Dehydration-rehydration Vesicles Research Articles

Daptomycin Liposomes Exhibit Enhanced Activity against Staphylococci Biofilms Compared to Free Drug.

The purpose of the present study was to investigate the anti-staphylococcal activity of liposomal daptomycin against four biofilm-producing S. aureus and S. epidermidis clinical strains, three of which are methicillin-resistant. Neutral and negatively charged daptomycin-loaded liposomes were prepared using three methods, namely, thin-film hydration (TFH), a dehydration-rehydration vesicle (DRV) method, and microfluidic mixing (MM); moreover, they were characterized for drug encapsulation (EE%), size distribution, zeta-potential, vesicle stability, drug release, and drug integrity. Interestingly, whilst drug loading in THF and DRV nanosized (by extrusion) vesicles was around 30-35, very low loading (~4%) was possible in MM vesicles, requiring further explanatory investigations. Liposomal encapsulation protected daptomycin from degradation and preserved its bioactivity. Biofilm mass (crystal violet, CV), biofilm viability (MTT), and growth curve (GC) assays evaluated the antimicrobial activity of neutral and negatively charged daptomycin-liposomes towards planktonic bacteria and biofilms. Neutral liposomes exhibited dramatically enhanced inhibition of bacterial growth (compared to the free drug) for all species studied, while negatively charged liposomes were totally inactive. Biofilm prevention and treatment studies revealed high antibiofilm activity of liposomal daptomycin. Neutral liposomes were more active for prevention and negative charge ones for treating established biofilms. Planktonic bacteria as well as the matured biofilms of low daptomycin-susceptible, methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE) strains were almost completely eradicated by liposomal-daptomycin, indicating the need for their further exploration as antimicrobial therapeutics.

Engineered versus hybrid cellular vesicles as efficient drug delivery systems: a comparative study with brain targeted vesicles.

Herein we elaborated on methods to load cellular vesicles (CVs) and to incorporate cholesterol (Chol) and PEG lipids in their membrane, for enhancing the potential of such engineered CVs (e-CVs) as drug carriers. Hybrids formed by fusion between PEGylated liposomes (PEG-LIP) and CVs were evaluated as alternatives to e-CV, for the first time. Freeze-thawing cycles (FT) and incubation protocols were tested, and vesicle fusion was monitored by FRET dilution. B16F10, hCMEC/D3, and LLC cells were used for e-CV or hybrid development, and FITC-dextran as a model hydrophilic drug. Results show that dehydration rehydration vesicle (DRV) method is optimal for highest CV loading and integrity, while optimal protocols for Chol/PEG enrichment were identified. FT was found to be more efficient than incubation for hybrid formation. Interestingly, despite their high Chol content, CVs had very low integrity that was not increased by enrichment with Chol, but only after PEG coating; e-CVs demonstrated higher integrity than hybrids. Vesicle uptake by hCMEC cells is in the order: LIP < e-CVs < Hybrids ≤ CVs (verified by confocal microscopy); the higher PEG content of e-CVs is possibly the reason for their reduced cell uptake. While CV and hybrid uptake are highly caveolin-dependent, e-CVs mostly follow clathrin-dependent pathways. In vivo and ex vivo results show that brain accumulation of hybrids is only slightly higher that of CVs, indicating that the surface PEG content of hybrids is not sufficient to prevent uptake by macrophages of the reticuloendothelial system. Taking together with the fact that subjection of CVs to FT cycles reduced their cellular uptake, it is concluded that PEGylated e-CVs are better than hybrids as brain-targeted drug carriers.

Hypericin inclusion complexes encapsulated in liposomes for antimicrobial photodynamic therapy.

