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We report on the combined magnetization and electron paramagnetic resonance characterization of a novel Ti‐O organic–inorganic gel hybrid and the related electron–hole generation process upon UV illumination. We find that electrons are injected into the conduction band of the Ti‐O framework, photoreducing TiIVto TiIII. TiIIIsites are mainly located on the surface, owing to the nanometric dimensions of the inorganic component. Surprisingly, the symmetry of the TiO6octahedra depends on the level of illumination: in the lightly UV‐exposed samples TiIIIis sited in the weakly distorted TiO6octahedra to which methanoate groups are bonded, as suggested by electron spin echo envelope modulation (ESEEM) experiments. Extensive illumination causes structural rearrangements, leading to enhanced tetragonal TiO6distortion and shifting the TiIIIinteraction towards the hydroxide groups or water. The results provide clear evidence for an interfacial charge transfer between the quantum‐size TiO lattice and coordinated species upon in situ and ex situ UV illumination at temperatures from room temperature to 5 K.

Copper doped sodium titanate nanotubes and nanoribbons were prepared viatwo different doping methods called in situand ex situ. In the applied in situmethod, titanate nanotubes were grown from anatase TiO 2doped with Cu 2+, while in the ex situmethod, titanate nanotubes and nanoribbons were exposed to aqueous solution of Cu 2+species. By correlating XRD, electron microscopy, magnetic susceptibility, and electron paramagnetic resonance (EPR) measurements, we found that in the samples prepared viathe ex situdoping method, sub-10 nm CuO nanoparticles grow on the inner/outer surface of nanotubes/nanoribbons where the Neel transition temperature is strongly suppressed. In fact, they behave as superparamagnets with a blocking temperature of around 50 K. Strong evidence that Cu 2+species that form complexes between titanate layers coordinate with Na +ions comes from the pulsed EPR data.

We report on the synthesis of titania-based nanotubes and nanoribbons prepared by hydrothermal methods, as well as evaluating their structure and adsorption properties. Nanotubes were found to be hollow scrolls with a typical outer diameter of about 10 nm, inner diameter 4−5 nm and length of several hundred nm. The nanoribbons are highly crystalline, typically 40 nm in cross-section and up to a few μm in length. All these samples were exposed to a NO2 atmosphere in order to test their adsorption properties using electron paramagnetic resonance (EPR). The nanotubes were found to have high active surface areas of about 250 m²/g. When exposed to NO2 gas, NO2 molecules tend to physisorb on the nanotube surface via the nonbonding py orbital of the oxygen atoms. In contrast nanoribbons have much smaller active surface areas (~30 m²/g). The dominant EPR signal now comes from NO molecules adsorbed on Na⁺ ions. These results show that titania-based nanotubes and nanoribbons may represent an attractive option for the removal and catalysis of nitrogen oxides in future.

Hybrid organic−inorganic layered perovskites (RnNH3)2MnCl4 of lyophilic aliphatic cations RnNH3⁺ (Rn = CnH2n+1; n = 2, 9) have been studied for their potential as functional additives in poly(dimethylsiloxane) (PDMS). The evolution of the microstructure for various nanocomposite assemblies was characterized by means of X-ray diffraction and electron microscopy. Composites consisted of exfoliated MnCl4 layers and enhanced aspect ratio crystallites composed of bundles of a few MnCl4 sheets have been prepared. Such local morphology characteristics give rise to an inhomogeneous microstructure with enhanced surface areas and pronounced structural defects, suggestive of partial polymer intercalation only for the longer chain systems. The degree to which the two chemical realms interact at the interfacial regions does not affect the static magnetic properties. The composites display two-dimensional Heisenberg antiferromagnetic behavior (TN ≤ 42 K) similar to that in bulk powders. Electron paramagnetic resonance finds that the low-temperature spin dynamics of dispersed MnCl4 layers are determined by the magnons and static solitons as in bulk materials. On the other hand, size-dependent features and local scale imperfections in the morphology, arising from the composite formation, soften the low-energy magnetic excitations of the pinned solitons. The interaction of PDMS and organocation chains affects the dynamics and the orientational ordering of the longer, linear alkylammonium molecules.

