Lipid Membrane Of Liposomes Research Articles

An in vitro digestion study of encapsulated lactoferrin in rapeseed phospholipid–based liposomes

Effectiveness of liposomes elaborated with rapeseed phospholipid (RP) extracted from a residue of oil processing, stigmasterol (ST) and/or hydrogenated phosphatidylcholine (HPC) for the encapsulation lactoferrin (LF) was studied; lipid membrane of liposomes was characterized (bilayer size, chain conformational order, lateral packing, lipid phase, and morphology) and the protection offered to the encapsulated LF during in vitro digestion was determined. Liposomes composed of RP+STLC(low concentration) showed spherical and irregular vesicles without perforations. Lamellar structure was organized in a liquid–ordered phase with a potential orthorhombic packing. Stability and size of the liposomes were more affected by gastric digestion than intestinal digestion; 67–80% of the initially encapsulated LF remained intact after gastric digestion whereas the percentage was reduced to 16–35% after intestinal digestion. Our results shows that liposomes elaborated with RP, properly combined with other lipids, can be a useful oral delivery system of molecules sensitive to digestive enzymes.

Phospholipid prodrug conjugates of insoluble chemotherapeutic agents for ultrasound targeted drug delivery.

The hydrophobicity and high potency of many therapeutic agents makes them difficult to use effectively in clinical practice. This work focuses on conjugating phospholipid tails (2T) onto podophyllotoxin (P) and its analogue (N) using a linker and characterizing the effects of their incorporation into lipid-based drug delivery vehicles for triggered ultrasound delivery. Differential Scanning Calorimetry results show that successfully synthesized lipophilic prodrugs, 2T-P (~28 % yield) and 2T-N(~26 % yield), incorporate within the lipid membranes of liposomes. As a result of this, increased stability and incorporation are observed in 2T-P and 2T-N in comparison to the parent compounds P and N. Molecular dynamic simulation results support that prodrugs remain within the lipid membrane over a relevant range of concentrations. 2T-N's (IC50: 20 nM) biological activity was retained in HeLa cells (cervical cancer), whereas 2T-P's (IC50: ~4 µM) suffered, presumably due to steric hindrance. Proof-of-concept studies using ultrasound in vitro microbubble and nanodroplet delivery vehicles establish that these prodrugs are capable of localized drug delivery. This study provides useful information about the synthesis of double tail analogues of insoluble chemotherapeutic agents to facilitate incorporation into drug delivery vehicles. The phospholipid attachment strategy presented here could be applied to other well suited drugs such as gemcitabine, commonly known for its treatment of pancreatic cancer.

Preparation, Characterization, and in Vitro/in Vivo Evaluation of Paclitaxel-Bound Albumin-Encapsulated Liposomes for the Treatment of Pancreatic Cancer.

Paclitaxel (PTX)-loaded liposomes were developed with the goal of enhancing the effects of cancer treatment. Although loading substances into the lipid membrane of liposome cause some destabilization of the lipid membrane, PTX was nearly exclusively embedded in the lipid membrane of liposomes, due to its low water solubility. Hydrophobic drugs can be encapsulated into the inner core of bovine serum albumin (BSA)-encapsulated liposomes (BSA-liposome) via noncovalent binding to albumin. Since PTX is able to noncovalently bind to albumin, we attempted to prepare PTX-loaded BSA-liposome (PTX–BSA-liposome). The amount of PTX loaded in the BSA-liposome could be increased substantially by using ethanol, since ethanol increases PTX solubility in BSA solutions via prompting the binding PTX to BSA. On the basis of the results of transmission electron microscopy and small-angle X-ray scattering, PTX–BSA-liposome formed unilamellar vesicles that were spherical in shape and the PTX was encapsulated into the inner aqueous core of the liposome as a form of PTX–BSA complex. In addition, the PTX–BSA-liposome, as well as nab-PTX, showed cytotoxicity against human pancreatic cancer cells, AsPC-1 cells, in a PTX concentration-dependent manner. The in vivo antitumor effect of PTX–BSA-liposomes was also observed in a mouse model that had been subcutaneously inoculated with pancreatic cancer cells by virtue of its high accumulation at the tumor site via the enhanced permeability retention effect. These results suggest that PTX–BSA-liposomes have the potential for serving as a novel PTX preparation method for the treatment of pancreatic cancer.

