Development of a liposome formulation for D-cycloserine local delivery

Multilamellar liposomes loaded with D-cycloserine (D-CS) were prepared by a thin layer evaporation technique, followed by freezing and thawing cycles. Charged components and bioadhesive material, such as distearolylphosphatitylethanolamine covalently coupled with methoxypolyethyleneglycol, were used to prepare liposomes with different physico-chemical and technological properties. Negatively charged liposomes showed higher D-CS encapsulation efficiency (about 37%, w/w) than neutral and positively charged liposomes (about 5 and 17%, w/w, respectively). All formulations showed in vitro, after a burst effect, a prolonged release of the encapsulated drug. Lipid vesicles made of dipalmitoylphosphatidylcholine (DPPC) were used as a biomembrane model to evaluate in vitro the interaction of D-CS with biological membranes. Differential scanning calorimetry was used as a simple and noninvasive technique of analysis. D-CS was distributed in the aqueous compartments of liposomes for interaction with the phospholipid polar head-groups (enhancement of Delta H value). However, due to its high diffusibility the drug was also able to freely permeate through DPPC liposomes, altering during this passage the hydrophobic domains of the bilayers. Stability studies were performed at different temperatures and pH values to assay the integrity of the drug during the liposome production steps. D-CS was rapidly degraded at acidic pH, but no significant hydrolysis was observed at pH 7.4 after 7 days.     

Effect of liposome-treated red blood cells in an anemic rat model

Context: Liposomes have been shown to improve human red blood cell (RBC) in vitro quality by minimizing membrane damage occurring during 42-d hypothermic storage. Small animal models are necessary to evaluate novel blood products and guide future clinical studies.

Objectives: The aim of this study was to assess the effect of liposome treatments on rat RBC hypothermic storage lesion (HSL) and to examine in vivo outcomes of transfusing liposome treated RBCs in a rat model.

Materials and methods: Unilamellar liposomes were synthesized which contained saturated (DPPC:CHOL, 7:3?mol%), unsaturated (DOPC:CHOL, 7:3?mol%), saturated charged (DPPC:CHOL:PS, 6:3:1?mol%), and unsaturated charged (DOPC:CHOL:PS, 6:3:1?mol%) phospholipids. After liposome treatment, rat RBC quality was assessed by percent hemolysis, deformability, aggregation, hematological indices, microvesiculation, and cholesterol/phospholipid concentrations. An anemic rat model of myocardial ischemia and reperfusion (I/R) was used to evaluate the outcomes of transfusing liposome-treated RBCs.

Results: All four liposome treatments resulted in significant decreases in hemolysis, with the most prominent effect seen with DOPC-liposomes (DOPC: 1.6?±?0.1% versus control: 3.1?±?0.2%, p?=?0.015). RBCs treated with uncharged liposomes had lower hemolysis compared with charged liposomes (3.4?±?0.2% versus 3.9?±?0.4%, p?=?0.010). The in vivo study showed no significant difference in the hemoglobin levels and infarct size (53.3?±?13.1% versus 45.3?±?8.4%, p?=?0.223) between liposome and control groups.

Discussion and conclusion: Liposome treatment improved in vitro quality of stored rat RBCs. However, the changes observed in vitro were not sufficient to improve the in vivo outcomes of myocardial I/R in anemic rats transfused with liposome-treated RBCs.     

Hyaluronic acid modified pH-sensitive liposomes for targeted intracellular delivery of doxorubicin

Context: Surface-modified pH-sensitive liposomal system may be useful for intracellular delivery of chemotherapeutics.

Objective: Achieving site-specific targeting with over-expressed hyaluronic acid (HA) receptors along with using pH sensitive liposome carrier for intracellular drug delivery was the aim of this study.

Materials and methods: Stealth HA-targeted pH-sensitive liposomes (SL-pH-HA) were developed and evaluated to achieve effective intracellular delivery of doxorubicin (DOX) vis–a-vis enhanced antitumor activity.

