Nose to brain delivery in rats: Effect of surface charge of rhodamine B labeled nanocarriers on brain subregion localization.

Nose to brain delivery and nanotechnology are the combination of innovative strategies for molecules to reach the brain and to bypass blood brain barriers. In this work we investigated the fate of two rhodamine B labeled polymeric nanoparticles (Z-ave <250nm) of opposite surface charge in different areas of the brain after intranasal administration in rats. A preliminary screening was carried out to select the suitable positive (chitosan/poly-l-lactide-co-glycolide) nanocarrier through photon correlation spectroscopy and turbiscan. Physico-chemical and technological characterizations of poly-l-lactide-co-glycolide (negative) and chitosan/poly-l-lactide-co-glycolide (positive) fluorescent labeled nanoparticles were performed. The animals were allocated to three groups receiving negative and positive polymeric nanoparticles via single intranasal administration or no treatment. The localization of both nanocarriers in different brain areas was detected using fluorescent microscopy. Our data revealed that both nanocarriers reach the brain and are able to persist in the brain up to 48h after intranasal administration. Surface charge influenced the involved pathways in their translocation from the nasal cavity to the central nervous system. The positive charge of nanoparticles slows down brain reaching and the trigeminal pathway is involved, while the olfactory pathway may be responsible for the transport of negatively charged nanoparticles, and systemic pathways are not excluded.

Chitosan amphiphile coating of peptide nanofibres reduces liver uptake and delivers the peptide to the brain on intravenous administration.

The clinical development of neuropeptides has been limited by a combination of the short plasma half-life of these drugs and their ultimate failure to permeate the blood brain barrier. Peptide nanofibres have been used to deliver peptides across the blood brain barrier and in this work we demonstrate that the polymer coating of peptide nanofibres further enhances peptide delivery to the brain via the intravenous route. Leucine(5)-enkephalin (LENK) nanofibres formed from the LENK ester prodrug - tyrosinyl(1)palmitate-leucine(5)-enkephalin (TPLENK) were coated with the polymer - N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ) and injected intravenously. Peptide brain delivery was enhanced because the GCPQ coating on the peptide prodrug nanofibres, specifically enables the peptide prodrug to escape liver uptake, avoid enzymatic degradation to non-active sequences and thus enjoy a longer plasma half life. Plasma half-life is increased 520%, liver AUC0-4 decreased by 54% and brain AUC0-4 increased by 47% as a result of the GCPQ coating. The increased brain levels of the GCPQ coated peptide prodrug nanofibres result in the pharmacological activity of the parent drug (LENK) being significantly increased. LENK itself is inactive on intravenous injection.

Optimisation of synthetic vector systems for cancer gene therapy - the role of the excess of cationic dendrimer under physiological conditions.

We have previously demonstrated in a therapeutic study that a single systemic course of DAB-Am16 dendriplexes loaded with plasmid expressing TNFα over a period of time of 10 days led to a regression of 100% of tumours and to long term cures of up to 80% of animals. However, the formulation had a relatively low colloidal stability requiring administration soon after nanoparticle preparation. Similar to other cationic polyplex and dendrimer DNA delivery systems, DAB-AM16 dendrimer formulations contained a substantial proportion of free polymer; this free polymer is present independently of the specific polymer:DNA ratio and increases with increasing proportion of polymer (N:P charge ratio) in the formulation. It has previously been shown for this and other systems that the excess of polymer plays a role in promoting the transfection efficiency of synthetic vectors. This has been linked to effects of the polymer on the efficiency of intracellular processing, e.g. endosomal release. However, the free polymer may have additional effects that are relevant to the efficiency of the formulation. This study therefore considered the effect of free dendrimer on the colloidal stability of the complexes, the interaction of the complex with the formulation medium, and with biological components, i.e. electrolytes and serum proteins after administration. Analysis of the total potential of interaction shows that, even at high N:P ratios, the excess of free dendrimer in the medium is not enough to induce the aggregation of the formulation due to depletion forces. This finding is unusual and can be attributed to the particularly low Mw of these dendrimers (1.6 kDa). On the other hand, formulations are highly sensitive to the strength of the dendrimer:DNA interactions. These can be controlled by the degree of protonation (α) of the dendrimer which is strongly dependent on bulk pH. Modulation of the protonation level to α≥0.4 allows reproducible production of colloidally stable particles. Finally, we have demonstrated that electrolytes and proteins present in physiological media play a crucial role to favour the efficiency of these synthetic vectors reducing the toxicity associated with their cationic groups.