1H NMR Determination of Incorporated Porphyrin Location in Lipid Membranes of Liposomes

Abstract Lipid-membrane-incorporated symmetrical and unsymmetrical porphyrins were prepared with relatively high concentrations via exchange from cyclodextrins to liposomes. We determined the location of these porphyrins in the lipid membranes from chemical shift changes of the lipid signals in the 1H NMR spectra. Symmetrical tetraphenyl porphyrin accumulates in the hydrophobic core of the lipid membranes. In contrast, several unsymmetrical porphyrins with more polar group(s), such as pyridine and pyridinium moieties, are located close to the liposome surface.

Targeted anion transporter delivery by coiled-coil driven membrane fusion.

Synthetic anion transporters (anionophores) have potential as biomedical research tools and therapeutics. However, the efficient and specific delivery of these highly lipophilic molecules to a target cell membrane is non-trivial. Here, we investigate the delivery of a powerful anionophore to artificial and cell membranes using a coiled-coil-based delivery system inspired by SNARE membrane fusion proteins. Incorporation of complementary lipopeptides into the lipid membranes of liposomes and cell-sized giant unilamellar vesicles (GUVs) facilitated the delivery of a powerful anionophore into GUVs, where its anion transport activity was monitored in real time by fluorescence microscopy. Similar results were achieved using live cells engineered to express a halide-sensitive fluorophore. We conclude that coiled-coil driven membrane fusion is a highly efficient system to deliver anionophores to target cell membranes.

The engineering of doxorubicin-loaded liposome-quantum dot hybrids for cancer theranostics

Many studies have recently attempted to develop multifunctional nanoconstructs by integrating the superior fluorescence properties of quantum dots (QD) with therapeutic capabilities into a single vesicle for cancer theranostics. Liposome-quantum dot (L-QD) hybrid vesicles have shown promising potential for the construction of multifunctional nanoconstructs for cancer imaging and therapy. To fulfil such a potential, we report here the further functionalization of L-QD hybrid vesicles with therapeutic capabilities by loading anticancer drug doxorubicin (Dox) into their aqueous core. L-QD hybrid vesicles are first engineered by the incorporation of TOPO-capped, CdSe/ZnS QD into the lipid bilayers of DSPC:Chol:DSPE-PEG2000, followed by Dox loading using the pH-gradient technique. The loading efficiency of Dox into L-QD hybrid vesicles is achieved up to 97%, comparable to liposome control. All these evidences prove that the incorporation of QD into the lipid bilayer does not affect Dox loading through the lipid membrane of liposomes using the pH-gradient technique. Moreover, the release study shows that Dox release profile can be modulated simply by changing lipid composition. In conclusion, the Dox-loaded L-QD hybrid vesicles presented here constitute a promising multifunctional nanoconstruct capable of transporting combinations of therapeutic and diagnostic modalities.

Influence of Polyelectrolyte Chemical Structure on their Interaction with Lipid Membrane of Zwitterionic Liposomes

In this paper we extend our previous experimental work on interaction between polyelectrolytes and liposomes. First, the adsorption of chitosan and alkylated chitosan (cationic polyelectrolytes) with different alkyl chain lengths on lipid membranes of liposomes is examined. The amount of both chitosans adsorbed remains the same even if more alkylated polysaccharide has to be added to get saturation if compared with unmodified chitosan. It is demonstrated that alkyl chains do not specifically interact with the lipid bilayer and that electrostatic interaction mechanism governs the chitosan adsorption. The difference observed between unmodified and alkylated chitosans behavior to reach the plateau can be interpreted in terms of a competition between electrostatic polyelectrolyte adsorption on lipid bilayer and hydrophobic autoassociation in solution (which depends on the alkyl chain length). Second, interaction of liposomes with hyaluronan (HA) and alkylated hyaluronan (anionic polyelectrolytes) is analyzed. The same types of results as discussed for chitosan are obtained, but in this case, autoassociation of alkylated HA only occurs in the presence of salt excess. Finally, a first positive layer of chitosan is adsorbed on the lipid membrane, followed by a second negative layer of HA at three different pHs. This kind of multilayer decoration allows the control of the net charge of the composite vesicles. A general conclusion is that whatever the pH and, consequently, the initial charge of the liposomes, chitosan adsorption gives positively charged composite systems, which upon addition of hyaluronan, give rise to negatively charged composite vesicles.

