Katarina Edwards

Chitosan as a nonviral gene delivery system. Structure–property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo

Chitosan is a natural cationic linear polymer that has recently emerged as an alternative nonviral gene delivery system. We have established the relationships between the structure and the properties of chitosan-pDNA polyplexes in vitro. Further, we have compared polyplexes of ultrapure chitosan (UPC) of preferred molecular structure with those of optimised polyethylenimine (PEI) polyplexes in vitro and after intratracheal administration to mice in vivo. Chitosans in which over two out of three monomer units carried a primary amino group formed stable colloidal polyplexes with pDNA. Optimized UPC and PEI polyplexes protected the pDNA from serum degradation to approximately the same degree, and they gave a comparable maximal transgene expression in 293 cells. In contrast to PEI, UPC was non toxic at escalating doses. After intratracheal administration, both polyplexes distributed to the mid-airways, where transgene expression was observed in virtually every epithelial cell, using a sensitive pLacZ reporter containing a translational enhancer element. However, the kinetics of gene expression differed – PEI polyplexes induced a more rapid onset of gene expression than UPC. This was attributed to a more rapid endosomal escape of the PEI polyplexes. Although this resulted in a more efficient gene expression with PEI polyplexes, UPC had an efficiency comparable to that of commonly used cationic lipids. In conclusion, this study provides insights into the use of chitosan as a gene delivery system. It emphasises that chitosan is a nontoxic alternative to other cationic polymers and it forms a platform for further studies of chitosan-based gene delivery systems.

Cryo transmission electron microscopy of liposomes and related structures

Abstract Cryo-transmission electron microscopy, c-TEM, has during the last 10 years contributed significantly to the understanding of the numerous, and often complex, structures formed by amphiphilic molecules in dilute aqueous solutions. In particular, the method has evolved as an important tool for the investigation of liposomes. In this review, we discuss and show examples of how the technique has been utilised to gain new information on the form and structure of liposomes, as well as on the morphological changes taking place upon encapsulation of drugs or interactions with surfactants, DNA and other polyelectrolytes. Several examples where c-TEM has been successfully employed to visualise related amphiphilic structures, such as thread-like micelles and particles of reversed phases, are also presented. In addition, we discuss recent developments concerning sample preparation and interpretation of images, as well as possible artefacts and their origin.

Liposomes, Disks, and Spherical Micelles: Aggregate Structure in Mixtures of Gel Phase Phosphatidylcholines and Poly(Ethylene Glycol)-Phospholipids

Poly(ethylene glycol) (PEG) decorated lipid bilayers are widely used in biomembrane and pharmaceutical research. The success of PEG-lipid stabilized liposomes in drug delivery is one of the key factors for the interest in these polymer/lipid systems. From a more fundamental point of view, it is essential to understand the effect of the surface grafted polymers on the physical-chemical properties of the lipid bilayer. Herein we have used cryo-transmission electron microscopy and dynamic light scattering to characterize the aggregate structure and phase behavior of mixtures of PEG-lipids and distearoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The PEG-lipids contain PEG of molecular weight 2000 or 5000. We show that the transition from a dispersed lamellar phase (liposomes) to a micellar phase consisting of small spherical micelles occurs via the formation of small discoidal micelles. The onset of disk formation already takes place at low PEG-lipid concentrations (<5 mol %) and the size of the disks decreases as more PEG-lipid is added to the lipid mixture. We show that the results from cryo-transmission electron microscopy correlate well with those obtained from dynamic light scattering and that the disks are well described by an ideal disk model. Increasing the temperature, from 25 degrees C to above the gel-to-liquid crystalline phase transition temperature for the respective lipid mixtures, has a relatively small effect on the aggregate structure.

Effect of polyethyleneglycol-phospholipids on aggregate structure in preparations of small unilamellar liposomes

Phospholipids with covalently attached poly(ethylene glycol) (PEG lipids) are commonly used for the preparation of long circulating liposomes. Although it is well known that lipid/PEG-lipid mixed micelles may form above a certain critical concentration of PEG-lipid, little is known about the effects of PEG-lipids on liposome structure and leakage at submicellar concentrations. In this study we have used cryogenic transmission electron microscopy to investigate the effect of PEG(2000)-PE on aggregate structure in preparations of liposomes with different membrane compositions. The results reveal a number of important aggregate structures not documented before. The micrographs show that enclosure of PEG-PE induces the formation of open bilayer discs at concentrations well below those where mixed micelles begin to form. The maximum concentration of PEG-lipid that may be incorporated without alteration of the liposome structure depends on the phospholipid chain length, whereas phospholipid saturation or the presence of cholesterol has little or no effect. The presence of cholesterol does, however, affect the shape of the mixed micelles formed at high concentrations of PEG-lipid. Threadlike micelles form in the absence of cholesterol but adapt a globular shape when cholesterol is present.

