than the diameter of the vesicles, a series of novel structures comprising mostly of tubules, vesicles inside tubules and concentric structures can be formed. The microtubules (up to 80 microm in length) form as a result of the pressure exerted on polyhedral niosomes, this leading to the fusion of many vesicles, the relative shear giving movement of the vesicles giving rise to the formation of three distinctive structures, namely tubules, vesicle within tubule and concentric ("whorl") morphologies. The entrapment efficiency of the tubules has been studied using a model solute 5(6)-carboxyfluorescein (CF), as has the effect of shear stress this and compaction pressure on the release of the entrapped solute. Deformation of the structures affects their ability to retain entrapped solute. Tubular structures heated above their transition temperatures reversibly transform into discrete vesicular structures.

Chemistry, Pharmaceutical↗

PubMed2003

Dendriplexes and their characterisation.

The interaction of DNA with partial dendrimers (dendritic polylysine containing seven lysines and eight terminal amino groups with or without a lipidic core) was studied. Compact complexes were formed which we term "dendriplexes". Agarose gel electrophoresis and exclusion of ethidium bromide confirmed the interaction. All the dendrons formed compact complexes above a 2:1 (+/-) charge ratio in water and HBSS. Photon correlation spectroscopy, electron microscopy and zeta potential measurements were used to determine, respectively, the particle size, shape and surface charge of the dendriplexes. The z-average diameter of the dendriplexes were found to be 60-70 nm irrespective of the dendron used and the zeta potential varied from 10 to 35 mV at a 3:1 (+/-) charge ratio depending on the dendron. The protection of the DNA component of these dendriplexes from nuclease degradation was confirmed by DNase protection assays.

Chemical Phenomena↗

PubMed2003

Formulation and stability of surface-tethered DNA-gold-dendron nanoparticles.

The formulation of plasmid DNA on 100 nm gold nanoparticles surface-tethered via cationic dendrons, and the behaviour of the complex in cell culture media, is described in this communication. Adsorption of dendrons onto gold nanoparticles in water resulted in the generation of positively charged nanoparticles with a corresponding small increase in particle size. Addition of plasmid DNA did not markedly reduce the surface potential but resulted in a approximately 10-20% increase in hydrodynamic diameter. More dramatic effects were seen in the presence of cell culture media that, overall, drastically increased the apparent size of the gold-dendron-DNA nanoparticles and reduced the surface potential of the colloids, the presence of serum components partially ameliorating these effects possibly due to steric stabilisation. Release of the surface-tethered DNA was reduced in cell culture media compared to water. This reduced detachment of DNA coupled with the flocculation of the carrier which would likely inhibit endocytosis, demonstrates the importance of testing drug delivery systems with relevant physiologically based fluids prior to their use in vivo studies.

Adsorption↗

PubMed2003

Dendrisomes: cationic lipidic dendron vesicular assemblies.

A new lipidic cationic polylysine dendron was prepared by solid-phase peptide synthesis. Its behaviour in aqueous media and its ability, with and without cholesterol, to form higher order structures, "dendrisomes", was studied to further our understanding of how dendrons interact with drug molecules and may be utilised as drug carriers. Dynamics simulations of the dendron show their flexibility. Incorporation of cholesterol increases the hydrodynamic diameter of the aggregates from 311 to 556 nm but does not affect their positive zeta potential (of the order of +50 mV). The dendrisomes encapsulated penicillin G (6.15% w/w) compared to only 1.4% w/w entrapment in REV liposomes of 1:1 distearoyl phosphatidylcholine:cholesterol. Cholesterol, however, decreases the entrapment efficiency. Electrostatic forces and H-bonding between the negatively charged drug and dendron amino groups are likely to be key in determining these interactions.

Chemical Phenomena↗