Association of vasoactive intestinal peptide with polymer-grafted liposomes: Structural aspects for pulmonary delivery

A polymer-grafted liposomal formulation that has the potential to be developed for aerosolic pulmonary delivery of vasoactive intestinal peptide (VIP), a potent vasodilatory neuropeptide, is described. As VIP is prone to rapid proteolytic degradation in the microenvironment of the lung a proper delivery system is required to increase the half-life and bioavailability of the peptide. Here we investigate structural parameters of unilamellar liposomes composed of palmitoyl-oleoyl-phosphatidylcholine, lyso-stearyl-phosphatidylglycerol and distearyl-phosphatidyl-ethanolamine covalently linked to polyethylene glycol 2000, and report on VIP-lipid interaction mechanisms. We found that the cationic VIP is efficiently entrapped by the negatively charged spherical liposomes and becomes converted to an amphipathic α-helix. By fluorescence spectroscopy using single Trp-modified VIP we could show that VIP is closely associated to the membrane. Our data suggest that the N-terminal random-coiled domain is embedded in the interfacial headgroup region of the phospholipid bilayer. By doing so, neither the bilayer thickness of the lipid membrane nor the mobility of the phospholipid acyl chains are affected as shown by small angle X-ray scattering and electron spin resonance spectroscopy. Finally, in an ex vivo lung arterial model system we found that liposomal-associated VIP is recognized by its receptors to induce vasodilatory effects with comparable high relaxation efficiency as free VIP but with a significantly retarded dilatation kinetics. In conclusion, we have designed and characterized a liposomal formulation that is qualified to entrap biologically active VIP and displays structural features to be considered for delivery of VIP to the lung.     

Detection of liposomal cholesterol and monophosphoryl lipid A by QS-21 saponin and Limulus polyphemus amebocyte lysate

Liposomes containing cholesterol (Chol) have long been used as an important membrane system for modeling the complex interactions of Chol with adjacent phospholipids or other lipids in a membrane environment. In this study we utilize a probe composed of QS-21, a saponin molecule that recognizes liposomal Chol and causes hemolysis of erythrocytes. The interaction of QS-21 with liposomal Chol results in a stable formulation which, after injection into the tissues of an animal, lacks toxic effects of QS-21 on neighboring cells that contain Chol, such as erythrocytes. Here we have used liposomes containing different saturated phospholipid fatty acyl groups and Chol, with or without monophosphoryl lipid A (MPLA), as model membranes. QS-21 is then employed as a probe to study the interactions of liposomal lipids on the visibility of membrane Chol. We demonstrate that changes either in the mole fraction of Chol in liposomes, or with different chain lengths of phospholipid fatty acyl groups, can have a substantial impact on the detection of Chol by the QS-21. We further show that liposomal MPLA can partially inhibit detection of the liposomal Chol by QS-21. The Limulus amebocyte lysate assay is used for binding to and detection of MPLA. Previous work has demonstrated that sequestration of MPLA into the liposomal lipid bilayer can block detection by the Limulus assay, but the binding site on the MPLA to which the Limulus protein binds is unknown. Changes in liposomal Chol concentration and phospholipid fatty acyl chain length influenced the detection of the liposome-embedded MPLA.     

Conformational state and functional activity of NADPH-dependent cytochrome P-450 reductase isolated and incorporated into phospholipid bilayers

It was shown that the alpha-helix content in both isolated and incorporated into phospholipid bilayer NADPH-dependent cytochrome P-450 reductase is 20%. NADPH or dithionite reduction of the flavoprotein is not followed by conformational changes. The incorporation of the NADPH-dependent cytochrome P-450 reductase molecule into the phospholipid bilayer does not affect its catalytic properties. It was found that the protein does not interact with the phospholipid bilayer of phosphatidyl choline liposomes but incorporates readily into the liposomal membrane from a microsomal phospholipid mixture with a binding constant of 17.4 microM.     