The naturally occurring anthraquinone derivative hypericin is a highly potent photosensitiser. Several in vitro studies show high phototoxicity of the pigment towards gram-positive bacteria. Nevertheless, the highly lipophilic nature and poor bioavailability prevent its application in daily clinical practice thus leading to a limited therapeutic value of hypericin. Liposomal encapsulation could help overcome these limitations and would make hypericin available for daily clinical practice. The use of liposomes as carriers for hypericin in antimicrobial photodynamic therapy (aPDT) is quite new. The aim of this work was to improve the photodynamic efficiency of the previously mentioned carriers by entrapping hypericin in the aqueous compartment of the liposomes. Therefore, a water-soluble inclusion complex of hypericin and (2-hydroxypropyl)-beta-cyclodextrin (Hyp-HPβCD) was prepared. After encapsulation of the inclusion complex into DSPC and DSPC/DPPC/DSPE-PEG liposomes with the dehydration-rehydration vesicle (DRV) method, the formulations were physicochemical characterised. The photodynamic efficiency towards the gram-positive model strain Staphylococcus saprophyticus subsp. bovis. was tested on planktonic cells as well as on biofilms. DSPC liposomes achieved a 4.1log reduction and the DSPC/DPPC/DSPE-PEG liposomes a 2.6log reduction in growth of planktonic bacteria, while Hyp-HPβCD showed total eradication. Even bacterial cells growing in a biofilm could be treated effectively in vitro.

Photodynamic therapy – hypericin tetraether liposome conjugates and their antitumor and antiangiogenic activity

Photodynamic therapy (PDT) is an established noninvasive tumor treatment. The hydrophobic natural occurring pigment hypericin shows a lot of attractive properties for the application in PDT. Hence, the administration to biological systems or patients requires the formulation in drug carriers enabling sufficient bioavailability. Therefore, free hypericin was encapsulated by the thin film hydration method or a hypericin-hydroxypropyl-β-cyclodextrin inclusion complex (Hyp-HPβCD) was incorporated by dehydration-rehydration vesicle method in either conventional or ultra-stable tetraether lipid (TEL) liposomes. The hydrodynamic diameter of the prepared nanoformulations ranged between 127 and 212 nm. These results were confirmed by atomic force microscopy. All liposomes showed a good stability under physiological conditions. TEL liposomes which tend to build more rigid bilayers, generate higher encapsulation efficiencies than their conventional counterparts. Furthermore, the suitability for intravenous application was confirmed by hemocompatibility studies resulting in a hemolytic potential less than 20% and a coagulation time less than 50 sec. The uptake of liposomal hypericin into human ovarian carcinoma cells (SK-OV-3) was confirmed using confocal microscopy and further characterized by pathway studies. It was demonstrated that the lipid composition and intraliposomal hypericin localization influenced the anti-vascular effect in the chorioallantoic membrane (CAM). While hypericin TEL liposomes exhibit substantial destruction of the microvasculature drug-in-cyclodextrin TEL liposomes showed no effect. Nevertheless, both formulations yielded severe photocytotoxicity in SK-OV-3 cells in a therapeutic dosage range. Conclusively, hypericin TEL liposomes would be perfectly suited for anti-vascular targeting while Hyp-HPβCD TEL liposomes could deliver the photosensitizer to the tumor site in a more protected manner.

Preparation and characterization of physicochemical properties of N, N-diethyl-meta-toluamide niosomes.

Introduction:The insect repellent compounds are used to protect humans, animals and plants against insect bites. Aromatic amides have insect repellent effects. N,N-diethyl-meta-toluamide (DEET) (C12 H17 NO) is one of the best insect repellents has been used for many years. DEET is a colorless, odorless liquid that is approximately insoluble in water and soluble in glycerin, ethanol, and isopropyl alcohol. Due to the solubility problem of DEET, its topical formulations usually have alcoholic bases, but these kind of formulations increase skin permeation and also systemic absorption of DEET, which leads to some toxic effects. The main goal of this study was to prepare the formulation of DEET niosomes in a topical dosage form with suitable stability properties.Materials and Methods:Three different methods were used to prepare niosome formulations: Dehydration rehydration vesicle method, direct mixing method, homogenizer method. Sorbitan surfactants, cholesterol, polyoxyethylenecetyl, phosphate buffer (pH 7.4), and charge inductive compounds like cetyltrimethylammonium bromide were used to provide a net negative charge to the final membrane structure. A high-performance liquid chromatography method was then used for the determination of the loaded DEET.Results:A large number of niosomes were multi-layered and have a spherical shape. In comparison, syringe method against direct mixing is more appropriate because of creation MLV and uniform niosomes but the best method is homogenizer method. Drug entrapment was between 14% and 21% in selected formulation.Conclusion:According to this study, homogenizing method can be used for formulation of DEET in niosome form in topical formulations.

Antimicrobial properties of liposomal azithromycin for Pseudomonas infections in cystic fibrosis patients.