We studied lipid aggregate penetration through nanoporous, semipermeable barriers by direct transport measurements in vitro and with the confocal laser scanning microscopy of the skin in vivo. We found that it is necessary to use mixed lipid bilayers with a low resistance to permeabilization and high flexibility to overcome narrow, normally confining pores. Partial molecular demixing in the stressed vesicle bilayer serves both purposes. An aggregate comprising a suitable blend of amphipats (Transfersome, Tfs) is, therefore, extremely deformable and easily crosses even very narrow pores (rTfs ≥ 10rpore, and possibly more). Each such vesicle then behaves as a responsive, self-optimizing, nanorobotic transport device. The mixed micelles with identical components or the simple vesicles (liposomes) with a similar size as that of unusually deformable vesicles do not share this quality. Liposomes only traverse barriers when rlipos ≤ 1.5rpore; they clog narrower pores, unless they get fragmented in/before the orifice. Mammalian skin is perforated by a very large number (≥10⁷ cm^-2) of very narrow (rpore ~ 0.3 nm) intercellular hydrophilic pores. These can be widened into the barrier-spanning, hydrophilic transcutaneous pathways (rpathway ~ 20−30 nm) by ultradeformable vesicles. Mixed micelles or liposomes do not activate such pores because they are respectively too small or too undeformable (κlipos > 10κTfs) and large (2rlipos/nm ≥ 45 ≫ 20) for the purpose. The outer two-thirds of the skin barrier also contain fewer but wider openings (rpore ≥ 3 μm), which encircle groups of cells in the stratum corneum. The resulting sparse, low-resistance intercluster pathway can accommodate various sufficiently small aggregates (ra ≤ 2 μm), including liposomes and micelles. All the tested lipidic particles can, therefore, reach locally ~60% of the skin barrier depth, on the average. Ultradeformable vesicles move through the skin most uniformly and to the greatest relative depth, however. Locally or near the skin surface the distribution of different lipid aggregates that penetrate a barrier can be similar.

We studied aggregate transport through semipermeable, nano-porous barriers experimentally and theoretically. By measuring and modeling the effect of hydration gradient across such barriers, spontaneous transbarrier transport of suitable lipid aggregates in vesicular form was proven to be driven by partial aggregate dehydration at the application site. By generalizing the Onsager transport model we derived a set of equations that rationalize all pertinent observations. Dehydration-induced vesicle motion starts with a lag time. This corresponds to the time needed to reach the limiting vesicle hydration; both are proportional to the starting excess water volume and decrease with increasing relative humidity at application site. The rate of transbarrier transport is insensitive to these parameters but increases with vesicle deformability and volume exchange capability. Both these properties depend on membrane composition. Reversible demixing of bilayer components is the cause of nonlinear bilayer characteristics and also potentially affects the effective membrane hydrophilicity. High hydrophilicity of vesicle surface and extreme aggregate shape adaptability together are necessary for successful material transport across the skin. This demonstrates the significance of basic biophysical investigations for better understanding of biological systems and for the practical use of artificial, nature-inspired carriers in drug delivery.