Liposomal 1,25 (OH)2 vitamin D3 compounds block proliferation and induce differentiation in myelomonocytic leukaemia cells.

The vitamin D3 derived hormone 1.25 (OH)2 vitamin D3 (1,25 D3) is able to induce growth arrest and differentiation in myelomonocytic leukaemia cells. In order to allow for specific delivery to leukaemic cells the lipophilic compound was incorporated into the lipid membranes of liposomes. Liposomal 1.25 D3 reduced proliferation as measured by 3H-thymidine incorporation in HL60 leukaemia cells by up to 60%. When liposomes were prepared at different concentrations of 1,25 D3 65% inhibition was achieved at 48 nM. The MC 1288 stereoisomer of 1,25 D3 was more potent and had the same activity at 4.8 nM. The effect of the liposomal compounds was specific to myeloid cells as they reduced proliferation in myelomonocytic HL60, monoblastic U937 and monocytic Mono Mac 6 cells but not in the T-cell lines Jurkat and Molt 4. The antiproliferative effect of liposomal 1,25 D3 was associated with an induction of differentiation since treated HL60 cells showed a monocytic morphology, increased expression of CD14 and decreased expression of CD33. When peripheral blood leukaemic cells from M4 and M5 acute myeloid leukaemia (AML) patients were admixed with liposomal compounds an antiproliferative effect was seen in all five cases, including the two cases where free compounds led to enhanced growth. Liposomal delivery of 1,25 (OH)2 vitamin D3 may offer a novel approach to treatment of myelomonocytic leukaemia.

Delivery of liposome-sequestered hydrophobic proteins to lysosomes of normal and Batten disease cells.

We have developed a method to deliver hydrophobic proteins such as ATP synthase subunit c and ubiquitin to lysosomes of PMN (polymorphonucleocytes) and fibroblasts. ATP synthase subunit c is stored in the lysosomes of various tissues in late infantile and juvenile forms of neuronal ceriod lipofuscinosis, also called Batten disease (BD). Whether this protein storage is due to an abbreviation in protein or in the lysosomal hydrolases of BD is still not clear. We have sequestered this protein and ubiquitin in the lipid membrane of liposomes. The liposomes coated with autologous heat-aggregated IgG or apolipoprotein E when presented to the PMN and fibroblasts, respectively, accumulated in the lysosomes. Both normal and BD PMN degraded 125I-ubiquitin; the rate of degradation was, however, slower by Batten PMN. These studies indicate that a hydrophobic molecule such as subunit c can be delivered to PMN and fibroblasts, and the sequestered proteins are accessible to lysosomal hydrolases. Therefore, this technique can be used to study the metabolism of highly hydrophobic proteins by lysosomes, especially the biochemical mechanism(s) of subunit c storage in BD.

