Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo

A liposomal delivery system that coordinates the release of irinotecan and floxuridine in vivo has been developed. The encapsulation of floxuridine was achieved through passive entrapment while irinotecan was actively loaded using a novel copper gluconate/triethanolamine based procedure. Coordinating the release rates of both drugs was achieved by altering the cholesterol content of distearoylphosphatidylcholine (DSPC)/distearoylphosphatidylglycerol (DSPG) based formulations. The liposomal retention of floxuridine in plasma after intravenous injection was dramatically improved by decreasing the cholesterol content of the formulation below 20 mol%. In the case of irinotecan, the opposite trend was observed where increasing cholesterol content enhanced drug retention. Liposomes composed of DSPC/DSPG/Chol (7:2:1, mole ratio) containing co-encapsulated irinotecan and floxuridine at a 1:1 molar ratio exhibited matched leakage rates for the two agents so that the 1:1 ratio was maintained after intravenous administration to mice. The encapsulation of irinotecan was optimal when copper gluconate/triethanolamine (pH 7.4) was used as the intraliposomal buffer. The efficiency of irinotecan loading was approximately 80% with a starting drug to lipid molar ratio of 0.1/1. Leakage of floxuridine from the liposomes during irinotecan loading at 50 degrees C complicated the ability to readily achieve the target 1:1 irinotecan/floxuridine ratio inside the formulation. As a result, a procedure for the simultaneous encapsulation of irinotecan and floxuridine was developed. This co-encapsulation method has the advantage over sequential loading in that extrusion can be performed in the absence of chemotherapeutic agents and the drug/drug ratios in the final formulation can be more precisely controlled.     

The encapsulation and release of guanosine from PEGylated liposomes

The encapsulation and release kinetics of guanosine from liposomes and polyethylene glycol (PEG)-modified liposomes are reported. Specifically, the influence of PEG chain length, PEGylation level, lipid type, drug-loading level, temperature, and solution conditions (i.e., salt and pH effects) on the rate and mechanism for release have been determined. Increasing PEGylation significantly reduced the guanosine release kinetics; this is more significant for greater molecular weight PEG and is correlated with the PEG layer thickness. Further, the mechanism for guanosine release changed from diffusion to interfacial control as the PEG level increased. The interfacial structure introduced by PEG also increased the activation energy required for guanosine transport across the lipid bilayer from 14 to 22 kJmol^?1. Findings from this study provide further insight into optimizing the formulation of Stealth liposomes.     

Induced drug release from lipid vesicles in serum by pH-change

Drugs can be released from lipid vesicles by pH-change in calf, horse or human serum when pH-sensitive trigger molecules are incorporated in the vesicle lipid bilayer. The lipid composition is so chosen that the drug release is best performed at 37 C. Specific drug targeting is envisaged to loci of the body with lower than physiological pH, such as primary or metastatic tumors.Recipient of Senior International Fellowship FO6-TW 00325 of the Fogarty International Center, National Institute of Health.     

Glucose-sensitivity of liposomes incorporating conjugates of glucose oxidase and poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecylacrylate)

Liposomes, which release their contents in response to the concentration of glucose, were prepared by modifying the liposomal surface with the conjugate of poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecylacrylate) (P(NIPAM-co-MAA-co-ODA)) and glucose oxidase (GOD). The maximum enzymatic activity of copolymer conjugated GOD (Polym-GOD) was observed around pH 5.0 and the value was about 40% of that of native GOD. Nine lysine residues per GOD molecule, on average, were found to be covalently attached to the copolymers. Egg phosphatidylcholine liposomes bearing Polym-GOD released their contents in response to the concentration of glucose and the sensitivity was higher than dipalmitoylphosphatidylcholine liposomes.     

