Large Multilamellar Liposomes Research Articles

LARGE MULTILAMELLAR NEUTRAL LIPOSOMES FORM A STABLE LAYER ON ARTICULAR CARTILAGE SURFACE UNDER EX-VIVO PHYSIOLOGICAL COMPRESSION

Purpose: Surface active phospholipids (SAPLs) including dipalmitoylphosphatidylcholine (DPPC) play a vital role in the natural boundary lubricant of articular cartilage and are degraded during progression of osteoarthritis (OA). MM-II is a suspension of large multilamellar neutral liposomes composed of two phospholipids, dimyristoylphosphatidylcholine (DMPC) and DPPC which is administered via intra-articular (IA) administration and is currently in a phase 2b clinical study. In a series of ex-vivo cartilage-on-cartilage friction and wear tests, MM-II liposomes were shown to lubricate cartilage and reduce hence friction and wear.

Local and systemic effects following intraarticular injection of MM-II liposomes. Results from two nonclinical toxicity studies in rabbits and dogs

Purpose: MM-II is a suspension of large multilamellar liposomes composed of two phospholipids, DMPC and DPPC, for intra-articular administration, under development for treatment of osteoarthritic pain. Due to their unique characteristics, MM-II liposomes are retained on the cartilage surface, providing long-term lubrication and leading to a reduction in wear of the cartilage. The purpose of these preclinical studies was to evaluate the toxicity of MM-II, when administered via intra-articular (IA) injection into the stifle joint of rabbits and dogs. Methods: In two GLP compliant studies, Rabbits or Beagle dogs were administered IA injection of placebo, saline or low (22 mg/kg and 20 mg/kg for rabbits and dogs respectively), mid (44 mg/kg and 39 mg/kg for rabbits and dogs respectively) and high (66 mg/kg and 59 mg/kg for rabbits and dogs respectively) doses of MM-II. Animals were assessed post dose for 4 days, 14 weeks, or 18 weeks. Assessment of toxicity was based on mortality, clinical observations, body weight change, food consumption, ophthalmic observations, electrocardiographic (ECG) measurements (dogs only), and clinical and anatomic pathology. As part of this study, blood samples were collected for toxicokinetic (TK) evaluation and synovial fluid samples were collected for clinical pathology evaluations. Results: Rabbits: No MM-II-related mortality, MM-II-related clinical observations; effects on qualitative food consumption; findings during ophthalmic examinations; effects in hematology, coagulation, clinical chemistry, or urinalysis test results; or organ weight effects or macroscopic findings were noted. MM-II related findings were limited to a non-adverse moderate increase in body weights for the females at all dose levels and reversible, non-adverse microscopic findings of synovial hyperplasia and mixed cell inflammation in the injected stifle of all groups administered MM-II. After MM-II administration, DMPC exposure, as assessed by mean Cmax and AUC0-72, was higher than its endogenous baseline level and the placebo and saline control groups. After reaching Cmax, DMPC concentrations declined largely, through 144 or 552 hours post dosing. DPPC exposure was relatively similar to its endogenous baseline level and to their levels in the placebo and saline control groups. Dogs: No MM-II-related mortality, MM-II-related clinical observations; effects on qualitative food consumption; findings during ophthalmic examinations; effects in hematology, coagulation, clinical chemistry, or urinalysis test results; or organ weight effects or macroscopic findings were noted. MM-II related findings were limited to reversible, transient, non-adverse, minimally to mildly higher synovial fluid white and red blood cell counts and microscopic findings of synovial hyperplasia and mixed cell inflammation in the injected stifle of all groups administered MM-II. After MM-II administration, DMPC exposure as assessed by mean Cmax and AUC0-72 was higher than its endogenous baseline levels and the placebo and saline control groups. After reaching Cmax, mean DMPC concentrations declined largely through 312 or 552 hours post dosing. DPPC exposure was relatively similar to its endogenous baseline level and to their levels in the placebo and saline control groups. Conclusions: In both studies, MM-II related findings were considered non-adverse at all dose levels. The NOAEL was determined as 59 mg/kg in the dogs study and 66 mg/kg in the rabbit study. Toxicokinetic findings in both studies indicated that DMPC exposure appears to be impacted by MM-II administration, but DPPC is not. The highest doses employed in these studies provide a maximal local safety margin of 11.9-fold for dogs and 22.9-fold for MM-II in rabbits of the highest dose to be administered in MM-II phase 2 clinical trial. To conclude, MM-II was found safe with no adverse events and with a NOAEL of highest dose employed in the study.