This work was carried out to construct a novel liposomal azithromycin formulation and examine its antimicrobial effects against Pseudomonas aeruginosa. The liposomal azithromycin formulation was prepared by the dehydration-rehydration vesicle method and its characterizations were tested. The MIC and the MBC of the liposomal formulation were determined by the microbroth dilution method. Liposomal azithromycin activity against biofilm-forming P. aeruginosa was assessed using a Calgary biofilm device. The effect of subinhibitory concentrations of liposomal azithromycin on bacterial virulence factors and motility studies was tested on P. aeruginosa strains. The bacteria and liposome interactions were studied using flow cytometry analysis. The toxicities of the liposomal formulation on erythrocytes and A549 lung cells were evaluated in vitro. The average diameter of the liposomal azithromycin was 406.07 ± 45 nm and the encapsulation efficiency was 23.8% ± 0.2%. The MIC and MBC values of liposomal azithromycin were significantly lower than those of free azithromycin. The liposomal azithromycin significantly reduced the bacteria in the biofilm and attenuated the production of different virulence factors; it also reduced the different patterns of bacterial motilities. By flow cytometry analysis data, it was shown that there are interactions of liposomes with the bacterial membranes. No significant haemolysis or cell toxicity was observed with the liposomal formulation. The results of this research indicate that this novel liposomal azithromycin formulation could be a useful therapy to enhance the safety and efficacy of azithromycin against P. aeruginosa-infected persons.

Increasing the stability of curcumin in serum with liposomes or hybrid drug-in-cyclodextrin-in-liposome systems: A comparative study

Curcumin (CURC) was incorporated in liposomes as free drug or after formation of hydropropyl-β- or hydroxypropyl-γ-cyclodextrin (HPβCD or HPγCD) complexes prepared by coprecipitation and characterized by X-ray diffractometry. Liposomes encapsulating CURC as free drug or CD-complexes (hybrid formulations) were prepared by the dehydration–rehydration vesicle (DRV) method followed by extrusion, and characterized for size, zeta-potential and CURC loading. CURC stability (at 0.01 and 0.05mg/mL) in 80% (v/v) fetal bovine serum (FBS) was evaluated at 37°C. Results demonstrate that HPβCD stabilizes CURC more than HPγCD, but liposome encapsulation provides substantially more protection, than CDs. CURC stabilization is similar, when encapsulated as free compound or CD-complex. However, the last method increases CURC loading by 23 times (depending on the lipid composition of liposomes and the CD used), resulting in higher solubility. The stability profile of CURC in serum depends on the composition of liposomes and CURC concentration, since at lower concentrations larger CURC fractions are protected due to protein binding. Compared to the corresponding CD complexes, hybrid formulations provide intermediate CURC solubility (comparable to HPβCD) but profoundly higher stabilization.

Study of the Charge Distribution on Liposome Particles Aerosolized by Air-Jet Atomization

Air-jet atomization is a common technique used for the generation of therapeutic aerosols from liposome suspensions for drug delivery to the lungs. Although the technique does not use an electric field, the aerosols generated by this technique are still charged, and this may affect respiratory drug deposition. In this study, the charge distribution of liposomes aerosolized by an air-jet atomizer was measured using a tandem differential mobility analyzer (TDMA) technique. The liposomes, composed of a mixture of two amphiphilic lipids and cholesterol, were synthesized by the dehydration-rehydration vesicle method. The effect of the precursor suspension properties, such as medium composition, pH, conductivity, and lipid mass concentration, on the charge distribution of the liposome aerosols was studied. Results showed that the atomized liposomes have a bipolar charge distribution, and the number-fraction of charged liposome aerosols was influenced strongly by properties of the precursor solution under investigation. Liposomes synthesized in deionized water were observed to carry much higher charges than those synthesized in phosphate-buffered saline (PBS). Increasing the lipid mass concentration in the precursor suspension resulted in a decrease in the charge on the aerosols. Thus, the precursor suspension properties--composition, pH, and conductivity--can be used to control the magnitude of charge on liposome aerosols and to synthesize engineered liposome particles for the pulmonary delivery of drugs with controlled alveolar deposition and controlled delivery to alveolar macrophages.