The stability of various aggregates in the form of lipid bilayer vesicles was tested by three different methods before and after crossing different semi-permeable barriers. First, polymer membranes with pores significantly smaller than the average aggregate diameter were used as the skin barrier model; dynamic light scattering was employed to monitor vesicle size changes after barrier passage for several lipid mixtures with different bilayer elasticities. This revealed that vesicles must adapt their size and/or shape, dependent on bilayer stability and elasto-mechanics, to overcome an otherwise confining pore. For the mixed lipid aggregates with highly flexible bilayers (Transfersomes®), the change is transient and only involves vesicle shape and volume adaptation. The constancy of ultradeformable vesicle size before and after pores penetration proves this. This is remarkable in light of the very strong aggregate deformation during an enforced barrier passage. Simple phosphatidylcholine vesicles, with less flexible bilayers, lack such capability and stability. Conventional liposomes are therefore fractured during transport through a semi-permeable barrier; as reported by other researchers, liposomes are fragmented to the size of a narrow pore if sufficient pressure is applied across the barrier; otherwise, liposomes clog the pores. The precise outcome depends on trans-barrier flux and/or on relative vesicle vs. pore size. Lipid vesicles applied on the skin behave accordingly. Mixed lipid vesicles penetrate the skin if they are sufficiently deformable. If this is the case, they cross inter-cellular constrictions in the organ without significant composition or size modification. To prove this, we labelled vesicles with two different fluorescent markers and applied the suspension on intact murine skin without occlusion. The confocal laser scanning microscopy (CLSM) of the skin then revealed a practically indistinguishable distribution of both labels in the stratum corneum, corroborating the first assumption. To confirm the second postulate, we compared vesicle size in the starting suspension and in the blood after non-invasive transcutaneous aggregate delivery. Size exclusion chromatograms of sera from the mice that received ultradeformable vesicles on the skin were undistinguishable from the results measured with the original vesicle suspension. Taken together, the results support our previous postulate that ultradeformable vesicles penetrate the skin intact, that is, without permanent disintegration.

The longitudinal relaxation rate of the first stable alanine radical, SAR1, was studied by employing pulse EPR technique over a wide temperature interval (5–290 K). The complex nonexponential recovery of the longitudinal magnetization in this temperature interval has been described with two characteristic relaxation times, 1/T*1aas the faster component and 1/T*1bas the slower component, respectively. It was shown that 1/T*1ais strongly affected by the CH3group dynamics of the SAR1 center. The complete temperature dependence of 1/T*1awas described by invoking several relaxation mechanisms that involve hindered motion of the CH3group from classical rotational motion to coherent rotational tunneling. It was shown that all relevant relaxation mechanisms are determined by a single correlation time with the potential barrier (ΔE/k=1570 K). On the other hand the temperature dependence of 1/T*1bis related to the motional dynamics of the neighborly NH3and CH3groups. We found a larger average potential barrier for this motion (ΔE/k=2150 K) corresponding to smaller tunneling frequencies of the neighbor groups.

We have investigated the molecular conformations of a lipopolymer with a polyoxazoline headgroup at air/water interfaces as a function of lateral area per molecule with X-ray and neutron reflectometry. The polymer 1,2-dioctadecanyl-sn-glycero-3-poly(2-methyl-2-oxazoline), PMO−(C18)2, forms stable surface monolayers. Pressure/area isotherms around room temperature show a plateau region, indicative of a phase transition whose origin was examined. For data evaluation, a novel approach was used that acts on explicit quasi-molecular ensemble conformations of the polymer [Politsch et al., preceding paper in this issue]. At lower surface pressure, the polymer density distribution exhibits a maximum near the interface, indicative of attractive interaction between the predominantly hydrophilic polymer chains and the hydrophobic surface. Across the plateau region of the isotherm, a change in the volume density distribution of the alkyl chains was observed which is indicative of a partial immersion of the lipid moieties into the aqueous subphase. In contrast, no major structural change across the phase transition was detected in the polymer volume density profiles which comply with scaling predictions at both sides of the phase transition if deviations due to nonidealities are neglected. We interpret these observations as an alkyl chain ordering induced by the steric interference between the PMO:  Immersion of alkyl chains into the subphase relaxes the strain on the hydrophobic anchors which derives from a reduction of the configurational entropy of the PMO chains due to their confinement to the interface.

We present a new data refinement technique for the evaluation of X-ray and neutron reflectometry measurements of interface layers comprising linear molecules. The new method is particularly well-suited&sbd;but not limited to&sbd;modeling the structure of surface-grafted linear polymers organized in “polymer brushes”. In this paper, we discuss technical details of the data inversion technique and demonstrate its capabilities by recovering the structure of a simulated molecular ensemble for which synthetic data sets have been constructed. In the following paper, we utilize this machinery to reveal the nature of a phase transition pertinent to surface-anchored lipopolymers at high molecular densities.

