Enhanced loading efficiency and retention of 225Ac in rigid liposomes for potential targeted therapy of micrometastases

Targeted alpha-particle emitters are promising therapeutics for micrometastatic disease. Actinium-225 has a 10-day half-life and generates a total of four alpha-particles per parent decay rendering (225)Ac an attractive candidate for alpha-therapy. For cancer cells with low surface expression levels of molecular targets, targeting strategies of (225)Ac using radiolabeled carriers of low specific radioactivities (such as antibodies) may not deliver enough alpha-particle emitters at the targeted cancer cells to result in killing. We previously proposed and showed using passive (225)Ac entrapment that liposomes can stably retain encapsulated (225)Ac for long time periods, and that antibody-conjugated liposomes (immunoliposomes) with encapsulated (225)Ac can specifically target and become internalized by cancer cells. However, to enable therapeutic use of (225)Ac-containing liposomes, high activities of (225)Ac need to be stably encapsulated into liposomes. In this study, various conditions for active loading of (225)Ac in preformed liposomes (ionophore-type, encapsulated buffer solution, and loading time) were evaluated, and liposomes with up to 73 +/- 9% of the initial activity of (225)Ac (0.2-200 microCi) were developed. Retention of radioactive contents by liposomes was evaluated at 37 degrees C in phosphate buffer and in serum-supplemented media. The main fraction of released (225)Ac from liposomes occurs within the first two hours of incubation. Beyond this two hour point, the encapsulated radioactivity is released from liposomes slowly with an approximate half-life of the order of several days. In some cases, after 30 days, (225)Ac retention as high as 81 +/- 7% of the initially encapsulated radioactivity was achieved. The (225)Ac loading protocol was also applied to immunoliposome loading without significant loss of targeting efficacy. Liposomes with surface-conjugated antibodies that are loaded with (225)Ac overcome the limitations of low specific activity for molecular carriers and are expected to be therapeutically useful against tumor cells having a low antigen density.     

LaPO4 nanoparticles doped with actinium-225 that partially sequester daughter radionuclides

Nanoscale materials have been envisioned as carriers for various therapeutic drugs, including radioisotopes. Inorganic nanoparticles (NPs) are particularly appealing vehicles for targeted radiotherapy because they can package several radioactive atoms into a single carrier and can potentially retain daughter radioisotopes produced by in vivo generators such as actinium-225 ((225)Ac, t(1/2) = 10 d). Decay of this radioisotope to stable bismuth-209 proceeds through a chain of short-lived daughters accompanied by the emission of four α-particles that release >27 MeV of energy. The challenge in realizing the enhanced cytotoxic potential of in vivo generators lies in retaining the daughter nuclei at the therapy site. When (225)Ac is attached to targeting agents via standard chelate conjugation methods, all of the daughter radionuclides are released after the initial α-decay occurs. In this work, (225)Ac was incorporated into lanthanum phosphate NPs to determine whether the radioisotope and its daughters would be retained within the dense mineral lattice. Further, the (225)Ac-doped NPs were conjugated to the monoclonal antibody mAb 201B, which targets mouse lung endothelium through the vasculature, to ascertain the targeting efficacy and in vivo retention of radioisotopes. Standard biodistribution techniques and microSPECT/CT imaging of (225)Ac as well as the daughter radioisotopes showed that the NPs accumulated rapidly in mouse lung after intravenous injection. By showing that excess, competing, uncoupled antibodies or NPs coupled to control mAbs are deposited primarily in the liver and spleen, specific targeting of NP-mAb 201B conjugates was demonstrated. Biodistribution analysis showed that ～30% of the total injected dose of La((225)Ac)PO(4) NPs accumulated in mouse lungs 1 h postinjection, yielding a value of % ID/g >200. Furthermore, after 24 h, 80% of the (213)Bi daughter produced from (225)Ac decay was retained within the target organ and (213)Bi retention increased to ～87% at 120 h. In vitro analyses, conducted over a 1 month interval, demonstrated that ～50% of the daughters were retained within the La((225)Ac)PO(4) NPs at any point over that time frame. Although most of the γ-rays from radionuclides in the (225)Ac decay chain are too energetic to be captured efficiently by SPECT detectors, appropriate energy windows were found that provided dramatic microSPECT images of the NP distribution in vivo. We conclude that La((225)Ac)PO(4)-mAb 201B conjugates can be targeted efficiently to mouse lung while partially retaining daughter products and that targeting can be monitored by biodistribution techniques and microSPECT imaging.     

