Sequential administration of PEG-Span 80 niosome enhances anti-tumor effect of doxorubicin-containing PEG liposome.

To improve the anti-tumor effect of polyethylene glycol-modified liposome containing doxorubicin (DOX-PEG liposome), the effect of sequential administration of PEG-Span 80 niosome was investigated for Colon-26 cancer cells (C26)-bearing mice. The concept of the current study is as follows: Since both particulates would be accumulated in the tumor tissue due to the enhanced permeability and retention (EPR) effect, PEG-Span 80 niosome, mainly composed of synthetic surfactant (Span 80), would interact with DOX-PEG liposome and be a trigger to induce the release of DOX from the liposome within the tumor tissue, leading to the improvement of anti-tumor effect of DOX-PEG liposome. To find out an adequate liposome for this strategy, several PEG liposomes with different compositions were examined in terms of drug release enhancement and it was found that PEG-Span80 niosome could significantly enhance the release of calcein and DOX from a PEG liposome composed of 90% hydrogenated soybean phosphatidylcholine (HSPC) and 10% cholesterol. The sequential administration of PEG-Span 80 niosome at 24 or 48 h after dosing of DOX-PEG liposome provided a higher anti-tumor effect than the single dose of DOX-PEG liposome in the C26-bearing mice. Particularly, the 24 h-later dosing of PEG-Span 80 niosome has been found to be more effective than the 48 h-later dosing. It was also confirmed that the coexistence of PEG-Span 80 niosome with DOX-PEG liposome in 50% serum or in 50% supernatant of tumor tissue homogenate significantly increased DOX release from PEG liposome, suggesting that DOX release from DOX-PEG liposome within tumor tissue would be enhanced via the interaction with PEG-Span 80 niosome. This strategy would lead to the safer and more inexpensive chemotherapy, since it could make it possible to provide the better anti-tumor effect by utilizing the lower dose of DOX.

Colloidal drug delivery systems: current status and future directions.

In this paper, we provide an overview an extensive range of colloidal drug delivery systems with special focus on vesicular and particulates systems that are being used in research or might be potentially useful as carriers systems for drug or active biomolecules or as cell carriers with application in the therapeutic field. We present some important examples of commercially available drug delivery systems with applications in research or in clinical fields. This class of systems is widely used due to excellent drug targeting, sustained and controlled release behavior, higher entrapment efficiency of drug molecules, prevention of drug hydrolysis or enzymatic degradation, and improvement of therapeutic efficacy. These characteristics help in the selection of suitable carrier systems for drug, cell, and gene delivery in different fields.

Retinoic acid liposome-hydrogel: preparation, penetration through mouse skin and induction of F9 mouse teratocarcinoma stem cells differentiation

Retinoic acid (RA), a metabolite of retinol, is one of the most biologically active forms of retinoid and plays vital roles in embryonic development and in the regulation of cell proliferation and differentiation. Knowing that liposomes simulate cell membranes and that hydrogel is an ideal delivery vehicle for topical medicine, liposome-hydrogel is a novel preparation that has synergistic advantages over each component separately. Our objective was to investigate the characteristics of RA liposome-hydrogel. For quality control of the RA-loaded liposomes, we measured their morphology, particle size, Zeta-potential, and entrapment efficiency. Then we determined the viscosity of RA liposome-hydrogel. Next, the diffusion through mouse skin was explored, followed by investigation of the mRNA expression levels of Ker18, REX1, and α-FP using Q-PCR. The results showed that RA liposome-hydrogel penetrates the mouse skin effectively. The permeation rates were: Qn (%) of RA liposome-hydrogel < Qn(%) of RA-loaded liposome < Qn (%) of RA. The mRNA expression levels were dose-dependent and the effective dose decreased between vehicles due to their different release rates. F9 mouse teratocarcinoma stem cells were an ideal model to explore the mechanism of RA liposome-hydrogel in stem cell differentiation.

