High Sensitivity Differential Scanning Calorimetry Study of DNA-Cationic Liposome Complexes

PURPOSE: To investigate plasmid DNA interactions with liposomes prepared from dimyristoylglyceroethylphosphocholine (EDMPC) and DOPE using high sensitivity differential scanning calorimetry (HSDSC). MATERIALS AND METHODS: Large unilamellar liposomes of EDMPC with DOPE (mol ratio 0-50%) were prepared. Plasmid DNA was added to give a final DNA/lipid (-/+) charge ratio of 0.5. Samples were placed into an HSDSC and cooled to 3 degrees C, held isothermally for 30 min and then the temperature was ramped to 120 degrees C at a rate of 1 degree C/min. RESULTS: On heating EDMPC liposomes, the main phase transition occurred at 21.2 degrees C, with a low temperature shoulder on the endothermic peak. At low DOPE concentrations the main phase transition temperatures and enthalpies of transition were lower than for pure EDMPC, with a peak corresponding to a pure EDMPC phase occurring at DOPE concentrations of 12-17 mol%. At 50 mol%, no main transition endotherm was observed. DNA solution produced two endothermic peaks with numerous 'satellite' peaks indicating thermal denaturation. DNA binding to EDMPC changed the shape of the thermogram, indicating alteration in lipid packing within the bilayer. DNA induced demixing in the bilayers of DOPE-containing liposomes. CONCLUSION: HSDSC provided information for characterizing liposome formulations and DNA interactions with such vesicles.     

Nanostructured lipid matrices for improved microencapsulation of drugs

Miconazole (MCZ) has very low solubility in both water and oil. Permeation rates through shed snakeskin from an aqueous suspension and a mineral oil suspension were 0.5 microg/cm(2)/h and almost none, respectively. When hydrogenated phosphatidylcholine (HPC) was added to mineral oil and heated to 95 degrees C, the solubility of MCZ increased in proportion to the HPC concentration. DSC measurements also indicated an interaction between them. Thus, a gel formed by hydrogenated phospholipid and mineral oil, as vehicle was prepared. The solubility of MCZ in the gel was around 1% and the permeation rate was 1.3 microg/cm(2)/h, which was about 2.5 times that from an aqueous suspension. As an alternative approach, a skin permeation enhancer, dodecyl 2-(N,N-dimethyl amino)propionate (DDAIP) was applied 2 h before a skin permeation study. The permeation from an aqueous suspension became 11 times that of the suspension without DDAIP pretreatment. The concentration of MCZ in the skin increased 8-fold, indicating that the enhancement effect involved high partition of MCZ into the skin. On the other hand, when a gel formulation was used, pretreatment with DDAIP was not as effective as incorporation of DDAIP in the gel formulation. Following pretreatment, permeation was only two times that of the gel without DDAIP pretreatment, and half that of the water suspension with DDAIP pretreatment. This suggested that release from the gel was the rate-limiting step with the gel formulation. When DDAIP was added to the gel, the permeation rate of MCZ was 3.3 microg/cm(2)/h. It was also a release limited type permeation. The gel with DDAIP is potentially a useful formulation, because of relatively high permeation while possibly avoiding overdosing.     

Synergistic absorption enhancement of salmon calcitonin and reversible mucosal injury by applying a mucolytic agent and a non-ionic surfactant

Using high sensitivity differential scanning calorimetry (HSDSC), the phase transitions of dimyristoylphosphatidylcholine (DMPC) liposomal bilayers and their interaction with the model steroid beclometasone dipropionate (BDP) were found to be dependent on the method of liposome manufacture. Ethanol-based proliposomes produced liposomes having no phospholipid pretransition, a main transition of high enthalpy and a low onset temperature, and a very low incorporation of the steroid (maximum 1 mol%). This was attributed to an alcohol-induced interdigitation of the bilayers, which was not apparently reversed by flushing the liposome dispersion with nitrogen in an attempt to remove ethanol. For liposomes manufactured by thin film or particulate-based proliposome methods, 1–2.5 mol% steroid was optimal for incorporation within bilayers, although the nature of the steroid interaction with the bilayers differed between the two methods. For liposomes manufactured by the thin film method, a higher steroid concentration resulted in a broadened main transition and a reduced melting cooperativity. This suggests that BDP formed separate domains within the bilayers which caused non-ideal mixing and phase separation at 5 mol% steroid. This observation was absent for liposomes generated from particulate-based proliposomes, indicating separate steroid domains were not formed and subsequent non-ideal mixing and phase separation did not occur. In addition, liposomes generated from particulate-based proliposomes showed reduced pretransition and main transition enthalpies. These differences were attributed to the employment of sucrose to manufacture the particulate-based proliposomes. This study has shown that the thermal behaviour of liposomes and their interaction with beclometasone dipropionate were dependent on the method of liposome manufacture. Moreover, particulate-based proliposomes may provide a reasonable alternative to the conventional thin film method in producing liposomes incorporating this steroid.     