Radiotoxicity of bismuth-213 bound to membranes of monolayer and spheroid cultures of tumor cells

Monoclonal antibody 13A to murine CD44 was used to bind the alpha-particle emitter 213Bi to cell surfaces of cultured EMT-6 or Line 1 tumor cells. Data on kinetics and saturation of binding, cell shape and nuclear size were used to calculate the absorbed dose to the nuclei. Treatment of monolayer cells with [213Bi]MAb 13A produced a classical exponential survival curve with no apparent shoulder. Microdosimetry analyses indicated that 1.4-1.7 Gy produced a 37% surviving fraction (D0). Multicellular spheroids were shown to bind [213Bi]MAb 13A mainly on the outer cell layer. Relatively small amounts of activity added to the spheroids resulted in relatively large absorbed doses. The result was that 3-6-fold less added radioisotope was necessary to kill similar fractions of cells in spheroids than in monolayer cells. These data are consistent with the interpretation that the alpha particles from a single 213Bi atom bound to one cell can penetrate and kill adjacent cells. Flow cytometry was used to sort cells originating from the periphery or from the interior of spheroids. Cells from the outside of the [213Bi]MAb 13A exposed spheroids had a lower surviving fraction per administered activity than cells from the interior. Cells were killed efficiently in spheroids up to 20-30 cells in diameter. The data support the hypothesis that alpha-particle emitters should be very efficient at killing cells in micrometastases of solid tumors.     

Monte Carlo modeling of the effects of injected short-, medium- and longer-range alpha-particle emitters on human marrow at various ages

The effects of injected short-, medium- and longer-range alpha-particle emitters ((149)Tb, (211)At/(211)Po and (213)Bi/(213)Po, respectively) on the total hemopoietic stem cell population of active normal bone marrow in humans of various ages has been estimated using Monte Carlo modeling. The fraction of the normal hemopoietic stem cells that are hit and survive has been calculated as a first step toward estimating the risk of development of therapy-induced leukemia. The fraction was lowest for the shorter-range alpha-particle emitter ((149)Tb) and highest for the longer-range alpha-particle emitter ((213)Bi/(213)Po), with the value for the medium-range alpha-particle emitter (211)At/(211)Po being intermediate between these. There was little variation in the data with the age of the subject within each alpha-particle emitter. This lack of age dependence provides reassurance that the fraction of cells hit in any subject of any age with normal marrow can be estimated by modeling newborn marrow (which requires little computing time) despite age-related differences in microarchitecture.     

Comparison of the radiotoxicity of two alpha-particle-emitting immunoconjugates,terbium-149 and bismuth-213, directed against a tumor-specific,exon 9 deleted (d9) E-cadherin adhesion protein

