Polycation liposome enhances the endocytic uptake of photosensitizer into cells in the presence of serum

To construct a novel drug delivery carrier that possesses high therapeutic efficacy with low dosage, we designed polyethylenimine-modified liposome (polycation liposome, PCL) and examined the entrapment of photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), for antiangiogenic photodynamic therapy (PDT). Photosensitizer entrapped in PCLs showed enhanced phototoxicity for a human vascular endothelial cell line, ECV304, in comparison with that for nonmodified control liposome. Interestingly, phototoxicity of control liposomal BPD-MA was suppressed in the presence of serum, but PCL maintained the phototoxicity in the presence of serum following PCL-mediated PDT treatment due to the stability of PCL and the reduced detachment of encapsulated photosensitizer from liposome to serum. In fact, PCL enhanced the uptake level of BPD-MA to ECV304 cells despite the presence or absence of serum. Since polycation modification enhances bioavailability of the liposomal photosensitizer and this property is maintained in the presence of serum, PCL would be useful for antiangiogenic PDT.     

Studies on curcumin and curcuminoids. XXIX. Photoinduced cytotoxicity of curcumin in selected aqueous preparations.

Natural curcumin was evaluated as a potential photosensitizer for oral applications. The photocytotoxicity of curcumin on salivary gland acinar cells (SM 10-12) was investigated in five aqueous preparations consisting of 5% DMSO, non-ionic micelles, cyclodextrin, liposomes, or a hydrophilic polymer. The difference in phototoxic effects between natural curcumin and synthetic curcumin was examined. Cytotoxicity in SM 10-12 cells exposed to curcumin in the concentration range 0.4-13.5 microM was investigated by MTT test, a fluorescence-staining microscopic test, and by Western immunoblotting techniques. The potential formation of a photoreaction product, hydrogen peroxide, was evaluated by a fluorescence assay. The light source was a halogen lamp used in the dental clinic, emitting mainly in the blue part of the spectrum. The phototoxic effect on SM 10-12 cells was dependent on curcumin concentration, the light dose and the type of preparation. Natural and synthetic curcumin induced phototoxicity to the same extent. Significant effects on the cells were obtained at low curcumin concentrations (< or =0.5 microM) and at a low light dose (< or =6 J cm(-2)), after 3 h incubation. Neither the activation of caspases-3, -7, -8 or -9, nor the formation of hydrogen peroxide could be detected in cells exposed to curcumin and light. The liposome preparation was the most efficient vehicle for curcumin to induce cell death. The phototoxic effect induced by curcumin is highly dependent on the type of preparation. Curcumin might be a potential photosensitizer in the treatment of oral lesions and cancers provided careful selection of the vehicle.     

Optimization on condition of glycyrrhetinic acid liposome by RSM and the research of its immunological activity

The aim of this study is to prepare glycyrrhetinic acid liposome (GAL) and optimize the preparation condition and to investigate further whether liposome could promote the immunological activity of glycyrrhetinic acid (GA). GAL was prepared using a film-dispersion method and the preparation conditions of GAL were optimized with response surface methodology (RSM). Moreover, GAL prepared under the optimal preparation conditions was added into chicken's T and B lymphocytes in vitro. The optimal preparation conditions for GAL by response surface methodology was as follows: ratio 9:1, soybean phospholipid cholesterol (w/w) 2.5:1 and water bath temperature 31 °C. Under these conditions, the experimental encapsulation efficiency of GAL was 83.46 ± 0.55%, which was close with the predicted value. Therefore, the optimized preparation condition is very reliable. The results showed that GAL could significantly promote T and B lymphocytes proliferation singly or synergistically with PHA and LPS and the concentration of immunoglobulins G (IgG) and immunoglobulins M (IgM). These results indicated that liposome could significantly improve the immunological activity of GA and drug action of GA. GAL demonstrates the significant immunological activity, which provides the theoretical basis for the further experiment in vivo.     

椒莪脂质体的制备及其质量标准的建立

目的:将椒莪油制成脂质体,优选制备工艺,建立质量标准。方法:采用薄膜超声法制备椒莪脂质体,通过正交实验优选处方和制备工艺,HPLC、GC建立其质量标准。结果:最佳处方为卵磷脂:胆固醇7:1,卵磷脂:油3.5:1;HPLC法测定脂质体中莪术油的含量,建立标准曲线,回归方程为Y=14958X+16795,r=0.9996;椒目仁油的测定方法同前文报道。得到的脂质体形态均一,包封率在75%左右。结论:建立的制备工艺简单,便于操作;检测方法的精密度、回收率均符合要求。     

Liposome mediated gene transfer

Liposomes, artificial membrane vesicles, are being intensively studied for their usefulness as delivery vehicles in vitro and in vivo. Substantial progress has been made in the development of procedures for liposome preparation, targeting and delivery of contents. The broad flexibility now available in the design of the structure and composition of liposomes, coupled to recent reports of liposome mediated gene transfer in animals, suggest that liposome technology is now poised to be utilized in the creation of custom-designed cell-type-specific gene transfer vehicles.     