Intraarticular injection of MM-II liposomes lubricates cartilage in vivo and reduces friction and wear in ex-vivo cartilage models

Purpose: MM-II is a suspension of large multilamellar liposomes for intra-articular administration, under development for treatment of osteoarthritic pain. Due to their unique characteristics, MM-II liposomes are retained on the cartilage surface, providing long-term lubrication and leading to a reduction in wear of the cartilage. As previously reported in a first-in-human clinical trial, MM-II, when injected intraarticularly, was shown to reduce pain up to 3 months post injection. This study aims to illustrate MM-II’s mechanism of action by studying the localization of the liposomes in the joint following an intraarticular injection into rabbits and using 2 ex-vivo cartilage models for evaluation of MM-II’s lubrication capabilities and its effect on reduction of friction and wear of the cartilage.

Intraarticular injection of MM-II, a suspension of empty large liposomes, reduces cartilage degeneration in rat osteoarthritis model

Purpose: MM-II is a suspension of large multilamellar liposomes composed of two phospholipids, dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) which is applied via intra-articular (IA) administration. In-vivo biodistribution studies and ex-vivo wear tests have shown that MM-II liposomes are retained on the cartilage surface, providing lubrication, and leading to a reduction in wear of the cartilage. In a first-in-man study, a single injection of MM-II was demonstrated to lower pain in osteoarthritis (OA) patients for up to 3 months. Given the lubricating properties, cartilage wear reduction, and pain-lowering effect of MM-II, we hypothesized that IA administration of MM-II may also reduce OA progression. The purpose of the current study was to evaluate the effects of three consecutive IA injections of MM-II with dosing intervals of one per week, on cartilage degeneration in a rat meniscal-tear-induced model of osteoarthritis. Methods: Male Lewis rats underwent a unilateral medial meniscal tear (MMT) on study day 0. The rats (n=15 per group) were treated by the IA route on study days 7, 14, and 21 with MM-II (50μl/knee) or a vehicle control. Additional group consisted of untreated disease controls. Four weeks after surgery, joints were processed for histological analysis following staining with toluidine blue. Endpoints for evaluation of therapeutic efficacy included histopathologic examination of chondrocyte death/cartilage degeneration. General cartilage degeneration includes chondrocyte death/loss, proteoglycan (PG) loss, and collagen loss or fibrillation. Substantial Cartilage Degeneration was identified by chondrocyte and proteoglycan loss extending through greater than 50% of the cartilage thickness and was measured by ocular micrometer. Gait analysis was performed at day 24 to confirm expected animal mobility post-surgery. Results: Weekly IA treatment with MM-II (5.35 mg/knee) resulted in significant beneficial effects on cartilage damage and collagen degeneration in the rat meniscal-tear-induced model of OA as determined by evaluation of knee histopathology including significant reductions in substantial cartilage degeneration widths (38% reduction), summed cartilage degeneration scores (33%), total joint scores (25-26%), and combined severe to mild collagen degeneration widths (32%) as compared to the vehicle control. MM-II treatment also resulted in significant reductions in subchondral bone sclerosis (16%) and osteophyte scores (16%) as compared to vehicle control rats. Rats treated with MM-II had a statistically significant increase in synovial inflammation as compared to the vehicle control group with all animals showing minimal to mild synovitis. Animal body weight gain and mobility post-surgery (day 24 gait scores) in rats treated with MM-II did not differ statistically from the placebo control group. All animals survived to study termination. Conclusions: Weekly IA treatment with MM-II was shown to lower OA disease progression as manifested by significant beneficial effects on cartilage damage and collagen degeneration in the rat MMT model of OA as determined by evaluation of knee histopathology. The minimal to mild synovitis associated with the injected liposomes is consistent with the reversible synovial hyperplasia seen in the toxicity studies and is expected from IA-injected particulate agents that lodge in the synovium. The cartilage damage improvements together with the gait data indicates that MM-II treatment was overall beneficial in the rat MMT model and has the potential to be a disease modifying OA therapy in addition to its pain lowering ability.