We investigated the effects of the alpha-particle emitters (149)Tb and (213)Bi coupled to a tumor-specific antibody targeting the mutated delta 9 E-cadherin (d9 E-Cad) on single cells and cell pellets. The d9 mutation of the adhesion molecule E-cadherin is found in 10% of diffuse-type gastric cancers and is not expressed in normal tissue. Human breast cancer cells (MDA-MB-435S) transfected with d9 E-Cad or the wild-type E-cadherin gene were used to study the effects of anti-d9 E-Cad MAb coupled to (149)Tb and (213)Bi ((149)Tb-d9 MAb and (213)Bi-d9 MAb). The density of binding sites determined on transfected MDA tumor cells by Scatchard analysis and flow cytometry varied from 4 x 10(4) to 6 x 10(4) antigens per cell. Internalization of radioimmunoconjugates by cells expressing d9 E-Cad was less than 10% of bound antibody within 240 min. The effect of the radioimmunoconjugates on cell suspensions and cell pellets was quantified by [(3)H]thymidine incorporation, and the dose to the cell nuclei was determined using microdosimetric calculations. (149)Tb and (213)Bi immunoconjugates affected cells in suspension similarly. Significant differences in the proliferation capacity of d9 E-cadherin- and wild-type E-cadherin-expressing cells were observed at activity concentrations around 185 kBq/ml, corresponding to antibody concentrations between 200 ng/ml and 1000 ng/ml. Proliferation after incubation with (213)Bi-d9 MAb was 50% greater in pelleted wild-type E-Cad-expressing cells compared to wild-type E-Cad cells in suspension. In contrast, the proliferation of pelleted d9 E-Cad cells was similar to that of d9 E-Cad cells in suspension. For (149)Tb-d9 MAb, no significant difference was found between pelleted cells and cells in suspension for low activity concentrations. However, at high activity concentrations, (149)Tb-d9 MAb had only a small effect on pelleted cells. These in vitro studies demonstrate different effects of (149)Tb and (213)Bi conjugated to a tumor-specific antibody toward single cells and tumor cell pellets. Microdosimetric simulation of single cell survival after alpha-particle irradiation modeled the experimental results with reasonable accuracy.     

Streptavidin in antibody pretargeting. 5. chemical modification of recombinant streptavidin for labeling with the alpha-particle-emitting radionuclides 213Bi and 211At

We are investigating the use of recombinant streptavidin (rSAv) as a carrier molecule for the short-lived alpha-particle-emitting radionuclides 213Bi ( t 1/2 = 45.6 min) and 211At ( t 1/2 = 7.21 h) in cancer therapy. To utilize rSAv as a carrier, it must be modified in a manner that permits rapid chelation or bonding with these short-lived radionuclides and also modified in a manner that diminishes its natural propensity for localization in the kidney. Modification for labeling with (213)Bi was accomplished by conjugation of rSAv with the DTPA derivative p-isothiocyanato-benzyl-CHX-A' (CHX-A'), 3a. Modification for direct labeling with 211At was accomplished by conjugation of rSAv with an isothiocyanatophenyl derivative of a nido-carborane (nCB), 3b, or an isothiocyanatophenyl-dPEG/decaborate(2-) derivative, 3c. After conjugation of the chelating or bonding moiety, rSAv was further modified by reaction with an excess (50-100 equivalents) of succinic anhydride. Succinylation of the lysine amines has previously been shown to greatly diminish kidney localization. rSAv modified by conjugation with 3a and succinylated rapidly radiolabeled with 213Bi (<5 min), providing a 72% isolated yield. 211At labeling of modified rSAv was accomplished in aqueous solution using chloramine-T as the oxidant. Astatination of rSAv conjugated with 3b and succinylated occurred very rapidly (<1 min), providing a 50% isolated radiochemical yield. Astatination of rSAv conjugated with 3c and succinylated was also very rapid (<1 min) providing 66-71% isolated radiochemical yields. Astatination of succinylated rSAv, 2a, which did not have conjugated borane cage moieties, resulted in a much lower radiolabeling yield (18%). The 213Bi or 211At-labeled modified rSAv preparations were mixed with the corresponding 125 I-labeled rSAv, and dual-label in vivo distributions were obtained in athymic mice. The in vivo data show that 213Bi-labeled succinylated rSAv [ 213Bi] 6a has tissue concentrations similar to those of 125 I-labeled modified rSAv [ 125 I] 6b, suggesting that (213)Bi is quite stable toward release from the chelate in vivo. In vivo data also indicate that the (211)At-labeled rSAv conjugated with 3b or 3c and succinylated are stable to in vivo deastatination, whereas succinylated rSAv lacking a boron cage moiety is subject to some deastatination. The modified rSAv conjugated with nido-carborane derivative 3b has a higher retention in many tissues than rSAv without the carborane conjugated. Interestingly, the rSAv conjugated with 3c, which also contains an m-dPEG 12 moiety, has significantly decreased concentrations in blood and other tissues when compared with those of direct-labeled rSAv, suggesting that it may be a good candidate for further study. In conclusion, rSAv that has been modified with CHX-A' and succinylated (i.e., 5a) may be useful as a carrier of 213Bi. The encouraging results obtained with the PEGylated decaborate(2-) derivative 3c and succinylated (i.e., 5c) suggests that its further study as a carrier of 211At in pretargeting protocols is warranted.     