Delivery of peptides to the blood and brain after oral uptake of quaternary ammonium palmitoyl glycol chitosan nanoparticles.

The clinical development of therapeutic peptides has been restricted to peptides for non-CNS diseases and parenteral dosage forms due to the poor permeation of peptides across the gastrointestinal mucosa and the blood-brain barrier. Quaternary ammonium palmitoyl glycol chitosan (GCPQ) nanoparticles facilitate the brain delivery of orally administered peptides such as leucine(5)-enkephalin, and here we examine the mechanism of GCPQ facilitated oral peptide absorption and brain delivery. By analyzing the oral biodistribution of radiolabeled GCPQ nanoparticles, the oral biodistribution of the model peptide leucine(5)-enkephalin and coherent anti-Stokes Raman scattering microscopy tissue images after an oral dose of deuterated GCPQ nanoparticles, we have established a number of facts. Although 85-90% of orally administered GCPQ nanoparticles are not absorbed from the gastrointestinal tract, a peak level of 2-3% of the oral GCPQ dose is detected in the blood 30 min after dosing, and these GCPQ particles appear to transport the peptides to the blood. Additionally, although peptide loaded nanoparticles from low (6 kDa) and high (50 kDa) molecular weight GCPQ are taken up by enterocytes, polymer particles with a polymer molecular weight greater than 6 kDa are required to facilitate peptide delivery to the brain after oral administration. By examining our current and previous data, we conclude that GCPQ particles facilitate oral peptide absorption by protecting the peptide from gastrointestinal degradation, adhering to the mucus to increase the drug gut residence time and transporting GCPQ associated peptide across the enterocytes and to the systemic circulation, enabling the GCPQ stabilized peptide to be transported to the brain. Orally administered GCPQ particles are also circulated from the gastrointestinal tract to the liver and onward to the gall bladder, presumably for final transport back to the gastrointestinal tract.

The complexation between novel comb shaped amphiphilic polyallylamine and insulin: towards oral insulin delivery.

Novel amphiphilic polyallylamine (PAA) were previously synthesised by randomly grafting palmitoyl pendant groups and subsequent quaternising with methyl iodide. The ability of these self-assembled polymers to spontaneously form nano-complexes with insulin in pH 7.4 Tris buffer was evaluated by transmittance study, hydrodynamic size and zeta potential measurements. The transmission electron microscopy images showed that non-quaternised polymer complexes appeared to form vesicular structures at low polymer:insulin concentrations. However, at higher concentrations they formed solid dense nanoparticles. The presence of quaternary ammonium moieties resulted in insulin complexing on the surface of aggregates. All polymers exhibited high insulin complexation efficiency between 78 and 93%. Incubation with trypsin, alpha-chymotrypsin and pepsin demonstrated that most polymers were able to protect insulin against enzymatic degradation by trypsin and pepsin. Quaternised polymers appeared to have better protective effect against trypsinisation, possibly due to stronger electrostatic interaction with insulin. Interestingly, non-quaternised polymers significantly enhanced insulin degradation by alpha-chymotrypsin. All polymers were less cytotoxic than PAA, with the quaternised polymers exhibiting up to 15-fold improvement in the IC(50) value. Based on these results, quaternised palmitoyl graft polyallylamine polymers showed promising potential as oral delivery systems for insulin.

Cancer and the blood-brain barrier: 'Trojan horses' for courses?

The blood-brain barrier (BBB) limits the bioavailability of most bioactive molecules and drugs in the CNS, leaving clinicians with only a few options for pharmacotherapy. In this issue Regina et al. demonstrate that a 'Trojan horse' drug conjugate, acting as a substrate of a physiological BBB receptor that facilitates transcytosis, significantly improves drug transport into the CNS. Specifically, the low-density lipoprotein receptor-related protein (LRP) is used to carry a conjugate of paclitaxel and Angiopep-2, an aprotinin-derived peptide, across the BBB. Interestingly, in its conjugated form paclitaxel circumvents the efflux pumps at the BBB but still retains its activity against microtubules. Importantly, the authors were able to demonstrate improved therapeutic efficacy of this approach in orthotopic models of primary and metastatic brain cancer. This proof-of-principle study thus represents a milestone for drug delivery across the BBB but also a starting point for studies exploring wider applicability and potential limitations of the approach.

