Mixed Liposomes Research Articles

Effects of a Delocalizable Cation on the Headgroup of Gemini Lipids on the Lipoplex-Type Nanoaggregates Directly Formed from Plasmid DNA

Lipoplex-type nanoaggregates prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, with a gemini cationic lipid (CL) [1,2-bis(hexadecyl imidazolium) alkanes], referred as (C16Im)2Cn (where Cn is the alkane spacer length, n = 2, 3, 5, or 12, between the imidazolium heads) and DOPE zwitterionic lipid, have been analyzed by zeta potential, gel electrophoresis, SAXS, cryo-TEM, fluorescence anisotropy, transfection efficiency, fluorescence confocal microscopy, and cell viability/cytotoxicity experiments to establish a structure-biological activity relationship. The study, carried out at several mixed liposome compositions, α, and effective charge ratios, ρeff, of the lipoplex, demonstrates that the transfection of pDNA using CLs initially requires the determination of the effective charge of both. The electrochemical study confirms that CLs with a delocalizable positive charge in their headgroups yield an effective positive charge that is 90% of their expected nominal one, while pDNA is compacted yielding an effective negative charge which is only 10-25% than that of the linear DNA. SAXS diffractograms show that lipoplexes formed by CLs with shorter spacer (n = 2, 3, or 5) present three lamellar structures, two of them in coexistence, while those formed by CL with longest spacer (n = 12) present two additional inverted hexagonal structures. Cryo-TEM micrographs show nanoaggregates with two multilamellar structures, a cluster-type (at low α value) and a fingerprint-type, that coexist with the cluster-type at moderate α composition. The optimized transfection efficiency (TE) of pDNA, in HEK293T, HeLa, and H1299 cells was higher using lipoplexes containing gemini CLs with shorter spacers at low α value. Each lipid formulation did not show any significant levels of toxicity, the reported lipoplexes being adequate DNA vectors for gene therapy and considerably better than both Lipofectamine 2000 and CLs of the 1,2-bis(hexadecyl ammnoniun) alkane series, recently reported.

Combinatorial Synthesis and High-Throughput Screening of Alkyl Amines for Nonviral Gene Delivery

Efficient delivery of plasmid DNA and siRNA into cells is essential for biological and biomedical research. Although significant efforts have been made to develop efficient nonviral vectors, such as cationic lipids and polymers, most of the vectors require multistep synthesis, which complicates both fast structural optimizations and combinatorial synthesis of such vectors. Here, we present a facile, single-step method based on an alkylation of amines, allowing for the fast parallel synthesis of libraries of cationic lipid-like molecules (lipidoids). We exploited the method to synthesize 200 lipidoids, which were screened for their transfection efficiency in HEK293T cells. The screen resulted in about 2% of new lipidoids capable of efficient cell transfection similar or higher than the efficiency of Lipofectamine 2000. In addition, we observed an enhancement of cellular transfection by combining single- with double-chain lipidoids, which was attributed to the different roles of the single- and double-tailed lipids in the mixed liposomes.

Morphological and Nanomechanical Behavior of Supported Lipid Bilayers on Addition of Cationic Surfactants

The addition of surfactants to lipid bilayers is important for the modulation of lipid bilayer properties (e.g., in protein reconstitution and development of nonviral gene delivery vehicles) and to provide insight on the properties of natural biomembranes. In this work, the thermal behavior, organization, and nanomechanical stability of model cationic lipid-surfactant bilayers have been investigated. Two different cationic surfactants, hexadecyltrimethylammonium bromide (CTAB) and a novel derivative of the amino acid serine (Ser16TFAc), have been added (up to 50 mol %) to both liposomes and supported lipid bilayers (SLBs) composed by the zwitterionic phospholipid DPPC. The thermal phase behavior of mixed liposomes has been probed by differential scanning calorimetry (DSC), and the morphology and nanomechanical properties of mixed SLBs by atomic force microscopy-based force spectroscopy (AFM-FS). Although DSC thermograms show different results for the two mixed liposomes, when both are deposited on mica substrates similar trends on the morphology and the mechanical response of the lipid-surfactant bilayers are observed. DSC thermograms indicate microdomain formation in both systems, but while CTAB decreases the degree of organization on the liposome bilayer, Ser16TFAc ultimately induces the opposite effect. Regarding the AFM-FS studies, they show that microphase segregation occurs for these systems and that the effect is dependent on the surfactant content. In both SLB systems, different microdomains characterized by their height and breakthrough force Fb are formed. The molecular organization and composition is critically discussed in the light of our experimental results and literature data on similar lipid-surfactant systems.