Ovalbumin encapsulation into liposomes results in distinct degrees of oral immunization in mice

Oral administration of protein antigens, such as ovalbumin, may result in induction of either tolerance or immunization. To avoid oral tolerance, there are new strategies to protect the antigens from degradation within the gastrointestinal tract and to allow them to reach inductive immunological sites. One such strategy is the usage of liposomes. Different parameters may influence the stability of liposomes in the gastrointestinal tract. Herein, we studied the immunological consequences of oral administration of liposome-encapsulated ovalbumin in different strains of mice using different liposomes. Our data demonstrated that ovalbumin liposomes improved the induction of oral immunization and the degree of improvement depended on the liposome type and on the strain of mice used. The mechanism responsible for this differential effect of liposomes depended on the site of antigen release and absorption. Therefore, some liposomes might be suitable as adjuvants for oral immunization, others for oral tolerance induction.     

Purification and reconstitution into liposomes of an integral membrane protein conferring immunity to colicin A

Abstract The immunity protein to colicin A protects producing cells from the action of this pore-forming toxin. It is located into the cytoplasmic membrane. This protein has been 'tagged' with an epitope from the colicin A protein for which a monoclonal antibody is available. The fusion protein (named VL1) has been purified after extraction from the membrane in two steps using a chromatofocusing and an immunoadsorbant chromatography. The purified protein has then been reconstituted into lipid vesicles.     

Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient

This study examines a new method for the remote loading of doxorubicin into liposomes. It was shown that doxorubicin can be loaded to a level of up to 98% into large unilamellar vesicles composed of egg phosphatidylcholine/cholesterol (7/3 mol/mol) with a transmembrane phosphate gradient. The different encapsulation efficiencies which were achieved with ammonium salts (citrate 100%, phosphate 98%, sulfate 95%, acetate 77%) were significantly higher as compared to the loading via sodium salts (citrate 54%, phosphate 52%, sulfate 44%, acetate 16%). Various factors, including pH-value, buffer capacity, solubility of doxorubicin in different salt solutions and base counter-flow, which likely has an influence on drug accumulation in the intraliposomal interior are taken into account. In contrast to other methods, the newly developed remote loading method exhibits a pH-dependant drug release property which may be effective in tumor tissues. At physiological pH-value doxorubicin is retained in the liposomes, whereas drug release is achieved by lowering the pH to 5.5 (approximately 25% release at 25 °C or 30% at 37 °C within two h). The DXR release of liposomes which were loaded via a sulfate gradient showed a maximum of 3% at pH 5.5.     

Rapid measurement of drug release from temperature-sensitive liposomes by electron paramagnetic resonance and radioisotope techniques

We have developed two new methods for quantifying drug release from temperature-sensitive liposomes. Large unilamellar vesicles were made by the reverse phase evaporation process. They contained a water-soluble electron paramagnetic resonance probe, trimethyl-4-amino-2,2,6,6-tetramethyl piperidine N-oxyl and the radioisotope cytosine-[³H]1-β-D-arabinofuranoside in their aqueous compartment. Release of the electron paramagnetic resonance probe was measured by placing the liposomes in a solution of a spin label quenching agent, potassium ferricyanide, and monitoring the reduction in signal strength. The measurement of radioisotope release involved rapid ultracentrifugation of the liposomes after which the supernatant was tested for the presence of radioactivity. Both methods were found to be rapid and convenient ways of measuring drug release from temperature-sensitive liposomes and both methods gave comparable results. The radioisotope assay provides a direct measurement of drug leakage, whereas the electron spin resonance assay provides a continuous marker for liposome stability as a function of temperature.     