Formation of transition metal–doxorubicin complexes inside liposomes

Doxorubicin complexation with the transition metal manganese (Mn(2+)) has been characterized, differentiating between the formation of a doxorubicin-metal complex and doxorubicin fibrous-bundle aggregates typically generated following ion gradient-based loading procedures that rely on liposome encapsulated citrate or sulfate salts. The physical and chemical characteristics of the encapsulated drug were assessed using cryo-electron microscopy, circular dichroism (CD) and absorbance spectrophotometric analysis. In addition, in vitro and in vivo drug loading and release characteristics of the liposomal formulations were investigated. Finally, the internal pH after drug loading was measured with the aim of linking formation of the Mn(2+) complex to the presence or absence of a transmembrane pH gradient. Doxorubicin was encapsulated into either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol (Chol) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Chol liposomes, where the entrapped salts were citrate, MnSO(4) or MnCl(2). In response to a pH gradient or a Mn(2+) ion gradient, doxorubicin accumulated inside to achieve a drug-to-lipid ratio of approximately 0.2:1 (wt/wt). Absorbance and CD spectra of doxorubicin in the presence of Mn(2+) suggested that there are two distinct structures captured within the liposomes. In the absence of added ionophore A23187, drug loading is initiated on the basis of an established pH gradient; however, efficient drug uptake is not dependent on maintenance of the pH gradient. Drug release from DMPC/Chol is comparable regardless of whether doxorubicin is entrapped as a citrate-based aggregate or a Mn(2+) complex. However, in vivo drug release from DSPC/Chol liposomes indicate less than 5% or greater than 50% drug loss over a 24-h time course when the drug was encapsulated as an aggregate or a Mn(2+) complex, respectively. These studies define a method for entrapping drugs possessing coordination sites capable of complexing transition metals and suggest that drug release is dependent on lipid composition, internal pH, as well as the nature of the crystalline precipitate, which forms following encapsulation.

Phospholipid membrane permeability of peptide nucleic acid.

Phospholipid vesicles (liposomes) as membrane models have been used to study the penetration properties of peptide nucleic acid (PNA), a new DNA analog in which the nucleobases are attached to a pseudo‐peptide backbone. The liposomes were characterised by carboxyfluorescein efflux, light‐scattering and cryogenic transmission electron microscopy. The liposome structure was found not to be affected by the incorporation of PNA or an oligonucleotide. Two 10‐mer fluorescein‐labelled PNAs were found to have low efflux rates (half‐times of 5.5 and 11 days), comparable to a 10‐mer oligonucleotide (half‐time of 7 days). We conclude that passive diffusion of unmodified PNA is not an effective way of transport into biological cells.

Physiochemical characterization of the Alzheimer's disease‐related peptides Aβ1–42Arctic and Aβ1–42wt

The amyloid β peptide (Aβ) is crucial for the pathogenesis of Alzheimers disease. Aggregation of monomeric Aβ into insoluble amyloid fibrils proceeds through several soluble Aβ intermediates, including protofibrils, which are believed to be central in the disease process. The main reason for this is their implication in familial Alzheimers disease with the Arctic amyloid precursor protein mutation (E693G). This mutation gives rise to early onset Alzheimers disease, and synthetic Aβ1–40Arctic displays an enhanced rate of protofibril formation in vitro[Nilsberth C, Westlind‐Danielsson A, Eckman CB, Condron MM, Axelman K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG, Naslund J & Lannfelt L. (2001) Nat Neurosci4, 887–893]. To increase our understanding of the mechanisms involved in Aβ aggregation, especially Aβ monomer oligomerization into protofibrils and protofibril fibrillization into fibrils, the kinetics of Aβ1–42wt and Aβ1–42Arctic aggregation were examined under different physiochemical conditions, such as concentration, temperature, ionic strength and pH. We used size exclusion chromatography for this purpose, where monomers are separated from protofibrils, and fibrils are separated from protofibrils in a centrifugation step. The Arctic mutation significantly accelerated both Aβ1–42wt protofibril formation and protofibril fibrillization. In addition, we demonstrated that two distinct chemical processes – monomer oligomerization and protofibril fibrillization – were affected differently by changes in the micro‐environment and that the Arctic mutation alters the peptide response to such changes.