Colorimetric and Fluorometric Detection of Neomycin Based on Conjugated Polydiacetylene Supramolecules

Utilizing the colorimetric and fluorogenic changes, a system based on polydiacetylenes (PDAs) is developed for the detection of neomycin. The PDA supramolecules polymerized from the mixed liposome composed of N-(3-hydroxyphenyl)pentacosa-10,12-diynamide (PCDA-AP) and pentacosa-10,12-diynoic acid (PCDA) at an optimized ratio of 1:9 display a unique colorimetric change (blue to red) and fluorescent enhancement in the presence of neomycin. The detection limit for neomycin is estimated to be 2.55 × 10(-7) M by the fluorogenic method. The optical changes induced by neomycin can be attributed to the disruption of the hydrogen bonding between phenol and carboxylic acid from PCDA-AP and PCDA.

Colorimetric and Fluorometric Assays Based on Conjugated Polydiacetylene Supramolecules for Screening Acetylcholinesterase and Its Inhibitors

Polydiacetylene supramolecules (PDAs) are unique sensing materials. Upon environmental stimulation, blue PDAs can undergo a colorimetric transition from blue to red accompanied by fluorescence enhancement. In this paper, we report a new PDA system polymerized from a mixed liposome comprising 2-(2-(2-hydroxyethoxy)ethoxy)ethyl pentacosa-10,12-diynoate and pentacosa-10,12-diynoic acid at a 3:7 ratio. The PDA system provided new colorimetric and fluorometric assay methods for screening acetylcholinesterase and its inhibitors through three processes. First, myristoylcholine reacted with PDAs, which then underwent colorimetric and fluorometric transition. Second, acetylcholinesterase catalyzed the hydrolysis of myristoylcholine into tetradecanoic acid, which reduced the myristoylcholine concentration and led to faded color and fluorescence. Third and last, acetylcholinesterase inhibitors retarded the activity of acetylcholinesterase, thereby inducing the recovery of color and fluorescence.

Octreotide labeled aggregates containing platinum complexes as nanovectors for drug delivery

The synthesis, formulation and a complete physico-chemical characterization, by dynamic light scattering and small angle neutron scattering techniques, of new liposomal aggregates obtained by co-assembling an amphiphilic molecule containing a platinum complex, Peg1500 -Lys(Pt-aminoEtGly)-Lys(C18)2, (abbreviated as (C18)2-PKAG-Pt), with a second amphiphilic monomer, (C18H37)2NCO(CH2)2CO(AdOO)5-Oct ((C18)2 L5-Oct), containing the octreotide bioactive peptide, is reported. Liposomes of (C18)2-PKAG-Pt present a radius of 48 nm, whereas the mixed aggregates (C18)2-PKAG-Pt/(C18)2L5-Oct at 90/10 M ratio give larger liposomes with a radius of 84 nm. In both cases, the bilayer thickness is ~5.3 nm. Encapsulation of doxorubicin in mixed liposomes is also obtained by using the pH gradient method. The obtained liposomes could represent a new target selective cargo system for delivery of cisplatin based drugs and/or doxorubicin on cells overexpressing the sstr2 and sstr5 somatostatin receptors.