The THE INFLUENCE OF CHLORPROMAZINE HYDROCHLORIDE ON THE SIZE OF SMALL UNILAMELLAR DIMYROSTYLPHOSPHATIDYLCHOLINE LIPOSOMES AS REVEALED BY LIGHT SCATTERING PHOTOMETRY

Objectives: This study aims to investigate the possible influence of the model, cationic, surface-active solute chlorpromazine hydrochloride (CPZ-HCl) on the size of small unilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes as a function of temperature and CPZ-HCl concentration, below and above the critical micelle concentration (CMC).
 Methods: Small unilamellar DMPC liposomes were prepared by dissolving DMPC in chloroform and the solvent was rota-evaporated in a water bath adjusted at 40 °C. The lipid film was then dispersed in 0.1 M KCl solution adjusted at pH 6.2 to form large multilamellar liposomes which are then sonicated and fractionated via Sepharose 2B-Cl gel. The elution profile was followed spectrophotometrically at λ 260 nm. Combined fractions from the trailing edge of the included peak which is due to small unilamellar liposomes, were used as a source throughout this study. The SOFICA light scattering photometer (Model 42000) was used to determine the weight average liposomes weight (Lw) of small unilamellar DMPC liposomes. The Lw was determined in the absence and presence of CPZ-HCl both above and below the CMC over the temperature range of 25 °C to 40 °C.
 Results: The Lw was observed to increase linearly in the absence and presence of CPZ-HCl.
 The Lw was observed to increase linearly in the absence of CPZ-HCl, from 1.88×106+0.02 g/mol at 25 °C to 3.25×106+0.03 g/mol at 40 °C. Similarly, the Lw was observed to increase linearly in the present of CPZ-HCl, for example at 18 mmol drug concentration, the Lw increases from 11×106+0.04 g/mol at 25 °C to 13.75×106+0.03 g/mol at 40 °C. When the data are presented as a function of CPZ-HCl concentration, a gradual increase in Lw was observed below the CMC. Little increase in Lw however, was observed at post-micellar concentrations of 14 mmol and 18 mmol.

Mechanism and kinetics of the loss of poorly soluble drugs from liposomal carriers studied by a novel flow field-flow fractionation-based drug release −/transfer-assay