High-resolution 3D isotropic MR imaging of mouse flank tumours obtained in vivo with solenoid RF micro-coil.

The investigation of mouse flank tumours by magnetic resonance imaging (MRI) is limited by the achievable spatial resolution, which is generally limited by the critical problem of signal-to-noise ratio. Sensitivity was improved by using an optimized solenoid RF micro-coil, built into the animal cradle. This simple design did not require extensive RF engineering expertise to construct, yet allowed high-resolution 3D isotropic imaging at 60 x 60 x 60 microm(3) for a flank tumour in vivo, revealing the heterogeneous internal structure of the tumour. It also allowed dynamic contrast enhanced (DCE) experiments and angiography (MRA) to be performed at 100 x 100 x 100 microm(3) resolution. The DCE experiments provided an excellent example of the diffusive spreading of contrast agent into less vascularized tumour tissue. This work is the first step in using high-resolution 3D isotropic MR to study transport in mouse flank tumours.

PEI-based vesicle-polymer hybrid gene delivery system with improved biocompatibility.

Wider use of the transfection agent polymer polyethylenimine (PEI) in vivo has been hampered by its toxicity. In order to examine whether material combining properties of polymers and lipid type of carriers would have improved characteristics, four PEI derivatives were synthesised: The methylation of the branched PEI (25 kDa) created a permanently charged quaternary ammonium derivative. Acylation of these backbones using pendant palmitic acid chains created amphiphilic PEI variants which formed nanoparticles or vesicles. Finally hydrophilic groups were added to the polymer backbone by PEGylation. The materials were characterised and their in vitro and in vivo properties were tested. The modifications improved the materials biocompatibility markedly when compared to the starting material but also reduced transfection efficiency. The material bearing ammonium and palmitoyl groups was 10x less toxic while retaining about 30% of the transfection efficiency in vitro. After intravenous administration in a mouse model the materials also gave rise to GFP transgene expression in the liver. The synthetic strategy altered complex physicochemistry and improved biocompatibility while maintaining in vitro gene expression for most formulations. The strategy of combination of complementary properties of cationic lipids and polymers into a hybrid material may also be applicable to other materials.

Gene delivery with synthetic (non viral) carriers.

Non-viral gene delivery involving the use of cationic polymer and cationic lipid based carriers still continues to enjoy a high profile due to the safety advantages offered by these systems when compared with viruses. However, there are still problems associated with the use of these agents, notably their comparatively low efficiency and the inability to target gene expression to the area of pathology. On intravenous administration gene expression is found predominantly in the first capillary bed encountered-the lung endothelium. The clinical use of non-viral gene delivery systems in cystic fibrosis or cancer has involved their direct application to the site of pathology due to the targeting difficulties experienced. For gene expression to occur genes must be transported to the interior of the cell nucleus and a number of biological barriers to effective gene delivery have been identified. These may be divided into extracellular such as the targeting barrier mentioned above and intracellular such as the need for endosomal escape after endocytosis and the inefficient trafficking of genes to the nucleus. Targeting ligands have been used with moderate success to overcome the targeting barrier while endosomal escape and nuclear targeting peptides are some of the strategies, which have been employed to overcome the problems of endosomal escape and nuclear trafficking. It is hoped that the next generation of carriers will incorporate mechanisms to overcome these barriers thus improving the efficacy of such materials.

Quaternary ammonium palmitoyl glycol chitosan--a new polysoap for drug delivery.

A new polysoap, quaternary ammonium palmitoyl glycol chitosan (GCPQ, M(w)=178,000 g mole(-1)) with drug solubilising potential has been synthesised and characterised. In solution hydrophobic domains of GCPQ polymeric micelles were identified by the hypsochromic shift in the lambda(max) of methyl orange and by the increase in the ratio of the fluorescence emission intensity of the third and first pyrene vibronic peaks (I(3)/I(1)). At high aqueous concentrations (>10 mg ml(-1)) GCPQ presents as a gel which solubilises pyrene (2.5 mM, normal solubility in water approximately 2 microM) on probe sonication. Dilution of the gel to a liquid solution of polymeric micelles (< or =3.75 mg ml(-1) of GCPQ), results in the observation of fluorescent pyrene excimers (excited dimers) and a high excimer to monomer fluorescence ratio (I(E)/I(M)). However, attempts to solubilise pyrene at a concentration of 2.5 mM in a liquid solution of GCPQ (3.75 mg ml(-1)) results in a reduced I(E)/I(M) value and pyrene precipitation. Viscometry measurements show a more compact conformation for the polymer solubilising pyrene than the polymer alone. The polymer is non-haemolytic when present as the liquid solution and relatively non cytotoxic. In conclusion, a new biocompatible polysoap (potential drug solubiliser) is described which forms hydrophobic domains in solution and shows hysteresis in its solubilisation of pyrene.