Supramolecular design of biocompatible nanocontainers based on amphiphilic derivatives of a natural compound isosteviol

Two diterpenoid surfactants with ammonium head groups and bromide (S1) or tosylate (S2) counterions have been synthesized. Exploration of these biomimetic species made it possible to demonstrate that even minor structural changes beyond their chemical nature may dramatically affect their solution behavior. While their aggregation thresholds differ inconsiderably, morphological behavior and affinity to lipid bilayer are strongly dependent on the counterion nature. Compound S2 demonstrates properties of typical surfactants and forms small micelle-like aggregates above critical micelle concentration. For surfactant S1, two critical concentrations and two types of aggregates occur. Structural transitions have been observed between small micelles and aggregates with higher aggregation numbers and hydrodynamic diameter of ca. 150 nm. Unlike S2, surfactant S1 is shown to integrate with liposomes based on dipalmitoylphosphatidylcholine, resulting in a decrease of the temperature of the main phase transition. Both surfactants demonstrate an effective complexation capacity toward oligonucleotide (ONu), which is supported by recharging the surfactant-ONu complexes and the ethidium bromide exclusion at a low N/P ratio. Meanwhile, a very weak complexation of plasmid DNA with the surfactants has been revealed in the gel electrophoresis experiment. The DNA transfer to bacterial cells mediated by the surfactant S1 is shown to depend on the protocol used. In the case of the electroporation, the inhibition of the cell transformation occurs in the presence of the surfactant, while upon the chemical treatment no surfactant effect has been observed. The variability in the morphology, the biocompatibility, the nanoscale dimension and the high binding capacity toward the DNA decamer make it possible to nominate the designed surfactants as promising carriers for biosubstrates or as a helper surfactant for the mixed liposome-surfactant nanocontainers.

Studies of mixed liposomes with novel sorbyl functionalized head group lipids

Three novel polymerizable amphiphiles with a sorbyl-substituted head group were synthesized and systematically characterized. These amphiphiles are neutral in charge. None of these molecules forms vesicles by itself, presumably due to lack of amphiphilicity and/or extensive head group interaction. Therefore, mixed vesicles were formed with other fluid lipids such as DPenPC, eggPC, or DOPC. We investigated the properties of these mixtures in both vesicles and Langmuir films. The Langmuir isotherms show formation of monolayers by all three molecules. However, the isotherms for mixed monolayers suggest that two components are largely immiscible to the mixing lipid. Under polymerization conditions, mixed vesicles of these amphiphiles form oligomers, suggesting that in spite of a larger head group, they form mesophases.

Synthesis and physicochemical characterization of pyrrolidinium based surfactants

Three new pyrrolidinium based surfactants were synthesized and characterized as pure aggregate components and in mixtures with 1,2-dimyristoyl-sn-glycero-3-phosphocholine to understand how the molecular structure of the cationic amphiphile and its mole percentage might affect the physicochemical properties of the resulting aggregates. The thermotropic behavior of the mixed liposomes was investigated by differential scanning calorimetry on multilamellar vesicles, whereas their size and surface potential were investigated on large unilamellar vesicles by dynamic laser light scattering and fluorescence experiments, respectively. The presence of either methoxy or hydroxy groups in the positions 3 and 4 of the pyrrolidinium ring as well as the presence of a second alkyl chain on pyrrolidinium nitrogen, controls the aggregates features.

