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Chemistry and Physics of Lipids 165 (2012) 438– 453

Contents lists available at SciVerse ScienceDirect

Chemistry and Physics of Lipids

jou rn al hom epa ge : www.elsev ier .com/ locate /chemphys l ip

ipospheres and pro-nano lipospheres for delivery of poorly water solubleompounds

nna Elgart, Irina Cherniakov, Yanir Aldouby, Abraham J. Domb, Amnon Hoffman ∗

nstitute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

r t i c l e i n f o

rticle history:vailable online 9 February 2012

eywords:ipospheresro-nano lipospheresolid lipid nano-particlesrug deliveryral drug delivery

a b s t r a c t

Lipospheres are a drug encapsulation system composed of water dispersible solid microparticles of par-ticle size between 0.01 and 100 �m in diameter with a solid hydrophobic lipid core stabilized by a layerof phospholipid molecules embedded in their surface. The bioactive compound is dissolved or dispersedin the solid lipid matrix of the internal core. Since lipospheres were introduced in the beginning of the1990s, they have been used for the delivery of multiple types of drugs by various routes of administration.Later, a self-assembling pro-nano lipospheres (PNL) encapsulation system was developed for oral drugdelivery. Lipospheres have several advantages over other delivery systems, such as better physical stabil-ity, low cost of ingredients, ease of preparation and scale-up, high dispersibility in an aqueous medium,

cular drug deliveryarenteral drug delivery

high entrapment of hydrophobic drugs, controlled particle size, and extended release of entrapped drugafter administration, from a few hours to several days.

This review article focuses on updated information on several aspects of lipospheres and PNL, includingpreparation techniques, physicochemical properties and in vitro evaluation methods. Additionally, itcovers lipospheres and PNL utilization for oral, ocular, and parenteral delivery, with special attention to

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unique considerations an

. Introduction

The introduction of combinatorial chemistry accompanied bydvances in in vitro high throughput screening methods hasesulted in the rapid identification of many highly potent but poorlyater soluble drug candidates. In fact, to date, more than 40% ofew chemical entities are lipophilic and exhibit poor water sol-bility (Lipinski et al., 2001). Development of such poorly wateroluble compounds towards clinically available drugs presents a

reat challenge facing the pharmaceutical scientists. Consequently,he understanding that the development of new active compoundslone is not enough to guarantee adequate pharmacotherapy of

Abbreviations: AFM, atomic force microscopy; AUC, area under the curve;CS, biopharmaceutical classification system; BS, bile salt; CNS, central nervousystem; CsA, cyclosporine A; DDS, drug delivery system; DG, diglyceride; DSC,ifferential scanning calorimetry; EE, entrapment efficiency; FA, fatty acid; GI,astro-intestinal; HLB, hydrophilic lipophilic balance; LC, loading capacity; LD,aser diffraction; MG, monoglyceride; NMR, nuclear magnetic resonance; PCS,hoton correlation spectroscopy; PE, phosphatidylethanolamine; PNL, pro-nano

ipospheres; RES, reticuloendothelial system; SEM, scanning electron microscopy;LN, solid lipid-based nanoparticle; TEM, transmission electron microscopy; TG,riglyceride; TNBS, trinitrobenzenesulfonic acid; XRD, X-ray diffractometry.∗ Corresponding author at: Department of Pharmaceutics, School of Pharmacy,

he Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 91120, Israel.el.: +972 2 6757014; fax: +972 2 6757246.

E-mail address: amnonh@ekmd.huji.ac.il (A. Hoffman).

009-3084/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.chemphyslip.2012.01.007

ects for each route of administration.© 2012 Elsevier Ireland Ltd. All rights reserved.

various disease states became widely accepted. Promising resultsobtained in in vitro studies very often are not corroborated by suc-cessful in vivo data. Multiple reasons stand behind these in vivoresults. Some drugs do not reach sufficient plasma concentrationsdue to limited solubility, poor absorption and extensive first passmetabolism. Some are characterized by unpredictable fluctuationsin plasma drug levels and thus lack effective dose–response cor-relation. Poor water solubility might exclude the possibility for IVadministration as well. Other drugs are distributed to additionaltissues besides the site of action and cause harsh adverse effectsor toxicity. Toxicity and lack of therapeutic effect might also resultfrom a drug’s decomposition during its voyage from the intestinallumen to the systemic blood circulation.