Liposomes represent a versatile drug formulation approach e.g. for improving the water-solubility of poorly soluble drugs but also to achieve drug targeting and controlled release. For the latter applications it is essential that the drug remains associated with the liposomal carrier during transit in the vascular bed. A range of in vitro test methods has been suggested over the years for prediction of the release of drug from liposomal carriers. The majority of these fail to give a realistic prediction for poorly water-soluble drugs due to the intrinsic tendency of such compounds to remain associated with liposome bilayers even upon extensive dilution. Upon i.v. injection, in contrast, rapid drug loss often occurs due to drug transfer from the liposomal carriers to endogenous lipophilic sinks such as lipoproteins, plasma proteins or membranes of red blood cells and endothelial cells.Here we report on the application of a recently introduced in vitro predictive drug transfer assay based on incubation of the liposomal drug carrier with large multilamellar liposomes, the latter serving as a biomimetic model sink, using flow field-flow fractionation as a tool to separate the two types of liposomes. By quantifying the amount of drug remaining associated with the liposomal drug carrier as well as that transferred to the acceptor liposomes at distinct times of incubation, both the kinetics of drug transfer and release to the water phase could be established for the model drug p-THPP (5,10,15,20-tetrakis(4-hydroxyphenyl)21H,23H-porphine). p-THPP is structurally similar to temoporfin, a photosensitizer which is under clinical evaluation in a liposomal formulation. Mechanistic insights were gained by varying the donor-to-acceptor lipid mass ratio, size and lamellarity of the liposomes. Drug transfer kinetics from one liposome to another was found rate determining as compared to redistribution from the outermost to the inner concentric bilayers, such that the overall process could be adequately described by a single 1st order kinetic model. By varying the donor-to-acceptor lipid mass ratio in the range 1:1 to 1:10, a correlation was established between donor-to-acceptor-lipid mass ratio and transfer kinetics, which is regarded essential for scaling to physiological lipid mass ratios. By applying the assay to a series of structurally related model compounds of different bilayer affinity, transfer and release kinetics were established over the whole expected range of liposome bilayer associated drugs in terms of water solubility and lipophilicity. A very rapid transfer and considerable release from liposomes to the water phase was observed for the more water-soluble compounds Sudan II (clogP 5.45) and Sudan III (clogP 6.83). For the more lipophilic compounds, the rate of transfer from the donor liposomes followed the rank order Sudan IV (fastest)>Oil Red O>Sudan Black>p-THPP (slowest). For an equimolar donor-to-acceptor lipid mass ratio, half-lifes of transfer in the range of 12min (Sudan IV) up to 1.5h (p-THPP) were determined. In essence, the results presented here allow for both, mechanistic insights and predictions of drug loss from liposomal carriers upon exposure to biological sinks, which appear more realistic than the commonly employed in vitro release tests.

Atmospheric-pressure guided streamers for liposomal membrane disruption

The potential to use liposomes (LIPs) as a cellular model in order to study interactions of cold atmospheric-pressure plasma with cells is herein investigated. Cold atmospheric-pressure plasma is formed by a dielectric-barrier discharge reactor. Large multilamellar vesicle liposomes, consisted of phosphatidylcholine and cholesterol, are prepared by the thin film hydration technique, to encapsulate a small hydrophilic dye, i.e., calcein. The plasma-induced release of calcein from liposomes is then used as a measure of liposome membrane integrity and, consequently, interaction between the cold atmospheric plasma and lipid bilayers. Physical mechanisms leading to membrane disruption are suggested, based on the plasma characterization including gas temperature calculation.

Quality of liposomal phospholipids at different stages in the manufacturing process

We report here studies of the effects of liposome preparation methods on measures of the quality of phosphatidylcholine used, this being the main component of the liposomal bilayer and extremely unstable to the temperatures of manufacturing processes containing oxygen and transition metal ions. One process destructive for this phospholipid is peroxidation, the level of which is generally assessed in terms of the malondialdehyde concentration and the Klein oxidation index. Extrusion and homogenization of large multilamellar liposomes were found to be the least damaging methods of preparing liposomes, as compared with sonication. Attempts to introduce an antioxidant (vitamin E) did not lead to any significant improvement in the parameters assessed.

Insertion of semifluorinated diblocks on DMPC and DPPC liposomes. Influence on the gel and liquid states of the bilayer

Differential Scanning Calorimetry (DSC) was used to study the effect of the incorporation of a series of semifluorinated diblocks F n H m (F 6H 10, F 6H 16, F 8H 14, F 8H 16, F 8H 18 and F 8H 20) on the gel and liquid states of the bilayer of large multilamellar DMPC and DPPC liposomes. The presence of the F n H m diblocks affects slightly the T m of the main gel–liquid transitions of DMPC and DPPC, but is accompanied by the appearance of a second transition in the calorimetric traces whose T m is mainly determined by the length of the F n segment. The DSC results are consistent with the previously established conclusion that the F n segments of the diblocks form a central layer in the core of the lipid bilayer, with the H m segments being interdigitated with the lipid chains. The DSC traces suggest that the structure of the fluorinated liposomes is a double bilayer at 3:4 and 1:2 and a trilayer at 2:1 lipid/F n H m molar ratios. At temperatures between the two phase transitions T m ’s, the fluorinated liposomes are neither in a gel-like or a liquid-like state but rather posses both characteristics.