Interactions of surfactants (edge activators) and skin penetration enhancers with liposomes

Incorporating edge activators (surfactants) into liposomes was shown previously to improve estradiol vesicular skin delivery; this phenomenon was concentration dependent with low or high concentrations being less effective. Replacing surfactants with limonene produced similar behaviour, but oleic acid effects were linear with concentration up to 16% (w/w), beyond which it was incompatible with the phospholipid. This present study thus employed high sensitivity differential scanning calorimetry to probe interactions of additives with dipalmitoylphosphatidylcholine (DPPC) membranes to explain such results. Cholesterol was included as an example of a membrane stabiliser that removed the DPPC pre-transition and produced vesicles with a higher transition temperature (T(m)). Surfactants also removed the lipid pre-transition but reduced T(m) and co-operativity of the main peak. At higher concentrations, surfactants also formed new species, possibly mixed micelles with a lower T(m). The formation of mixed micelles may explain reduced skin delivery from liposomes containing high concentrations of surfactants. Limonene did not remove the pre-transition but reduced T(m) and co-operativity of the main peak, apparently forming new species at high concentrations, again correlating with vesicular delivery of estradiol. Oleic acid obliterated the pre-transition. The T(m) and the co-operativity of the main peak were reduced with oleic acid concentrations up to 33.2mol%, above which there was no further change. At higher concentrations, phase separation was evident, confirming previous skin transport findings.     

Study of the influence of the penetration enhancer isopropyl myristate on the nanostructure of stratum corneum lipid model membranes using neutron diffraction and deuterium labelling

In order to elucidate the mode of action of the lipophilic penetration enhancer isopropyl myristate (IPM) on a molecular scale, we investigated oriented quaternary stratum corneum (SC) lipid model membranes based on ceramide AP, cholesterol, palmitic acid and cholesterol sulfate containing 10 wt% IPM by means of neutron diffraction. Our results indicate that IPM affects the lamellar lipid assembly in terms of bilayer perturbation and disordering. Phase segregation occurred, indicating that IPM is not likely to mix properly with the other SC lipids due to its branched structure. We used selective deuterium labelling to localize the penetration enhancer, and could successfully prove the presence of IPM in the two coexisting lamellar phases. We conclude that IPM's mode of action as penetration promoter is presumably based on incorporation into the SC lipid matrix, extraction of certain SC lipids into a separate phase and perturbation of the multilamellar lipid assembly.     

Modes of action of terpene penetration enhancers in human skin; Differential scanning calorimetry,small-angle X-ray diffraction and enhancer uptake studies

The mechanisms through which the terpenes, d-limonene, 1–8-cineole and nerolidol, increase the permeability of human stratum corneum (s.c.) and the mechanisms underlying propylene glycol (PG)/terpene synergy were investigated using differential scanning calorimetry (DSC), small-angle X-ray diffraction (SAXD) and enhancer uptake studies. DSC experiments identified two major lipid transitions at 72° and 83°C. d-Limonene reduced the temperatures of both transitions by approx. 20°C without affecting their enthalpies (ΔH). 1–8-Cineole also reduced the temperatures of both transitions by approx. 20°C but also reduced ΔH for the first major lipid transition; ΔH for the second was unaffected. d-Limonene increased the combined entropy change (ΔS) associated with both lipid transitions by 11% whereas 1–8-cineole decreased ΔS by 32%. The decrease in ΔS produced by 1–8-cineole provides evidence that this enhancer is lipid disruptive at normal skin temperature. Nerolidol reduced the transition temperatures of both major lipid transitions by approx. 4°C and also decreased their cooperativity. Reduced bilayer cooperativity indicates that this enhancer also disrupts the intercellular lipids. Lack of a clear baseline prevented accurate measurement of ΔH and ΔS values following nerolidol treatment. SAXD experiments showed that d-limonene and 1–8-cineole act to reduce the intensity of lipid based reflections. Decreases in reflection intensities may be linked to a disruption of lipid packing within the bilayers and/or to a disturbance in the stacking of the bilayers. Treatment with nerolidol did not markedly reduce the intensities of the bilayer based reflections. Uptake studies revealed that large quantities of terpenes can be accommodated by the s.c. (mean uptake of d-limonene, 1–8-cineole and nerolidol was 8.90%, 26.2% and 39.6% w/w dry s.c.). The possibility that terpene enhancers pool in the s.c. is discussed. DSC and SAXD investigations provided fragmented evidence that PG/terpene synergy may produce enhanced lipid bilayer disruption. Enhancer uptake studies showed that PG does not significantly increase terpene delivery to the s.c. above that provided by application of neat terpenes.     