Evaluation of tetraether lipid-based liposomal carriers for encapsulation and retention of nucleoside-based drugs

Although liposomal nanoparticles are one of the most versatile class of drug delivery systems, stable liposomal formulation of small neutral drug molecules still constitutes a challenge due to the low drug retention of current lipid membrane technologies. In this study, we evaluate the encapsulation and retention of seven nucleoside analog-based drugs in liposomes made of archaea-inspired tetraether lipids, which are known to enhance packing and membrane robustness compared to conventional bilayer-forming lipids. Liposomes comprised of the pure tetraether lipid generally showed improved retention of drugs (up to 4-fold) compared with liposomes made from a commercially available diacyl lipid. Interestingly, we did not find a significant correlation between the liposomal leakage rates of the molecules with typical parameters used to assess lipophilicity of drugs (such logD or topological polar surface area), suggesting that specific structural elements of the drug molecules can have a dominant effect on leakage from liposomes over general lipophilic character.     

Effects of phospholipid hydrolysis on the aggregate structure in DPPC/DSPE-PEG2000 liposome preparations after gel to liquid crystalline phase transition

Upon storage of phospholipid liposome samples, lysolipids, fatty acids, and glycerol-3-phosphatidylcholine are generated as a result of acid- or base-catalyzed hydrolysis. Accumulation of hydrolysis products in the liposome membrane can induce fusion, leakage, and structural transformations of the liposomes, which may be detrimental or beneficial to their performance depending on their applications as, e.g., drug delivery devices. We investigated in the present study the influence of phospholipid hydrolysis on the aggregate morphology of DPPC/DSPE-PEG2000 liposomes after transition of the phospholipid membrane from the gel phase to liquid crystalline phase using high performance liquid chromatography (HPLC) in combination with static light scattering, dynamic light scattering, and cryo-transmission electron microscopy (cryo-TEM). The rates of DPPC hydrolysis in DPPC/DSPE-PEG2000 liposomes were investigated at a pH of 2, 4, or 6.5 and temperatures of 22 degrees C or 4 degrees C. Results indicate that following phase transition, severe structural reorganizations occurred in liposome samples that were partially hydrolyzed in the gel phase. The most prominent effect was an increasing tendency of liposomes to disintegrate into membrane discs in accordance with an increasing degree of phospholipid hydrolysis. Complete disintegration occurred when DPPC concentrations had decreased by, in some cases, as little as 3.6%. After extensive phospholipid hydrolysis, liposomes and discs fused to form large bilayer sheets as well as other more complex bilayer structures apparently due to a decreased ratio of lysolipid to palmitic acid levels in the liposome membrane.     

Interaction of Paclitaxel with Phospholipid Bilayers

Abstract

Paclitaxel is an effective anticancer drug. Recently, paclitaxel encapsulated in liposomes was promoted as a better tolerated pharmaceutical formulation than that currently in use. The data presented in this study show the effects of paclitaxel on phospholipid bilayers. Experiments involving the phospholipid head group probe CAT-16 show significant disordering of the interfacial region. The pretransition was abolished and the main phase transition temperature in paclitaxel loaded liposomes was reduced. 2T II values of 7-NSA and 16-NSA spin probes reporting from the middle and from the core of the phospholipid bilayer, respectively, show that the presence of paclitaxel eliminated the pretransition (from Lβ/ to Pβ/) while inducing a slight reduction in the main (Pβ/ to Lα) phase transition temperature; in the same temperature interval, the central resonance line width δ H O displayed a greater rate of spin label reorientation in paclitaxel loaded bilayers. Further data are presented clearly demonstrating that the presence of paclitaxel in liposomal membrane increases the solubility of hydrophobic compounds. Differential scanning calorimetry was used to confirm that the presence of paclitaxel stabilized the lamellar structure of the bilayer and increased the transition temperature from lamellar Lα phase to hexagonal H II phase of TPE liposomes. The encapsulation of paclitaxel in liposomes depends on phospholipid characteristics; more drug is contained in the bilayer of liposomes containing unsaturated fatty acid chains and phosphorylcholine headgroups, such as DEPC and egg PC.     