Solubilization of lecithin vesicles by C12E8 : structural transitions and temperature effects

Abstract The structural transitions induced by addition of the nonionic surfactant C 12 E 8 (octaethylene glycol n -dodecyl monoether) to small unilamellar lecithin vesicles has been studied by means of static and dynamic light scattering as well as with cryo-transmission electron microscopy. At low surfactant concentration a slight swelling of the vesicles, due to the incorporation of surfactant monomers into the vesicle membrane, can be seen. Large unilamellar vesicles begin to form as the surfactant concentration exceeds 30 mol%. The vesicle size increases with C 12 E 8 concentration until about 40 mol% has been added. At this point the intensity of light scattered at 90° starts to decrease and dynamic light scattering indicates bimodal size distributions. The cryo-TEM micrographs show cylindrical micelles in coexistence with bilayer sheets. At C 12 E 8 concentrations above 70 mol%, the lipid vesicles are completely solubilized into mixed micelles that gradually become spherical and decrease in size with surfactant concentration. Depending on the C 12 E 8 concentration the formation of the lipid/surfactant mixed aggregates may take from less than a second to more than an hour. Increasing the temperature shifts the lipid/surfactant molar ratios at which the different amphiphilic structures start to appear toward lower values.

Effects of PEG-lipids on permeability of phosphatidylcholine/cholesterol liposomes in buffer and in human serum.

The permeability of liposomal membranes was studied as a function of the amount of incorporated PEG-lipid. The fluorescent dyes ethidium, propidium and 5(6)-carboxy fluorescein were used as markers for measurements of spontaneous leakage. The results show that addition of up to 8 mol% of PEG(2000)-DSPE into liposomal membranes of DSPC/Cho and EPC/Cho reduces the permeability of carboxyfluorescein in buffer solution. In contrast, the leakage of the more amphiphilic dye ethidium was not to any measurable extent affected by PEG-lipid inclusion. Another important difference was that ethidum leakage showed a clear dependence on temperature whereas leakage of carboxyfluorescein from pegylated liposomes did not. We conclude that the mechanisms by which the two dyes permeate the liposomal bilayer are qualitatively different. Both ethidium and carboxyfluorescein did interact with human serum components in a way that made measurements in serum unreliable. The more hydrophilic ethidium analogue propidium was shown not to interact with human serum components to any detectable extent. This made propidium suitable for permeability determinations in human serum. It was found that liposomes composed of pure EPC or EPC with 5 mol% DSPE-PEG, displayed a dramatic increase in permeability when subjected to a medium composed of 20% human serum in buffer. Addition of 40 mol% cholesterol to the EPC bilayers reduced the observed release rate in human serum substantially, whereas no stabilizing effect was observed upon PEG-lipid inclusion.

Formation of drug-arylsulfonate complexes inside liposomes: a novel approach to improve drug retention.

The development of procedures to enhance drug retention in liposomes is important in order to achieve therapeutically optimized rates of drug release from liposomal carriers. In this study, the ability of lipophilic weak base drugs to complex with arylsulfonates resulting in formation of intravesicular precipitates is investigated as a means to enhance drug retention. It is shown that the arylsulfonates benzenesulfonate and hydroxybenzenesulfonate (HBS) induce precipitation of ciprofloxacin and vinorelbine, two representative weak base drugs that are difficult to retain in liposomal systems. The most complete precipitation was observed at pH values corresponding to charge neutralization of the drug-arylsulfonate complex. HBS is shown to be a much more effective precipitating agent than benzenesulfonate. It is also shown that vinorelbine and ciprofloxacin can be loaded into large unilamellar vesicles (LUV) containing the calcium salt of HBS using an ionophore-based loading method. Following drug loading, the formation of intravesicular drug-arylsulfonate precipitates of vinorelbine and ciprofloxacin was observed by cryo-electron microscopy. In vitro release experiments showed substantial improvements in drug retention for both vinorelbine and ciprofloxacin when HBS was present as compared to standard loading procedures employing MgSO4 as the entrapped solute. In vivo release experiments for vinorelbine in NuNu mice indicated a half-time for release for HBS-containing LUV of approximately 30 h, compared to 6.4 h for LUV loaded employing MgSO4. It is suggested that encapsulation procedures employing HBS in the internal medium can improve the retention of drugs that are difficult to retain in liposomes, possibly leading to enhanced therapeutic properties.