Interaction of phloretin and 6-ketocholestanol with DPPC-liposomes as phospholipid model membranes

Phloretin and 6-ketocholestanol are penetration enhancers for percutaneous delivery of certain topically applied drugs. In the present study some physicochemical experiments have been performed to elucidate the mechanism of action of phloretin and 6-ketocholestanol. The penetration enhancing effect of phloretin and 6-ketocholestanol is believed to be due to their increase of the fluidity of the intercellular lipid bilayers of the stratum corneum. Phospholipid vesicles were chosen as a simple model to represent these bilayers. The effect of phloretin and 6-ketocholestanol on phase transition temperature and enthalpy was studied using differential scanning calorimetry. Beside of that the size of liposomes was monitored when the amount of penetration enhancer in the liposome preparation was changed. Addition of increasing amounts of phloretin and 6-ketocholestanol to the bilayer resulted in lowering of phase transition temperatures and increasing the enthalpy. Additionally the size of the liposomes was increased when penetration enhancer was added. The results suggest that phloretin as well as 6-ketocholestanol would interact with stratum corneum lipids in a similar manner, both reduce the diffusional resistance of the stratum corneum to drugs with balanced hydrophilic-lipophilic characteristics.     

Mechanism of action of Azone as a percutaneous penetration enhancer: Lipid bilayer fluidity and transition temperature effects

Azone is an effective penetration enhancer for the percutaneous delivery ofcertain topically applied drugs. Fundamental physicochemical experiments have been performed to elucidate the mechanism of action of Azone. The penetration enhancing effect of Azone is believed to be due to its increasing the fluidity of the intercellular lipid bilayers of the stratum corneum. Phospholipid vesicles were chosen as a simple model to represent these bilayers. The effect of Azone on phase transition temperature and lipid fluidity was studied using turbidity and fluorescent probe (pyrene excimer) technique. Addition of increasing amounts of Azone to the bilayer resulted in lowering of phase transition temperature, shown by turbidity of vesicle suspensions, and an increase in lipid fluidity, shown by changes in pyrene fluorescence. The results suggest that Azone would interact with striatum corneum lipids in a similar manner, thereby reducing the diffusional resistance of the stratum corneum to drugs with balanced hydrophilic-lipophilic characteristics.     

Dimethylamino Acid Esters as Biodegradable and Reversible Transdermal Permeation Enhancers: Effects of Linking Chain Length,Chirality and Polyfluorination

Purpose  Series of N,N-dimethylamino acid esters was synthesized to study their transdermal permeation-enhancing potency, biodegradability and reversibility of action. Effects of chirality, linking chain length and polyfluorination were investigated. Materials and Methods   In vitro activities were evaluated using porcine skin and four model drugs—theophylline, hydrocortisone, adefovir and indomethacin. Biodegradability was determined using porcine esterase, reversibility was measured using electrical resistance. Results  No differences in activity were found between (R), (S) and racemic dodecyl 2-(dimethylamino)propanoate (DDAIP). Substitution of hydrocarbon tail by fluorocarbon one resulted in loss of activity. Replacement of branched linking chain between nitrogen and ester of DDAIP by linear one markedly improved penetration-enhancing activity with optimum in 4–6C acid derivatives. Dodecyl 6-(dimethylamino)hexanoate (DDAK) was more potent than clinically used skin absorption enhancer DDAIP for theophylline (enhancement ratio of DDAK and DDAIP was 17.3 and 5.9, respectively), hydrocortisone (43.2 and 11.5) and adefovir (13.6 and 2.8), while DDAIP was better enhancer for indomethacin (8.7 and 22.8). DDAK was rapidly metabolized by porcine esterase, and displayed low acute toxicity. Electrical resistance of DDAK-treated skin barrier promptly recovered to control values. Conclusion  DDAK, highly effective, broad-spectrum, biodegradable and reversible transdermal permeation enhancer, is promising candidate for future research.     