Transfer of phospholipid and protein into the envelope of gram-negative bacteria by liposome fusion

A liposome-bacterial fusion system was developed in order to introduce preformed terminal complement complexes, C5b-9, into the outer membrane of Gram-negative bacteria. Liposomes were prepared from a total phospholipid extract of Salmonella minnesota Re595. Fusion between liposomes and Salmonella sp. or Escherichia coli 17 was dependent on time, temperature, pH, and Ca2+ and PO4- concentration. Only Salmonella sp. with attenuated LPS core regions were able to fuse efficiently with liposomes. It was demonstrated that fusion of liposomes with S. minnesota Re595 or E. coli 17 under optimum conditions resulted in (i) quantitative transfer of the self-quenching fluorescent membrane probe octadecyl rhodamine B chloride from the liposomal bilayer to the bacterial envelope, (ii) transfer of radiolabeled liposomal phospholipid to the bacterial outer membrane and its subsequent translocation to the cytoplasmic membrane, demonstrated by isolation of the bacterial membranes following fusion, and (iii) delivery of liposome-entrapped horseradish peroxidase (HRP) to the periplasmic space, confirmed by a chemiluminescent assay. Following fusion of liposomes incorporating C5b-9 complexes with S. minnesota Re595 or E. coli 17, immunological analysis of the isolated membranes revealed C5b-9 complexes located exclusively in the outer membrane.     

Biophysical characterization of gold nanoparticles-loaded liposomes

Gold nanoparticles were prepared and loaded into the bilayer of dipalmitoylphosphatidylcholine (DPPC) liposomes, named as gold-loaded liposomes. Biophysical characterization of gold-loaded liposomes was studied by transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) spectroscopy as well as turbidity and rheological measurements. FTIR measurements showed that gold nanoparticles made significant changes in the frequency of the CH₂ stretching bands, revealing that gold nanoparticles increased the number of gauche conformers and create a conformational change within the acyl chains of phospholipids. The transmission electron micrographs (TEM) revealed that gold nanoparticles were loaded in the liposomal bilayer. The zeta potential of DPPC liposomes had a more negative value after incorporating of Au NPs into liposomal membranes. Turbidity studies revealed that the loading of gold nanoparticles into DPPC liposomes results in shifting the temperature of the main phase transition to a lower value. The membrane fluidity of DPPC bilayer was increased by loading the gold nanoparticles as shown from rheological measurements. Knowledge gained in this study may open the door to pursuing liposomes as a viable strategy for Au NPs delivery in many diagnostic and therapeutic applications.     

Membrane fusion activity of purified SipB, a Salmonella surface protein essential for mammalian cell invasion

An early event in Salmonella infection is the invasion of non-phagocytic intestinal epithelial cells. The pathogen is taken up by macropinocytosis, induced by contact-dependent delivery of bacterial proteins that subvert signalling pathways and promote cytoskeletal rearrangement. SipB, a Salmonella protein required for delivery and invasion, was shown to localize to the cell surface of bacteria invading mammalian target cells and to fractionate with outer membrane proteins. To investigate the properties of SipB, we purified the native full-length protein following expression in recombinant Escherichia coli. Purified SipB assembled into hexamers via an N-terminal protease-resistant domain predicted to form a trimeric coiled coil, reminiscent of viral envelope proteins that direct homotypic membrane fusion. The SipB protein integrated into both mammalian cell membranes and phospholipid vesicles without disturbing bilayer integrity, and it induced liposomal fusion that was optimal at neutral pH and influenced by membrane lipid composition. SipB directed heterotypic fusion, allowing delivery of contents from E. coli-derived liposomes into the cytosol of living mammalian cells.     