Rifampicin-loaded liposomes for the passive targeting to alveolar macrophages: in vitro and in vivo evaluation

Mycobacterium avium complex (MAC), the most frequent cause of opportunistic nontuberculous pulmonary infection, is made up of a group of intracellular pathogens that are able to survive and multiply inside lung alveolar macrophages. As nebulized liposomes are reported to be effective to target antibacterial agents to macrophages, in this work we have prepared and characterized re-dispersible freeze-dried rifampicin (RFP)-loaded vesicles by using soy lecithin (SL) and a commercial, enriched mixture of soy phosphatidylcholine (Phospholipon 90, P90) with or without cholesterol. The obtained results showed that RFP could be loaded stably in SL vesicles only when cholesterol was not present in the film preparation, whereas with P90 vesicles, the highest stability was obtained with formulations prepared with P90/cholesterol 7:1 or 4:1 molar ratios. RFP-liposome aerosols were generated using an efficient high-output continuous-flow nebulizer, driven by a compressor. After the experiments, nebulization efficiency (NE%) and nebulization efficiency of the encapsulated drug (NEED%) were evaluated. The results of our study indicated that nebulization properties and viscosity of formulations prepared with the low-transition-temperature phospholipids, SL and P90, are affected by vesicle composition. However, all formulations showed a good stability during nebulization and they were able to retain more than 65% of the incorporated drug. The effect of liposome encapsulation on lung levels of RFP following aerosol inhalation was determined in rats. The in vitro intracellular activity of RFP-loaded liposomes against MAC residing in macrophage-like J774 cells was also evaluated. Results indicated that liposomes are able to inhibit the growth of MAC in infected macrophages and to reach the lower airways in rats.     

Characterization of a novel pH-sensitive peptide that enhances drug release from folate-targeted liposomes at endosomal pHs

Although liposomes have proven useful for the delivery of drugs and gene therapy vectors, their potencies are often compromised by poor unloading following uptake into their target cells. We have consequently explored the properties of a novel 29-residue amphipathic peptide that was designed by arrangement of hydrophobic and hydrophilic residues to disrupt liposomes at lower peptide concentrations than previously tested peptides. The peptide was indeed found to promote pH-dependent liposome unloading with improved efficiency. A peptide of the same sequence, but half the length, however, promoted pH-dependent permeabilization only at much higher concentrations. Further characterization of the longer peptide revealed that release of liposome contents (i) occurred at a pH of ∼6, (ii) became less efficient as the size of the encapsulated cargo increased, and (iii) was moderately suppressed in cholesterol-containing liposomes. Use of this peptide to enhance the cytotoxicity of cytosine arabinoside encapsulated in folate-targeted liposomes demonstrated an increase in drug potency of ∼30-fold. Gene expression by a serum-stable folate-targeted liposomal vector was also measurably enhanced by inclusion of the peptide. We conclude that intracellular unloading of liposomal contents can be significantly improved by co-encapsulation of an optimally designed, pH-sensitive peptide.     

Simple and rapid determination of irinotecan and its metabolite SN-38 in plasma by high-performance liquid-chromatography: application to clinical pharmacokinetic studies

Irinotecan (CPT-11) is an anticancer agent widely employed in the treatment of colorectal carcinoma. A simple, rapid and sensitive high-performance liquid chromatographic method for the simultaneous determination of CPT-11 and its metabolite SN-38 in plasma, and their preliminary clinical pharmacokinetics are described. Both deproteinisation of plasma specimens (100 μl) and addition of the internal standard, camptothecin (CPT), are achieved by incorporating to samples 100 μl of a solution of CPT (1 μg/ml) in acetonitrile–1 mM orthophosphoric acid (90:10); 200 μl of this acidified acetonitrile solution, drug-free, is also added to accomplish complete deproteinisation: this procedure reduces sample preparation time to a minimum. After deproteinisation, samples are treated with potassium dihydrogenphosphate (0.1 M) and injected into a Nucleosil C₁₈ (5 μm, 250×4.0 mm) column. Mobile phase consists of potassium dihydrogenphosphate (0.1 M)–acetonitrile (67:33), at a flow-rate of 1 ml/min. CPT-11, SN-38 and CPT are detected by fluorescence with excitation wavelength set at 228 nm and emission wavelengths of CPT-11, SN-38 and CPT fixed, respectively, at 450, 543 and 433 nm. The limits of quantitation for CPT-11 and SN-38 are 1.0 and 0.5 ng/ml, respectively. This method shows good precision: the within day relative standard deviation (RSD) for CPT-11 (1–10 000 ng/ml) is 5.17% (range 2.15–8.27%) and for SN-38 (0.5–400 ng/ml) is 4.33% (1.32–7.78%); the between-day RSDs for CPT-11 and SN-38, in the previously described ranges, are 6.82% (5.03–10.8%) and 4.94% (2.09–9.30%), respectively. Using this assay, plasma pharmacokinetics of CPT-11, SN-38 and its glucuronidated form, SN-38G, have been determined in one patient receiving 200 mg/m² of CPT-11 as a 90 min intravenous infusion. The peak plasma concentration of CPT-11 at the end of the infusion is 3800 ng/ml. Plasma decay is biphasic with a terminal half-life of 11.6 h. The volume of distribution at steady state (V_ss) is 203 l/m², and the total body clearance (Cl) is 14.8 l/h·m². The maximum concentrations of SN-38 and SN-38G reach 28.9 and 151 ng/ml, respectively.     