Abstract

Cryoelectron microscopy has been used to study the reorganization of unilamellar cationic lipid vesicles upon the addition of DNA. Unilamellar DNA-coated vesicles, as well as multilamellar DNA lipid complexes, could be observed. Also, DNA induced fusion of unilamellar vesicles was found. DNA appears to adsorb to the oppositely charged lipid bilayer in a monolayer of parallel helices and can act as a molecular “glue” enforcing close apposition of neighboring vesicle membranes. In samples with relatively high DNA content, there is evidence for DNA-induced aggregation and flattening of unilamellar vesicles. In these samples, multilamellar complexes are rare and contain only a small number of lamellae. At lower DNA contents, large multilamellar CL-DNA complexes, often with >10 bilayers, are formed. The multilamellar complexes in both types of sample frequently exhibit partially open bilayer segments on their outside surfaces. DNA seems to accumulate or coil near the edges of such unusually terminated membranes. Multilamellar lipid-DNA complexes appear to form by a mechanism that involves the rupture of an approaching vesicle and subsequent adsorption of its membrane to a “template” vesicle or a lipid-DNA complex.

The dynamics of the structural changes in γ-irradiated ammonium dihydrogen arsenate (ADA) on going from the paraelectric to the antiferroelectric phase has been studied using the AsO4-4 centre as a probe. Evidence was found for the existence of fully ordered low temperature clusters which can be considered as a local manifestation of the central mode. The lifetime of these clusters, which are highly mobile, is longer than 10-6 sec. No evidence was found, however, for the ferroelectric Slater "top" and "bottom" configurations besides the four antiferroelectric ones.

We describe the synthesis of lipopolymers suitable for conformation and mobility studies by neutron scattering methods and 2H-NMR spectroscopy. Starting with 2-methyl-2-oxazoline MeOx monomers, we developed two synthetic routes for the preparation of 2-trideuteromethyl-2-oxazoline D3-MeOx. Both rely on cheap and easily available deuterated sources, but the yield of the products and their degree of deuteration as well as efficiency differ considerably. Using a lipid initiator, we efficiently introduced the lipid end-group into the polymer. Sequential addition of deuterated and non-deuterated monomers led to the segmentally deuterated poly(acetyliminoethylene) lipopolymers [diblock or triblock copolymers consisting of poly(acetyliminoethylene) and poly(trideuteroacetyliminoethylene)]. We also achieved good control of the position and length of the blocks with a narrow molecular mass distribution. Two sets of substances were synthesized differing in molecular mass and degree of deuteration. The physical properties of these substances are described separately.

Solvated phospholipids exhibit a rich polymorphism which depends not only on the lipid chemical structure but also on the characteristics of the bathing solution [1]. This is due to the strong and influential interactions between the lipid headgroups and the solvent or solute molecules. Particularly efficient in this respect are various cationic molecules or molecules with a high propensity to hydrogen bond formation which all bind avidly to the lipid phosphate groups. The di- and polyvalent cations, for example, are electrostatically attracted by the negative electronic cloud around the (typically ionized) PO4--group; protons donating or accepting molecules, such as the amino-compounds or the molecules with the readily accessible OH-residues, bind directly to the OH-groups on the phosphates via H-bonds. Water and various alcohols are the most prominent examples for this latter type of interaction.

Pyroelectric infrared detectors are efficient and cheap sensors for many applications. In order to acquire a common optic mode rejection capability, the double serial or parallel construction of active electrodes has been employed. The latter type is difficult to construct due to connection between the electrodes. The usual solution is to mount the two separate thin crystalline active elements on the separate holders. This technique lacks uniformity and precise cancellation of common mode. We have found the way to construct and fabricate the double parallel detector formed on a single piece of thin pyroelectric material. In this paper we describe the technology as well as the results of the detector parameter measurements.

Using the large (≈ 100 GHz) hyperfine coupling of the Tl2+ (6s1) ion as a spin probe, we have investigated the evolution of glassy behavior in (NH4) xRb1-xH2 PO4 single crystals. Also reported is a microwave dielectric loss determination of the phase diagram of (ND4) xRb1-xD2AsO4. The glass formation temperature, Tc, is found to be significantly higher and the glass phase range narrower in the deuterated arsenate system than in the corresponding protonated arsenate system. Both of these phenomena are tentatively interpreted in terms of a random bond version of the Ising model in a transverse tunneling field.