Spectrophotometric measurement of intraliposomal trehalose

Trehalose, a non-reducing glucose disaccharide found at high concentrations in many species of anhydrobiotic organisms, shows significant promise in protecting cellular viability and structural integrity during freezing and desiccation. As mammalian cell membranes are impermeable to trehalose, extensive efforts have been taken to introduce trehalose into mammalian cells. In this study, we report on the characterization of trehalose-containing liposomes, with focus on the entrapment of trehalose inside liposomes, as the first step in establishing liposomes as a delivery system in the biopreservation field. Liposomes were synthesized by hydrating a phospholipid/cholesterol lipid bilayer with 200-400 mM trehalose buffer and repeatedly extruding the lipid suspension to form unilamellar vesicles. The trehalose content of the liposomal lysate was determined spectrophotometrically using a commercial kit Megazyme and confirmed with HPLC measurements. The number of liposomes was calculated from the phosphate content of the liposomal preparation and an estimated number of lipid molecules in a 401+/-8 nm liposome. Based on an intraliposomal trehalose content, the calculated liposomal encapsulation efficiency of 200 mM trehalose liposomes was of 92+/-0.7%. This value was in agreement with the 300 and 400 mM trehalose liposomes (91.1+/-8.2% and 102.1+/-9.4%, respectively). The Megazyme method for trehalose measurement is an inexpensive and sensitive technique that does not require specialized instrumentation or extensive technical expertise. Therefore, it can be used to enhance current efforts in the development of alternative strategies for the cryo- and lyoprotection of mammalian cells.     

The antimicrobial peptide trichogin and its interaction with phospholipid membranes.

The interaction of the antimicrobial peptide trichogin GA IV with phospholipid bilayers has been studied. A series of analogs of trichogin was synthesized in which the nitroxide spin label, 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidino-1-oxyl (TOAC), replaced one of the three alpha-aminoisobutyric acid (Aib) residues in the sequence. These modified peptides were used to assess the location of different residues of the peptide in a phospholipid bilayer composed of egg phosphatidylcholine containing 0.4 mol% of a fluorescently labelled phospholipid. We demonstrate that the substitution of Aib residues with TOAC does not alter the manner in which the peptide affects membrane curvature or induces vesicle leakage. The proximity of the nitroxide group on the peptide to the 4,4-difluoro-4-bora-3a,4a-diaza-S-indacene (BODIPY) fluorophore attached to the phospholipid was estimated from the extent of quenching of the fluorescence. By this criterion it was concluded that the peptide penetrates into the bilayer and that Aib4 is the most deeply inserted of the Aib residues. The results suggest that the helix axis of the peptide is oriented along the plane of the membrane. All of the peptides were shown to raise the bilayer to the hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine, indicating that they promote positive membrane curvature. This is a property observed with peptides that do not penetrate deeply into the bilayer or are oriented along the bilayer normal. We also demonstrate trichogin-promoted leakage of the aqueous contents of liposomes. These results indicate that the peptides cause bilayer destabilization. The extent of leakage induced by trichogin is very sensitive to the peptide to lipid ratio over a narrow range.     

Effects of phospholipid hydrolysis on the aggregate structure in DPPC/DSPE-PEG2000 liposome preparations after gel to liquid crystalline phase transition

Upon storage of phospholipid liposome samples, lysolipids, fatty acids, and glycerol-3-phosphatidylcholine are generated as a result of acid- or base-catalyzed hydrolysis. Accumulation of hydrolysis products in the liposome membrane can induce fusion, leakage, and structural transformations of the liposomes, which may be detrimental or beneficial to their performance depending on their applications as, e.g., drug delivery devices. We investigated in the present study the influence of phospholipid hydrolysis on the aggregate morphology of DPPC/DSPE-PEG₂₀₀₀ liposomes after transition of the phospholipid membrane from the gel phase to liquid crystalline phase using high performance liquid chromatography (HPLC) in combination with static light scattering, dynamic light scattering, and cryo-transmission electron microscopy (cryo-TEM). The rates of DPPC hydrolysis in DPPC/DSPE-PEG₂₀₀₀ liposomes were investigated at a pH of 2, 4, or 6.5 and temperatures of 22 °C or 4 °C. Results indicate that following phase transition, severe structural reorganizations occurred in liposome samples that were partially hydrolyzed in the gel phase. The most prominent effect was an increasing tendency of liposomes to disintegrate into membrane discs in accordance with an increasing degree of phospholipid hydrolysis. Complete disintegration occurred when DPPC concentrations had decreased by, in some cases, as little as 3.6%. After extensive phospholipid hydrolysis, liposomes and discs fused to form large bilayer sheets as well as other more complex bilayer structures apparently due to a decreased ratio of lysolipid to palmitic acid levels in the liposome membrane.     