Results: The in vitro release studies demonstrated that the release of DOX from SL-pH-HA was pH-dependent, i.e. faster at mildly acidic pH ～5, compared to physiological pH ～7.4. SLpH-HA was evaluated for their cytotoxicity potential on CD44 receptor expressing MCF-7 cells. The half maximal inhibitory concentration (IC50) of SL-pH-HA and SL-HA were about 1.9 and 2.5?μM, respectively, after 48?h of incubation. The quantitative uptake study revealed higher localization of targeted liposomes in the receptor positive cells, which was further confirmed by fluorescent microscopy. The antitumor efficacy of the DOX-loaded HA-targeted pH-sensitive liposomes was also verified in a tumor xenograft mouse model.

Discussion: DOX was efficiently delivered to the tumor site by active targeting via HA and CD44 receptor interaction. The major side-effect of conventional DOX formulation, i.e. cardiotoxicity was also estimated by measuring serum enzyme levels of LDH and CPK and found to be minimized with developed formulation. Overall, HA targeted pH-sensitive liposomes were significantly more potent than the non-targeted liposomes in cells expressing high levels of CD44.

Conclusion: Results strongly implies the promise of such liposomal system as an intracellular drug delivery carrier developed for potential anticancer treatment.     

Polymer coated liposomes for use in the oral cavity – a study of the in vitro toxicity,effect on cell permeability and interaction with mucin

In this study we investigated the in vitro toxicity, impact on cell permeability and mucoadhesive potential of polymer-coated liposomes intended for use in the oral cavity. A TR146 cell line was used as a model. The overall aim was to end up with a selection of safe polymer coated liposomes with promising mucoadhesive properties for drug delivery to the oral cavity. The following polymers were tested: chitosan, low-methoxylated pectin (LM-pectin), high-methoxylated pectin (HM-pectin), amidated pectin (AM-pectin), Eudragit, poly(N-isopropylacrylamide-co-methacrylic acid) (p(NIPAAM-co-MAA)), hydrophobically modified hydroxyethyl cellulose (HM-HEC), and hydrophobically modified ethyl hydroxyethyl cellulose (HM-EHEC). With chitosan as an exception, all the systems exhibited no significant effect on cell viability and permeability at the considered concentrations. Additionally, all the formulations showed to a varying degree an interaction with mucin (BSM type I-S); the positively charged formulations exhibited the strongest interaction, while the negatively and neutrally charged formulations displayed a moderate or low interaction. The ability to interact with mucin makes all the liposomal formulations promising for oromucosal administration. Although the chitosan-coated liposomes affected the cell viability, this formulation also influenced the cell permeability, which makes it an interesting candidate for systemic drug delivery from the oral cavity.     

Effects of liposomal encapsulation on the antioxidant activity of lipophilic prodrugs of idebenone

Context: Increasing the lipophilicity and/or amphiphilicity of drugs is a potential strategy to improve loading and retention in lipid-based carriers, such as liposomes or lipid nanoparticles.

Objective: Idebenone (IDE), an antioxidant compound structurally related to coenzyme Q, or amphiphilic prodrugs of IDE with lipoamino acids, were loaded in neutral or negatively charged SUVET unilamellar liposomes to achieve a controlled release.

Methods: Technological properties of these systems in the presence of loaded drugs were evaluated in terms of vesicle size, homogeneity, and surface charge, as well as in vitro drug release. The effect of liposomal carrier on the in vitro antioxidant activity of the prodrugs was evaluated from using different biochemical assays on murine astrocyte cultures.

Results and discussion: Although a good loading efficiency was obtained, liposomes were not able to release efficiently the encapsulated drugs, at least in the in vitro serum-free conditions used for the biological tests. However, in some cases, such as in the comet assay, encapsulation of IDE prodrugs in liposomes allowed for the improvement of their protective activity, compared to the free compounds, against the oxidative damage induced on cultured astrocytes.