Interaction between X-ray and alpha-particle damage in V79 cells

V79 cells have been exposed to X-rays or 238Pu alpha-particles or to X-rays following priming alpha-particle doses of 0.5, 2 or 2.5 Gy. The survival curve for exposure to alpha-particles was exponential with a D0 of 0.89 Gy. Following exposure to priming alpha-particle doses the resulting X-ray survival curves had the same slope as the single dose X-ray curve, but a reduced shoulder. For alpha-particle priming doses of 0.5 and 2 Gy this reduction was the same as for the same X-ray doses. 2.5 Gy alpha-particles reduced the subsequent X-ray curve Dq to almost zero. alpha-particles do cause damage capable of interacting with X-ray damage.     

Absence of a dose-rate effect in the transformation of C3H 10T1/2 cells by alpha-particles

The findings of Hill et al. (1984) on the greatly enhanced transformation frequencies at very low dose rates of fission neutrons induced us to perform an analogous study with alpha-particles at comparable dose rates. Transformation frequencies were determined with gamma-rays at high dose rate (0.5 Gy/min), and with alpha-particles at high (0.2 Gy/min) and at low dose rates (0.83-2.5 mGy/min) in the C3H 10T1/2 cell system. alpha-particles were substantially more effective than gamma-rays, both for cell inactivation and for neoplastic transformation at high and low dose rates. The relative biological effectiveness (RBE) for cell inactivation and for neoplastic transformation was of similar magnitude, and ranged from about 3 at an alpha-particle dose of 2 Gy to values of the order of 10 at 0.25 Gy. In contrast to the experiments of Hill et al. (1984) with fission neutrons, no increased transformation frequencies were observed when the alpha-particle dose was protracted over several hours.     

The effect of 238Pu alpha-particles on the mouse fibroblast cell line C3H 10T1/2: characterization of source and RBE for cell survival

Considerable interest has been aroused in recent years by reports that the transforming and carcinogenic effectiveness of low doses of high LET radiations can be increased by reducing the dose rate, especially for transformation of 10T1/2 cells in vitro by fission-spectrum neutrons. We report on conditions which have been established for irradiation of 10T1/2 cells with high LET monoenergetic alpha-particles (energy of 3.2 MeV, LET of 124 keV microns-1) from 238Pu. The alpha-particle irradiator allows convenient irradiation of multiple dishes of cells at selectable high or low dose rates and temperatures. The survival curves of irradiated cells showed that the mean lethal dose of alpha-particles was 0.6 Gy and corresponded to an RBE, at high dose rates, of 7.9 at 80 per cent survival and 4.6 at 5 per cent survival, relative to 60Co gamma-rays. The mean areas of the 10T1/2 nuclei, perpendicular to the incident alpha-particles, was measured as 201 microns2, from which it follows that, on average, only one in six of the alpha-particle traversals through a cell nucleus is lethal. Under the well-characterized conditions of these experiments the event frequency of alpha-particle traversals through cell nuclei is 9.8 Gy-1.     

Synthesis, conjugation, and radiolabeling of a novel bifunctional chelating agent for (225)Ac radioimmunotherapy applications

225Ac (t(1/2) = 10 days) is an alternative alpha-emitter that has been proposed for radioimmunotherapy (RIT) due to its many favorable properties, such as half-life and mode of decay. The factor limiting use of (225)Ac in RIT is the lack of an acceptably stable chelate for in vivo applications. Herein is described the first reported bifunctional chelate for (225)Ac that has been evaluated for stability for in vivo applications. The detailed synthesis of a bifunctional chelating agent 2-(4-isothiocyanatobenzyl)-1,4,7,10,13, 16-hexaazacyclohexadecane- 1,4,7,10,13,16-hexaacetic acid (HEHA-NCS) is reported. This ligand was conjugated to three monoclonal antibodies, CC49, T101, and BL-3 with chelate-to-protein ratios between 1.4 and 2. The three conjugates were radiolabeled with (225)Ac, and serum stability study of the [(225)Ac]BL-3-HEHA conjugate was performed.     