A promising strategy to overcome these obstacles is thedevelopment of suitable drug delivery systems (DDS). The under-standing that the in vivo fate of the drug is dictated not onlyby the drug itself, but also by the mode of administration andthe carrier system which should enable an optimal drug releaseprofile according to the therapy requirements is crucial for suchdevelopment (Mehnert and Mader, 2001). One of the most popularpharmaceutical approaches to overcome these obstacles is the useof various nano-dispersion systems as carriers of drug substances.

Though the concept of the essential scientific field of moderntimes – nanotechnology – was introduced in 1959 by Feynmanin his famous lecture “There’s plenty room at the bottom”, theprimary development of nanotechnology occurred only in the

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ineteen eighties and the early nineties. The invention of thecanning tunneling microscope (STM) by Binnig and Rohrer isonsidered by some to be the actual beginning in the developmentf nanotechnologies. There are many variations of the definitionf the term “nanotechnology” according to the field in whichanotechnology is applied. The US National Science Foundationefined nanotechnology as science, engineering, and technologyonducted at the nano-scale of approximately 1–100 nm.

The application of nanotechnology in drug delivery systems is aery popular approach in the pharmaceutical industry to improvehe bioavailability of drugs. There are a number of areas in whichanotechnology is being applied in drug delivery, e.g. improving theioavailability of poorly water soluble drugs (Hu et al., 2004; Li et al.,009; Zhang et al., 2011; Manjunath and Venkateswarlu, 2006),rug targeting i.e. transporting therapeutic agents to a specific cellr tissue (Wong et al., 2007) and controlled release delivery systemsYang et al., 1999; zur Muhlen et al., 1998).

Lipospheres are lipid-based water dispersible solid particles ofarticle size between 0.01 and 100 �m in diameter composed of aolid hydrophobic lipid core (triglycerides), stabilized by a layerf phospholipid molecules embedded in their surface. The lipo-pheres are suitable for oral, parenteral and topical drug delivery ofioactive compounds and are designed to overcome the drawbacksssociated with traditional colloidal systems such as emulsions,iposomes and polymeric nanoparticles (Domb et al., 1996; Maniart al., 1991).

The internal core contains the bioactive compound dissolved orispersed in the solid fat matrix (Bekerman et al., 2004).Various

ipospheres have been used for the controlled delivery of differ-nt types of drugs including anti-inflammatory compounds, localnesthetics, antibiotics, and anticancer agents, as well as carriersf vaccines and adjuvants (Domb, 2006; Amselem, 1996; Amselemt al., 1992b,a).

Similar systems based on solid fats and phospholipids haveeen described, as well as solid lipid nanospheres (SLN) which aressentially nano-size lipospheres. All of the above were extensivelyeviewed elsewhere (Domb, 2006; Muller et al., 2000).

Passive and active targeting of nano-lipospheres is also possibleased on two different approaches. Firstly, nano-lipospheres woulde able to deliver a concentrated dose of drug in the vicinity ofhe tumor via the enhanced permeability (passive targeting) andetention effect. Secondly, active targeting to various tissues maye achieved via utilization of ligands on the surface of nanoparticles.

n addition, nano-lipospheres would reduce the drug’s presence inealthy tissues by limiting drug distribution to the target organIrache et al., 2011). The broad subject of targeting, and especiallyctive and carrier mediated targeting of nanoparticles is beyond thecope of this review and is extensively reviewed elsewhere (Peert al., 2007; Chrastina et al., 2011; Shapira et al., in press).