Conclusions: Experimental in vitro data suggested that the high affinity shown by these lipophilic IDE derivatives for the liposomal carriers negatively affect their biological activity.     

Ethanol-based proliposome delivery systems of paclitaxel for in vitro application against brain cancer cells

In this study the anticancer activity of paclitaxel-loaded nano-liposomes on glioma cell lines was investigated. Soya phosphatidylcholine:cholesterol (SPC:Chol), hydrogenated soya phosphatidylcholine:cholesterol (HSPC:Chol) or dipalmitoylphosphatidylcholine:cholesterol (DPPC:Chol) in 1:1?mole ratio were used to prepare ethanol-based proliposomes. Following hydration of proliposomes, the size of resulting vesicles was subsequently reduced to nanometer scale via probe-sonication. The resulting formulations were characterized in terms of size, zeta potential and morphology of the vesicles, and entrapment efficiency of paclitaxel (PX) as well as the final pH of the preparations. DPPC-liposomes entrapped 35–92% of PX compared to 27–74% and 25–60% entrapped by liposomes made from SPC and HSPC formulations respectively, depending on drug concentration. The entrapment efficiency of liposomes was dependent on the lipid bilayer properties and ability of PX to modify surface charge of the vesicles. In vitro cytotoxicity studies revealed that PX-liposome formulations were more selective at inhibiting the malignant cells. The cytotoxicity of PX-liposomes was dependent on their drug-entrapment efficiency. This study has shown PX-liposomes generated from proliposomes have selective activity against glioma cell lines, and the synthetic DPPC phospholipid was most suitable for maximized drug entrapment and highest activity against the malignant cells in vitro.     

Generation of fatty acids from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cardiolipin liposomes that stabilize recombinant human serum albumin

Abstract

Context: At elevated temperatures, studies have shown that serum albumin undergoes irreversible changes to its secondary structure. Anionic fatty acids and/or anionic surfactants have been shown to stabilize human serum albumin (HSA) against thermal denaturation through bridging hydrophobic domains and cationic amino acids residues of the protein.

Objective: As albumin can readily interact with a variety of liposomes, this study proposes that cardiolipin delivered via 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes can improve the thermal stability of recombinant HSA produced in Saccharomyces cerevisiae (ScrHSA) in a similar manner to anionic fatty acids.

Materials and methods: Thermal stability and structure of ScrHSA in the absence and presence of DPPC/cardiolipin liposomes was assessed with U/V circular dichroism spectropolarimetry and protein thermal stability was confirmed with differential scanning calorimetry.

Results: Although freshly prepared DPPC/cardiolipin liposomes did not improve the stability of ScrHSA, DPPC/cardiolipin liposomes incubated at room temperature for 7?d (7dRT) dramatically improved the thermal stability of the protein. Mass spectrometry analysis identified the presence of fatty acids in the 7dRT liposomes, not identified in freshly prepared liposomes, to which the improved stability was attributed.

Discussion and conclusion: The generation of fatty acids is attributed to either the chemical hydrolysis or oxidative cleavage of the unsaturated acyl chains of cardiolipin. By modulating the lipid composition through the introduction of lipids with higher acyl chain unsaturation, it may be possible to generate the stabilizing fatty acids in a more rapid manner.     

Liposomal Anticancer Drugs as Agents to be used in Combination with other Anticancer Agents: Studies on a Liposomal Formulation with two Encapsulated Drugs