Thermolabile fusogenic liposomes were devised based on the stoichiometric 1/2 mixtures of dipalmitoylphosphatidylcholine (DPPC) and elaidic acid (ELA) and from the similar stoichiometric mixtures of DPPC, dipalmitoylphosphatidylglycerol (DPPG) and elaidoyl alcohol (EL-OH) or palmitelaidoyl alcohol (PEL-OH). The resulting vesicle suspensions are fusogenic in the region of hyperthermia (≥ 42°C) and can be targeted selectively to the heated tumor tissue. Incorporation of DPPG or fatty alcohols into the vesicle membranes also leads to a non-specific, temporary vesicle material accumulation in the lung, however, probably due to platelet activation. Vesicle material accumulation in A-431 tumors, xenotransplanted in nude mice, after 30 min of local hyperthermia (42°C) is 4-fold higher for the DPPC/ELA (1/2), 2.8-fold higher for the DPPC/DPPG/EL-OH (0.8/0.2/2) and 3.7-fold higher for the DPPC/ELA/EL-OH (1/1/1) mixtures than for similar vesicles used at the physiological temperature. Extension of hyperthermia to 60 min induces a 7.8-fold relative material accumulation in the tumor tissue when the thermolabile, fusogenic DPPC/ELA/EL-OH (1/1/1) vesicles are used. Simple DPPC vesicles only reach concentrations in the heated tumor or muscle tissue that are 1.85-fold and 1.38-fold higher than in the normothermic control, respectively. This is probably a consequence of simple vasodilatation. In vitro experiments revealed that the adsorption of serum proteins to the vesicle membrane decreases the chain-melting phase transition temperature and the transition enthalpy of vesicle suspension. Adsorption is most prominent at the chain-melting phase transition temperature of the mixed lipid bilayers, which is also the critical temperature for the induction of liposome fusion. This hampers the practical use of the resulting vesicle suspensions in vivo. The serum-induced decrease of the chain-melting phase transition temperature, which is likely to change as a function of time in vivo depends on the lipid composition and on the local surface charge density of vesicles. Incorporation of ELA and DPPG concentrations above 15 mol-%, for example, reduce the extent of protein adsorption onto vesicles. This has to be borne in mind when devising vesicles for practical applications.

Based on the concept of rational membrane design it is proposed how to develop liposomes suitable for the targeting in vitro and in vivo. Such targeting can rely on the chemical or physical 'pointers'. Hyperthermia is a particularly convenient example of the latter, suggesting that thermolabile fusogenic liposomes should be devised and used for the agent delivery into cells. The resulting efficacy depends on the extent to which the phase characteristics of the fusogenic lipid membrane are made to fit the delivery-triggering signal: increasing the temperature and/or lowering the pH-value in the investigated system are most useful for this purpose, the role of membrane defects, such as may arise during the liposome manufacturing process or in the vicinity of lipid phase transitions also being of extreme importance. Thermolabile fusogenic liposomes prepared from the stoichiometric 1/2 mixture of diacylphos-?hatidylcholines with appropriate fatty acids implement these requirements. 'heir temperature dependence permitting physical targeting and their pH-dependence being suitable for the 'chemical targeting' to the acidic surrounding. Dipalmitoylphosphatidylcholine/elaidic acid mixture is, perhaps, the best such combination for the use in vivo. The efficacy of fusion between the corresponding lipid vesicles and cells in vitro as well as the delivery of radioactive labels into the heated tumours (but not in the normal muscle tissue with the inpenetrable micro-vasculature) provide compelling evidence for this.