Preparation and characterization of galactose-modified liposomes by a nonaqueous enzymatic reaction

In this study, NOH (NOH?=?N-octadecyl-4-[(D-galactopyranosyl)oxy]-2,3,5,6-tetrahydroxy hexanamide) was enzymatically synthesized as a targeting molecule and incorporated into liposomes to prepare a liposome surface modified with galactose. Glycyrrhetinic-acid-loaded liposome (GA-LP) and glycyrrhetinic-acid-loaded liposome surface modified with galactose (NOH-GA-LP) were prepared by the ethanol-injection method. NOH-GA-LP was characterized by morphology, particle size, zeta potential, encapsulation efficiency, release in vitro, and stability. The size of spherical particles was in the range of 179-211?nm. Spherical particles exhibit a positive electrical charge (38.7 mV) and possess high encapsulation efficiency (91.3%) and show sustained release (72% over 48 hours) in vitro. This novel approach for the liposome surface modified with galactose by enzymatic synthesis is expected to provide potential application as a drug carrier for active targeted delivery to hepatocytes.     

Gene delivery into hepatocytes with the preS/liposome/DNA system

Gene delivery into human hepatocytes remains a critical issue for the development of liver-directed gene therapy. Gene delivery based on non-viral vectors is an attractive approach relative to viral vectors. In this report, novel delivery system of preS/liposome/DNA virus-like particle (VLP) was developed for gene transfection into hepatocytes in vivo and in vitro. Plasmid pCMVbeta, expressing beta-galactosidase, was encapsulated with cationic liposome, and then the histidine-tagged preS domain of hepatitis B virus was coated on the surface of liposome/DNA to form preS/liposome/ DNA VLP. Transfection efficiencies of preS/liposome/DNA, liposome/DNA, naked DNA and preS were analyzed using several different human cell lines. The highest transfection efficiency was found using preS/liposome/DNA VLP as the transfection reagent in human hepatocyte (HH) cell line. Results show that preS domain of hepatitis B virus coated on liposome/DNA can be used for highly efficient gene transfection into human hepatocytes. Moreover, the target characteristic of preS/liposome/DNA was analyzed in vivo. After preS/liposome/DNA VLP was injected into immunocompromised (Nude) mice via the tail vein, most of beta-galactosidase was expressed in the liver; however, no significant target expression was found with the injection of liposome/ DNA or naked DNA. Our results show that preS/liposome/DNA VLP can be used as a novel liver-specific gene delivery system.     

Antigen-presenting liposomes are effective in treatment of recurrent herpes simplex virus genitalis in guinea pigs.

The therapeutic and immunologic effects of a liposome preparation containing both a macrophage activator, muramyl-tripeptide-phosphatidylethanolamine, and a recombinant antigen, glycoprotein D of herpes simplex virus type 1, have been investigated. This preparation was tested in vitro for the ability to stimulate peripheral blood lymphocytes and in vivo for the control of recurrent herpes genitalis in guinea pigs. Our results show that the liposome-antigen-adjuvant preparation is capable of enhancing antigen-specific lymphocyte stimulation, which may be related to the observed 75% suppression of the frequency and severity of reactivation of recurrent herpes simplex virus type 2 genitalis compared with that of placebo controls.     

Determinants of liposome partitioning in aqueous two-phase systems: Evaluation by means of a factorial design

This article evaluates the influence of five parameters on liposome partitioning in aqueous two-phase systems (ATPSs), composed of poly(ethyleneglycol) (PEG)/dextran (Dx), using the factorial experimental design together with a multiple regression. Mathematical models to quantify the influence of these parameters, individually and/or jointly, on liposome partitioning in ATPS were developed. The models were statistically tested and verified by experimentation. This approach was then used to define the conditions for the preferential accumulation of liposomes in the top PEG-rich phase. The models predicted a significant effect of liposome surface charge, PEG molecular weight, phase-forming polymer concentration, and phosphate ion concentration on the partition behavior of liposomes. For negatively charged liposomes, it was found that the smaller the molecular weight of PEG and polymer concentration and the larger the phosphate ion concentration, the greater the partition coefficient of the liposomes. No significant effect of pH, at the range of 6-8, on liposome partitioning was noted. This approach has led to the development of an optimal two-phase system where 90% of negatively charged liposomes accumulated in the PEG phase. In addition to the general scientific value of this research, it has a technological importance as ATPSs may be useful for removing the unentrapped drug from liposomes during their preparation for pharmaceutical applications. (c) 1996 John Wiley & Sons, Inc.     