Abstract

When considering the use of combination therapies with liposomal anticancer agents several approaches can be defined. One approach could rely on administration of one liposomal formulation with more than one entrapped cytotoxic drug. This study focuses on an assessment of a liposomal formulation containing vincristine and mitoxantrone. Distearoyl phosphatidylcholine (DSPC)/Cholesterol (Choi) (55:45 molar ratio) liposomes were loaded with vincristine using transmembrane pH gradients. These systems were subsequently incubated with mitoxantrone to effect uptake of the second drug. Retention of both drugs was determined in vitro and in vivo. In vitro drug release indicated >95% retention of mitoxantrone and approximately 75% retention of vincristine when liposomes were prepared with an initial interior pH of 2.0. In vivo results however, demonstrated that greater than 80% of the encapsulated vincristine was released within 1 hour following i.v. administration. The instability of a liposomal formulation containing two anticancer drugs following i.v. administration may be a consequence of a combination of factors including drug-loading induced collapse of the transmembrane pH gradient, loss due to osmotic effects and an associated insertion of serum proteins into the bilayer, as well as the presence of a large biological “sink” which can alter the transbilayer drug gradient in favor of drug release.     

Vincristine-sulphate–loaded liposome-templated calcium phosphate nanoshell as potential tumor-targeting delivery system

Vincristine-sulfate–loaded liposomes were prepared with an aim to improve stability, reduce drug leakage during systemic circulation, and increase intracellular uptake. Liposomes were prepared by the thin-film hydration method, followed by coating with calcium phosphate, using the sequential addition approach. Prepared formulations were characterized for size, zeta potential, drug-entrapment efficiency, morphology by transmission electron microscopy (TEM), in vitro drug-release profile, and in vitro cell cytotoxicity study. Effect of formulation variables, such as drug:lipid ratio as well as nature and volume of hydration media, were found to affect drug entrapment, and the concentration of calcium chloride in coating was found to affect size and coating efficiency. Size, zeta potential, and TEM images confirmed that the liposomes were effectively coated with calcium phosphate. The calcium phosphate nanoshell exhibited pH-dependent drug release, showing significantly lower release at pH 7.4, compared to the release at pH 4.5, which is the pH of the tumor interstitium. The in vitro cytotoxicity study done on the lung cancer cell line indicated that coated liposomes are more cytotoxic than plain liposomes and drug solution, indicating their potential for intracellular drug delivery. The cell-uptake study done on the lung cancer cell line indicated that calcium-phosphate–coated liposomes show higher cell uptake than uncoated liposomes.     

Effect of lyophilization on liposomal encapsulation of salmon calcitonin

Purpose: The intent of this work was to assess the impact of lyophilization on the encapsulation of salmon calcitonin (sCT) into liposomes.

Methods: Four different liposomal formulations were investigated, i.e. DPPC:Chol:DSPE-PEG₂₀₀₀ (75:20:5 and 65:30:5) and DPPC:Chol (80:20 and 66.7:33.3). Lipid films were prepared and hydrated with loading buffer containing sCT and different concentrations of the cryoprotectant, trehalose dihydrate. The liposomes were lyophilized, reconstituted and extruded to obtain small unilamellar vesicles. Non-encapsulated sCT was separated by gel filtration. Non-lyophilized formulations and liposomes lyophilized without the cryoprotectant were used as controls. Liposomes were analyzed for particle size, polydispersity index, zeta-potential and encapsulation efficiency. ³¹P-NMR (phosphorous nuclear magnetic resonance spectroscopy) was performed on selected formulations.

Results: Post-lyophilization, no significant change in particle sizes and zeta-potentials were noted, regardless of the presence or absence of the cryoprotectant. Encapsulation efficiencies, however, increased following lyophilization, in both PEGylated (lyophilization control batch) and non-PEGylated liposomes (cryoprotectant batches only). ³¹P-NMR revealed the presence of two distinct vesicle populations – liposomes and micelles – in PEGylated formulation. The presence of micelles might be responsible for the observed encapsulation enhancement of sCT in the PEGylated formulation.

Conclusions: Lyophilization resulted in an increase in encapsulation efficiency of sCT in PEGylated liposomes, even in the absence of a cryoprotectant, due to presence of micellar vesicles.     