Liposome fusogenicity and entrapment efficiency of antigen determine the Th1/Th2 bias of antigen-specific immune response

Liposomes, either alone or in combination with additional immunostimulatory molecules, could be used for the delivery of antigens as vaccine adjuvants. The aim of this study was to investigate the influence of (a) composition (fusogenicity and charge) of large multilamellar liposomes, (b) antigen entrapment efficiency into cationic liposomes and (c) addition of immunostimulatory peptidoglycan monomer (PGM) into liposomal formulations on intensity and direction of antigen-specific humoral immune response. Ovalbumin (OVA) was used as a model antigen and liposomal formulations were tested in a well defined experimental mice model. It was shown that, by means of controlling ionic strength of the media, entrapment efficiency of OVA was enhanced and this lead to Th1 biased immune response. Also, by varying liposome composition and increasing fusogenicity of liposomes immune response was directed toward Th1. Addition of immunostimulatory PGM into liposomal formulation resulted in a switch toward Th2 type immune response.

IL-12 delivered intratumorally by multilamellar liposomes reactivates memory T cells in human tumor microenvironments

Using a novel loading technique, IL-12 is reported here to be efficiently encapsulated within large multilamellar liposomes. The preclinical efficacy of the cytokine loaded liposomes to deliver IL-12 into human tumors and to reactive tumor-associated T cells in situ is tested using a human tumor xenograft model. IL-12 is released in vivo from these liposomes in a biologically active form when injected into tumor xenografts that are established by the subcutaneous implantation of non-disrupted pieces of human lung, breast or ovarian tumors into immunodeficient mice. The histological architecture of the original tumor tissue, including tumor-associated leukocytes, tumor cells and stromal cells is preserved anatomically and the cells remain functionally responsive to cytokines in these xenografts. The local and sustained release of IL-12 into the tumor microenvironment reactivates tumor-associated quiescent effector memory T cells to proliferate, produce and release IFN-gamma resulting in the killing of tumor cells in situ. Very little IL-12 is detected in the serum of mice for up to 5 days after an intratumoral injection of the IL-12 liposomes. We conclude that IL-12 loaded large multilamellar liposomes provide a safe method for the local and sustained delivery of IL-12 to tumors and a therapeutically effective way of reactivating existing tumor-associated T cells in human solid tumor microenvironments. The potential of this local in situ T cell re-stimulation to induce a systemic anti-tumor immunity is discussed.

Formation of homogeneous unilamellar liposomes from an interdigitated matrix

Phospholipid–ethanol–aqueous mixtures containing bilayer-forming lipids and 20–50 wt.% of water form viscous gels. Further hydration of these gels results in the formation of liposomes whose morphology depends upon the lipid type. Upon hydration of gels containing mixtures of the lipids 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), small homogeneous and unilamellar liposomes were produced. In contrast, hydration of gels containing only POPC resulted in formation of large multilamellar liposomes. Likewise, mulitlamellar liposomes resulted when this method was applied to form highly fusogenic liposomes comprised of the novel negatively charged N-acyl-phosphatidylethanolamine (NAPE) mixed with di-oleoyl-phosphatidylcholine (DOPC) (7:3) [T. Shangguan, C.C. Pak, S. Ali, A.S. Janoff, P. Meers, Cation-dependent fusogenicity of an N-acyl phosphatidylethanolamine, Biochim. Biophys. Acta 1368 (1998) 171–183]. In all cases, the measured aqueous entrapment efficiencies were relatively high. To better understand how the molecular organization of these various gels affects liposome morphology, we examined samples by freeze-fracture transmission electron microscopy and X-ray diffraction. We found that phospholipid–ethanol–water gels are comprised of highly organized stacks of lamellae. A distinct feature of the gel samples that result in small unilamellar liposomes is the combination of acyl chain interdigitation and net electrostatic charge. We speculate that the mechanism of unilamellar liposome formation proceeds via formation of stalk contacts between neighboring layers similar to membrane hemifusion intermediates, and the high aqueous entrapment efficiencies make this liposome formation process attractive for use in drug delivery applications.