Micelles as intermediates in the preparation of protein-liposome conjugates

A method for preparing protein-liposome conjugates based on micelles as intermediates was developed. Ovalbumin was thiolated with 2-IT and conjugated to the surface of micelles composed of a maleimide-derivatized active lipid and a micelle-forming lipid. These micelles were then incubated with liposomes, allowing the micelle components to exchange into the liposome bilayers. Using this technique we were able to demonstrate that it was possible to saturate the surface of the micelle with protein and use this property to control the level of conjugation. Titration of these protein-micelle conjugates into liposome solutions resulted in reproducible batches of protein-liposome conjugates. Chemical cross-linking could be observed in some cases; however, this was controllable through selection of reagent concentrations. The effects of parameters such as thiolation levels, micelle lipid composition, active lipid structure, micelle-forming lipid structure, and micelle/liposome/protein ratios were examined. The method represents a general approach to the preparation of well defined and reproducible protein-liposome-based drug formulations.     

β-Lactoglobulin improves liposome’s encapsulation properties for vitamin E delivery

Vitamin E (VE) or α-tocopherol is the major fat-soluble antioxidant in the human body. It is a sensitive, easily oxidized in the air, molecule, so it must be protected from pro-oxidant elements which could affect its physiological benefits. Encapsulation constitutes a promising approach to maintain VE native properties over time and increase its concentration in aqueous media. Liposomes have been studied as sustained delivery systems, being biodegradable, non-toxic and non-immunogenic. A new liposome/β-lactoglobulin (β-Lg) formulation has been developed and characterized as a possible stable delivery system for VE. β-Lg has been selected due to its property to bind a variety of hydrophobic molecules. The aim of this study was the preparation of β-Lg-liposome formulation and the determination of VE encapsulation efficiency, in order to develop a new more efficient carrier for VE in aqueous media.     

A tumor vasculature targeted liposome delivery system for combretastatin A4: Design, characterization, and in vitro evaluation

The objective of this study was to develop an efficient tumor vasculature targeted liposome delivery system for combretastatin A4, a novel antivascular agent. Liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, distearoyl phosphoethanolamine-polyethylene-glycol-2000 conjugate (DSPE-PEG), and DSPE-PEG-maleimide were prepared by the lipid film hydration and extrusion process. Cyclic RGD (Arg-Gly-Asp) peptides with affinity for αvβ3-integrins expressed on tumor vascular endothelial cells were coupled to the distal end of PEG on the liposomes sterically stabilized with PEG (long circulating liposomes, LCL). The liposome delivery system was characterized in terms of size, lamellarity, ligand density, drug loading, and leakage properties. Targeting nature of the delivery system was evaluated in vitro using cultured human umbilical vein endothelial cells (HUVEC). Electron microscopic observations of the formulations revealed presence of small unilamellar liposomes of ∼120 nm in diameter. High performance liquid chromatography determination of ligand coupling to the liposome surface indicated that more than 99% of the RGD peptides were reacted with maleimide groups on the liposome surface. Up to 3 mg/mL of stable liposomal combretastatin A4 loading was achieved with ∼80% of this being entrapped within the liposomes. In the in vitro cell culture studies, targeted liposomes showed significantly higher binding to their target cells than non-targeted liposomes, presumably through specific interaction of the RGD with its receptors on the cell surface. It was concluded that the targeting properties of the prepared delivery system would potentially improve the therapeutic benefits of combretastatin A4 compared with nontargeted liposomes or solution dosage forms.     