Small Cationic DDA:TDB Liposomes as Protein Vaccine Adjuvants Obviate the Need for TLR Agonists in Inducing Cellular and Humoral Responses

Most subunit vaccines require adjuvants in order to induce protective immune responses to the targeted pathogen. However, many of the potent immunogenic adjuvants display unacceptable local or systemic reactogenicity. Liposomes are spherical vesicles consisting of single (unilamellar) or multiple (multilamellar) phospholipid bi-layers. The lipid membranes are interleaved with an aqueous buffer, which can be utilised to deliver hydrophilic vaccine components, such as protein antigens or ligands for immune receptors. Liposomes, in particular cationic DDA:TDB vesicles, have been shown in animal models to induce strong humoral responses to the associated antigen without increased reactogenicity, and are currently being tested in Phase I human clinical trials. We explored several modifications of DDA:TDB liposomes - including size, antigen association and addition of TLR agonists – to assess their immunogenic capacity as vaccine adjuvants, using Ovalbumin (OVA) protein as a model protein vaccine. Following triple homologous immunisation, small unilamellar vesicles (SUVs) with no TLR agonists showed a significantly higher capacity for inducing spleen CD8 IFNγ responses against OVA in comparison with the larger multilamellar vesicles (MLVs). Antigen-specific antibody reponses were also higher with SUVs. Addition of the TLR3 and TLR9 agonists significantly increased the adjuvanting capacity of MLVs and OVA-encapsulating dehydration-rehydration vesicles (DRVs), but not of SUVs. Our findings lend further support to the use of liposomes as protein vaccine adjuvants. Importantly, the ability of DDA:TDB SUVs to induce potent CD8 T cell responses without the need for adding immunostimulators would avoid the potential safety risks associated with the clinical use of TLR agonists in vaccines adjuvanted with liposomes.

Comparison of liposome based antigen delivery systems for protection against Leishmania donovani

Liposomes have been widely exploited as antigen delivery systems for a variety of diseases including leishmaniasis. These vesicles can be prepared in various ways which may affect the immunogenicity of the encapsulated antigens. In this study we compared the vaccine potentiality of three cationic formulations with Leishmania donovani promastigote membrane antigens (LAg) and the best vesicle was evaluated for long-term protection against experimental visceral leishmaniasis. We immunized mice with LAg encapsulated in multilamellar vesicles (MLV), dehydration–rehydration vesicles (DRV) and reverse-phase evaporation vesicles (REV) and challenged them with parasites ten days after vaccination. LAg in MLV or DRV induced almost complete protection, while LAg alone or entrapped in REV exhibited partial resistance. Protection observed with antigen incorporated MLV or DRV was predominantly Th1 as evidenced by elicitation of significantly high DTH, IgG2a antibodies and IFN-γ. MLV encapsulated LAg demonstrated durable cell-mediated immunity and mice challenged ten weeks after vaccination could also resist experimental challenge strongly. Field trials of L. donovani vaccine were unsatisfactory mainly due to lack of an appropriate adjuvant. Cationic MLV when used as adjuvant with protein antigens induced sustained Th1 immunity. Adjuvant potential of cationic MLV can be utilized to design subunit vaccines.

The role of liposome charge on immune response generated in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63)

Liposomes as a lipid-based system have been shown to be an effective adjuvant formulation. In this study, the role of liposome charge in induction of a Th1 type of immune response and protection against leishmaniasis in BALB/c mice was studied. Liposomes containing rgp63 were prepared by Dehydration-Rehydration Vesicle (DRV) method. Neutral liposomes consisted of dipalmitoylphosphatidylcholine and cholesterol. Positively and negatively charged liposomes were prepared by adding dimethyldioctadecylammonium bromide (DDAB) or dicetyl phosphate (DCP) to the neutral liposome formulation, respectively. Female BALB/c mice were immunized subcutaneously with negatively, positively charged or neutral liposomes encapsulated with rgp63, rgp63 in soluble form or PBS, three times in 3 week intervals. The extent of protection and type of immune response generated were studied in different groups of mice. The group of mice immunized with rgp63 encapsulated in neutral liposomes showed a significantly ( P < 0.01) smaller footpad swelling upon challenge with Leishmania major compared with positively or negatively charged liposomes. The mice immunized with neutral liposomes also showed a significantly ( P < 0.01) the lowest splenic parasite burden, the highest IgG2a/IgG1 ratio and IFN-γ production and the lowest IL-4 level compared to the other groups. The results indicated that a Th1 type of immune response was induced in mice immunized with neutral liposomes more efficiently than positively charged liposomes and conversely negatively charged liposomes induced a Th2 type of immune response.