Liposomal immunostimulatory DNA sequence (ISS-ODN): an efficient parenteral and mucosal adjuvant for influenza and hepatitis B vaccines

Synthetic oligodeoxynucleotides (ODNs) containing immunostimulatory sequences (ISS-ODN, also known as CpG-ODNs) have been shown to display in experimental models potent Th1-biassed immunoadjuvant activity upon parenteral or mucosal co-administration with a variety of antigens. In an attempt to potentiate adjuvant activity, and to reduce dose and number of administrations, ISS-ODN was entrapped (up to 90% efficiency) in large (1.5 μm) multilamellar liposomes using a simple and fast (5 min) procedure. Mice were vaccinated once or twice intramuscularly (i.m.) or intranasally (i.n.) with subunit influenza vaccines (consisting of the viral hemagglutinin and neuraminidase, HN) or with hepatitis B surface antigen particles (HBsAg), either non-encapsulated or liposome-encapsulated, together with free or liposomal ISS-ODN (5–25 μg per dose). At 3–12 weeks post-vaccination, the humoral (systemic, mucosal) and cellular responses and protective immunity were assessed. Vaccine formulations containing liposomal ISS-ODN co-administered with either soluble antigen or liposomal antigen (in the same vesicles or in separate vesicles) were up to 30 times more effective than formulations containing un-encapsulated ISS-ODN in inducing: (a) antigen-specific serum and mucosal IgG2a and IgA antibodies; (b) splenocyte proliferative response, cytotoxic activity and IFNγ production; (c) a DTH response; and (d) protection against virus challenge. The response was Th1-dominant in the influenza model and a mixed Th1+Th2 response in the hepatitis B model. No adverse reactions were noted. Thus, liposomal encapsulation of ISS-ODN further enhances its inherent adjuvant activity.

Calcium alginate gel-entrapped liposomes

We developed a novel method to prepare calcium alginate gel-entrapped large multilamellar vesicle liposomes. The calcium alginate-entrapped liposomes showed both calcein release and insulin release in a calcium-dependent manner. Relatively slow kinetic component of calcein release was observed in the presence of 100 mM CaCl 2 but not in 100 mM EDTA, a strong calcium chelator, after addition of detergent. We attribute the component to diffusion of calcein from calcium alginate gel particles.

The influence of the vehicle on the synthesis of porphyrins after topical application of 5-aminolaevulinic acid. Implications in cutaneous photodynamic sensitization

Background The optimal vehicle to ensure adequate penetration of 5‐aminolaevulinic acid (ALA) for its use in photodynamic therapy (PDT) of skin lesions has not been determined. Objectives We aimed to study the effects of ALA in various vehicle formulations [saline lotion with and without dimethylsulphoxide (DMSO), cream, liposomes and vaseline] after topical application in a murine subcutaneous adenocarcinoma model. Methods The effect of DMSO on porphyrin synthesis and ALA penetration through the skin was studied by measuring the uptake of 14C label from ALA, ALA and porphobilinogen accumulation, and some haem enzyme activities. The tissue distribution and kinetics of porphyrin synthesis after topical application of ALA entrapped in large multilamellar liposomes was also determined. Results ALA in saline lotion, alone or with 10% DMSO, proved to be the most efficient vehicle for tumour porphyrin accumulation (mean ± SD 1·75 ± 0·25 and 2·09 ± 0·39 µg g−1, respectively), whereas cream and liposomes induced lower levels and identical porphyrin accumulation (0·60 µg g−1). Using ALA + DMSO saline lotion, a higher porphyrin accumulation was found in skin overlying the tumour tissue and in the first 2 mm of tumour, probably due to increased ALA penetration, or greater interconversion to porphyrins, or greater retention of ALA and/or porphyrins. Conclusions These findings reinforce the importance of the vehicle in topical ALA‐based PDT, and explain the mechanism of action of DMSO in enhancing protoporphyrin IX biosynthesis in superficial lesions.

The influence of the vehicle on the synthesis of porphyrins after topical application of 5-aminolaevulinic acid. Implications in cutaneous photodynamic sensitization.