Synthesis and Enhancing Effect of Dodecyl 2-(N,N-Dimethylamino)propionate on the Transepidermal Delivery of Indomethacin,Clonidine, and Hydrocortisone

The biodegradable transdermal penetration enhancer, dodecyl 2-(N,N-dimethylamino)propionate (II; DDAIP), was prepared by reacting dodecyl 2-bromopropionate (I), obtained by reaction of n-dodecanol with 2-bromopropionyl halogenide, with dimethylamine. The penetration enhancing effects of DDAIP on the transport of indomethacin, clonidine, and hydrocortisone across shed snake skin (Elaphe obsoleta) were evaluated. Azone and lauryl alcohol, a possible decomposition product of DDAIP, were used as standard enhancers for comparison. In terms of flux, DDAIP showed 4.7 and 7.5 times the promoting effect for indomethacin compared to azone and lauryl alcohol, respectively. With clonidine this effect was 1.7 and 3.1 times, whereas with hydrocortisone it was 2.4 and 2.8 times higher, respectively. In vitro biodegradability of DDAIP was demonstrated in the presence of porcine esterase. The results indicate that DDAIP increases markedly the transepidermal delivery of several types of drug substances.     

Deoxycholate alters the order of acyl chains in freeze-thaw extrusion vesicles of L-α-dipalmitoyl phosphatidylcholine: Study of the 1,6-diphenyl-1,3,5-hexatriene steady-state fluorescence anisotropy

The destabilization of phosphatidylcholine bilayer membranes by the bile salt sodium deoxycholate (DOC) was studied from steady-state fluorescence anisotropy measurements. Freeze-thaw extrusion vesicles (FATVETs) of L-α-dipalmitoyl phosphatidylcholine (DPPC) composition were prepared by sequential extrusion through polycarbonate membranes and characterized for their overall inner volume, average size and size distribution, and lamellarity. Interactions between acyl chains in the lipid matrix, which reflect in the rotational diffusion motion in the 1,6-diphenyl-1,3,5-hexatriene (DPH) molecules, are perturbed by the presence of bile salt in the medium (even at low concentrations) below and above the main transition phase temperature of pure DPPC bilayers. Its effects on the lipid matrix are clearly reflected in the DPH steady-state fluorescence anisotropy (rs) measurements. The resolution of r_s into its static (r_∞) and dynamic component (rd) show that DOC affects both the amplitude and the velocity of DPH movements. However, at temperatures below the gel ↔ liquid crystal phase transition point, the static component (that reflects chain order) is more markedly affected than is the dynamic component (which reflects bilayer fluidity). Thus, at 31°C, angle θ, a measure of the amplitude of DPH oscillations, rises from 23° to 52° over the DOC concentration range from zero to 2.0 mM (equivalent to an effective molar ratio in the bilayer of R_e^25° = 0.12); at 45°C, however, it varies from 62° to 70°. These changes in the bilayer packing status may be responsible for the alteration in the retention ability of liposomal formulations of the cytostatic agent 5-fluorouracil at sub-solubilizing concentrations of deoxycholate.     

A calorimetric study of phosphocholine membranes mixed with desmopressin and its diacylated prodrug derivative (DPP)

The influence of the water-soluble peptide, desmopressin (DDAVP) and its dipalmitoylated prodrug derivative (DPP) on the thermal behaviour of three different saturated phosphatidylcholine lipid membranes was investigated by differential scanning calorimetry. For lipid membranes composed of dimyristoyl, dipalmitoyl and distearoyl phosphatidylcholines the addition of DDAVP at concentrations of up to 10 mol% resulted in an insignificant change in the thermodynamic phase behaviour. In contrast, the dipalmitoylated DPP prodrug caused major changes on the lipid membrane phase behaviour manifested as a drastic decrease in the heat capacity peak height and a concomitant broadening of the main phase transition as well as a decrease in the transition enthalpy. In addition, the main phase transition temperature was slightly decreased and the pre-transition of the three phosphatidylcholines was abolished when DPP was present.     