Lipospheres have several advantages over other particulateelivery systems such as emulsions, liposomes and microspheres,

ncluding: improved drug stability, formulation stability, the abil-ty to freeze dry and reconstitute, the possibility for controlledrug release, high drug payload, controlled particle size and thevoidance of carrier toxicity and the presence of organic sol-ents. Advantages of the use of lipospheres for oral administrationnclude the possibility for drug protection from hydrolysis, as

ell as increased drug bioavailability and prolonged plasma lev-ls (Souto and Muller, 2007). In addition, the matrix is composedf physiological components and/or excipients of accepted statuse.g. GRAS status), which reduces the risk for acute/chronic tox-city (Domb, 2006). On the other hand, the disadvantages of such

elivery systems are associated mostly with their preparation tech-iques involving high pressure and rapid temperature changes,nd include high pressure induced drug degradation, lipid crystal-ization, gelation phenomena and co-existence of several colloidal

s of Lipids 165 (2012) 438– 453 439

species (Mehnert and Mader, 2001). Today, several techniques areemployed to produce lipospheres, such as high pressure homog-enization, hot and cold homogenization, solvent emulsificationevaporation, etc. (Souto and Muller, 2007). An alternative methodis in situ preparation of lipospheres with a particle size below100 nm. This method was developed by using a dispersible pre-concentrate system (Bekerman et al., 2004). This delivery system,termed pro-nano liposphere (PNL), is based on a solution containingthe drug, triglyceride, phospholipid and other additives in a mix-ture of common surfactants, and an organic solvent that is misciblewith all components. This solution spontaneously forms nanopar-ticles when gently mixed in an aqueous media, such as the upperGI lumen content.

This review will focus on updated information on the prepa-ration, physicochemical properties and in vitro evaluation oflipospheres and PNL as carrier systems for poor water-solubledrugs. These lipid dispersions can be used for different routes ofadministration. The peroral route is the most preferred mode ofdrug administration and the parenteral route is the most chal-lenging one. Thus, the center of attention of this review will benano-dispersion systems for parenteral and peroral administra-tion of poor water-soluble compounds. Nevertheless, ocular andCNS-targeted administrations will be discussed as well.

2. Preparation of lipospheres and PNL

The internal hydrophobic core of lipospheres is composed oflipids, mainly solid triglycerides, while the surface activity of lipo-sphere particles is provided by the surrounding phospholipid layer.The clear advantage of lipospheres is the fact that the lipid core con-sists of physiological naturally occurring biodegradable lipids, thusminimizing the danger of acute and chronic toxicity.

The lipid that constitutes the core component of the lipospheresand PNL is solid at room temperature, and might melt, or stay solidat body temperature, depending on the particle design. By utilizingsolid lipid as a core, several setbacks associated with the usage ofliquid or semi-liquid lipid core might be reduced or avoided, i.e.inherent instability and irreversible drug/excipient precipitation(Tang et al., 2008).

Usually, the oil, which has a maximum solubilizing potential forthe drug under investigation, is initially selected with the intentionof achieving the maximal drug loading in the lipospheres. Con-currently, the selected oil should be able to yield particles withnano-range size. Hence, the choice of the oily phase is often a com-promise between its ability to solubilize the drug and its ability tofacilitate formation of a nano-encapsulation system with desiredcharacteristics (Date et al., 2010).

The neutral lipids that are usually utilized for the hydrophobiccore of the liposphere formulations are tricaprin, trilaurin, tris-tearin, stearic acid, ethyl stearate, and hydrogenated vegetableoil. Modified or hydrolyzed vegetable oils have also been widelyused since these excipients exhibit better drug solubility proper-ties. They offer formulative and physiological advantages and theirdegradation products resemble the natural end products of intesti-nal digestion (Gursoy and Benita, 2004).

The choice of an appropriate surfactant for the liposphere for-mulations is often dictated by safety considerations. Emulsifiers ofnatural origin are preferred since they are considered to be saferthan the synthetic surfactants. Non-ionic surfactants are less toxicthan ionic surfactants but they may lead to reversible changesin the permeability of the intestinal lumen. The acceptability of

the selected surfactant for the desired route of administration andits regulatory status (e.g., generally regarded as safe [GRAS] sta-tus) must also be considered (Gursoy and Benita, 2004). It shouldbe noted that the surfactants are not innocuous and they have