O ácido retinóico (RA) é um metabolito de retinol. Ele também é uma das formas mais biologicamente ativas de retinóide. Desempenha papel vital no desenvolvimento embrionário e na regulação da proliferação e diferenciação celular. Sabendo-se que lipossomas simulam a membrana das células e que hidrogel é um sistema ideal para o medicamento tópico, o lipossoma-hidrogel é uma nova preparação, que apresenta vantagens sinérgicas em relação a cada um dos componentes separados. Nosso objetivo foi investigar as características de RA lipossoma-hidrogel. A fim de controlar a qualidade do lipossoma carregado com RA, medimos morfologia, tamanho das partículas, potencial zeta e eficiência de retenção. Em seguida, determinou-se a viscosidade de RA lipossoma-hidrogel. Em seguida, avaliou-se a sua difusão através da pele de camundongos, seguida da investigação dos níveis da expressão de mRNA de Ker18, REX e de α-FP, utilizando-se Q-PCR. Os resultados mostraram que RA lipossoma-hidrogel pode penetrar na pele do camundongo de forma eficaz. As taxas de permeação foram: Qn (%) de RA lipossoma-hidrogel<Qn(%) de lipossoma RA- carregado <Qn (%) de RA. Os níveis de expressão de mRNA foram dependentes de dose e a dose efetiva diminuiu entre os veículos devido às diferentes taxas de liberação, As células estaminais de teratocarcinoma F9 de camundongo mostraram-se como modelo ideal para explorar o mecanismo de diferenciaçãode células tronco pelo RA lipossoma-hidrogel.

Enhanced anti-inflammatory activity of carbopol loaded meloxicam nanoethosomes gel.

The aim of the current investigation is to develop nanoethosomes for transdermal meloxicam delivery. The ethosomes were prepared by varying the variables such as concentrations of phospholipids 90G, ethanol, and sonication time while entrapment efficiency, vesicle size and transdermal flux were the chosen responses. Results indicate that the nanoethosomes of meloxicam provides lesser vesicles size, better entrapment efficiency and improved flux for transdermal delivery as compared to rigid liposomes. The optimized formulation (MCEF-OPT) obtained was further evaluated for an in vivo anti-inflammatory activity in rats. Optimized nanoethosomal formulation with vesicles size of 142.3nm showed 78.25% entrapment efficiency and achieved transdermal flux of 10.42µg/cm(2)/h. Nanoethosomes proved to be significantly superior in terms of, amount of drug permeated into the skin, with an enhancement ratio of 3.77 when compared to rigid liposomes. In vivo pharmacodynamic study of carbopol(®) loaded nanoethosomal gel showed significant higher percent inhibition of rat paw edema compared with oral administration of meloxicam. Our results suggest that nanoethosomes are an efficient carrier for transdermal delivery of meloxicam.

Thermolabile liposomes: a controlled release delivery tool in diagnosis/therapy in experimental pulmonary ɶdema.

Liposomes, usually assembled from organic/synthetic lipidic compounds, are biocompatible, biodegradable, non-toxic, and do not induce immune response. Due to their structural versatility in terms of size, composition, surface charge, bilayer fluidity and ability to encapsulate drugs regardless of their solubility, liposomes enable the production of a vast number and type of formulations with potential clinical use. They can be administered through several routes of administration (e.g. i.v., i.m., oral, nasal, etc.). The use of liposomes enables the variation and control retention of drugs in biologic fluids, enhancing blood circulation and specific compartments residence. They can be tailored to target specific tissues and cells. They can play a very important role for imaging diagnosis and/or therapy. After an extensive literature review of the subject, we selected a particular area of potential clinical application: pulmonary ɶdema. This clinical entity has a variety of possible etiologies, conducing to two main types of edema: cardiogenic and non-cardiogenic. At the moment a dedicated technique for the early diagnosis/therapy of this pathology is lacking. We propose a new methodology using a specially designed GUV formulation, encapsulating chosen radiotracers labeled with 99mTc. The aim of the work has been successfully achieved in an experimental animal model of cardiogenic pulmonary oedema. Experiments using an animal model of non-cardiogenic pulmonary oedema are in course (simultaneous study with two different drugs), using the same GUV methodology. Preliminary results are very promising.

Complexation of siRNA and pDNA with cationic liposomes: the important aspects in lipoplex preparation.