Synthesis and in vitro Antiviral Properties of Amphiphilic Dinucleoside Phosphate Derivatives of 2′,3-dideoxycytidine (ddC)

N4-hexadecyl-5′-0-(4-monomethoxytrityl)-2′-deoxycytidine-3′-hydrogenphosphonate and 5′-0-(4-monomethoxytrityl)-2′-deoxythymidine-3′-0-hydrogenphosphonate were condensed with 2′,3′-dideoxycytidine (ddC) according to the hydrogenphosphonate method to yield N4-hexadecyl-2′-deoxycytidylyl-(3′-5′)-2′,3′-dideoxycytidine (N4-hexadecyldC-ddC) and 2′-deoxythymidylyl-(3′-5′)-N4-palmitoyl-2′,3′-dideoxycytidine (dT-N4-palmddC). N4-palmitoyl-2′,3′-dideoxycytidine (N4-palmddC) was synthesized by reacting palmitic anhydride with ddC. Both dinucleoside phosphates have amphiphilic properties and represent a new class of ddC derivatives in which in the case of the dinucleosides, the ddC-5′-monophosphate is masked with lipophilic residues of variable stability. The ddC derivatives can be solubilized in water by micelle formation and, because they have lipophilic residues, they can be incorporated into the lipid membranes of liposomes. The ddC derivatives were shown to have antiviral activities comparable to those of AZT and ddC when tested in vitro against HIV-1-infected HeLa and H9 cells as well as infected human monocytes/macrophages.

Synthesis, and Some Properties of, Amphiphilic Dinucleoside Phosphate Derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT)

N4-hexadecyl-5′-0-(4-monomethoxytrityl)-2′-deoxycytidine-3′-hydrogenphosphate was reacted with 3′-azido-2′,3′-dideoxythymidine (AZT) according to the hydrogenphosphate method to yield N4-hexadecyl-2′-deoxycytidylyl-(3′-5)-3′-azido-2′,3′-dideoxythymidine. N4-palmitoyl-5′-O-(4-monomethoxytrityl)-2′-deoxycytidine-3′-(2-chlorophenyl)-phosphate was condensed to AZT using the triester method to give N4-palmitoyl-2′-deoxycytidylyl-(3′-5′)-3,-azido-2′,3′-dideoxythymidine. Both dinucleosidephosphates have amphiphilic properties and represent a new class of AZT derivatives in which the polar AZT-5′-monophosphate is masked with lipophilic deoxycytidine residues of variable stability. The AZT derivatives are water soluble, by forming micelles, and as a result of their amphiphilic nature, they can be incorporated into the lipid membranes of liposomes. In contrast to the micellar drug preparations, the liposomal formulations were shown to exert no lytic activity on human erythrocytes. Both AZT derivatives have anti HIV-1 activity in vitro.

Muramyl Tripeptide Phosphatidylethanolamine Encapsulated in Liposomes Stimulates Monocyte Production of Tumor Necrosis Factor and Interleukin-1 In Vitro

Muramyl tripeptide phosphatidylethanolamine (MTP-PE), a synthetic lipophilic analogue of muramyl dipeptide (MDP), can be incorporated into the lipid membrane of liposomes. Liposomes containing MTP-PE (L-MTP-PE) stimulated monocytes to selectively kill tumors, but not normal cells in vitro. Furthermore, the activation of monocyte tumoricidal function was demonstrated following the i.v. infusion of L-MTP-PE in a phase I trial with cancer patients. The purpose of this study was to determine the mechanism by which L-MTP-PE activates monocytes. Monocyte tumoricidal function is linked to both interleukin-1 (IL-1) and tumor necrosis factor (TNF). Therefore, normal human monocytes were incubated for various times with L-MTP-PE, empty liposomes, or medium in the presence or absence of gamma interferon (IFN-gamma). The supernatants were removed and assayed for TNF and IL-1 using the L929 and D10.G4.1 assays, respectively. TNF was detected after a 4 hr incubation with L-MTP-PE but not with empty liposomes or medium. TNF secretion peaked at 8 hr and was sustained for up to 72 hr. A 4-fold increase in TNF mRNA levels was demonstrated after 8 hr. An increased level of IL-1 beta mRNA was detected after a 4 hr incubation, but only low level IL-1 secretion was detected in monocytes incubated with L-MTP-PE. Adherent monocytes were frozen and thawed to release intracellular IL-1. Intracellular IL-1 was significantly increased in monocytes incubated with L-MTP-PE. Intracellular IL-1 levels peaked by 8 hr and decreased by 72 hr. Activators were then assayed in the presence or absence of IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)