Interactions of VIP with rigid phospholipid bilayers: implications for vasoreactivity

The purpose of this study was to determine whether vasoactive intestinal peptide (VIP), a pleiotropic amphipathic peptide, interacts with rigid liposomes composed of gel phase phospholipids. We found that incubation of VIP with small unilamellar gel phase liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and egg phosphatidylglycerol (ePG) for 2h at room temperature had no significant effects on VIP secondary structure. Moreover, suffusion of VIP (0.01, 0.1 and 1.0nmol) incubated in saline or with DPPC/ePG liposomes (size, 30 and 100nm) for 2h at room temperature or 4 degrees C onto the intact hamster cheek pouch microcirculation elicited a similar concentration-dependent vasodilation except for 0.01nmol VIP (P<0.05). By contrast, incubation of VIP with gel phase liposomes overnight at 4 degrees C significantly potentiated vasodilation evoked by all three concentrations of the peptide in comparison to aqueous VIP (P<0.05). VIP-induced vasodilation was liposome size-independent. The ratio of VIP to phospholipids in DPPC/ePG liposomes was concentration-independent. Collectively, these data indicate that short-term interactions of VIP with rigid phospholipid bilayers are limited resulting in only modest effects on VIP vasoreactivity in vivo.     

The incorporation of rifampicin into multilayer and monolayer vesicles (liposomes) of different phosholipid composition

The incorporation of rifampicin into multilayer phospholipid vesicles depending on the concentration of antibiotic and phospholipid content was studied. The extent of incorporation of rifampicin into monolayer vesicles (liposomes), obtained by the homogenization of multilamellar vesicles, was determined by the method of gel filtration. It was found that rifampicin better penetrates and is retained in membranes consisting of a mixture of phosphatidylcholine and cardiolipin, the maximum incorporation of rifampicin into liposomes being 17%. It was shown by 31P NMR spectroscopy that, during the interaction of rifampicin with the phospholipid membrane, the bilayer packing of phospholipids is destroyed.     

Chitosan-reduced gold nanoparticles: a novel carrier for the preparation of spray-dried liposomes for topical delivery

Exposure of skin to various chemical and physical agents results in excessive stress to the outermost cell layer of the skin, causing different degenerative effects that can be minimized by using antioxidant formulations. The major challenge, in this regard, is to develop a formulation, which can prevent photodegradation of the actives, thus allowing a significant amount to be deposited at the site. In recent decades, liposomal formulations have been extensively employed to overcome the barrier properties of the skin and photodegradation of actives. In the present study, chitosan-reduced gold nanoparticles were investigated for its potential as a carrier to prepare liposomes by a spray-drying method. Liposomes so obtained were characterized for phospholipid recovery, diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, particle size, zeta potential, encapsulation efficiency, and deposition of drug and gold nanoparticles in the rat skin. Further, a liposomal gel formulation was prepared using Carbopol? 980 NF (Noveon Systems, Kochi, India) and evaluated for drug deposition in the skin. Antioxidant activity of vitamin C encapsulated in gold liposomes was determined on a human leukemia (HL-60) cell line. The use of gold nanoparticles as a carrier showed improved phospholipid recovery and thus overcomes the liposome scalability problem. DRIFT spectra confirmed the presence of phospholipid in the formulation. Liposomal gel showed improved drug deposition, as compared to control and marketed preparations. A more interesting contribution of the chitosan-reduced gold nanoparticles was an enhanced antioxidant activity seen in case of the vitamin C-loaded gold liposomal formulation. Liposomal formulation was found to be stable for 3 months at 30°C and 65% relative humidity.     