Zn phthalocyanines loaded into liposomes: Characterization and enhanced performance of photodynamic activity on glioblastoma cells

Photodynamic therapy (PDT) is considered a promising strategy for cancer treatment. PDT utilizes light in combination with a photosensitizer (PS) to induce several phototoxic reactions. Phthalocyanines (Pcs), a second generation of photosensitizers, have been studied in several cancer models. Among these, Pcs, have become of interest for the treatment of glioblastomas which are one of the most common and aggressive forms of tumors of the central nervous system. Due to the lipophilic nature of Pcs and their limited solubility in water, Pcs can be loaded in liposomes. In this work, we characterized liposomes of ZnPc and TAZnPc and their effectiveness to photoinactivate glioblastoma cells, was evaluated. Both Pcs show an increase in their photosensitizing activity when they were administrated in Dipalmitoylphosphatidylcholine-cholesterol liposomes compared to Pcs administrated in dimethylformamide.     

Drug loading into and drug release from pH- and temperature-responsive cylindrical hydrogels

Hydrogels that undergo deformation upon appropriate changes in pH or temperature have considerable promise as drug delivery vehicles. Drug uptake in swelling and nonswelling cylindrical hydrogels and drug release from these into a target fluid are investigated here. A mathematical model for hydrogel-solution composite, a composite of a distributed parameter system (cylindrical hydrogel) and a lumped parameter system (surrounding solution), is developed. The polymer network displacement in a swelling/deswelling hydrogel is described by a stress diffusion coupling model. The analytical solution for network displacement is used to predict solvent intake by swelling hydrogels, solvent efflux from deswelling hydrogels, and changes in pressure, porosity, and effective drug diffusivity. These in turn influence drug uptake during and after hydrogel swelling and drug release from hydrogel during and after deswelling. Numerical results illustrate benefits of hydrogel swelling for drug loading and merits of different modes of drug release. Drug uptake and drug release by temperature-responsive hydrogels are compared with those by hydrogels not subject to deformation.     

Study of the release of a microencapsulated acid dye in polyamide dyeing using mixed cationic liposomes

The main objective of this work was to increase the retarding effect of the acid dye Telon^® Blue RR (C.I. Acid Blue 62; DyStar, Frankfurt, Germany) release on polyamide fibres dyeing by encapsulation of the dye in liposomes as an alternative to synthetic auxiliaries, in order to reduce effluent pollution. The retarding effect achieved with the use of mixed cationic liposomes of dioctadecyldimethylammonium bromide (DODAB)/soybean lecithin (containing a 10% molar fraction of DODAB) was better in comparison with either pure soybean lecithin liposomes or synthetic auxiliaries. The retarding effect of liposomes on the dye release was analysed through changes in the absorption and fluorescence spectra of the acid dye at different conditions. The effect of temperature (in the range of 25 °C - 70 °C) on the spectroscopic behaviour of the dye in the absence and in presence of polyamide was also studied, in order to simulate the dyeing conditions. Exhaustion curves obtained in dyeing experiments showed that, below 45 °C, the retarding effect of the mixed liposomes (lecithin/DODAB (9:1)) was similar to that of the auxiliaries, but better than the one of pure lecithin liposomes. At higher temperatures (above 45 °C), the system lecithin/DODAB presents a better performance, achieving a higher final exhaustion level when compared with the commercial leveling agent without losing the smoothing effect of lecithin.     