Interactions of chlorpromazine and imipramine with artificial membranes investigated by equilibrium dialysis, dual-wavelength photometry, and fluorimetry.

Binding of chlorpromazine and imipramine to liposomes of various synthetic lipids was investigated by equilibrium dialysis, dual-wavelength photometry, and fluorimetry. As proved by equilibrium dialysis, liposomes of all investigated lipids bound chlorpromazine to a greater extent than imipramine. At temperatures above the lipid phase transition the membranes bound more of both drugs than they did below. These results were confirmed by dual-wavelength photometry at low drug concentrations. Chlorpromazine and imipramine fluidized lipid bilayers below their transition temperatures. Less imipramine bound to positively charged liposomes as compared with membranes of zwitterionic phosphocholine. This emphasizes the importance of ionic interactions for the binding of imipramine to lipid membranes. Chlorpromazine binding, however, was little affected by ionic interactions. Both chlorpromazine and imipramine had the same effect on the fluorescence of 1-anilino-8-naphthalene-sulfonate (a probe for the polar part of the membrane), whereas the fluorescence of perylene (a probe for the inner hydrocarbon region of bilayers) was quenched by chlorpromazine but not by imipramine. From the results obtained with the three complementary methods it is concluded that chlorpromazine binds to the surface of membranes and also penetrates into the inner hydrocarbon phase of the bilayer, whereas imipramine only binds near the surface of the liposomes.     

Ligand effects on membrane lipids associated with sodium, potassium-activated adenosine triphosphatase: comparative spin probe studies with rat brain and heart enzyme preparations

Physical properties of membrane lipids associated with rat or dog brain and heart Na+, K+-ATPase preparations were compared using an electron spin resonance probe, 5-doxyl stearate. The degree of acyl chain order of the membrane lipids was greater for brain enzyme than for heart enzyme preparations; membrane lipids in the rat heart enzyme preparations were the most disordered. In the absence of added ligands, membrane lipids did not appear to undergo a detectable temperature-dependent rearrangement or structural transition. An apparent transition was observed in the simultaneous presence of Na+, Mg2+, and ATP. These ligands increased lipid order at temperatures above the structural transition, but not below it. In the presence of the above ligands, K+ caused a marked decrease in the transition temperature in the rat brain enzyme preparations, but only a modest decrease in rat heart enzyme preparations. Arrhenius plots of rat brain and heart Na+, K+-ATPase activity revealed a break point roughly corresponding to respective membrane lipid transition temperatures observed in the presence of Na+, K+, Mg2+, and ATP. A low concentration of ouabain (1 microM) failed to affect either the lipid transition temperature estimated by the spin probe or the value of lipid order of the rat brain enzyme preparations observed in the presence of Na+, Mg2+, and ATP, but markedly reduced the effect of K+ to lower the transition temperature observed in the presence of the above ligands. A high concentration (100 microM) of ouabain which was needed to completely inhibit rat heart enzyme eliminated the thermally induced structural rearrangement observed in the presence of Na+, Mg2+, and ATP, apparently through a nonspecific lipid perturbation. These results indicate that differences in the physical properties of the membrane lipids per se are unlikely to account for the low affinity of rat heart Na+, K+-ATPase for ouabain and also suggest that the use of high concentrations of ouabain required to completely inhibit Na+, K+-ATPase activity may cause nonspecific changes in addition to inhibition of Na+, K+-ATPase or the sodium pump.     

Interaction of miltefosine with intercellular membranes of stratum corneum and biomimetic lipid vesicles

Miltefosine (MT) is an alkylphospholipid approved for breast cancer metastasis and visceral leishmaniasis treatments, although the respective action mechanisms at the molecular level remain poorly understood. In this work, the interaction of miltefosine with the lipid component of stratum corneum (SC), the uppermost skin layer, was studied by electron paramagnetic resonance (EPR) spectroscopy of several fatty acid spin-labels. In addition, the effect of miltefosine on (i) spherical lipid vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and (ii) lipids extracted from SC was also investigated, by EPR and time-resolved polarized fluorescence methods. In SC of neonatal Wistar rats, 4% (w/w) miltefosine give rise to a large increase of the fluidity of the intercellular membranes, in the temperature range from 6 to about 50°C. This effect becomes negligible at temperatures higher that ca. 60°C. In large unilamelar vesicles of DPPC no significant changes could be observed with a miltefosine concentration 25% molar, in close analogy with the behavior of biomimetic vesicles prepared with bovine brain ceramide, behenic acid and cholesterol. In these last samples, a 25 mol% molar concentration of miltefosine produced only a modest decrease in the bilayer fluidity. Although miltefosine is not a feasible skin permeation enhancer due to its toxicity, the information provided in this work could be of utility in the development of a MT topical treatment of cutaneous leishmaniasis.     