Liposomes made of dipalmitoylphosphatidylcholine (DPPC2), dipalmitoyl-phosphatidylglycerol (DPPG), and different long-chain fatty alcohols were investigated with respect to their colloidal stability, chain-melting phase transition temperature, and temperature dependent inter-vesicle fusion. In particular, the practical usefulness of the stoichiometric 1/2 (mol/mol) mixtures of the phospholipids and fatty alcohols, mainly elaidoyl alcohol (EL-OH) were studied. The mole fraction of DPPG in the bilayers of such vesicles affects crucially the colloidal stability of the resulting lipid suspensions; at least 15 mol-% of DPPG (relative to DPPC) must be incorporated into the bilayers in order to make the liposome suspension colloidally sufficiently stable at room temperature. The corresponding DPPC/DPPG/EL-OH (0.85/0.15/2) mixed lipid vesicles undergo a lamellar-gel to inverted hexagonal (HIT) phase transition at 52.7°C, however, and then fuse and aggregate massively. The related phase transition temperature of the DPPC/DPPG/palmitelaidoyl alcohol (0.85/0.15/2) mixture is 48.4°C. This indicates that the chain-melting phase transition temperature of the investigated lipid mixtures is rather sensitive to the alcohol chain-length. This transition temperature is independent, however, of the bulk proton concentration in the pH region between 4.9 and 7.2. Stoichiometric 1/2 mixtures of phospholipids and EL-OH have a high propensity for the inter-vesicle fusion at 42°C and neutral pH. The reason for such fusion 10°C below the lamellar-to-nonlamellar phase transition temperature are the defects that are generated during the chain-melting of the (partly segregated) phospholipid component at 42°C; the proximity of the lamellar to non-lamellar phase transition temperature of the phospholipid/fatty alcohol (1/2) complex at 52°C also plays an important role.

Statement of problem. Acid etching creates retentive microcraters on enamel surfaces. Designing of a partial denture often involves reshaping the supporting and retentive teeth by grinding the enamel. Unfortunately, both these procedures damage the enamel surface. In vivo such surface damage takes several months to recover. Purpose. This study evaluated the effect of 1-minute etching, prolonged etching, and grinding on the permeability of dental enamel for water-soluble molecules. Material and methods. With the electron paramagnetic resonance and a two-chamber diffusion cell, the influence of etching and grinding on the diffusion of spin label molecules through the enamel was studied quantitatively. The enamel permeability was measured in 30 sound enamel samples, of which 10 samples were exposed to 1-minute etching with 37% phosphoric acid, 10 samples were etched for 5 minutes, and 10 samples were ground with a diamond bur. Results and Conclusions. All procedures significantly increased the permeability of dental enamel. These results demonstrate that in vivo the acid-etched and ground dental enamel surfaces are less protected and consequently, unless the tooth is properly protected, are more susceptible to carious lesions. Therefore ground or accidentally etched enamel should be protected. (J Prosthet Dent 1997;77:578-82)

Electron paramagnetic resonance (EPR) measurements have been carried out over the frequency range of 1–370 GHz on single crystals of potassium peroxychromate (K3CrO8) with the view of examining the current models of exchange narrowing of EPR signals in solids. K3CrO8has a simple (tetragonal) lattice structure, can be grown as single crystals pure or diluted with an isostructural diamagnetic host K3NbO8, and its paramagnetism can be described by a very simple (S=12,I= 0) spin Hamiltonian. The measurements were made at various orientations of single crystals in the Zeeman field, with emphasis on the principal directions of theg-tensor. For essentially all orientations, the linewidth decreases monotonically for measurements at resonance frequencies, ω0, from 1 to about 100 GHz, and then starts to increase at higher ω0. In order to delineate the spin exchange effects from other sources of line broadening, the measurements were repeated with a diluted spin system, K3NbO8containing ≅0.5 mole % of K3CrO8, representing the broadening effect of all the magnetic field dependent terms, such as the broadening due to theg-strain and sample holder/waveguide magnetization at the high field utilized, up to 14 T. Using these data, the K3CrO8linewidths were analyzed in terms of the current models of spin exchange narrowing in three-dimensional systems. A reasonably good agreement was found with the Anderson–Weiss model, when modified for various line broadening effects. The accuracy of the analysis procedure was confirmed by the comparison of the presently determined values of the exchange constant,J,and the dipolar field,Hp,with their values obtained by dc magnetic susceptibility measurements and theoretical analysis, respectively; the agreement was within 5% forJ(=1.35 K) and about 25% forHp(160 G). However, some deviations and unusual splittings were noted in measurements at 370 GHz, whose origin remains unclear.