In the last two decades, cationic liposomes have been investigated as vehicles for nucleic acids [plasmid DNA (pDNA) and small interfering RNA (siRNA)] delivery in vitro and in vivo. The formation of cationic liposomes-nucleic acids complexes, termed lipoplexes, depends on a number of experimental variables. The quality of the nucleic acid and the cationic liposome as well as the selection of diluents for diluting the concentrated stocks strongly affect the resulting lipoplexes and their efficiency of gene-expression or gene-silencing effect following transfection. In addition, the molar ratio of cationic lipid nitrogen (N) to siRNA or pDNA phosphate (P) (N/P ratio) influences the final characteristics of the lipoplexes, such as size, surface zeta potential, and reproducibility, thereby reflecting their efficiency following transfection. The methods presented in this chapter could be helpful to obtain reliable and reproducible lipoplexes and experimental results.

Design of a modern liposome and bee venom formulation for the traditional VIT-venom immunotherapy

Traditional venom immunotherapy uses injections of whole bee venom in buffer or adsorbed in Al (OH)3 in an expensive, time-consuming way. New strategies to improve the safety and efficacy of this treatment with a reduction of injections would, therefore, be of general interest. It would improve patient compliance and provide socio-economic benefits. Liposomes have a long tradition in drug delivery because they increase the therapeutic index and avoid drug degradation and secondary effects. However, bee venom melittin (Mel) and phospholipase (PLA2) destroy the phospholipid membranes. Our central idea was to inhibit the PLA2 and Mel activities through histidine alkylation and or tryptophan oxidation (with pbb, para-bromo-phenacyl bromide, and/or NBS- N-bromosuccinimide, respectively) to make their encapsulations possible within stabilized liposomes. We strongly believe that this formulation will be nontoxic but immunogenic. In this paper, we present the whole bee venom conformation characterization during and after chemical modification and after interaction with liposome by ultraviolet, circular dichroism, and fluorescence spectroscopies.

DNA-based therapeutics and DNA delivery systems: a comprehensive review.

The past several years have witnessed the evolution of gene medicine from an experimental technology into a viable strategy for developing therapeutics for a wide range of human disorders. Numerous prototype DNA-based biopharmaceuticals can now control disease progression by induction and/or inhibition of genes. These potent therapeutics include plasmids containing transgenes, oligonucleotides, aptamers, ribozymes, DNAzymes, and small interfering RNAs. Although only 2 DNA-based pharmaceuticals (an antisense oligonucleotide formulation, Vitravene, (USA, 1998), and an adenoviral gene therapy treatment, Gendicine (China, 2003), have received approval from regulatory agencies; numerous candidates are in advanced stages of human clinical trials. Selection of drugs on the basis of DNA sequence and structure has a reduced potential for toxicity, should result in fewer side effects, and therefore should eventually yield safer drugs than those currently available. These predictions are based on the high selectivity and specificity of such molecules for recognition of their molecular targets. However, poor cellular uptake and rapid in vivo degradation of DNA-based therapeutics necessitate the use of delivery systems to facilitate cellular internalization and preserve their activity. This review discusses the basis of structural design, mode of action, and applications of DNA-based therapeutics. The mechanisms of cellular uptake and intracellular trafficking of DNA-based therapeutics are examined, and the constraints these transport processes impose on the choice of delivery systems are summarized. Finally, the development of some of the most promising currently available DNA delivery platforms is discussed, and the merits and drawbacks of each approach are evaluated.

Recent advances with liposomes as pharmaceutical carriers.

Liposomes - microscopic phospholipid bubbles with a bilayered membrane structure - have received a lot of attention during the past 30 years as pharmaceutical carriers of great potential. More recently, many new developments have been seen in the area of liposomal drugs - from clinically approved products to new experimental applications, with gene delivery and cancer therapy still being the principal areas of interest. For further successful development of this field, promising trends must be identified and exploited, albeit with a clear understanding of the limitations of these approaches.

The effect of pH on the electrophoretic behaviour of a new class of liposomes: arsonoliposomes.

Herein we report the effect of pH on the surface charge of a new class of liposomes: arsonoliposomes. Plain or mixed arsonoliposomes with cholesterol (Chol) and distearoyl-phosphatidylcholine (DSPC) in 1:1 molar ratio were prepared with lauryl-(C12), myristoyl-(C14) and palmitoyl-(C16) acyl side chain arsonolipids. The one step hydration method was used for vesicle preparation and zeta potential measurements were performed in the pH range from 3 to 9. The results revealed that these lipids hold a negative surface charge at all pH values investigated. The presence of cholesterol in 1:1 molar ratio results in higher zeta potential compared with plain arsonoliposomes with the exception of palmitoyl-(C16) acyl chain arsonolipids in neutral and slightly basic pH values. Oppositely, the DSPC (1:1 molar ratio) containing arsonoliposomes had lower values of zeta potential compared with plain arsonoliposomes. Concluding, the experimental results reveal that zeta potential of arsonoliposomes is indeed modified when the vesicles are incubated in environments with different acidity. In most cases these changes are in accordance with the ionization pattern of the arsonolipid headgroup, while some peculiar deviations may be connected with the known difference in the structure between some of the vesicle types studied.