Ribbon-type and cluster-type lipoplexes constituted by a chiral lysine based cationic gemini lipid and plasmid DNA

Lipoplexes constituted by plasmid DNA pEGFP-C3 (pDNA) or linear double-stranded calf thymus DNA (ctDNA) and mixed cationic liposomes consisting of several percentages of the cationic lysine derived lipid C6(LL)2 and the zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) have been analyzed by both experimental and theoretical approaches. Experimental studies, consisting of electrophoretic mobility/zeta potential, small angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), negatively stained transmission electron microscopy (NS-TEM), and GelRed f1uorescence intercalation assays, have been carried out at several liposome and lipoplex compositions, defined in terms of cationic lipid molar fraction and either the mass or charge ratios of the lipoplex. The electrochemical study confirms that, in the presence of the mixed lipids and in contrast with what has usually been found for linear DNA, the plasmid DNA is compacted with a large number of its Na+ counterions, thus yielding a much lower effective negative charge (q−pDNA) than that for ctDNA (q−ctDNA), as reported recently by us (J. Am. Chem. Soc., 2011) for other lipoplexes. This finding is revealed as crucial for an optimum and efficient lipoplex preparation, since a lower effective negative charge implies a lower quantity of cationic lipid and, accordingly, a potential lower cytotoxicity. TEM experiments reveal a complex scenario of multilamellar nanostructures, from ribbon-type (typically present for chiral lipids) to cluster-type structures (usually found in cationic lipid/DOPE systems), the composition of the mixed liposome playing an important role in the final morphology of the lipoplex. SAXS diffractograms confirm the existence of these two types of multilamellar structures through a deconvolution process of the first peak of diffractograms into two overlapping bands. On the other hand, a theoretical complexation model is employed to determine the net charge of the lipoplexes studied in this work. The model allows analysis and comparison of the electrochemical behaviour of lipoplexes containing linear DNA vs. those constituted by a supercoiled DNA, confirming the experimental findings.

Effect of anionic-lipid-molecule geometry on the structure and properties of liposome-polycation complexes

The influence of anionic amphiphilic compounds that have different geometric shapes and become incorporated into bilayers on the polycation-mixed liposome interaction and the structure and properties of the resulting complexes is analyzed. Phosphatidylserine, cardiolipin, and sodium dodecyl phosphate are used as anionic lipids, and poly(N-ethyl-4-vinylpyridinium bromide) and polylysine are used as polycations. Polycation adsorption on the surfaces of all examined types of liposomes is accompanied by the neutralization of their charge, an increase in the size of particles of the systems, and quenching of fluorescence labels. Liposomes whose membranes contain incorporated cylindrical phosphatidylserine molecules retain their integrity during contact with polycations. The resulting complexes quantitatively dissociate into initial components during an increase in the salt concentration in the surrounding solution. In the case of liposomes with asymmetric anionic lipids, that is, cardiolipin and sodium dodecyl phosphate, the conditions of retaining the membrane integrity and reversible complexation are fulfilled only at relatively low molar fractions of both lipids. The obtained data witness the decisive effect of the geometry of anionic lipid molecules on the stability of complexes formed from mixed liposomes and polycations.

Characterization of cationic liposomes. Influence of the bilayer composition on the kinetics of the liposome breakdown

The cationic large unilamellar mixed liposomes from 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and didodecyldimethylammonium bromide (DDAB) or dioctadecyldimethylammonium bromide (DODAB) were prepared. The influence of the addition of Triton X-100 (TX-100) or octaethylene glycol mono-n-dodecylether (C 12E 8) on the membrane integrity was investigated turbidimetrically. The stability of the liposomal systems was estimated by monitoring fluorimetrically at 25 °C the rate of spontaneous and surfactant-induced release of entrapped 5(6)-carboxyfluorescein (CF). In order to evaluate the interaction of the cationic DODAB guest with the host POPC membrane, the main phase transition temperatures ( T m) were determined by electron paramagnetic resonance spectroscopy (EPR). All the results obtained show that the presence of DODAB and DDAB stabilizes the POPC liposomes. The extent of stabilization depends on the concentration and nature of the cationic guest.