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avorable and/or unfavorable biological effects depending upon thehemical nature and the concentration of the surfactant. Manyonionic surfactants, such as Cremophor EL (polyethylene glycolPEG]-35-castor oil), have the ability to enhance permeability andptake of drugs that are susceptible to P-glycoprotein-mediatedfflux. Vitamin E-TPGS (d-�-tocopheryl polyethylene glycol 1000uccinate) is a good example, as it increases the absorption fluxf amprenavir (Yu et al., 1999), and has been characterized as annhibitor of P-gp-mediated drug transport in the human intesti-al Caco-2 cell monolayers and in other cell lines (Dintaman andilverman, 1999). It has also been shown to enhance the bioavail-bility of cyclosporine in human volunteers (Chang et al., 1996)nd of colchicine in rats when the drugs were administered orallyBittner et al., 2002; Cornaire et al., 2004). However, these surfac-ants can also have structure-dependent, concentration-dependentnd route of administration-dependent adverse effects. For exam-le, Cremophor EL can cause anaphylactic shock and histamineelease on parenteral administration (ten Tije et al., 2003), whereast is well tolerated upon oral administration (Pouton and Porter,008). Certain surfactants might cause irritation to the GI mucosand skin at higher concentrations (Date et al., 2010). Surfactantsypically used in a liposphere preparation may include sorbitanerivatives, Tween and Span; PEGilated fats, Chremophor EL andipoPeg.

The choice of organic solvents depends on the route of adminis-ration and naturally is more limited for ophthalmic and parenteralreparations. Organic solvents such as ethanol, propylene glycolPG), and polyethylene glycol (PEG) are suitable for oral delivery,nd they enable the dissolution of large quantities of either theydrophilic surfactant or the hydrophobic drug in the lipid base. Onhe other hand, alcohols and other volatile co-solvents have the dis-dvantage of evaporating into the shells of the soft gelatin, or hard,ealed gelatin capsules leading to drug precipitation. Alcohol-freeormulations that have been designed to overcome this obsta-le (Constantinides, 1995) have limited lipophilic drug dissolutionbility (Gursoy and Benita, 2004). Triacetin is a suitable organic sol-ent since it is miscible in the oil/lipid phases and it can be used toolubilize a hydrophobic drug (Patel and Sawant, 2009).

The phospholipids used to form the surrounding layer ofipospheres are usually pure egg phosphatidylcholine, soybeanhosphatidylcholine, dimyristoyl phosphatidylglycerol and phos-hatidylethanolamine.

.1. Homogenization methods:

.1.1. High shear homogenization methodHigh Shear homogenization and ultrasound are dispersing tech-

iques which were initially used for the production of nano-sizedarticulate systems. However, the presence of microparticles andetal contamination problems were often associated with thisethod, which led to the development of more sophisticated pro-

uction methods (Mehnert and Mader, 2001).

.1.2. High pressure homogenization (HPH) methodThe initial step involves drug incorporation into bulk lipid by

issolving or dispersing the drug in lipid melt. A high pressure100–2000 bar) homogenizer further pushes this liquid through aarrow gap, upon rapid acceleration to a very high velocity. Theesulting shear stress and cavitation forces disrupt the particles andeduce them down to a submicron size (Mehnert and Mader, 2001).

.1.3. Hot homogenization method

In this method, the active agent is dissolved or dispersed in

he melted solid carrier, i.e. tristearin or polycaprolactone, and hot buffer solution is added at once along with the phospho-ipid powder. The hot mixture is homogenized for about 2–5 min

s of Lipids 165 (2012) 438– 453

using a homogenizer or ultrasound probe, after which a uniformemulsion is obtained. HPH of the obtained emulsion is then per-formed at a temperature above the melting point of the core lipid.Generally, higher temperatures result in reduced particle size;however, they may also increase the degradation rate of the drug.The obtained nano-emulsion is then rapidly cooled down to about20 ◦C by immersing the formulation flask in an ice/water bath whilehomogenization is continued to yield a uniform dispersion of solidlipospheres (Domb, 2006).

2.1.4. Cold homogenization methodThis method was developed in order to overcome several prob-

lems associated with the hot homogenization method, i.e. hightemperature induced drug degradation, complexity of the crys-tallization step leading to modifications of the drug, and drugdistribution to the aqueous phase. The initial step involves drugincorporation into bulk lipid by dissolving or dispersing the drugin lipid melt, which is then rapidly cooled, resulting in homoge-nous distribution of the drug in the lipid matrix. Next, The obtainedsolid lipid matrix is milled to micron-sized particles, and the parti-cles are dispersed in a chilled emulsifier solution. This suspensionis subjected to homogenization at low temperature to obtainednano-sized dispersion system (Mehnert and Mader, 2001).