Interaction of Polyethyleneglycol-Phospholipid Conjugates with Cholesterol-Phosphatidylcholine Mixtures: Sterically Stabilized Liposome Formulations

Purpose. The purpose of this study was to investigate polyethyleneglycol (PEG)-phosphatidylethanolamine (PE) conjugate interaction with cholesterol-phospholipid mixtures in an attempt to explain the effect of cholesterol on liposome circulation time. Methods. Differential scanning calorimetry, NMR, electron microscopy, dynamic light scattering and fluorescence spectroscopy were the major methods used. Results. Studies performed in the absence of cholesterol indicated the formation of three distinct physical states depending on the chain length of PEG in PEG-PE. Mixed micelle formation was observed at concentrations of PEG( 1,000)-DPPE above 7 mol% of lipid. Phase separated lamellae were observed at all concentrations of PEG( 12,000)-DPPE (Bedu-Addo et al. Pharm. Res. 13:710–717 (1996)). Upon incorporation of high concentrations of cholesterol >30 mol% into the lipid bilayer, the formation of phase separated lamellae was completely inhibited and the formation of mixed micelles significantly reduced. At high concentrations of PEG( 1,000)-PE, solubilization of the bilayer occurred with preferential solubilization of cholesterol over phosphati-dylcholine. Maximum steric stabilization (surface protection) was observed with low concentrations of short chain PEG-PE and high concentrations of cholesterol. Conclusions. The study provides a physical mechanism for the following observations: the blood circulation time is significantly increased or decreased with liposomes highly enriched with cholesterol or PEG-PE respectively.     

Evidence that Oleic Acid Exists in a Separate Phase Within Stratum Corneum Lipids

Oleic acid is known to be a penetration enhancer for polar to moderately polar molecules. A mechanism related to lipid phase separation has been previously proposed by this laboratory to explain the increases in skin transport. In the studies presented here, Fourier transform infrared spectroscopy (FT-IR) was utilized to investigate whether or not oleic acid exists in a separate phase within stratum corneum (SC) lipids. Per-deuterated oleic acid was employed allowing the conformational phase behavior of the exogenously added fatty acid and the endogenous SC lipids to be monitored independently of each other. The results indicated that oleic acid exerts a significant effect on the SC lipids, lowering the lipid transition temperature (T _m) in addition to increasing the conformational freedom or flexibility of the endogenous lipid alkyl chains above their T _m. At temperatures lower than T _m, however, oleic acid did not significantly change the chain disorder of the SC lipids. Similar results were obtained with lipids isolated from the SC by chloroform:methanol extraction. Oleic acid, itself, was almost fully disordered at temperatures both above and below the endogenous lipid T _m in the intact SC and extracted lipid samples. This finding suggested that oleic acid does exist as a liquid within the SC lipids. The coexistence of fluid oleic acid and ordered SC lipids, at physiological temperatures, is consistent with the previously proposed phase-separation transport mechanism for enhanced diffusion. In this mechanism, the enhanced transport of polar molecules across the SC can be explained by the formation of permeable interfacial defects within the SC lipid bilayers which effectively decrease either the diffusional path length or the resistance, without necessarily invoking the formation of frank pores.     

Studies on the aggregation and possible channel formation in membranes of a cyclic hexapeptide,cyclo (d‐Ala‐l‐Pro‐l‐Ala)2

Abstract: The interaction of zwitterionic lipid DMPC and DPPC with cyclic hexapeptide, cyclo (d ‐Ala‐l ‐Pro‐l ‐Ala)₂ was studied using circular dichroism (CD) and differential scanning calorimetry (DSC). Preliminary membrane conductance results showed that the peptide has a tendency to form channels inside the lipid bilayer. CD studies indicated that as the lipid/peptide (L/P) ratio (DMPC/peptide) was increased, the magnitude of the negative CD band having a λ_max around 200 nm decreased. At a L/P ratio of 210:1, this band disappeared completely, indicating dramatic conformational changes in the peptide on interaction with the lipid bilayer. Reduction of the phase transition temperature and the maximum heat capacity of the lipid bilayer (DPPC) for gel‐to‐liquid crystalline phase transition indicates a strong interaction of the peptide with the lipid bilayer.     