Novel camptothecin analogue (gimatecan)-containing liposomes prepared by the ethanol injection method

Small-sized liposomes have several advantages as drug delivery systems, and the ethanol injection method is a suitable technique to obtain the spontaneous formation of liposomes having a small average radius. In this paper, we show that liposomal drug formulations can be prepared in situ, by simply injecting a drug-containing lipid(s) organic solution into an aqueous solution. Several parameters should be optimized in order to obtain a final suitable formulation, and this paper is devoted to such an investigation. Firstly, we study the liposome size distributions determined by dynamic light scattering (DLS), as function of the lipid concentration and composition, as well as the organic and aqueous phases content. This was carried out, firstly, by focusing on POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) then on the novel L-carnitine derivative PUCE (palmitoyl-(R)-carnitine undecyl ester chloride), showing that it is possible to obtain monomodal size distributions of rather small vesicles. In particular, depending on the conditions, it was possible to achieve a population of liposomes with a mean size of 100 nm, when a 50 mM POPC ethanol solution was injected in pure water; in the case of 50 mM PUCE the mean size was around 30 nm, when injected in saline (0.9% NaCl). The novel anticancer drug Gimatecan, a camptothecin derivative, was used as an example of lipophilic drug loading by the injection method. Conditions could be found, under which the resultant liposome size distributions were not affected by the presence of Gimatecan, in the case of POPC as well as in the case of PUCE. To increase the overall camptothecin concentration in the final liposomal dispersion, the novel technique of "multiple injection method" was used, and up to a final 5 times larger amount of liposomal drug could be reached by maintaining approximately the same size distribution. Once prepared, the physical and chemical stability of the liposome formulations was satisfactory within 24, as judged by DLS analysis and HPLC quantitation of lipids and drug. The Gimatecan-containing liposomes formulations were also tested for in vitro and in vivo activity, against the human nonsmall cell lung carcinoma NCI-H460 and a murine Lewis lung carcinoma 3 LL cell lines. In the in vitro tests, we did not observe any improvement or reduction of the Gimatecan pharmacological effect by the liposomal delivery system. More interestingly, in the in vivo Lewis lung carcinoma model, the intravenously administration of liposomal Gimatecan formulation showed a mild but significant increase of Tumor Volume Inhibition with respect to the oral no-liposomal formulation (92% vs. 86 %, respectively; p < 0.05). Finally, our study showed that the liposomal formulation was able to realize a delivery system of a water-insoluble drug, providing a Gimatecan formulation for intravenous administration with a preserved antitumoral activity.     

Liposome-associated tumor necrosis factor retains bioactivity in the presence of neutralizing anti-tumor necrosis factor antibodies

Cell-associated TNF-alpha, either bound to its receptor on monocyte membranes or expressed as an integral membrane protein, can exert potent tumor cytolytic activity. We assessed the interaction of TNF with the lipid bilayer membrane system, liposomes, and the effects of membrane association on TNF bioactivity. High levels of TNF can be encapsulated within liposomes. At neutral pH, TNF binds to the surface of preformed liposomes (liposome-associated TNF), but does not partition into the lipid bilayer. TNF appears to bind to negatively charged phospholipid head groups of the outer membrane leaflet. Free TNF, liposome-associated TNF, and liposome-encapsulated TNF display comparable abilities to activate human peripheral blood monocytes and to lyse tumor cells. However, liposome-encapsulated TNF, as well as TNF bound to the outer surface of preformed liposomes, retains bioactivity in the presence of anti-TNF antibodies that neutralize free TNF. The interaction of liposomal TNF with cell surface TNF receptors thus appears to be preserved in the presence of neutralizing antibodies.     