Efficient bifunctional decadentate ligand 3p-C-DEPA for targeted α-radioimmunotherapy applications

A new bifunctional ligand 3p-C-DEPA was synthesized and evaluated for use in targeted α-radioimmunotherapy. 3p-C-DEPA was efficiently prepared via regiospecific ring opening of an aziridinium ion and conjugated with trastuzumab. The 3p-C-DEPA-trastuzumab conjugate was extremely rapid in binding (205/6)Bi, and the corresponding (205/6)Bi-3p-C-DEPA-trastuzumab complex was stable in human serum. Biodistribution studies were performed to evaluate in vivo stability and tumor targeting of (205/6)Bi-3p-C-DEPA-trastuzumab conjugate in tumor bearing athymic mice. (205/6)Bi-3p-C-DEPA-trastuzumab conjugate displayed excellent in vivo stability and targeting as evidenced by low organ uptake and high tumor uptake. The results of the in vitro and in vivo studies indicate that 3p-C-DEPA is a promising chelator for radioimmunotherapy of (212)Bi and (213)Bi.     

A retention mechanism for distribution of mitochondria during cell division in budding yeast.

Mitochondria are indispensable for normal eukaryotic cell function. As they cannot be synthesized de novo and are self-replicating, mitochondria must be transferred from mother to daughter cells. Studies in the budding yeast Saccharomyces cerevisiae indicate that mitochondria enter the bud immediately after bud emergence, interact with the actin cytoskeleton for linear, polarized movement of mitochondria from mother to bud, but are equally distributed among mother and daughter cells [1] [2] [3]. It is not clear how the mother cell maintains its own supply of mitochondria. Here, we found that mother cells retain mitochondria by immobilization of some mitochondria in the 'retention zone', the base of the mother cell distal to the bud. Retention requires the actin cytoskeleton as mitochondria colocalized with actin cables in the retention zone, and mutations that perturb actin dynamics or actin-mitochondrial interactions produced retention defects. Our results support the model that equal distribution of mitochondria during cell division is a consequence of two actin-dependent processes: movement of some mitochondria into the daughter bud and immobilization of others in the mother cell.     

Investigation of parameters influencing incorporation,retention and cellular cytotoxicity in liposomal formulations of poorly soluble camptothecin

Abstract

Improving tumor delivery of lipophilic drugs through identifying advanced drug carrier systems with efficient carrier potency is of high importance. We have performed an investigative approach to identify parameters that affect liposomes’ ability to effectively deliver lipophilic camptothecin (CPT) to target cells. CPT is a potent anticancer drug, but its undesired physiological properties are impairing its therapeutic use. In this study, we have identified parameters influencing incorporation and retention of lipophilic CPT in liposomes, evaluating the effect of lipid composition, lipid chemical structure (head and tail group variations, polymer inclusion), zeta potential and anisotropy. Polyethyleneglycol (PEG) surface decoration was included to avoid liposome fusing and increase the potential for prolonged in vivo circulation time. The in vitro effect of the different carrier formulations on cell cytotoxicity was compared and the effect of active targeting of one of the formulations was evaluated. We found that a combination of liposome surface charge, lipid headgroup and carbon chain unsaturation affect CPT incorporation. Retention in liposomes was highly dependent on the liposomal surroundings and liposome zeta potential. Inclusion of lipid tethered PEG provided stability and prevented liposome fusing. PEGylation negatively affected CPT incorporation while improving retention. In vitro cell culture testing demonstrated that all formulations increased CPT potency compared to free CPT, while cationic formulations proved significantly more toxic to cancer cells that healthy cells. Finally, antibody mediated targeting of one liposome formulation further enhanced the selectivity towards targeted cancer cells, rendering normal cells fully viable after 1 hour exposure to targeted liposomes.     