Characterisation of Chitosan-Coated Vesicles Encapsulating DNA Suitable for Gene Delivery

Encapsulation of DNA (varying type and molecular weight) in vesicles (dehydration-rehydration vesicles; DRV) prepared from zwitterionic phospholipids using the dehydration-rehydration method was determined to be in the range of 10-90%. Encapsulation was dependent on the amount of DNA added but not its molecular weight. Zeta potential measurements of the DRV suggested that DNA was associated with the vesicles exterior, while small angle neutron scattering (SANS) studies indicated that DNA was trapped in the interlamellar water space between lipid bilayers-as evidenced by a slight decrease in bilayer thickness and an increase in d-spacing. Circular dichroism measurements determined the conformation of the DNA in the DRV was not the usual B-form but rather a structure more reminiscent of the less hydrated, C-form, supporting the suggestion that DNA was present in the interlamellar water space. DRV were successfully coated with chitosan as evidenced by SANS studies and zeta potential measurements. The amount of chitosan coating the DRV [quantified by ultra violet (UV)/Vis spectroscopy] was dependent upon the starting concentration of chitosan and independent of the presence of DNA. Chitosan-coated DRV containing DNA exhibited an improved size stability while 'empty' uncoated vesicles were poorly stable. A coating of chitosan reduced the amount of DNA leaking from the vesicles.

Humoral Immune Response Against Epidermal Growth Factor Encapsulated in Dehydration Rehydration Vesicles of Different Phospholipid Composition

We investigated the influence of dehydration-rehydration vesicles (DRV) phospholipid composition and the addition of other components on human recombinant epidermal growth factor (hrEGF) encapsulation efficiency and its release from liposomes. Encapsulation of EGF into DRV composed of phosphatidylcholine with different unsaturation levels was around 20–35%. The best result was obtained with dipalmitoyl phosphatidylcholine: cholesterol (DPPC:Ch) liposomes (35%) corresponding to the lowest hrEGF release during one month of storage. Even with this phospholipid composition, modification of the DRV procedure by including an extrusion step did not improve hrEGF encapsulation efficiency, rendering less stable particles. The inclusion of recombinant P64k from Neisseria meningitidis (rP64k), as such or conjugated to hrEGF, decreased the encapsulation efficiency of the latter protein into DRV or freeze and thaw multilamellar vesicles (FATMLV). The hrEGF release from liposomes could be related to the interaction between this polypeptide and the bilayer, as evidenced by increased carboxyfluorescein release from hrEGF-DRV; less susceptibility to fluorescence quenching by acrylamide in the presence of liposomes; and a measurable decrease of phospholipid phase transition Δ enthalpy (ΔH). DRV comprising saturated phospholipids (DPPC:Ch or distearoyl phosphatidylcholine [DSPC]:Ch) and containing the conjugate EGF-P64k induced a more efficient immune response against hrEGF than unsaturated phospholipid and alum in terms of total IgG, IgG2a, and IgG2b subclasses and the ability of antibody to inhibit the interaction of the EGF receptor with hrEGF.

Effect of sugars on storage stability of lyophilized liposome/DNA complexes with high transfection efficiency

Cationic lipid-based gene delivery systems have shown promise in transfecting cells in vitro and in vivo. However, liposome/DNA complexes tend to form aggregates after preparation. Lyophilization of these systems, therefore, has become of increasing interest. In this study, we investigated the feasibility of preserving complexes as a dried preparation using a modified dehydration rehydration vesicle (DRV) method as a convenient and reliable procedure. We also studied storage stability of a lyophilized novel cationic gene delivery system incorporating sucrose, isomaltose and isomaltotriose. Liposomes were composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and L-dioleoylphosphatidylethanolamine (DOPE), plus sucrose, isomaltose or isomaltotriose. Lyophilized liposome/DNA complexes were stored at -20, 25, 40 and 50 degrees C and their stability was followed for 50 days. Liposome/DNA complexes with sucrose could be stored even at 50 degrees C without large loss of transfection efficiency. The transfection efficiency of formulations stored at various temperatures indicated that the stabilizing effect of sugars on plasmid DNA was higher in the following order: isomaltotriose<isomaltose<sucrose, which was inverse to the order of their glass transition temperature (T(g)) values. It was concluded that we could prepare novel lyophilized liposome/DNA complexes with high transfection efficiency and stability, which might be concerned that sucrose stabilized plasmid DNA in liposomes by directly interacting with plasmid DNA rather than by vitrifying to a high T(g) solid.