Structural characterization of liposomes made of diether archaeal lipids and dipalmitoyl-L-α-phosphatidylcholine

The physicochemical properties of binary lipid mixtures of diether C 25,25 lipids and dipalmitoyl-L-α-phosphatidylcholine (DPPC) were studied using photon correlation, fluorescence and electron paramagnetic resonance spectroscopy, and transmission electron microscopy. These two types of lipids can be mixed at all molar ratios to form unilamellar and multilamellar liposomes. Fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatrien in mixed liposomes indicates that the abrupt changes in order parameter in the hydrophobic part of bilayer membranes made of DPPC lipids disappears with increasing mol% C 25,25 lipids. Electron paramagnetic resonance spectroscopy shows that at temperatures below 50 °C, the interfacial regions of membrane bilayer of mixed liposomes is more fluid than for pure DPPC liposomes, while at higher temperatures, the impact of the long isoprenoid chains on the membrane stability becomes more pronounced. Photon correlation spectroscopy and transmission electron microscopy show that mixed liposomes do not fuse or aggregate, even after 41 days at 4 °C.

Thermotropic phase behaviour of mixed liposomes of archaeal diether and conventional diester lipids

The phase behaviour and phase stability of mixed liposomes prepared from diether polar lipids isolated from thermophilic Archaea and from conventional diester dipalymitoyl-l-α-phosphatidylcholine (DPPC) at different molar ratios were investigated by differential scanning calorimetry. The permeability of these mixed liposomes was investigated as a function of temperature, by measuring the fluorescence emission intensity for the release of calcein in the temperature range from 20 to 98 °C. Our data show that diether polar lipids isolated from hyperthermophilic Archaea can form mixed liposomes with synthetic α-DPPC. Liposomes prepared from pure archaeal lipids do not show the characteristic gel-to-liquid crystalline phase transition characteristics of DPPC liposomes in the temperature range from 0 to 100 °C. In the presence of 5 mol% archaeal lipids in mixed α-DPPC liposomes, the temperature of the phase transition decreases and the broadness of the peak increases, although the enthalpy change (ΔH) of the phase transition is not significantly influenced. At molar ratios of archaeal lipids greater than 50 mol% in these mixed liposomes, the typical gel-to-liquid crystalline phase transition disappears, which indicates that the lipids are predominantly in the liquid crystalline state. At ratios of archaeal lipids greater than 50 mol% in these mixed liposomes, their permeability for anionic calcein is the same as for liposomes made from 100 mol% archaeal lipids, across the whole temperature range.

DSPC/DLPC Mixed Films Supported on Silica: A QCM-D and Friction Force Study

The membrane properties of phospholipid mixtures supported on silica were studied by means of a quartz crystal microbalance with dissipation monitoring (QCM-D) technique, in situ soft-contact atomic force microscopy (AFM), and friction force microscopy (FFM). The phospholipids used in this study were di-stearoylphosphatidylcholine (DSPC) and dilauroylphosphatidylcholine (DLPC). The phospholipid films were prepared by a vesicle-fusion method, in which DSPC/DLPC mixed liposomes dispersed in an aqueous medium are adsorbed on silica and their structure is transformed into a bilayer on the substrate. The changes in QCM-D (frequency and dissipation) and friction responses of DSPC single systems (gel state at 25°C) are relatively large compared with those of DLPC single systems (liquid-crystalline state at 25°C) and those of mixed DSPC/DLPC systems. This suggests that (i) the gel-state DSPC liposomes are somewhat flattened on the silica, by keeping their solid-like molecular rigidity, whereas (ii) both the liquid-crystalline DLPC and mixed liposomes experience instantaneous structural transformation at the silica/water interface and form a normally flattened bilayer on the substrate. The friction force response is dependent on the phase state of the phospholipids, and the liquid-crystalline DLPC has a more significant impact on the overall membrane properties (i.e., the degree of swelling and the friction response on the surface) than does the gel-state DSPC.