Ciprofloxacin as Ocular Liposomal Hydrogel

The purpose of this study was to prepare and characterize an ocular effective prolonged-release liposomal hydrogel formulation containing ciprofloxacin. Reverse-phase evaporation was used for preparation of liposomes consisting of soybean phosphatidylcholine (PC) and cholesterol (CH). The effect of PC/CH molar ratio on the percentage drug encapsulation was investigated. The effect of additives such as stearylamine (SA) or dicetyl phosphate (DP) as positive and negative charge inducers, respectively, were studied. Morphology, mean size, encapsulation efficiency, and in vitro release of ciprofloxacin from liposomes were evaluated. For hydrogel preparation, Carbopol 940 was applied. In vitro transcorneal permeation through excised albino rabbit cornea was also determined. Optimal encapsulation efficiency of 73.04 ± 3.06% was obtained from liposomes formulated with PC/CH at molar ratio of 5:3 and by increasing CH content above this limit, the encapsulation decreased. Positively charged liposomes showed superior entrapment efficiency (82.01 ± 0.52) over the negatively charged and the neutral liposomes. Hydrogel containing liposomes with lipid content PC, CH, and SA in molar ratio 5:3:1, respectively, showed the best release and transcorneal permeation with the percentage permeation of 30.6%. These results suggest that the degree of encapsulation of ciprofloxacin into liposomes and prolonged in vitro release depend on composition of the vesicles. In addition, the polymer hydrogel used in preparation ensure steady and prolonged transcorneal permeation. In conclusion, ciprofloxacin liposomal hydrogel is a suitable delivery system for improving the ocular bioavailability of ciprofloxacin.     

Potential effect of cationic liposomes on interactions with oral bacterial cells and biofilms

Context: Although oral infectious diseases have been attributed to bacteria, drug treatments remain ineffective because bacteria and their products exist as biofilms. Cationic liposomes have been suggested to electrostatically interact with the negative charge on the bacterial surface, thereby improving the effects of conventional drug therapies. However, the electrostatic interaction between oral bacteria and cationic liposomes has not yet been examined in detail.

Objective: The aim of the present study was to examine the behavior of cationic liposomes and Streptococcus mutans in planktonic cells and biofilms.

Materials and methods: Liposomes with or without cationic lipid were prepared using a reverse-phase evaporation method. The zeta potentials of conventional liposomes (without cationic lipid) and cationic liposomes were ?13 and 8?mV, respectively, and both had a mean particle size of approximately 180?nm. We first assessed the interaction between liposomes and planktonic bacterial cells with a flow cytometer. We then used a surface plasmon resonance method to examine the binding of liposomes to biofilms. We confirmed the binding behavior of liposomes with biofilms using confocal laser scanning microscopy.

Results: The interactions between cationic liposomes and S. mutans cells and biofilms were stronger than those of conventional liposomes. Microscopic observations revealed that many cationic liposomes interacted with the bacterial mass and penetrated the deep layers of biofilms.

Discussion and conclusion: In this study, we demonstrated that cationic liposomes had higher affinity not only to oral bacterial cells, but also biofilms than conventional liposomes. This electrostatic interaction may be useful as a potential drug delivery system to biofilms.     

Which Liposomes to Bypass Multidrug Resistance? A Study of Doxorubicin-Loaded Vesicles

Abstract

Liposomes used as antitumoral drug carriers have recently been designated as potential tools to overcome multidrug resistance. In order to understand better the mechanism of such an effect, we have investigated the capacity of liposomes exhibiting a pH gradient to trap efficiently the antitumoral drug doxorubicin in conditions of high dilution such as those used for cell cultures treatment. A simple calculation described the transmembrane pH gradient and the thermodynamic equilibrium of the neutral form, on both sides of the membrane. It showed that liposomes, even efficiently loaded with doxorubicin in response to the pH gradient will lose most of their content upon dilution because of the neutral form physicochemical gradient. Using fluorescence properties of the drug, we found that liposomes made of egg yolk phosphatidylcholine (EPC), phosphatidyl serine (PS) and cholesterol in the ratio 10:1:4 rather closely fitted the situation predicted by the calculation and that the equilibrium state after dilution was reached within one hour. We also showed using liposomes made of dipalmitoyl phosphatidyl choline (DPPC) and cholesterol in the ratio 11:4 that the drastic leakage could be overcome by changing the physical state of the liposome membrane at 37°C.