The specificity of association between monovalent ions and a hydrophilic surface is probably determined by solvent-mediated interactions, as indicated both by experimental data for lipid bilayer membranes and by a non-local electrostatic model. The strength of association between such ions and a charged polar surface decreases with ion radius, owing to the better dielectric screening of the electrostatic surface potential at great separations. For non-charged hydrophilic surfaces the trend is reversed, the selectivity now being governed by the free energy of partial ion dehydration.

DOCSPER—A Synthetic Lipid Fit for In Vivo ApplicationDOCSPER [1,3-Dioleoyloxy-2-(N5-carbamoyl-spermine)-propane] is a cationic amphiphile consisting of a hydrophobic 1,3 dioleylglycerol moiety and threefold positively charged spermine head group (1). We optimised the 5-step-synthesis of the lipospermine and after up-scaling we have obtained sufficient amounts to initiate preclinical investigations. DOCSPER was tested for its ability to transfect eukaryotic cells in vitro. It has proven to possess high transfection efficiency in comparison to commercially available liposomal transfection agents. Furthermore, DOCSPER was extensively tested in several in vivo studies (23). These studies revealed a high transfection efficiency, whereas very low toxicity levels were detected. Thus, the results clearly indicate that the cationic lipid DOCSPER is a reliable, low-risk system for broad applications in gene therapy.Groth D. et al. Int J Pharm 1998; 162:143–157.Nikol S. et al. Int J Angiol 2000; 9:87–95.Armeanu S. et al. Mol Ther 2000; 1(4):366–375.

Targeted Delivery of Antisense Oligodeoxynucleotides In Vivo by Means of Coated Cationic LipoplexesEarlier we reported on the massive uptake of liposomes surface-modified with negatively charged aconitylated albumin (Aco-HSA) by liver endothelial cells (EC) in vivo. In the present work we apply this principle for in vivo delivery of antisense oligodeoxynucleotides (ODN) to these cells by means of coated cationic lipoplexes (CCL) (1). CCL were prepared by complexing ODN with the cationic lipid DOTAP and subsequent coating of the complex by neutral lipids including a lipid-anchored poly(ethylene glycol). Aco-HSA was covalently coupled.The Aco-HSA-CCLs were 160 nm in size, contained 1.03 ± 0.35 nmol ODN and 54 ± 18 µg Aco-HSA per µ mol total lipid. The Aco-HSA-CCLs were rapidly eliminated from plasma, 60 of the injected dose being recovered in the liver after 30 m. Within the liver, the EC accounted for two thirds of total liver uptake. Non-targeted CCLs were eliminated very slowly: after 30 m >90 of the particles was in the blood. Currently, we compare the encapsulation efficiency, stability and targetability of the CCL with stabilized antisense lipid particles (SALP) (2), while also the biological activity of these carriers is addressed. In conclusion our results demonstrate that antisense ODN can be targeted very efficiently to EC in vivo, employing plasma-stable CCL, surface modified with negatively charged albumin.ReferencesStuart DD, Allen TM. BBA 2000; 1463:219–229.Semple S. et al. BBA 2001; 1510: 152–166.

Lipid vesicles composed of phosphatidylcholine and suitable polyoxyethylene-derivat-ives of phosphatidylethanolamine (cryptosomes) remain in circulation 8-10 times longer than standard liposomes after an i.v. administration in mice. In contrast to previous belief, this longevity is not destroyed by the net charges on the lipid vesicle surface and is not a direct consequence of the high surface hydrophilicity; also bilayer fluidity is not an obstacle for the attainment of long circulation times. All these three factors, however, can affect the effectiveness of the drug encapsulation into lipid vesicles and the stability of the resulting carrier suspensions. Terminal head-group modifications, moreover, can affect the final carrier and drug distribution after vesicle applications in vivo and lead to accumulation in certain body subsites, such as the gut.