Triggered release of doxorubicin following mixing of cationic and anionic liposomes

In many applications, an ability of liposomes to retain drug and then rapidly release it at some later time would be of benefit. In this work, we investigate the ability of cationic large unilamellar vesicles (LUV) to promote rapid release of doxorubicin from anionic LUV. It is shown that the addition of cationic liposomes containing cholesterol, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) and the cationic lipid N,N-dioleyl-N,N-dimethylammonium chloride (DODAC) to doxorubicin-containing LUV composed of cholesterol, DOPE, DSPC and the anionic lipid dioleoyphosphatidylglycerol (DOPG) can result in release of more than 90% of the drug in times of 30 s or less. Further, it is shown that these release characteristics are exquisitely dependent on the presence of DOPE and cholesterol. In the absence of DOPE, much slower release rates are observed, with maximum release levels of 50% after a 2-h incubation at 20 °C. Remarkably, threshold levels of more than 10 mol% cholesterol are required before any appreciable release is observed. [³¹P]NMR spectroscopy and freeze-fracture electron microscopy studies reveal that systems giving rise to rapid release of doxorubicin exhibit limited formation of inverted hexagonal (H_II) phase, suggesting that these lipids facilitate drug release by formation of local regions of non-bilayer structure. It is concluded that drug release triggered by mixing anionic and cationic liposomes could be of utility in drug delivery applications.     

High-performance liquid chromatographic assay with fluorescence detection for the simultaneous measurement of carboxylate and lactone forms of irinotecan and three metabolites in human plasma

Irinotecan (CPT-11), a camptothecin analog, is metabolized to SN-38, an active topoisomerase I inhibitor, and inactive metabolites, including APC and SN-38 glucuronide (SN-38G). A high-performance liquid chromatographic assay method to simultaneously measure the lactone and carboxylate forms of CPT-11, SN-38, SN-38G, and APC in human plasma was developed. Chromatography was accomplished with a reversed-phase C(8) column and fluorescence detection. A gradient mobile phase system was used. The buffer for mobile phase A consisted of 0.75 M ammonium acetate, 5 mM tetrabutylammonium phosphate (pH 6.0), and acetonitrile (86:14, v/v). The buffer for mobile phase B was identical to mobile phase A with the exception of the concentration (50:50, v/v). Precipitation of plasma proteins was performed with cold methanol. The linear range of detection of the lactone and carboxylate forms of SN-38, SN-38G, and APC was 2-25 ng/ml, and 5-300 ng/ml for CPT-11. The limit of quantitation for the analytes ranged from 0.5 to 5 ng/ml. Analysis of patients' plasma samples obtained before and after CPT-11 administration showed that the assay is suitable for measuring lactone and carboxylate forms of CPT-11, SN-38, SN-38G, and APC in clinical studies.     

Kinetic analysis of product release and metal ions in a metallonuclease

Most nucleases rely on divalent cations as cofactors to catalyze the hydrolysis of nucleic acid phosphodiester bonds. Here both equilibrium and kinetic experiments are used to test recently proposed models regarding the metal ion dependence of product release and the degree of cooperativity between metal ions bound in the active sites of the homodimeric PvuII endonuclease. Equilibrium fluorescence anisotropy studies indicate that product binding is dramatically weakened in the presence of metal ions. Pre-steady state kinetics indicate that product release is at least partially rate limiting. Steady state and pre-steady state data fit best to models in which metals remain bound to the enzyme after the release of product. Finally, analysis of cooperative and independent binding models for metal ions indicates that single turnover kinetic data are consistent with little to no positive cooperativity between the two metal ions binding each active site.     