Referring to the drug release characteristics of other colloidal systems having demonstrated significant capacitiy to oververcome doxorubicin resistance, we concluded that liposomes made of lipids in gel (Lβ or Lβ') state at 37°C could be interesting tools in MDR bypass because they very efficiently retain their content under high dilution conditions.     

The influence of bile salts on the response of liposomes to ultrasound

Context: Triggering drug release from delivery vehicles with ultrasound has potential applications in targeted drug delivery. It was hypothesized that the addition of bile salts would increase the sensitivity of liposomes to ultrasound through creation of defects.

Objective: The aim of this study was to investigate whether incorporating bile salts into liposomes would lead to differential effects on their response to low and high frequency ultrasound.

Materials and methods: Cholate, chenodeoxycholate, ursodeoxycholate, glycocholate and taurocholate were the selected bile salts. Response to ultrasound was characterized by measuring the release of carboxyfluorescein (CF).

Results: At 30?kHz ultrasound, taurocholate containing liposomes were most responsive and released 70% (±2) CF after 30 seconds of sonication. Compared to this, liposomes that did not contain bile salts released just 7% (±2). At 1.1?MHz ultrasound, all liposome formulations were unresponsive. To increase the response of liposomes at 1.1?MHz ultrasound, a combination of membrane destabilizers were added to DSPC liposomes. DOPE, a hexagonal phase lipid was used in combination with taurocholate. Surprisingly, liposomes containing DOPE and taurocholate were more resistant to 1.1?MHz ultrasound than ones containing only DOPE.

Discussion: This suggests that the sensitivity of liposomes towards ultrasound may not simply be defined by a single membrane component but instead depends on the interaction between constituting lipid components. Furthermore, strategies other than membrane destabilization may be required to sensitize liposomes towards high frequency ultrasound.

Conclusion: Bile salts may be used to increase or decrease the sensitivity of liposomes to low frequency ultrasound.     

Characterization of the interaction between liposomal formulations and Pseudomonas aeruginosa

The interactions between three liposomal formulations and Pseudomonas aeruginosa cells were evaluated by a lipid mixing assay and electron paramagnetic resonance (EPR) spectroscopy. The effect of the bacteria on the liposomal phase characteristics, the release of the liposomes’ content, and the uptake rate of gentamicin by bacteria were monitored as a function of time, using EPR spectroscopy. The [16-DSA uptake]_Total from DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) liposomes reached 93?±?12% over a 3-hour assay period, of which 9% crossed the bacterial inner membrane. A small amount of 16-DSA uptake from DPPC/Chol (cholesterol) vesicles was found throughout the 3-hour period of time. Although DPPC/DMPG (dimyristoylphosphatidylglycerol) vesicles showed a smaller value of [16-DSA uptake]_Total with respect to that of DPPC vesicles, they appeared to be effective in disrupting the bacterial membrane, resulting in a greater accumulation of 16-DSA inside the inner membrane. Exposure to bacteria caused the DPPC/Chol, DPPC, and DPPC/DMPG formulations to release 4.6?±?1.5, 17.6?±?1.2, and 34?±?3.7% of their content, respectively. Time-dependent fluid regions were developed within the vesicles when mixed with bacteria, and their growth over time depended on liposomal formulations. Incubation of gentamicin with bacteria for 3 hours resulted in 87?±?3% of the drug crossing the bacterial inner membrane. In conclusion, interaction between the liposome drug carriers and the bacterial cells result in vesicle fusion, disruption of the bacterial membrane, release of the liposomal content in the close vicinity of the bacteria cells, and the subsequent intracellular uptake of the released liposomal content.     