Biodistribution characteristics of galactosylated emulsions and incorporated probucol for hepatocyte-selective targeting of lipophilic drugs in mice.

PURPOSE: Galactosylated emulsions containing cholesten-5-yloxy-N-(4-((1-imino-2-D-thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol) as a "homing device" were developed for hepatocyte-selective drug targeting. The targeting efficiency of galactosylated emulsions was evaluated by a distribution study in mice. METHODS: Soybean oil/EggPC/cholesterol (Chol) (weight ratio, 70:25: 5) (bare) emulsions and soybean oil/EggPC/Gal-C4-Chol (weight ratio, 70:25:5) (Gal) emulsions were prepared and labeled with [3H]cholesteryl hexadecyl ether (CHE). [14C]probucol as a model lipophilic drug was incorporated in the emulsions or EggPC/Chol/Gal-C4-Chol (Gal) liposomes. Their tissue and intrahepatic distribution were evaluated following intravenous injection in mice. RESULTS: After intravenous injection, Gal-emulsions were rapidly eliminated from the blood and accumulated in the liver, in contrast to the bare-emulsions. The liver uptake clearance of Gal-emulsions was 3.2- and 1.2-times greater than that of bare-emulsions and Gal-liposomes, respectively. The uptake ratio in liver parenchymal cells (PC) and nonparenchymal cells (NPC) of Gal-emulsions was higher than that of Gal-liposomes, being 7.4 and 3.0, suggesting that Gal-emulsions are an effective PC-selective carrier. The hepatic uptake of Gal-emulsions, but not that of bare-emulsions, was significantly inhibited by the pre-dosing of not only lactoferrin but also Gal-liposomes, suggesting asialoglycoprotein receptor-mediated endocytosis. Furthermore, [14C]probucol incorporated in Gal-emulsions was efficiently delivered to the liver compared with Gal-liposomes. CONCLUSION: Gal-emulsions have been proven to be an alternative carrier for hepatocyte-selective drug targeting.

Liposomally targeted cytotoxic drugs for the treatment of cancer.

Phospholipid spherules composed of lipid bilayer membranes entrapping a central aqueous core were first described more than 30 years ago (Bangham et al 1965). The term liposome was coined in 1968 (Sessa & Weissmann 1968) and the first suggestions that these vesicles might have potential as vehicles for targeted drug delivery for a range of diseases, including cancer, appeared shortly afterwards (Gregoriades et al 1974; Gregoriades 1976a, b). However, the process of turning this expectation into a clinical reality has suffered a number of setbacks and has taken more than a quarter of a century. In the process, new types of liposomes with favourable in-vivo pharmacokinetics and biodistribution patterns have been generated (Lasic & Papahadjopoulos 1995). Many of these preparations have been subjected to extensive examination and an increasing number of agents have entered clinical trials. In this review, we will trace the development of those liposomes that are currently undergoing (or are about to undergo) clinical evaluation.

Designing of 'intelligent' liposomes for efficient delivery of drugs.

The liposome- vesicles made by a double phospholipid layers which may encapsulate aqueous solutions- have been introduced as drug delivery vehicles due to their structural flexibility in size, composition and bilayer fluidity as well as their ability to incorporate a large variety of both hydrophilic and hydrophobic compounds. With time the liposome formulations have been perfected so as to serve certain purposes and this lead to the design of "intelligent" liposomes which can stand specifically induced modifications of the bilayers or can be surfaced with different ligands that guide them to the specific target sites. We present here a brief overview of the current strategies in the design of liposomes as drug delivery carriers and the medical applications of liposomes in humans.

Liposomes: applications in medicine.