Preliminary characterization of novel amino acid based polymeric vesicles as gene and drug delivery agents.

The amino acid homopolymers, poly-L-lysine and poly-L-ornithine, have been modified by the covalent attachment of palmitoyl and methoxypoly(ethylene glycol) (mPEG) residues to produce a new class of amphiphilic polymers-PLP and POP, respectively. These amphiphilic amino acid based polymers have been found to assemble into polymeric vesicles in the presence of cholesterol. Representatives of this new class of polymeric vesicles have been evaluated in vitro as nonviral gene delivery systems with a view to finding delivery systems that combine effective gene expression with low toxicity in vivo. In addition, the drug-carrying capacity of these polymeric vesicles was evaluated with the model drug doxorubicin. Chemical characterization of the modified polymers was carried out using (1)H NMR spectroscopy and the trinitrobenzene sulfonic acid (TNBS) assay for amino groups. The amphiphilic polymers were found to have an unreacted amino acid, palmitoyl, mPEG ratio of 11:5:1, and polymeric vesicle formation was confirmed by freeze-fracture electron microscopy and drug encapsulation studies. The resulting polymeric vesicles, by virtue of the mPEG groups, bear a near neutral zeta-potential. In vitro biological testing revealed that POP and PLP vesicle-DNA complexes are about one to 2 orders of magnitude less cytotoxic than the parent polymer-DNA complexes although more haemolytic than the parent polymer-DNA complexes. The polymeric vesicles condense DNA at a polymer:DNA weight ratio of 5:1 or greater and the polymeric vesicle-DNA complexes improved gene transfer to human tumor cell lines in comparison to the parent homopolymers despite the absence of receptor specific ligands and lysosomotropic agents such as chloroquine.

The effect of processing variables on the physical characteristics of non-ionic surfactant vesicles (niosomes) formed from a hexadecyl diglycerol ether.

Niosomes are vesicles formed by self-assembly of non-ionic surfactants. In this investigation, the effects of processing variables, particularly temperature and sonication, on the physical characteristics and phase transitional behaviour of two niosomal systems based on a hexadecyl diglycerol ether (C(16)G(2)) have been studied. Systems containing C(16)G(2), cholesterol and poly-24-oxyethylene cholesteryl ether (Solulan C24) in the molar ratios 91:0:9 and 49:49:2 were prepared by aqueous dispersion of films, followed by examination of 5(6)-carboxyfluorescein entrapment, particle size and morphology. The thermal behaviour was examined using high sensitivity differential scanning calorimetry (HSDSC) and hot stage microscopy, while the effects of sonication were studied in terms of size and morphology, both immediately after preparation and on storing for 1 h at room temperature and 60 degrees C. Polyhedral niosomes were formed from systems containing C(16)G(2) and Solulan C24 alone, while cholesterol-containing systems formed spherical vesicles mixed with tubular structures; the polyhedral systems were found to have a larger particle size and higher CF entrapment efficiency. HSDSC studies showed the polyhedral systems to exhibit an endotherm at 45.4 degrees C and a corresponding exotherm at 39.1 degrees C on cooling which were ascribed to a membrane phase transition; no equivalent transition was observed for the cholesterol containing systems. Hot stage microscopy showed the polyhedral vesicles to convert to spherical structures at approximately 48 degrees C, while on cooling the spherical vesicles split into smaller structures and reverted to the polyhedral shape at approximately 49 degrees C. Sonication resulted in the polyhedral vesicles forming spherical structures which underwent a particle size increase on storage at room temperature but not at 60 degrees C. The study suggests that the polyhedral vesicles undergo a reversible transition to spherical vesicles on heating or sonication and that this morphological change may be associated with a membrane phase transition.

Liposomes encapsulating polymeric chitosan based vesicles--a vesicle in vesicle system for drug delivery.