Biophysical studies on chitosan-coated liposomes

Liposomes have been used as delivery vehicles for stabilizing drugs, overcoming barriers to cellular and tissue uptake, and for directing their contents toward specific sites in vivo. Chitosan is a biological macromolecule derived from crustacean shells and has several emerging applications in drug development, obesity control, and tissue engineering. In the present work, the interaction between chitosan and dipalmitoyl phosphatidylcholine (DPPC) liposomes was studied by transmission electron microscopy (TEM), zeta potential, solubilization using the nonionic detergent octylglucoside (OG), as well as Fourier transform infrared (FTIR) spectroscopy and viscosity measurements. The coating of DPPC liposomes by a chitosan layer was confirmed by electron microscope images and the zeta potential of liposomes. Coating of liposome by chitosan resulted in an increase in liposomal size by addition of a layer of 92 ± 27.1 nm. The liposomal zeta potential became increasingly positive as chitosan concentration increased from 0.1 to 0.3% w/v, then it held at a relatively constant value. The amount of detergent needed to completely solubilize the liposomal membrane was increased after coating of liposomes with chitosan, indicating an increased membrane resistance to the detergent and hence a change in the natural membrane permeation properties. In the analysis of FTIR spectra of DPPC, the symmetric and antisymmetric CH₂ (at 2,800–3,000 cm⁻¹) bands and the C=O (at 1,740 cm⁻¹) stretching band were investigated in the absence and presence of the chitosan. It was concluded that appropriate combining of the liposomal and chitosan characteristics might be utilized for the improvement of the therapeutic efficacy of liposomes as a drug delivery system.     

Calorimetric study on pH-responsive block copolymer grafted lipid bilayers: rational design and development of liposomes

This study is focused on chimeric advanced drug delivery nanosystems and specifically on pH-sensitive liposomes, combining lipids and pH-responsive amphiphilic block copolymers. Chimeric liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and two different forms of block copolymers, i.e. poly(n-butylacrylate)-b-poly(acrylic acid) (PnBA-b-PAA) at 70 and 85% content of PAA at six different molar ratios, each form respectively. PAA block exhibits pH-responsiveness, because of the regulative group of –COOH. –COOH is protonated under acidic pH (pK_a ca. 4.2), while remains ionized under basic or neutral pH, leading to liposomes repulse and eventually stability. Lipid bilayers were prepared composed of DPPC and PnBA-b-PAA. Experiments were carried out using differential scanning calorimetry (DSC) in order to investigate their thermotropic properties. DSC indicated disappearance of pre-transition at all chimeric lipid bilayers and slight thermotropic changes of the main transition temperature. Chimeric liposomes have been prepared and their physicochemical characteristics have been explored by measuring the size, size distribution and ζ-potential, owned to the presence of pH-responsive polymer. At percentages containing medium to high amounts of the polymer, chimeric liposomes were found to retain their size during the stability studies. These results were well correlated with those indicated in the DSC measurements of lipid bilayers incorporating polymers in order to explain their physicochemical behavior. The incorporation of the appropriate amount of these novel pH-responsive block copolymers affects thus the cooperativity, the liposomal stabilization and imparts pH-responsiveness.     

Development of egg PC/cholesterol/α-tocopherol liposomes with ionic gradients to deliver ropivacaine

Context: Ropivacaine (RVC) is an aminoamide local anesthetic widely used in surgical procedures. Studies with RVC encapsulated in liposomes and complexed in cyclodextrins have shown good results, but in order to use RVC for lengthy procedures and during the postoperative period, a still more prolonged anesthetic effect is required.

Objective: This study therefore aimed to provide extended RVC release and increased upload using modified liposomes.

Materials and methods: Three types of vesicles were studied: (i) large multilamellar vesicle (LMV), (ii) large multivesicular vesicle (LMVV) and (iii) large unilamellar vesicle (LUV), prepared with egg phosphatidylcholine/cholesterol/α-tocopherol (4:3:0.07?mol%) at pH 7.4. Ionic gradient liposomes (inside: pH 5.5, pH 5.5?+?(NH₄)₂SO₄ and pH 7.4?+?(NH₄)₂SO₄) were prepared and showed improved RVC loading, compared to conventional liposomes (inside: pH 7.4).