Design of polymeric microparticles for pH-responsive and time-sustained drug release

We described the design of uniform microencapsulates with almost 100% encapsulation efficiency, synthesized without organic solvents, via microfluidic spray drying of water-based dispersions of pH-responsive methacrylic acid polymers (Eudragit^® L 30D-55). The effects of incorporating water-based network-forming materials in the formulations on pH-responsiveness and controlled release patterns of enteric microparticles were observed. Acid hydrolysed tetraethoxysilane (TEOS) was used to form an interpenetrating, rigid framework of silica, whereas Eudragit^® NE (a copolymer based on ethyl acrylate and methyl methacrylate) was added to produce a more flexible polymeric network. The spray-dried microparticles generally displayed crumbled or buckled morphologies dependent on drying temperatures, due to large hydrodynamic sizes of solutes in feed dispersions. The drug release kinetics of microparticles were sensitive to the type and the added amount of network-forming materials, due to different colloidal interactions between Eudragit^® L and either silica or the copolymer. This study demonstrated a strategy to design enteric microparticles with different microstructural properties and drug release behaviours through understanding of colloidal interactions between constituents of matrix materials.     

Development and modeling of arsenic-trioxide–loaded thermosensitive liposomes for anticancer drug delivery

In this article, a novel delivery system for the anticancer drug, arsenic trioxide (ATO), is characterized. The release of ATO from DPPC liposomes with MPPC lysolipid incorporated into the bilayer was measured. Upon heating the liposomes to 37°C, there was a 15–25% release over 24 hours. The ATO release from the DPPC and DPPC:MPPC (5%) systems leveled off after 10 hours at 37°C, whereas the DPPC:MPPC (10%) liposomes continue to release ATO over the 24-hour time span. Upon heating the liposomes rapidly to 42°C, the release rate was substantially increased. The systems containing lysolipids exhibited a very rapid release of a significant amount of arsenic in the first hour. In the first hour, the DPPC:MPPC (5%) liposomes released 40% of the arsenic and the DPPC:MPPC (10%) liposomes released 55% of the arsenic. Arsenic release from pure DPPC liposomes was comparable at 37 and 42°C, indicating that the presence of a lysolipid is necessary for a significant enhancement of the release rate. A coarse-grained molecular dynamics (CGMD) model was used to investigate the enhanced permeability of lysolipid-incorporated liposomes and lipid bilayers. The CG liposomes did not form a gel phase when cooled due to the high curvature; however, permeability was still significantly lower below the liquid-to-gel phase-transition temperature. Simulations of flat DPPC:MPPC bilayers revealed that a peak in the permeability did coincide with the phase transition from the gel to LC state when the lysolipid, MPPC, was present. No pores were observed in the simulations, so it is unlikely this was the permeability-enhancing mechanism.     

Calcium and magnesium induced changes in the relative fluidity of phosphatidylcholine liposomes

The effect of Ca²⁺ and Mg²⁺ on relative fluidity of phosphatidylcholine liposomes was studied by measuring the degree of chlorophyll fluorescence polarization. An increase in the degree of fluorescence polarization was observed on incubation of liposomes with different concentrations of Ca²⁺ or Mg²⁺. The results have been interpreted on the basis of increase in the size of liposomes which could be brought about by calcium or magnesium induced fusion of small unilamellar liposomes to form larger vesicles. Fusion of liposomes has also been confirmed by the experiments on efficiency of energy transfer from chlorophyll b to chlorophyll a, and transmission electron microscopy of liposomes before and after incubation with Ca²⁺ and Mg²⁺.