Effectiveness of liposomal paclitaxel against MCF-7 breast cancer cells

Paclitaxel is an effective chemotherapeutic agent that is widely used for the treatment of several cancers, including breast, ovarian, and non-small-cell lung cancer. Due to its high lipophilicity, paclitaxel is difficult to administer and requires solubilization with Cremophor EL (polyethoxylated castor oil) and ethanol, which often lead to adverse side effects, including life-threatening anaphylaxis. Incorporation of paclitaxel in dimyristoylphosphatidylcholine:dimyristoylphosphatidylglycerol (DPPC:DMPG) liposomes can facilitate its delivery to cancer cells and eliminate the adverse reactions associated with the Cremophor EL vehicle. Accordingly, the effectiveness of liposomal paclitaxel on MCF-7 breast cancer cells was examined. The results from this study showed that (i) the lipid components of the liposomal formulation were nontoxic, (ii) the cytotoxic effects of liposomal paclitaxel were improved when compared with those seen with conventional paclitaxel, and (iii) the intracellular paclitaxel levels were higher in MCF-7 cells treated with the liposomal paclitaxel formulation. The results of these studies showed that delivery of paclitaxel as a liposomal formulation could be a promising strategy for enhancing its chemotherapeutic effects.     

Elucidation of the Interaction Mechanism with Liposomes of gH625-Peptide Functionalized Dendrimers

We have demonstrated that amide-based dendrimers functionalized with the membrane-interacting peptide gH625 derived from the herpes simplex virus type 1 (HSV-1) envelope glycoprotein H enter cells mainly through a non-active translocation mechanism. Herein, we investigate the interaction between the peptide-functionalized dendrimer and liposomes composed of PC/Chol using fluorescence spectroscopy, isothermal titration calorimetry, and surface plasmon resonance to get insights into the mechanism of internalization. The affinity for the membrane bilayer is very high and the interaction between the peptide-dendrimer and liposomes took place without evidence of pore formation. These results suggest that the presented peptidodendrimeric scaffold may be a promising material for efficient drug delivery.     

Novel Camptothecin Analogue (Gimatecan)‐Containing Liposomes Prepared by the Ethanol Injection Method

Small‐sized liposomes have several advantages as drug delivery systems, and the ethanol injection method is a suitable technique to obtain the spontaneous formation of liposomes having a small average radius. In this paper, we show that liposomal drug formulations can be prepared in situ, by simply injecting a drug‐containing lipid(s) organic solution into an aqueous solution. Several parameters should be optimized in order to obtain a final suitable formulation, and this paper is devoted to such an investigation. Firstly, we study the liposome size distributions determined by dynamic light scattering (DLS), as function of the lipid concentration and composition, as well as the organic and aqueous phases content. This was carried out, firstly, by focusing on POPC (1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) then on the novel L‐carnitine derivative PUCE (palmitoyl‐(R)‐carnitine undecyl ester chloride), showing that it is possible to obtain monomodal size distributions of rather small vesicles. In particular, depending on the conditions, it was possible to achieve a population of liposomes with a mean size of 100 nm, when a 50 mM POPC ethanol solution was injected in pure water; in the case of 50 mM PUCE the mean size was around 30 nm, when injected in saline (0.9% NaCl). The novel anticancer drug Gimatecan, a camptothecin derivative, was used as an example of lipophilic drug loading by the injection method. Conditions could be found, under which the resultant liposome size distributions were not affected by the presence of Gimatecan, in the case of POPC as well as in the case of PUCE. To increase the overall camptothecin concentration in the final liposomal dispersion, the novel technique of “multiple injection method” was used, and up to a final 5 times larger amount of liposomal drug could be reached by maintaining approximately the same size distribution. Once prepared, the physical and chemical stability of the liposome formulations was satisfactory within 24, as judged by DLS analysis and HPLC quantitation of lipids and drug. The Gimatecan‐containing liposomes formulations were also tested for in vitro and in vivo activity, against the human nonsmall cell lung carcinoma NCI‐H460 and a murine Lewis lung carcinoma 3 LL cell lines. In the in vitro tests, we did not observe any improvement or reduction of the Gimatecan pharmacological effect by the liposomal delivery system. More interestingly, in the in vivo Lewis lung carcinoma model, the intravenously administration of liposomal Gimatecan formulation showed a mild but significant increase of Tumor Volume Inhibition with respect to the oral no‐liposomal formulation (92% vs. 86 %, respectively; p < 0.05). Finally, our study showed that the liposomal formulation was able to realize a delivery system of a water‐insoluble drug, providing a Gimatecan formulation for intravenous administration with a preserved antitumoral activity.