Gene vectors based on DOEPC/DOPE mixed cationic liposomes: a physicochemical study

A double approach, experimental and theoretical, has been followed to characterize from a physicochemical standpoint the compaction process of DNA by means of cationic colloidal aggregates. The colloidal vectors are cationic liposomes constituted by a mixture of a novel cationic lipid, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (DOEPC) and a zwitterionic lipid, the 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE). A wide variety of high precision experimental techniques have been used to carry out the analysis: electrophoretic mobility, small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM) and fluorescence spectroscopy (ethidium bromide intercalation assays). On the other hand, a theoretical model that considers the renormalization of charges of both the polyelectrolyte and the colloidal aggregates sheds light as well on the characteristics of the compaction process. This global information reveals that the compaction of DNA by the cationic liposomes is mostly driven by the strong electrostatic interaction among the positively charged surfaces of the colloidal aggregates and the negatively charged DNA, with a potent entropic component. DOEPC/DOPE liposomes are mostly spherical, with a mean diameter of around 100 nm and a bilayer thickness of 4.4 nm. From a morphological viewpoint, an appreciable amount of multilamellar structures has been found not only on the lipoplexes but also on the parent liposomes. The isoneutrality of the lipoplexes is found at liposome/DNA mass ratios that decrease with the molar fraction of cationic lipid in the mixed liposome (α). This liposome composition has a clear effect as well on the lipoplex structure, which goes from an inverted hexagonal phase (HII), usually related to improved cell transfection efficiency, at low cationic lipid molar fraction (α ≈ 0.2), to a lamellar structure (Lα) when the cationic lipid content in the mixed liposomes increases (α ≥ 0.4), irrespective of the lipoplex charge ratio. On the other hand, a theoretical complexation model is employed to determine the net charge of the lipoplexes studied in this work, by using renormalized charges. The model allows us to confirm and predict the experimental isoneutrality conditions as well as to determine the maximum magnitude of this charge as a function of the composition of the resulting lipoplexes.

Effect of Lipid Composition on the Structure and Theoretical Phase Diagrams of DC-Chol/DOPE-DNA Lipoplexes

Lipoplexes constituted by calf-thymus DNA (CT-DNA) and mixed cationic liposomes consisting of varying proportions of the cationic lipid 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and the zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine (DOPE) have been analyzed by means of electrophoretic mobility, SAXS, and fluorescence anisotropy experiments, as well as by theoretically calculated phase diagrams. Both experimental and theoretical studies have been run at several liposome and lipoplex compositions, defined in terms of cationic lipid molar fraction, α, and either the mass or charge ratios of the lipoplex, respectively. The experimental electrochemical results indicate that DC-Chol/DOPE liposomes, with a mean hydrodynamic diameter of around (120 ± 10) nm, compact and condense DNA fragments at their cationic surfaces by means of a strong entropically driven electrostatic interaction. Furthermore, the positive charges of cationic liposomes are compensated by the negative charges of DNA phosphate groups at the isoneutrality L/D ratio, (L/D)(ϕ), which decreases with the cationic lipid content of the mixed liposome, for a given DNA concentration. This inversion of sign process has been also studied by means of the phase diagrams calculated with the theoretical model, which confirms all the experimental results. SAXS diffractograms, run at several lipoplex compositions, reveal that, irrespectively of the lipoplex charge ratio, DC-Chol/DOPE-DNA lipoplexes show a lamellar structure, L(α), when the cationic lipid content on the mixed liposomes α ≥ 0.4, while for a lower content (α = 0.2) the lipoplexes show an inverted hexagonal structure, H(II), usually related with improved cell transfection efficiency. A similar conclusion is reached from fluorescence anisotropy results, which indicate that the fluidity on liposome and lipoplexes membrane, also related with better transfection results, increases as long as the cationic lipid content decreases.