The role of CpG ODN in enhancement of immune response and protection in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63) encapsulated in cationic liposome

CpG oligodeoxynucleotides (CpG ODN) are known to be a potent immunoadjuvant for a wide range of antigens. The aim of this study was to evaluate the role of CpG ODN co-encapsulated with rgp63 antigen in cationic liposomes (Lip-rgp63-CpG ODN) in immune response enhancement and protection in BALB/c mice against leishmaniasis. Lip-rgp63-CpG ODN prepared by using dehydration-rehydration vesicle (DRV) method significantly inhibited (P<0.001) Leishmania major infection in mice measured by footpad swelling compared to Lip-rgp63, rgp63 alone, rgp63 plus CpG ODN, PBS or control liposomes. The mice immunized with Lip-rgp63-CpG ODN also showed the lowest spleen parasite burden, highest IgG2a/IgG1 ratio and IFN-gamma production and the lowest IL-4 production compared to the other groups. The results indicate that co-encapsulation of CpG ODN in liposomes improves the immunogenicity of Leishmania antigen.

Intracellular distribution of fluorescent probes delivered by vesicles of different lipidic composition

In order to study mechanisms involved in liposome–cell interaction, this work attempted to assess the influence of vesicle composition on the delivery of liposomal content to Hela cells. In particular, to evaluate pH-sensitive properties and cell interaction of the prepared liposomes, the lipid formulations contained cholesterol (Chol) and they were varied by using phosphatidylcholines with different purity degree: soy lecithin (SL; 80% phosphatidylcholine), a commercial mixture of soy phosphatidylcholine (P90; 90% phosphatidylcholine) or dipalmitoylphosphatidylcholine (DPPC; 99% of purity). A second series of liposomes also contained stearylamine (SA). Dehydration-rehydration vesicles (DRV) were prepared and then sonicated to decrease vesicle size. Vesicle–cell interactions and liposomal uptake were examined by fluorescence microscopy using carboxyfluorescein (CF) and phosphatidylethanolamine-dioleoyl-sulforhodamine B (Rho-PE) as fluorescent markers. Fluorescence dequenching assay was used to study the influence of pH on CF release from the liposomal formulations. Liposome adhesion on the cell surface and internalization were strongly dependent on vesicle bilayer composition. SA vesicles were not endocytosed. DPPC/Chol liposomes were endocytosed but did not release their fluorescent content into the cytosol. SL/Chol and P90/Chol formulations displayed a diffuse cytoplasmic fluorescence of liposomal marker.

Comparison of vesicle based antigen delivery systems for delivery of hepatitis B surface antigen

There is a clinical need for a more effective vaccine against hepatitis B, and in particular vaccines that may be suitable for therapeutic administration. This study assesses the potential of cationic surfactant vesicle based formulations using two agents; the cationic amine containing [ N-( N′, N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) or dimethyl dioctadecylammonium bromide (DDA) with hepatitis B surface antigen (HBsAg). Synthetic mycobacterial cord factor, trehalose 6,6′-dibehenate (TDB) has been used as an adjuvant and the addition of 1-monopalmitoyl glycerol (C16:0) (MP) and cholesterol (Chol) to DDA–TDB is assessed for its potential to facilitate formation of dehydration–rehydration vesicles (DRV) at room temperature, and the effect of this on immune responses. A DRV formulation is directly compared to an adsorbed formulation of the same composition and preparation protocol (MP:dioleoyl phosphoethanolamine (DOPE):Chol:DC-Chol) and the direct substitution of MP with phosphatidylcholine (PC) is also compared in DRV antigen-entrapped formulations. MP and Chol were shown to facilitate the use of DDA–TDB in DRV formulations prepared at room temperature, whilst there was marginal alteration of immunogenicity (a reduction in HBsAg-specific IL-2). The HBsAg adsorbed DRV formulation was not significantly different from the HBsAg entrapped DRV formulation. Overall, DDA formulations incorporating TDB showed markedly increased antigen specific splenocyte proliferation and elicited cytokine production concomitant with a strong T cell driven response, delineating formulations that may be useful for further evaluation of their clinical potential.