Drug delivery systems comprising vesicles prepared from one amphiphile encapsulating vesicles prepared from a second amphiphile have not been prepared previously due to a tendency of the bilayer components of the different vesicles to mix during preparation. Recently we have developed polymeric vesicles using the new polymer-palmitoyl glycol chitosan and cholesterol in a 2:1 weight ratio. These polymeric vesicles have now been encapsulated within egg phosphatidylcholine (egg PC), cholesterol (2:1 weight ratio) liposomes yielding a vesicle in vesicle system. The vesicle in vesicle system was visualised by freeze fracture electron microscopy. The mixing of the different bilayer components was studied by monitoring the excimer fluorescence of pyrene-labelled polymeric vesicles after their encapsulation within egg PC liposomes or hexadecyl diglycerol ether niosomes. A minimum degree of lipid mixing was observed with the polymeric vesicle-egg PC liposome system when compared to the polymeric vesicle-hexadecyl diglycerol ether niosome system. The polymeric vesicle-egg PC vesicle in vesicle system was shown to retard the release of encapsulated solutes. 28% of 5(6)-carboxyfluorescein (CF) encapsulated in the polymeric vesicle compartment of the vesicle in vesicle system was released after 4 h compared to the release of 62% of encapsulated CF from plain polymeric vesicles within the same time period.

The encapsulation of bleomycin within chitosan based polymeric vesicles does not alter its biodistribution.

Polymeric vesicles have recently been developed from an amphiphilic chitosan derivative--palmitoyl glycol chitosan. Their potential as a drug delivery system was evaluated using the anti-cancer compound bleomycin as a model drug. Palmitoyl glycol chitosan (GCP41) was synthesised by conjugation of palmitoyl groups to glycol chitosan. Bleomycin-containing vesicles (669 nm diameter) were prepared from a mixture of GCP41 and cholesterol by remote loading. The vesicles were imaged by freeze-fracture electron microscopy and their in-vitro stability tested. Incubation of the larger vesicles with plasma in-vitro led to a reduction of mean size by 49%, a reaction not seen with control sorbitan monostearate niosomes (215 nm in size). They also showed a higher initial drug release (1 h), but GCP41 and sorbitan monostearate vesicles retained 62% and 63% of the encapsulated drug after 24h, respectively. The biodistribution of smaller vesicles (290 nm) prepared by extrusion through a 200-nm filter was also studied in male Balb/c mice. Encapsulation of bleomycin into polymeric vesicles did not significantly alter the pharmacokinetics of biodistribution of bleomycin in male Balb/c mice although plasma and kidney levels were slightly increased. It is concluded that the extruded GCP41 vesicles break down in plasma in-vivo and hence are unlikely to offer any therapeutic advantage over the free drug.

Niosomes and polymeric chitosan based vesicles bearing transferrin and glucose ligands for drug targeting.

PURPOSE: To prepare polymeric vesicles and niosomes bearing glucose or transferrin ligands for drug targeting. METHODS: A glucose-palmitoyl glycol chitosan (PGC) conjugate was synthesised and glucose-PGC polymeric vesicles prepared by sonication of glucose-PGC/cholesterol. N-palmitoylglucosamine (NPG) was synthesised and NPG niosomes also prepared by sonication of NPG/ sorbitan monostearate/ cholesterol/ cholesteryl poly-24-oxyethylene ether. These 2 glucose vesicles were incubated with colloidal concanavalin A gold (Con-A gold), washed and visualised by transmission electron microscopy (TEM). Transferrin was also conjugated to the surface of PGC vesicles and the uptake of these vesicles investigated in the A431 cell line (over expressing the transferrin receptor) by fluorescent activated cell sorter analysis. RESULTS: TEM imaging confirmed the presence of glucose units on the surface of PGC polymeric vesicles and NPG niosomes. Transferrin was coupled to PGC vesicles at a level of 0.60+/-0.18 g of transferrin per g polymer. The proportion of FITC-dextran positive A431 cells was 42% (FITC-dextran solution), 74% (plain vesicles) and 90% (transferrin vesicles). CONCLUSIONS: Glucose and transferrin bearing chitosan based vesicles and glucose niosomes have been prepared. Glucose bearing vesicles bind Con-A to their surface. Chitosan based vesicles are taken up by A431 cells and transferrin enhances this uptake.