Results and discussion: An high-performance liquid chromatography analytical method was validated for RVC quantification. The liposomes were characterized in terms of their size, zeta potential, polydispersion, morphology, RVC encapsulation efficiency (EE(%)) and in vitro RVC release. LMVV liposomes provided better performance than LMV or LUV. The best formulations were prepared using pH 5.5 (LMVV 5.5_in) or pH 7.4 with 250?mM (NH₄)₂SO₄ in the inner aqueous core (LMVV 7.4_in?+?ammonium sulfate), enabling encapsulation of as much as 2% RVC, with high uptake (EE(%) ～70%) and sustained release (～25?h).

Conclusion: The encapsulation of RVC in ionic gradient liposomes significantly extended the duration of release of the anesthetic, showing that this strategy could be a viable means of promoting longer-term anesthesia during surgical procedures and during the postoperative period.     

Correlation between octanol/water and liposome/water distribution coefficients and drug absorption of a set of pharmacologically active compounds

Abstract

Absorption and consequent therapeutic action are key issues in the development of new drugs by the pharmaceutical industry. In this sense, different models can be used to simulate biological membranes to predict the absorption of a drug. This work compared the octanol/water and the liposome/water models. The parameters used to relate the two models were the distribution coefficients between liposomes and water and octanol and water and the fraction of drug orally absorbed. For this study, 66 drugs were collected from literature sources and divided into four groups according to charge and ionization degree: neutral; positively charged; negatively charged; and partially ionized/zwitterionic. The results show a satisfactory linear correlation between the octanol and liposome systems for the neutral (R^2?=?0.9324) and partially ionized compounds (R^2?=?0.9367), contrary to the positive (R^2?=?0.4684) and negatively charged compounds (R^2?=?0.1487). In the case of neutral drugs, results were similar in both models because of the high fraction orally absorbed. However, for the charged drugs (positively, negatively, and partially ionized/zwitterionic), the liposomal model has a more-appropriate correlation with absorption than the octanol model. These results show that the neutral compounds only interact with membranes through hydrophobic bonds, whereas charged drugs favor electrostatic interactions established with the liposomes. With this work, we concluded that liposomes may be a more-appropriate biomembrane model than octanol for charged compounds.     

Inhibition by Liposomal Cholesterol of Streptococcus Pneumoniae Induced Lung Epithelial Cell Damage

Abstract

Streptococcus pneumoniae was shown to be capable of lysing A549 cells in culture. Membrane damage to cells as assessed by trypan blue exclusion increased with increasing concentration of bacteria. After 45 min of incubation with 7.5 × 10⁸ bacteria/ml less than 20% of A549 cells excluded trypan blue. The lytic activity of S. pneumoniae was inhibited by phosphatidylcholine liposomes containing cholesterol. Using an haemolysis assay and S. pneumoniae's culture filtrates, the efficiency of the anti-lytic activity of liposomes was found to be distearoylphosphatidylcholine (DSPC) > dipalmitoylphosphatidylcho-line (DPPC) > dimyristoylphosphatidylcholine (DMPC). Furthermore, the anti-lytic activity also depended on the cholesterol content in a non-trivial manner. There was no protection against haemolytic activity at cholesterol content of less than 20% for DSPC and 35 mole% for DPPC and DMPC liposomes respectively. Above these threshold values inhibition of lytic activity increased sharply. In agreement with the haemolysis results, A549 cells were protected by liposomes against the lytic activity of S. pneumoniae with the efficiency also being DSPC > DPPC > DMPC. Clearly the efficiency of liposomal cholesterol is increased with increasing gel to liquid crystalline phase transition temperature of the lipid matrix. The results suggest that liposomal cholesterol may be used to protect the host against cell damage caused by S. pneumoniae.