Study of the release of a microencapsulated acid dye in polyamide dyeing using mixed cationic liposomes

The main objective of this work was to increase the retarding effect of the acid dye Telon® Blue RR (C.I. Acid Blue 62; DyStar, Frankfurt, Germany) release on polyamide fibres dyeing by encapsulation of the dye in liposomes as an alternative to synthetic auxiliaries, in order to reduce effluent pollution. The retarding effect achieved with the use of mixed cationic liposomes of dioctadecyldimethylammonium bromide (DODAB)/soybean lecithin (containing a 10% molar fraction of DODAB) was better in comparison with either pure soybean lecithin liposomes or synthetic auxiliaries. The retarding effect of liposomes on the dye release was analysed through changes in the absorption and fluorescence spectra of the acid dye at different conditions. The effect of temperature (in the range of 25 °C - 70 °C) on the spectroscopic behaviour of the dye in the absence and in presence of polyamide was also studied, in order to simulate the dyeing conditions. Exhaustion curves obtained in dyeing experiments showed that, below 45 °C, the retarding effect of the mixed liposomes (lecithin/DODAB (9:1)) was similar to that of the auxiliaries, but better than the one of pure lecithin liposomes. At higher temperatures (above 45 °C), the system lecithin/DODAB presents a better performance, achieving a higher final exhaustion level when compared with the commercial leveling agent without losing the smoothing effect of lecithin.

Interaction and Transport of Poly(l-lysine) Dendrigrafts through Liposomal and Cellular Membranes: The Role of Generation and Surface Functionalization

Two generations of poly(l-lysine) dendrigrafts (DGLs) were studied with regard to their ability to interact with and translocate through liposomal and cellular membranes. Partial guanidinylation of the surface amino groups of the starting dendrigrafts afforded the guanidinylated derivatives whose membrane translocation properties were also assessed. Mixed liposomes, consisting of dihexadecyl phosphate, phosphatidylcholine, and cholesterol, were employed as model membranes, while A549 human lung carcinoma cells were used for cellular uptake studies. At high surface group/liposomal phosphate molar ratios and depending on the structure of the DGL, the interaction led to aggregation. Dendrigraft liposomal internalization was achieved, however, at low molar ratios. Thus translocation of the second generation dendrigrafts was rather limited at 25 degrees C, which, however, was enhanced when the bilayer was in the liquid-crystalline phase. In contrast, third-generation counterparts exhibited minor translocational ability. Furthermore, the introduction of a guanidinium group to dendrigrafts was found to enhance their transport through liposomal membranes. On the other hand, cellular uptake by A549 cells was monitored up to 3 h incubation time via fluorescence registration employing fluorescein-labeled dendrigrafts. The efficiency of dendrigraft internalization was enhanced by the presence of the guanidinium groups, while DGLs were preferentially localized in the nucleus and nuclear membrane, as revealed by fluorescence microscopy.

Cationic liposomes, loaded with m-THPC, in photodynamic therapy for malignant glioma

In this study we investigated the feasibility of mixed liposomes formed by dimyristoyl- sn-glycero-phosphatidylcholine (DMPC) and cationic gemini surfactant (Gemini 1) loaded with the chlorin m-tetrahydroxyphenylchlorin ( m-THPC), in photodynamic therapy (PDT) for glioma. To this aim, an in vitro study was carried out by employing various human glioblastoma cell lines (A172, DBTRG, LN229, U118). The following liposomal formulations were tested: (i) DMPC and Gemini 1; (ii) m-THPC in DMPC in the absence or (iii) in the presence of Gemini 1 in the molar ratio 8:2; 7:3, and 6:4. The presence of Gemini 1 significantly increased the intracellular uptake of chlorin in all cell tested although with a different extent: LN229 > U118 > A172 > DBTRG. The cytotoxicity of chlorin-loaded liposomes was then tested by cloning efficiency performed on different cultures, before and after irradiation with laser light at 652 nm, at a Fluence Rate of 200 mW/s for 100 s, with a total Fluence of 20 J/cm −2. In the absence of irradiation, the different liposomal formulations induced a cytotoxicity in less than 30% of glioblastoma cells. On the contrary, irradiation induced total destruction of all cultures treated with m-THPC/DMPC + Gemini 1 in the ratios 8:2, or 7:3, or 6:4.