Improved retention of idarubicin after intravenous injection obtained for cholesterol-free liposomes

To date there has been a focus on the application of sterically stabilized liposomes, composed of saturated diacylphospholipid, polyethylene glycol (PEG) conjugated lipids (5-10 mole%) and cholesterol (CH) (>30 mole%), for the systemic delivery of drugs. However, we are now exploring the utility of liposome formulations composed of diacylphospholipid conjugated PEG mixtures prepared in the absence of added cholesterol, with the primary objective of developing formulations that retain encapsulated drug better than comparable formulations prepared with cholesterol. In this report the stability of cholesterol-free distearoylphosphatidylcholine (DSPC):distearoylphosphatidylethanolamine (DSPE)-PEG(2000) (95:5 mol/mol) liposomes was characterized in comparison to cholesterol-containing formulations DSPC:CH (55:45 mol/mol) and DSPC:CH:DSPE-PEG(2000) (50:45:5 mol/mol/mol), in vivo. Circulation longevity of these formulations was determined in consideration of variables that included varying phospholipid acyl chain length, PEG content and molecular weight. The application of cholesterol-free liposomes as carriers for the hydrophobic anthracycline antibiotic, idarubicin (IDA), was assessed. IDA was encapsulated using a transmembrane pH gradient driven process. To determine stability in vivo, pharmacokinetic studies were performed using 'empty' and drug-loaded [(3)H]cholesteryl hexadecyl ether radiolabeled liposomes administered intravenously to Balb/c mice. Inclusion of 5 mole% of DSPE-PEG(2000) or 45 mole% cholesterol to DSPC liposomes increased the mean plasma area under the curve (AUC(0-24h)) 19-fold and 10-fold, respectively. Cryo-transmission electron micrographs of IDA loaded liposomes indicated that the drug formed a precipitate within liposomes. The mean AUC(0-4h) for free IDA was 0.030 micromole h/ml as compared to 1.38 micromole h/ml determined for the DSPC:DSPE-PEG(2000) formulation, a 45-fold increase, demonstrating that IDA was retained better in cholesterol-free compared to cholesterol-containing liposomes.     

Enhanced Gene Delivery and Expression in Human Hepatocellular Carcinoma Cells by Cationic Immunoliposomes

Abstract

A targeted vector allowing enhanced gene transfer to human hepatocellular carcinoma (HCC¹) cells in vitro was developed using cationic liposomes covalently conjugated with the mAb AF-20. This high affinity antibody recognizes a rapidly internalized 180 kDa cell surface glycoprotein which is abundantly expressed on the surface of human HCC and other cancer cells. Quantitative binding analysis of liposomes with target cells by flow cytometry showed specific association of mAb-targeted liposomes with human HCC cells. Using mAb-targeted cationic liposomes containing 20% DOTAP, in the presence or absence of serum, gene expression in HuH-7 cells was enhanced up to 40-fold as compared to liposomes conjugated with an isotype-matched non-relevant control antibody. Transfection specificity was not observed in a control cell line that does not express the antigen recognized by mAb AF-20. This study demonstrates that cationic liposome formulations can be targeted with monoclonal antibodies (mAbs) to enhance specific in vitro gene delivery and expression in the presence or absence of serum.     

Simultaneous interaction of monoclonal antibody-targeted liposomes with two receptors on K562 cells

We have investigated the interaction of targeted liposomes with human erythrocytes, and K562 cells, a human leukemic line which expresses both glycophorin A and Fc receptors. Liposomes conjugated to monoclonal anti-human glycophorin A bind to human erythrocytes in 80-fold greater amounts than liposomes conjugated to a non-specific monoclonal antibody. Binding is inhibited by soluble anti-glycophorin but not by its Fab fragment. In contrast, binding of antibody-conjugated liposomes to K562 cells is very high irrespective of the specificity of the antibody. Liposomes conjugated to a nonspecific monoclonal antibody interact with K562 cells via an Fc receptor, and binding is inhibited by soluble human IgG. Liposomes conjugated to anti-human glycophorin A interact with K562 cells via an Fc receptor and glycophorin A. Binding is not inhibited by either human IgG or anti-glycophorin Fab alone. Binding is only partially inhibited by anti-glycophorin, or by human IgG in the presence of anti-glycophorin Fab, and completely inhibited only by human IgG in the presence of anti-glycophorin. Simultaneous binding of targeted liposomes to two cell membrane antigens is therefore partially resistant to inhibition by single soluble ligands even when they are present in large excess. We conclude that simultaneous binding to more than one receptor may be of considerable advantage for in vivo applications of targeted liposomes.     