Liposomes are spherical lipid bilayers from 50 nm to 1000 nm in diameter that serve as convenient delivery vehicles for biologically active compounds. The field of liposome research has expanded considerably over the last 30 years. It is now possible to engineer a wide range of liposomes varying in size, phospholipid composition and surface characteristics to suit the specific application for which they are intended. This paper gives an overview of the main advances in liposome research from a point of view of their applications in medicine. Aqueous contrast enhancing agents entrapped in liposomal carriers can be targeted to the liver and spleen and distinctions can be made between normal and tumorous tissue using computed tomography. Topical application of liposomes has great potential in dermatology. Liposomes have been used to deliver anticancer agents in order to reduce the toxic effects of the drugs when given alone or to increase the circulation time and effectiveness of the drugs. From the original concept of encapsulating hemoglobin in an inert shell, liposome-encapsulated hemoglobin (LEH) has evolved into a fluid proven to carry oxygen, capable of surviving for reasonable periods in the circulation and amenable to large-scale production. Liposomes may be used to target specific cells by attaching amino acid fragments such as antibodies or proteins or appropriate fragments that target specific receptor sites. Liposomal DNA delivery vectors and further enhancements in the forms of LPDI and LPDII are some of the safest and potentially most versatile transfer vectors used to date. DNA vaccination and improved efficiency of gene therapy are just a few of the upcoming applications of liposomes.

[Liposomes as drug carrier systems. Preparation, classification and therapeutic advantages of liposomes]. / A liposzóma mint gyógyszerszállító rendszer. A liposzómák elóállítása, alapvetó típusaik és terápiás alkalmazásuk elónyei.

Bangham et al. (1965) created first the concept of the liposome as a microparticulate lipoidal vesicle separated from its aqueous environment by one or more lipid bilayers. Later Gregoriadis and Ryman (1972) suggested to use liposomes as drug carrier systems. Nowadays liposomes are under extensive investigation for improving the delivery of therapeutic agents, enzymes, vaccines and genetic materials. Liposomes offer an excellent opportunity to selective targeting of drugs which is expected to optimize the pharmacokinetical parameters, the pharmacological effect and to reduce the toxicity of the encapsulated drugs. To understand the system it is important to know the basic properties of these lipoidal vesicles. Our aim was to focus on the lipid composition and the method of liposome preparation what determine the liposomal membrane fluidity, permeability, vesicle size, charge density, steric hindrance and stability of the liposomes as principle factors those influence the fate of liposomes, their interactions with the blood components and other tissues after systemic administration or local use.

Liposomes: presentation and actual applicative trends in medicine.

In this paper the main aspects of characterization, handling and applications of liposomes are presented. In the last 25 years much attention has been focused to liposomal systems for optimization of the drug targeting. Several pathways to optimize the drug action of liposomes in various situations as cancer, microbial therapy, vaccines, oral therapy and diagnosis were tested. Certain applications of liposomes especially those implying the phagocytic cells sustain a real interest for industrial applications.

Efficacy of liposome encapsulated triethylenetetraamine hexaacetic acid (TTHA) against cadmium intoxication: role of lipid composition.

Efficacy of Triethylenetetraamine hexaacetic acid (TTHA) encapsulated in liposomes having different lipid compositions was examined in animals pre-exposed to cadmium. Mice were injected with cadmium as cadmium (II) chloride 0.5 mg/kg b. wt. intraperitoneally daily for five days. Four weeks after the last injection of cadmium they were administered three injections of TTHA encapsulated in liposomes composed of either phosphatidyl choline:cholesterol (PC:Chol) or sphingomyelin:cholesterol (SM:Chol) in 1:1 molar ratio at a gap of 48 h. Urinary and fecal elimination of cadmium and its distribution in liver, kidneys and spleen were examined. Treatment with TTHA encapsulated in liposomes mobilized higher amount of cadmium from liver and spleen. The overall efficiency for cadmium mobilization was better in TTHA encapsulated in SM:Chol liposome treated group which also led to enhanced excretion of cadmium through urine and feces. The results indicate that TTHA encapsulated in SM:Chol liposomes exhibited highest efficacy in mobilizing cadmium from the body of pre-exposed mice followed by PC:Chol liposomes and the free drug.