Stability evaluation of temoporfin-loaded liposomal gels for topical application

Temoporfin (mTHPC) is a potent second-generation synthetic photosensitizer. Topical delivery of mTHPC is of great interest for the photodynamic therapy of psoriasis and superficial skin cancer lesions. The aim of this study was to evaluate the stability of hydrophilic gels containing mTHPC-loaded liposomes. Two different mTHPC-loaded liposome dispersions, composed of 15 % (w/w) nonhydrogenated soybean lecithin of different phosphatidylcholine content, were prepared and incorporated (2:1 w/w) into hydrogels of different carbomer concentrations (1.5, 2.25, and 3%; w/w). Obtained liposomal hydrogels, containing 0.15% (w/w) mTHPC, 10% (w/w) phospholipids, and 0, 0.5, or 1% (w/w) carbomer, were analyzed for flow properties, liposome particle size, and polydispersity index (PDI), pH value, and mTHPC content after their preparation and at predetermined time intervals during 6 months of storage at 4 and 23°C. All hydrogels showed, during the whole period of investigation, adequate characteristics for topical application (i.e., they revealed shear-thinning plastic flow behavior). Rheological parameters, particle size, and PDI of liposomes in hydrogels, mTHPC content, and pH value did not show remarkable changes during the storage of gels, which could make them unacceptable for topical use. The obtained results indicated physical and chemical stability of liposomal gels containing mTHPC during 6 months of storage at both temperatures.     

Physicochemical evaluation of a stability-driven approach to drug entrapment in regular and in surface-modified liposomes

The traditional mode of encapsulating drugs in liposomes poses risks to drug stability, especially when recognition agents are attached to the liposomal surface to obtain targeted liposomes. To reduce such risks, we devised a simple, novel method to entrap drugs in liposomes, consisting of (i) preparation and lyophilization of drug-free regular and surface-modified liposomes and (ii) drug encapsulation in the course of liposome reconstitution through rehydration in an aqueous solution of the drug. In this paper, we report physicochemical studies in which we compared regular and surface-modified liposomes made by this novel approach (denoted N-liposomes) to respective liposomes made by the traditional mode (denoted T-liposomes). The studies were performed with fluorescein, sucrose, histidine, mitomycin C (MMC), and chloramphenicol (CAM) encapsulated (each) in regular and in bioadhesive liposomes, the latter having hyaluronic acid as the surface-bound ligand. Our major findings are as follows: (1) The drug-specific encapsulation efficiencies spanning the range of 10-90% were, excepting sucrose, either similar in the N- and T-liposomes or better in the N- than in the T-liposomes, for both regular and bioadhesive liposomes. (2) For all liposome types and methods of preparation, fluorescein, histidine, and MMC did not adsorb to the liposomal surface. Sucrose and MMC did adsorb to the liposomal surface irrespective of the liposome preparation mode, sucrose favoring bioadhesive over regular liposomes and MMC having the opposite trend. (3) For both regular and bioadhesive liposomes, the mechanism of drug efflux from the N-liposomes was found to be governed by a single rate constant, as previously found for the T-liposomes. The magnitudes obtained, ranging from 3.5(+/-0.2) x 10(-3) to 400(+/-17) x 10(-3) h(-1), were always drug specific and occasionally also liposome type (i.e., regular or bioadhesive) specific. For MMC and CAM, the novel approach rendered liposomes with improved sustained release. The results reported here attest, overall, to the potential of this novel approach, meriting further investigations. Studies currently underway with MMC indicate N-liposomes also have functional advantages.     

Mixed Liposome Approach for Ratiometric and Sequential Delivery of Paclitaxel and Gemcitabine

Paclitaxel (PTX) and gemcitabine (GEM) are often used in combination due to the synergistic anticancer effects. PTX and GEM combination showed a synergistic effect to SKOV-3 cells at a molar ratio of 1 to 1 and in PTX ? GEM sequence. Liposomes were explored as a carrier of PTX and GEM combination. We optimized the drug loading in liposomes varying the preparation method and co-encapsulated PTX and GEM in a single liposome preparation maintaining the maximum loading efficiency of each drug. However, drug release kinetics from the co-loaded liposomes (LpPG) was suboptimal because of the detrimental effect of PTX on GEM-release control. Instead, a mixture of LpP and LpG, which were separately optimized according to the desired release kinetics, achieved a greater cytotoxic effect than LpPG, due to the attenuation of GEM release relative to PTX. This study illustrates that co-encapsulation in a single carrier is not always desirable for the delivery of drug combinations, when the activity depends on the dosing sequence. These combinations may benefit from the mixed liposome approach, which offers greater flexibility in controlling the ratio and release kinetics of component drugs.     