Gradients across the outer skin layers may result in fields enforcing a lipid flow into or through the intact skin surface provided that lipids are applied in the form of special vesicles. The osmotic gradient, for example, which is created by the difference in the total water concentrations between the skin surface and the skin interior, provides one possible source of such driving force. It is sufficiently strong to push at least 0.5 mg of lipids per hour and cm2through the skin permeability barrier in the region of stratum corneum. The lipid concentration gradient, on the contrary, does not contribute much to the lipid penetration into dermis. Occlusion, therefore, is detrimental for the vesicle penetration into intact skin.

Delivery of the drug at a specific site (drug targeting) or controlled and prolonged release of the liposome-bound drug are the two major considerations for adding liposomes to the existing arsenal of drug delivery systems. In particular the concept of liposomal drug targeting has been evolving rapidly in the past 10 years with the development of 'second generation' carriers such as immunoliposomes (liposomes bearing covalently coupled antibodies as homing device) and, more recently, the long-circulating liposomes. In this contribution novel approaches in the field of liposomal drug targeting will be briefly described: (1) immunoliposomes for chemotherapy of intraperitoneal malignancies, such as ovarian carcinoma, (2) a new type of immunoliposomes for mediating the targeting of enzymes to be used for site-specific prodrug activation (immuno-enzymosomes), (3) long-circulating liposomes for the targeting of antibiotics to sites of bacterial infection, and (4) polyethyleneglycol (PEG)-modified proteoliposomes with the homing device coupled to the ends of the long PEG chains for achieving effective target binding along with prolonged circulation times.

Since the introduction of the first through the skin (TTS) therapeutic in 1980, a total of 34 TTS products have been marketed and numerous drugs have been tested by more than 50 commercial organisations for their suitability for TTS delivery. Most of the agents which have been tested have had low molecular weights, due to the impermeability of the skin barrier. This barrier resides in the outermost skin layer, the stratum corneum. It is mechanical, anatomical, as well as chemical in nature; laterally overlapping cell multi-layers are sealed by tightly packed, intercellular, lipid multi-lamellae. Chemical skin permeation enhancers increase the transport across the barrier by partly solubilising or extracting the skin lipids and by creating hydrophobic pores. This is often irritating and not always well-tolerated. The TTS approach allows drugs (< 400 kDa in size) to permeate through the resulting pores in the skin, with a short lag-time and subsequent steady-state period. Drug bioavailability for TTS delivery is typically below 50%, avoiding the first pass effect. Wider, hydrophilic channels can be generated by skin poration, with the aid of a small electrical current (> 0.4 mA/cm2) across the skin (iontophoresis) or therapeutic ultrasound (few W/cm2; sonoporation). High-voltage (> 150 V, electroporation) widens the pores even more and often irreversibly. These standard poration methods require experience and equipment and are therefore, not practical; at best, charged/small molecules (≤ 4000 kDa in size) can be delivered efficiently across the skin. In spite of the potential harm of gadget-driven skin poration, this method is used to deliver molecules which conventional TTS patches are unable to deliver, especially polypeptides. Lipid-based drug carriers (liposomes, niosomes, nanoparticle microemulsions, etc.) were proposed as alternative, low-risk delivery vehicles. Such suspensions provide an improved drug reservoir on the skin, but the aggregates remain confined to the surface. Conventional carrier suspensions increase skin hydration and/or behave as skin permeation enhancers. The recently developed carriers; Transferomes, comprise pharmaceutically-acceptable, established compounds and are thought to penetrate the skin barrier along the naturally occurring transcutaneous moisture gradient. Transfersomes are believed to penetrate the hydrophilic (virtual) channels in the skin and widen the former after non-occlusive administration. Both small and large hydrophobic and hydrophilic molecules are deliverable across the stratum after conjugation with Transfersomes. Drug distribution after transdermal delivery probably proceeds via the lymph. This results in quasi-zero order kinetics with significant systemic drug levels reached after a lag-time of up to a few hours. The relative efficiency of TTS drug delivery with Transfersomes is typically above 50 %; with the added possibility of regional drug targeting.