A non-covalently cross-linked chitosan based hydrogel.

Hydrogels are normally formed by the covalent cross-linking of linear polymers. In the case of chitosan based hydrogels this cross-linking is often achieved with glutaraldehyde, glyoxal or other reactive cross-linking agents. Such hydrogel materials have limited biocompatibility and biodegradability. However by the attachment of hydrophobic palmitoyl groups to glycol chitosan, a water soluble chitosan derivative, we have produced a version of the amphiphilic vesicle forming polymer-palmitoyl glycol chitosan (Uchegbu et al., 1998, J Pharm Pharmacol 58, 453-458). The level of palmitoylation in this variant of the polymer (GCP11), as determined by proton neutron magnetic resonance spectroscopy, is 19.62+/-2.42% (n=4). GCP11 has been used to prepare soft, slowly eroding hydrogels suitable for drug delivery by simply freeze-drying an aqueous dispersion of the polymer. Non-covalent cross-linking to form the gel matrix is achieved by the hydrophobic interactions of the palmitoyl groups. The resulting material, as examined by scanning electron microscopy, is porous and may be hydrated to up to 20x its weight in aqueous media without any appreciable change in volume-transforming from an opaque to a translucent solid. The slow erosion of this material in aqueous environments gives a biodegradable and ultimately more biocompatible material than covalently cross-linked hydrogels. Unlike most chitosan-based gels, the gel is hydrated to 20x its weight at alkaline pH but only 10x its weight at neutral and acid pH. This is as a result of the gradual erosion of the gel at lower pH values. Hydration is also reduced from 20x the dry gel weight in water to 10x the dry gel weight in the presence of dissolved salts such as sodium chloride. GCP11 hydrogels have been loaded to 0.1% w/w with a model fluorophore, rhodamine B, by simply freeze-drying an aqueous dispersion of GCP11 in the presence of a solution of rhodamine B dissolved in either water or phosphate buffered saline (PBS, pH=7.4). The release of this model fluorophore was retarded by between 8 and 12% when PBS was contained in the gel in accordance with the hydration profiles. Rhodamine B release was also reduced by between 13 and 25% in the presence of acid as a result of the reduced solubility of rhodamine B at acid pH.

Osmotic behaviour of polyhedral non-ionic surfactant vesicles (niosomes).

In addition to common spherical non-ionic surfactant vesicles (niosomes), disc-like, tubular, and polyhedral niosomes have also been reported. The permeability and osmotic activity of niosomes are important in determining their use as controlled-release drug-delivery systems. These properties have been compared for polyhedral niosomes prepared by hydrating a mixture of a hexadecyl diglycerol ether (C16G2), a poly(24)oxyethylene cholesteryl ether (Solulan C24), 91:9 or 98:2, and conventional spherical niosomes prepared from the same surfactants but with cholesterol. When subjected to osmotic gradients, polyhedral niosomes, the membranes of which are in the gel phase, swell and shrink less than their spherical counterparts and they are more permeable to the hydrophilic solute 5(6)-carboxyfluorescein. In 2 M NaCl the rate of release of carboxyfluorescein from polyhedral niosomes (both containing 9% Solulan C24) into either a hypotonic (water) or an isotonic medium (2 M NaCl) was low. This contrasted with similarly loaded spherical niosomes and polyhedral niosomes containing 2% Solulan C24, from which release was high in hypotonic media (e.g. water) but less in an isotonic medium (2 M NaCl). For both polyhedral and spherical niosomes encapsulating carboxyfluorescein (pKa = 6.4), release rates were higher at pH 8 than at pH 5. Polyhedral niosomes are thus, in general, less osmotically active than spherical niosomes because of their rigid but highly permeable membranes. The unusual polyhedral membrane impermeability to carboxyfluorescein co-entrapped with salt in hypotonic media is a function of Solulan C24 content, and is possibly a result of salting out of the polyoxyethylene chains; this is, therefore, a property that might be manipulated in the design of a drug-delivery system.

In vitro/in vivo characterisation of polyhedral niosomes.