Selective partitioning of cholesterol and a model drug into liposomes of varying size

The resistance of a lipid bilayer with respect to a bending deformation generally depends on the presence of membrane additives such as sterols, cosurfactants, peptides, and drugs. As a consequence, the partitioning of membrane additives into liposomes becomes selective with respect to liposome size; i.e., membrane rigidification depletes the membrane additives in the smaller (more strongly curved) liposomes. We have measured this liposome size-selective partitioning for two membrane additives - cholesterol and the porphyrin-based photosensitizer temoporfin - using asymmetrical flow field-flow fractionation (AF4) of liposomes and radioactive labeling of the membrane additive and lipid. The method yields either the molar cholesterol-to-lipid or the temoporfin-to-lipid ratio as a function of liposome size, from which we calculate the corresponding change of the membrane bending stiffness. For small unilamellar fluid-phase liposomes composed of palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylglycerol (POPG), we find that cholesterol rigidifies the host membrane in a manner consistent with previously reported measurements. In contrast, temoporfin softens this membrane. Partitioning results for gel-phase liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) are also curvature-sensitive but cannot be interpreted on the basis of the bending stiffness alone.     

Oxidative stress can affect the gene silencing effect of DOTAP liposome in an in vitro translation system

Oxidative stress can affect in vitro GFP expression through its control of the gene silencing effect of the liposome prepared by 1,2-dioleoyl-3-trimethyl-ammonium propane (DOTAP). The gene silencing effect of cationic DOTAP liposome in in vitro GFP expression, especially focusing on its translation process, and the effects of oxidative stress on its silencing effect were investigated. GFP expression, initiated by mRNA, was found to be thoroughly inhibited in the presence of DOTAP liposome at concentration of more than 2.5 mM, though its inhibitory effect was reduced in the presence of hydrogen peroxide. The analyses of (i) the interaction of mRNA with DOTAP, (ii) the chemical structure of DOTAP, and (iii) the membrane fluidity of DOTAP liposome imply the possible role of gene expression by the liposome membrane and stress conditions.     

The crossflow injection technique: an improvement of the ethanol injection method

A novel scalable liposome preparation technique for pharmaceutical application is presented. Previous experiments have shown that the concept of continuous crossflow injection is a promising approach. For the characterization of the process, we focus on the influencing parameters like the lipid concentration, the injection hole diameter, the injection pressure, the buffer flow rate, and system performance. These experiments demonstrate that the injection hole diameter and the system performance do not influence the vesicle forming process and that a minimum of buffer flow rate is required to affect batch homogeneity. In contrast, strongly influencing parameters are lipid concentration in combination with increasing injection pressures. After exceeding the upper pressure limit of the linear range, where injection velocities remain constant, the vesicle batches are narrowly distributed, also when injecting higher lipid concentrations. Reproducibility and scalability data show similar results with respect to vesicle size and size distribution and demonstrate the stability and robustness of the novel continuous liposome preparation technique.     

Experimental study of photodynamic effect on bacterial wound microflora

The in vitro study of the influence of photodynamic action (with the use of photosensitizer "Photosens", laser and non-laser irradiation) on the strains of the main representatives purulent wound microflora was carried out. Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis cultures have been isolated from wound secretions and identified. The photosensitizer was shown to produce no bactericidal effect by itself. Irradiation with laser and non-laser light sources induced a sharp decrease in the number of viable cells in the cultures under study. The irradiation of the photosensitizer led to its activation, manifested in bactericidal action. The results thus obtained confirm good prospect of using photodynamic therapy for the treatment of purulent wounds.     

Differential interactions of a biological photosensitizer with liposome membranes having varying surface charges

The present work demonstrates the interaction of promising cancer cell photosensitizer, harmane (HM), with liposome membranes of varying surface charges, dimyristoyl-l-α-phosphatidylcholine (DMPC) and dimyristoyl-l-α-phosphatidylglycerol (DMPG). Electrostatic interaction of the cationic probe (HM) with the surface charges of the lipids is responsible for differential modulation of the spectral properties of the drug in different lipid environments. Estimation of partition coefficient (K(p) (±10%) = 5.58 × 10(4) in DMPC and 3.28 × 10(5) in DMPG) of HM between aqueous buffer and lipid phases reflect strong binding interaction of the drug with both the lipids. Evidence for greater degree of partitioning of HM into DMPG membrane compared to DMPC membrane has been deduced and further substantiated from experimental studies such as steady-state fluorescence anisotropy, micropolarity determination. The molecular modeling investigation by docking simulation coupled with fluorescence quenching experiment has been exploited to substantiate the location of drug at the lipid head-group region. Modulation of the dynamical properties of the drug within the lipid environments has also been addressed. Rotational relaxation dynamics studies unravel the impartation of a significant degree of motional restriction on the probe molecule within the lipids and reinforce the differential interactions of HM with the two lipid systems along the lines of other findings. Fluorescence kinetics studies reveal a faster association (in terms of apparent rate constants describing the process of interaction) of the drug with DMPG membrane compared to DMPC. This result is argued in connection with the electrostatic interaction between the drug and the liposome surface charges.