Liposomas en dermatología / Liposomas in dermatology

Los liposomas son microvesículas esféricas compuestas por bicapas paralelas de fosfolípidos. Las diversas propiedades de los liposomas permiten su aplicación potencial en diversos campos de la medicina. Las interacciones de los liposomas con la piel son de particular importancia en dermatología, en especial como vehículos para diverso compuestos de uso tópico y por sus efectos directos sobrela piel. En este trabajo se realizauna revisión sobre la composición, estructura, técnicas de preparación y propiedades de los liposomas. Tambien se revisan sus aplicaciones en medicina general y en especial en dermatología por sus particulares interacciones con la piel

Anfotericina B liposomal / Liposomal amphotericin B

La anfotericina B es el antifúngico de elección en los pacientes inmunocomprometidos con micosis diseminadas. La mayor desventaja que presenta es su toxicidad: a) durante la administración: fiebre, escalofríos, convulsiones, vómitos, hipocaliemia, flebitis, "rash", broncoespasmo; b) toxicidad renal con aumento de urea y creatinina y c) toxicidad medular con anemia normocítica normocrómica. La toxicidad ha llevado a usar pre e intramedicaciones, a variar de 6 a 1 h. el tiempo de administración y a diluirla en Intralipid (MR) en vez de dextrosa. Aprovechando su lipofilia, se la ha incorporado a liposomas que estan constituidos por una o más bicapas fosfolipídicas y un centro acuoso. La anfotericina queda atrapada en la bicapa. En el torrente circulatorio los liposomas entran en contacto con las opsoninas que los presentan al sistema retículo endotelial (SRE) y con las proteínas de alta densidad con actividad fosfolipasa que disuelven la bicapa, liberándose la anfotericina B. Cuando los liposomas son incorporados al SRE, en el interior de las células se libera la droga. Los liposomas de gran tamaño se concentran en el SRE hepático, los más chicos llegan al hepatocito y los de mayor vida media a médula ósea. Al ser transportada dentro de los liposomas, la anfotericina ocasiona escasos efectos tóxicos, pudiéndose administrar hasta 5mg/kg/día con buena tolerancia. Los liposomas pueden ser multilamerales y sus capas estar constituidas por dimeristeril fosfatidilglicerol (DMPG) y dimeristerilfosfatidilcolina (DMPC) o ser unilamerales y la capa estar formada por diesteroilfosfatidilglicero (DEPG), fosfatidilcolina y colesterol, o por esteralilamina, fosfatidilcolina y colesterol, o sulfato de colesterol (AU)

Anfotericina B liposomal / Liposomal amphotericin B

La anfotericina B es el antifúngico de elección en los pacientes inmunocomprometidos con micosis diseminadas. La mayor desventaja que presenta es su toxicidad: a) durante la administración: fiebre, escalofríos, convulsiones, vómitos, hipocaliemia, flebitis, "rash", broncoespasmo; b) toxicidad renal con aumento de urea y creatinina y c) toxicidad medular con anemia normocítica normocrómica. La toxicidad ha llevado a usar pre e intramedicaciones, a variar de 6 a 1 h. el tiempo de administración y a diluirla en Intralipid (MR) en vez de dextrosa. Aprovechando su lipofilia, se la ha incorporado a liposomas que estan constituidos por una o más bicapas fosfolipídicas y un centro acuoso. La anfotericina queda atrapada en la bicapa. En el torrente circulatorio los liposomas entran en contacto con las opsoninas que los presentan al sistema retículo endotelial (SRE) y con las proteínas de alta densidad con actividad fosfolipasa que disuelven la bicapa, liberándose la anfotericina B. Cuando los liposomas son incorporados al SRE, en el interior de las células se libera la droga. Los liposomas de gran tamaño se concentran en el SRE hepático, los más chicos llegan al hepatocito y los de mayor vida media a médula ósea. Al ser transportada dentro de los liposomas, la anfotericina ocasiona escasos efectos tóxicos, pudiéndose administrar hasta 5mg/kg/día con buena tolerancia. Los liposomas pueden ser multilamerales y sus capas estar constituidas por dimeristeril fosfatidilglicerol (DMPG) y dimeristerilfosfatidilcolina (DMPC) o ser unilamerales y la capa estar formada por diesteroilfosfatidilglicero (DEPG), fosfatidilcolina y colesterol, o por esteralilamina, fosfatidilcolina y colesterol, o sulfato de colesterol