1-O-Alkylglycerol vesicles (Algosomes): their formation and characterization

1-O-alkylglycerols (ALKG) have exhibited several biological activities and a prominent effect on blood-brain barrier permeability. They have markedly improved brain uptake of cancerostatic agents. Since ALKG are amphiphilic, we explored their tendency to assemble into bilayer vesicles, which can be applied as carriers for drugs. Vesicles (Algosomes) were formed by film hydration method using ALKG (tetra-, penta-, hexa-, hepta-, octa- or nona-decylglycerols) in combination with cholesterol (CHOL) and dicetyl phosphate (DCP) (1-O-alkylglycerol:CHOL:DCP in 45:45:10 molar ratio). On microscopic examination, the algosomes were found to be conspicuously spherical and the dispersion was a mixture of multi-lamellar and small-unilamellar vesicles. Phase transition temperatures of 1-O-hexadecylglycerol (HXDG) and CHOL mixtures were tested by differential scanning calorimetry (DSC). The changes in phase transition temperatures indicate the vesicle forming tendency of ALKG in presence of CHOL. Alkyl chain length dependent variations in vesicle size, zeta-potential (ZP) and capture volume (CV) could not be observed. Vesicles of 1-O-tetradecylglycerol (TTDG) showed improvement in CV with increase in CHOL content from 15 to 55 mol%. However the vesicle size decreased. On challenging algosomes with hypertonic salt solution [potassium iodide (KI) in water], vesicle size decreased and thus algosomes were found to be osmotically sensitive. Algosome dispersions on addition of higher concentrations of KI (40-100 mM) brought about increases in vesicle size and at concentrations 60 mM and above showed aggregation. All vesicular dispersions were stable for only a few days.     

Encapsulation of plasmid DNA in stabilized plasmid-lipid particles composed of different cationic lipid concentration for optimal transfection activity

In previous work (Wheeler et al. (1999) Gene Therapy 6, 271-281) we have shown that plasmid DNA can be entrapped in "stabilized plasmid lipid particles" (SPLP) using low levels (5-10 mol%) of cationic lipid, the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), and a polyethyleneglycol (PEG) coating for stabilization. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). However, these SPLP exhibit low transfection potencies in vitro as compared to plasmid/cationic lipid complexes formed with liposomes composed of cationic and neutral lipid at a 1:1 lipid ratio. The objective of this study was to construct SPLPs with increased cationic lipid contents that result in maximum transfection levels. A phosphate buffer detergent dialysis technique is described resulting in formation of SPLP containing 7-42.5 mol% DODAC with reproducible encapsulation efficiency of up to 80%. An octanoyl acyl group was used as anchor for the PEG moiety (PEG-CerC8) permitting a quick exchange out of the SPLP to further optimize the in vitro and in vivo transfection. We have demonstrated that this technique can be used to encapsulate either linearized DNA or supercoiled plasmids ranging from 3-20 kb. The SPLP formed could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution of approximately 75 +/- 6 nm as determined by cryo-electron microscopy. The high plasmid-to-lipid ratio observed corresponded to one plasmid per particle. The SPLP consist of a lipid bilayer surrounding the plasmid DNA as visualized by cryo-electron microscopy. SPLP containing a range of DODAC concentrations were tested for in vitro and in vivo transfection. In vitro, in COS-7 cells transfection reached a maximum after 48 h. The transfection efficiency increased when the DODAC concentration in the SPLP was decreased from 42.5 to 24 mol% DODAC. Decreasing the cationic lipid concentration improved transfection in part due to decreased toxicity. In vivo studies using an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP showed maximum transfection activity for SPLP containing 24 mol% DODAC. Gene expression observed in tumor cells was increased by approximately one magnitude as compared to cationic lipid/DNA complexes. The SPLP were stable and upon storage at 4 degrees C no significant change in the transfection activity was observed over a one-year period. Thus this phosphate buffer detergent dialysis technique can be used to generate SPLP formulations containing a wide range of cationic lipid concentrations to determine optimal SPLP composition for high transfection activity and low toxicity.