Dependence of "macroscopic fluctuations" from cosmophysical factors. Spatial anisotropy

It was shown in experiments with collimators that the changes with time in the shape of histograms constructed from the measurements of the alpha-activity of 239Pu microsamples depend on the direction of the flow alpha-particles. If the flow of alpha-particles was directed to the Polar Star, the dependence of the probability of the realization of histograms of similar shape on time disappeared. The measurements of alpha-particle flows directed to the East and West revealed a high probability of the repeated realization of histograms of similar shape in 1436 min, i.e., in a sederal day. A comparison of "west" and "east" histograms showed a high probability of the realization of similar "west" histograms 718 min (half a sideral day) later than "east" histograms. The results suggest that the shape of histograms depends on the pattern of the starry sky and the space-time inhomogeneity corresponding to this pattern.     

Improved retention of idarubicin after intravenous injection obtained for cholesterol-free liposomes

To date there has been a focus on the application of sterically stabilized liposomes, composed of saturated diacylphospholipid, polyethylene glycol (PEG) conjugated lipids (5-10 mole%) and cholesterol (CH) (>30 mole%), for the systemic delivery of drugs. However, we are now exploring the utility of liposome formulations composed of diacylphospholipid conjugated PEG mixtures prepared in the absence of added cholesterol, with the primary objective of developing formulations that retain encapsulated drug better than comparable formulations prepared with cholesterol. In this report the stability of cholesterol-free distearoylphosphatidylcholine (DSPC):distearoylphosphatidylethanolamine (DSPE)-PEG₂₀₀₀ (95:5 mol/mol) liposomes was characterized in comparison to cholesterol-containing formulations DSPC:CH (55:45 mol/mol) and DSPC:CH:DSPE-PEG₂₀₀₀ (50:45:5 mol/mol/mol), in vivo. Circulation longevity of these formulations was determined in consideration of variables that included varying phospholipid acyl chain length, PEG content and molecular weight. The application of cholesterol-free liposomes as carriers for the hydrophobic anthracycline antibiotic, idarubicin (IDA), was assessed. IDA was encapsulated using a transmembrane pH gradient driven process. To determine stability in vivo, pharmacokinetic studies were performed using ‘empty’ and drug-loaded [³H]cholesteryl hexadecyl ether radiolabeled liposomes administered intravenously to Balb/c mice. Inclusion of 5 mole% of DSPE-PEG₂₀₀₀ or 45 mole% cholesterol to DSPC liposomes increased the mean plasma area under the curve (AUC_0-24h) 19-fold and 10-fold, respectively. Cryo-transmission electron micrographs of IDA loaded liposomes indicated that the drug formed a precipitate within liposomes. The mean AUC_0-4h for free IDA was 0.030 μmole h/ml as compared to 1.38 μmole h/ml determined for the DSPC:DSPE-PEG₂₀₀₀ formulation, a 45-fold increase, demonstrating that IDA was retained better in cholesterol-free compared to cholesterol-containing liposomes.     

The development and application of protein-liposome conjugation techniques

Numerous techniques have been developed over the past 10 years for the conjugation of proteins to liposomes. Early procedures involved coupling with reagents such as glutaraldehyde or EDCI. Subsequently, more sophisticated approaches involving selective bifunctional coupling agents have been developed. These later procedures are also much more efficient for coupling in aqueous media. The techniques of coupling have become more rigorous because investigators have recognized the inherent problems of producing, purifying and characterizing protein conjugated liposomes.

Protein-liposome coupling techniques were developed mainly for targeted drug delivery. The attachment of specific antibodies to the surface of the liposomes makes them able to bind to cells and to subsequently be internalised by the cells. Protein conjugated liposomes have also been used for various immunochemical and diagnostic purposes. These include the binding of labelled liposomes to cells and the agglutination of cells or latex particles by protein conjugated liposomes.