The optimal vehicle to ensure adequate penetration of 5-aminolaevulinic acid (ALA) for its use in photodynamic therapy (PDT) of skin lesions has not been determined. We aimed to study the effects of ALA in various vehicle formulations [saline lotion with and without dimethylsulphoxide (DMSO), cream, liposomes and vaseline] after topical application in a murine subcutaneous adenocarcinoma model. The effect of DMSO on porphyrin synthesis and ALA penetration through the skin was studied by measuring the uptake of 14C label from ALA, ALA and porphobilinogen accumulation, and some haem enzyme activities. The tissue distribution and kinetics of porphyrin synthesis after topical application of ALA entrapped in large multilamellar liposomes was also determined. ALA in saline lotion, alone or with 10% DMSO, proved to be the most efficient vehicle for tumour porphyrin accumulation (mean +/- SD 1.75 +/- 0.25 and 2.09 +/- 0.39 microg g-1, respectively), whereas cream and liposomes induced lower levels and identical porphyrin accumulation (0.60 microg g-1). Using ALA + DMSO saline lotion, a higher porphyrin accumulation was found in skin overlying the tumour tissue and in the first 2 mm of tumour, probably due to increased ALA penetration, or greater interconversion to porphyrins, or greater retention of ALA and/or porphyrins. These findings reinforce the importance of the vehicle in topical ALA-based PDT, and explain the mechanism of action of DMSO in enhancing protoporphyrin IX biosynthesis in superficial lesions.

Liposome-induced pulmonary hypertension: properties and mechanism of a complement-mediated pseudoallergic reaction.

Intravenous injection of liposomes can cause significant pulmonary hypertension in pigs, a vasoconstrictive response that provides a sensitive model for the cardiopulmonary distress in humans caused by some liposomal drugs. The reaction was recently shown to be a manifestation of "complement activation-related pseudoallergy" (CARPA; Szebeni J, Fontana JL, Wassef NM, Mongan PD, Morse DS, Dobbins DE, Stahl GL, Bünger R, and Alving CR. Circulation 99: 2302-2309, 1999). In the present study we demonstrate that the composition, size, and administration method of liposomes have significant influence on pulmonary vasoactivity, which varied between instantaneously lethal (following bolus injection of 5 mg lipid) to nondetectable (despite infusion of a 2,000-fold higher dose). Experimental conditions augmenting the pulmonary hypertensive response included the presence of dimyristoyl phosphatidylglycerol, 71 mol% cholesterol, distearoyl phosphatidylcholine, and hemoglobin in liposomes, increased vesicle size and polydispersity, and bolus injection vs. slow infusion. The vasoactivity of large multilamellar liposomes was reproduced with human C3a, C5a, and xenoreactive immunoglobulins, and it correlated with the complement activating and natural antibody binding potential of vesicles. Unilamellar, monodisperse liposomes with 0.19 +/- 0.10 microm mean diameter had no significant vasoactivity. These data indicate that liposome-induced pulmonary hypertension in pigs is multifactorial, it is due to natural antibody-triggered classic pathway complement activation and it can be prevented by appropriate tailoring of the structure and administration method of vesicles.

In Vivo Liver-Directed Gene Transfer in Rats and Pigs with Large Anionic Multilamellar Liposomes: Routes of Administration and Effects of Surgical Manipulations on Transfection Efficiency

Conventional large (500–800 nm) multilamellar liposomes encapsulating DNA have been used in vivo as gene vectors into rat and pig liver. By using the intraportal vein route, high dose DNA (10 mg/kg) provided low efficiency and transient luciferase gene expression in the liver. This gene expression was, however, increased by liver resection (> 50%), ischemia (20 min) or orthotopic transplantation. As evidenced by histochemical analysis of β-galactosidase expression, the gene transfection mainly ensued in Kupffer cells, but spleen and lung were contaminated. In comparison, injection into the bile duct of even 25-fold lower dose of liposome-encapsulated DNA (0.4 mg/kg) produced higher (100-fold) and long-lasting (during 6 days, at least) luciferase expression in rat liver. The gene expression was restricted to the liver and enhanced by liver resection. By this route, transgene-expressing cells were mainly hepatocytes. A treatment with colchicine prior to the administration of the vector allowed the persistence of relative high gene expression for at least 7 days. In pigs, qualitatively similar, but quantitatively less efficient gene expression was obtained by either the portal vein or the bile duct route. These results indicate that intrabile duct route might render large non-viral vectors applicable to gene transfer into the hepatocytes. The efficiency of liposome-mediated gene transfer into the liver can be increased by liver resection, ischemia or transplantation performed before DNA injection.