Heparin incorporating liposomes as a delivery system of heparin from PET-covered metallic stents: Effect on haemocompatibility

We investigate the possibility of coating polymer-covered stents with heparin-encapsulating liposomes for improving their haemocompatibility. Thin-film hydration (for multilamellar vesicles, MLV), and the dehydration–rehydration vesicle (DRV) methods are used for preparation of low-molecular weight heparin (LMWH)-encapsulating liposomes with varying lipid compositions. Liposomes are characterized for LMWH encapsulation and retention. For measurement of LMWH, a chromogenic technique is adjusted. For evaluation of heparin release from vesicles in platelet poor plasma (PPP) coagulation time is measured in presence of liposomal samples. Results reveal that LMWH encapsulation in liposomes is higher in DRV, however compositions with high encapsulation are leaky during buffer incubation. Most liposomes release LMWH slowly during plasma incubation (retention after 24 h ranges between 74% and 95%). Concerning the haemocompatibility of polyethylene terephthlate-covered stents after coating with LMWH-encapsulating liposomes, there is a marked increase (higher for DRV-coated stents compared to MLV) in plasma recalcification time compared to the control (plain blood) and reference (non-coated stent), which increases with blood–material contact time. This is probably due to LMWH release, demonstrating that encapsulated LMWH retains its biological functionality. Interestingly, the DRV-coated stents retained a high plasma recalcification time and a large number of liposomes on the stents (as proven by SEM studies) even after extensive washing (high shear conditions), proving that this method may be functional under high flow applying in vivo conditions.

A novel approach to develop β-galactosidase entrapped in liposomes in order to prevent an immediate hydrolysis of lactose in milk

A fractional factorial screening design and a response surface methodology were applied to optimize the entrapment of β -galactosidase in liposomes by dehydration-rehydration vesicle method. The effect of lipid (phosphatidylcholine and cholesterol), enzyme concentration, buffer pH, type and time of sonication, and sucrose concentration were the parameters selected for this study. The cholesterol:phosphatidylcholine (Ch:PC) ratio was the most important factor followed by type of sonicator and enzyme:lipid (E:L) ratio. The buffer pH, sucrose concentration and sonication time did not play an important role in the response. Two of the factors (Ch:PC and E:L ratios) were further studied in a 3 2 central composite design. The optimized liposomal formulation with an entrapment efficiency of 28% was obtained with Ch:PC and E:L ratios of 0.53 and 13.76, respectively, at pH 6 and employing a probe type sonicator.

Preparation and characterization of dehydration–rehydration vesicles loaded with aminoglycoside and macrolide antibiotics

Enhanced activity of liposomes-encapsulated antibiotics against clinical isolates of Pseudomonas aeruginosa has been documented with liposomes of low encapsulation efficiency. We sough to construct liposomes with high yield entrapment of aminoglycoside and macrolide antibiotics as well as favorable stability in storage and physiological conditions. Liposome-entrapped aminoglycosides (amikacin, gentamicin, tobramycin) and a macrolide (erythromycin) were prepared by a modified dehydration–rehydration vesicles (DRVs) method, and their particle size and entrapment efficiency were determined. We studied in vitro stability of these vesicles over a 48 h period at 4 and 37 °C in phosphate-buffered saline (PBS) and in plasma at 37 °C. The mean particle size of DRVs loaded with antibiotics varied from 163.37 ± 38.44 to 259.83 ± 11.80 nm with no significant difference in regard with the type of the antibiotics encapsulated. Encapsulation efficiency of DRVs loaded with amikacin, gentamicin, tobramycin, and erythromycin were 29.27 ± 1.17, 33 ± 0.76, 22.33 ± 1.48 and 32.06 ± 0.82% of initial amount of the drug, respectively. These vesicles were stable regardless of the experimental temperature. Indeed, the liposomes retained more than 75% of the initially encapsulated drugs for the study period of 48 h. DRVs incubated in plasma however, released more antibiotics than those incubated in PBS. In conclusion, using this modified DRV method, we obtained small sized vesicles with high yield entrapment for aminoglycoside and macrolide antibiotics. The technique may be utilized to overcome the low encapsulation efficiency associated with aminoglycoside and macrolide antibiotics.