Non-ionic surfactant vesicles (niosomes) formed by a hexadecyl diglycerol ether (C16G2) and a series of polyoxyethylene alkyl ethers exhibit a variety of shapes dependent on their membrane composition. These surfactants form with an equimolar amount of cholesterol a mixture of largely spherical and tubular niosomes. In the absence of cholesterol, they form faceted polyhedral structures. The physicochemical and biological differences between polyhedral and spherical/tubular niosomes were studied. Polyhedral niosomes undergo a reversible shape transformation into spherical structures on heating above their phase transition temperature (Tm). The viscosity of polyhedral niosomes at room temperature is higher than their spherical counterparts due to their faceted and relatively rigid shape, and is more dependent on temperature due to shape transformation. At room temperature, polyhedral niosomes possess more rigid gel phase membranes and are less osmotically sensitive; however, they are more permeable because of a lack of or low levels of cholesterol in their membranes. Polyhedral niosomes loaded with luteinising hormone releasing hormone (LHRH), nonetheless, slow the release of drug compared to solution, albeit to a small extent.

Preparation and in vitro/in vivo evaluation of luteinizing hormone releasing hormone (LHRH)-loaded polyhedral and spherical/tubular niosomes.

Niosomes are vesicles formed by the self-assembly of nonionic surfactants in aqueous dispersions. They can entrap drugs and have been used experimentally as sustained drug delivery systems. Apart from conventional spherical niosomes, various types of vesicle ultrastructures can be formed by varying the composition of the vesicle membrane. Hexadecyl diglycerol ether (C16G2), cholesterol, and poly-24-oxyethylene cholesteryl ether (Solulan C24) in the ratio 91:0:9 gave polyhedral niosomes, whereas spherical and tubular niosomes are produced at a composition ratio of 49:49:2. The mean size of both polyhedral and spherical/tubular niosomes were within the range of 6 to 9 microm. Both types of vesicle were visualized by cryo-scanning electron microscopy. The properties of the two forms of niosomes were studied using luteinizing hormone releasing hormone (LHRH) as a model peptide. Analysis by high-performance liquid chromatography demonstrated high entrapment of LHRH acetate in polyhedral niosomes when prepared by remote loading methods using pH or (NH4)2SO4 gradients; in contrast, only low entrapment was achieved by passive loading methods (direct hydration at pH 7.4 or pH 3.0, dehydration-rehydration, and reversed-phase evaporation). In vitro studies demonstrated that both polyhedral and spherical/tubular niosomes were more stable in 5% rat skeletal muscle homogenate than in rat plasma. Also, polyhedral niosomes released more radiolabeled LHRH ([125I]LHRH) than spherical/tubular niosomes in both muscle homogenate and plasma. In clearance experiments in the rat, following intramuscular injection, both polyhedral and spherical/tubular niosomes gradually released [125I]LHRH into the blood, but some radioactivity remained at the injection site for 25 and 49 h, respectively. In contrast, [125I]LHRH in phosphate buffered saline was completely cleared from the injection site at 2 h. The release of drug is sustained by both niosome formulations, but spherical/tubular niosomes possess more stable membranes than polyhedral niosomes due to the presence of cholesterol.

Polymeric chitosan-based vesicles for drug delivery.

A simple carbohydrate polymer glycol chitosan (degree of polymerization 800 approx.) has been investigated for its ability to form polymeric vesicle drug carriers. The attachment of hydrophobic groups to glycol chitosan should yield an amphiphilic polymer capable of self-assembly into vesicles. Chitosan is used because the membrane-penetration enhancement of chitosan polymers offers the possibility of fabricating a drug delivery system suitable for the oral and intranasal administration of gut-labile molecules. Glycol chitosan modified by attachment of a strategic number of fatty acid pendant groups (11-16 mol%) assembles into unilamellar polymeric vesicles in the presence of cholesterol. These polymeric vesicles are found to be biocompatible and haemocompatible and capable of entrapping water-soluble drugs. By use of an ammonium sulphate gradient bleomycin (MW 1400), for example, can be efficiently loaded on to these polymeric vesicles to yield a bleomycin-to-polymer ratio of 0.5 units mg(-1). Previously polymers were thought to assemble into vesicles only if the polymer backbone was separated from the membrane-forming amphiphile by a hydrophilic side-arm spacer. The hydrophilic spacer was thought to be necessary to decouple the random motion of the polymer backbone from the ordered amphiphiles that make up the vesicle membrane. However, stable polymeric vesicles for use in drug delivery have been prepared from a modified carbohydrate polymer, palmitoyl glycol chitosan, without this specific architecture. These polymeric vesicles efficiently entrap water-soluble drugs.