Hemodynamic changes induced by liposomes and liposome-encapsulated hemoglobin in pigs: a model for pseudoallergic cardiopulmonary reactions to liposomes. Role of complement and inhibition by soluble CR1 and anti-C5a antibody.

Intravenous administration of some liposomal drugs can trigger immediate hypersensitivity reactions that include symptoms of cardiopulmonary distress. The mechanism underlying the cardiovascular changes has not been clarified. Anesthetized pigs (n=18) were injected intravenously with 5-mg boluses of large multilamellar liposomes, and the ensuing hemodynamic, hematologic, and laboratory changes were recorded. The significant (P<0.01) alterations included 79+/-9% (mean+/-SEM) rise in pulmonary arterial pressure, 30+/-7% decline in cardiac output, 11+/-2% increase in heart rate, 236+/-54% increase in pulmonary vascular resistance, 71+/-27% increase in systemic vascular resistance, and up to a 100-fold increase in plasma thromboxane B2. These changes peaked between 1 and 5 minutes after injection, subsided within 10 to 20 minutes, were lipid dose-dependent (ED50=4. 5+/-1.4 mg), and were quantitatively reproducible in the same animal several times over 7 hours. The liposome-induced rises of pulmonary arterial pressure showed close quantitative and temporal correlation with elevations of plasma thromboxane B2 and were inhibited by an anti-C5a monoclonal antibody (GS1), by sCR1, or by indomethacin. Liposomes caused C5a production in pig serum in vitro through classic pathway activation and bound IgG and IgM natural antibodies. Zymosan- and hemoglobin-containing liposomes and empty liposomes caused essentially identical pulmonary changes. The intense, nontachyphylactic, highly reproducible, complement-mediated pulmonary hypertensive effect of minute amounts of intravenous liposomes in pigs represents a unique, unexplored phenomenon in circulation physiology. The model provides highly sensitive detection and study of cardiopulmonary side effects of liposomal drugs and many other pharmaceutical products due to "complement activation-related pseudoallergy" (CARPA).

Effect of Peg Homopolymer and Grafted Amphiphilic Peg-Palmityl on the Thermotropic Phase Behavior of 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine Bilayer

Phospholipids covalently attached to polyethylene glycol (PEG-PE) are routinely used for the preparation of long-circulating liposomes. The common preparation procedure for long-circulating liposomes involves use of organic solvent. Although there is a plethora of studies describing the interaction of PEG-PE with bilayers, little is known about the effects of PEG homopolymers and single chain amphiphilic PEG on liposome structure. In the present investigation the interaction of PEG homopolymer and amphiphilic PEG-palmityl conjugate with large multilamellar liposomes composed of 1,2-dipalmitoyl-sn-glycero-phosphocholine was investigated utilizing differential scanning calorimetry. Vesicle and aggregate sizes were determined by dynamic light scattering. DSC thermograms revealed interaction of PEG homopolymer with DPPC when the two are premixed in organic solvent. The data suggest that PEG interacts with the phospholipid acyl chains deep in the bilayer. Several questions are raised regarding the suitability of the current procedure for preparation of long-circulating liposomes which utilizes organic solvent. Incorporation of only 2 mol% 5 kDa PEG-palmityl conjugate completely solubilized DPPC liposomes. Packing geometry of the lipid anchor, irrespective of the polymer molecular weight, is suggested to be the primary factor for successful grafting of hydrophilic polymers on liposomes. Pure PEG-palmityl formed self-assembled organized structures of potential use in the delivery of poorly soluble drugs.