A typical example for the application of the cold homogenizationmethod is the preparation of dexamethasone (a poorly water sol-uble steroid) lipospheres. Dexamethasone (active ingredient) andtristearin (lipid core) are added to a glass flask which is then heatedto melt the mixture. Hot phosphate buffer solution is added alongwith egg phosphatidylcholine. The mixture is homogenized for afew minutes until a uniform milky-like formulation is obtained.The hot formulation is rapidly cooled to below 20 ◦C with contin-ued mixing to yield a thin dispersion. Submicron size lipospheresare prepared by extrusion through a submicron series of filters at atemperature of 5 ◦C above the melting point of the liposphere corecomposition. Particle size may be reduced to about 200 nm (Domb,2006).

2.2. Solvent emulsification/evaporation method

Alternatively, lipospheres might be prepared by a solvent tech-nique. The premise for this method is the emulsification of apolymeric solution in an aqueous continuous phase. In this case, theactive agent, the solid carrier and the phospholipid are dissolved inan organic solvent. The O/W emulsion is produced by the agita-tion of two immiscible liquids. This mixture is further emulsified inan aqueous phase by HPH or another homogenization technique.The drug substance is either dispersed in solution in the solventsystem or is captured in the dispersed phase of the emulsion. Agi-tation of the system is continued until the solvent partitions intothe aqueous phase. The organic solvent is then evaporated and theresulting solid is mixed in warm buffer solution until a homoge-neous dispersion of lipospheres is obtained. This process resultsin hardened lipospheres which contain the active moiety (Domb,2006).The mean particle size depends on the lipid concentrationin the organic phase, with inverse correlation between the lipidconcentration and the obtained particle size (Mehnert and Mader,2001). The main problem with this method is the use of an organicsolvent, which must be removed until its concentration is withinacceptable limits.

2.3. Supercritical fluid method

To avoid organic solvent contamination, the supercritical fluidmethod was explored. Here, the lipid and the drug are dissolved ina suitable organic solvent to form a solution, which is emulsified inan aqueous phase to form an emulsion containing a discontinuous

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hase of micelles comprised of organic solvent, drug and lipid.inally, the emulsion is treated with a supercritical fluid under suit-ble conditions, which results in extraction of the organic solventrom the micelles, and precipitation of solid composite lipid drugano-sized particles in the aqueous dispersion (Shukla et al., 2011).

.4. PNL preparation

An alternative method for in situ preparation of nanosize lipo-pheres of particle size below 100 nm is based on the PNL concept.n this system, the drug, triglyceride, phospholipid and other addi-ives are dissolved in a mixture of common surfactants with highnd low hydrophilic lipophilic balance (HLB) such as Tween andpan, respectively, and an organic solvent that is miscible with allomponents. This clear anhydrous solution spontaneously formsanoparticles when gently mixed in an aqueous solution. Thearticle size is controlled mainly by the formulation composi-ion. Cationoic or anionic nano-lipospheres can be obtained whendding to the solution a cationic or anionic lipid such as stearylmine, phosphatidilethanol amine, stearic acid or phosphadiliccid. This concept has been successfully applied in our group forhe improvement of the bioavailability of cyclosporine A (Bekermant al., 2004).

.5. Sterilization

Parenteral and ophthalmic DDS require sterilization. Sterileiposphere formulations may be prepared by aseptic production, fil-ration, �-irradiation and heating. Sterile filtration of the dispersionhould be performed in the hot stage of the preparation through

0.2 �m filter at a temperature of 5 ◦C above the melting pointf the liposphere core composites. Heat sterilization using a stan-ard autoclave cycle is also a reliable procedure commonly applied.owever, it might result in temperature induced changes of thehysical stability of the dispersion, as well as of the incorporatedrug. In our work, we have noticed that utilization of the heat ster-

lization method resulted in the decomposition of the formulationsnder investigation. �-irradiation sterilization of liposphere formu-

ations, on the other hand, did not affect their physical properties.or example, when liposphere formulations of 1:4:2 and 2:4:2upivacaine:tristearin:phospholipid (w/w, % ratio) were irradiatedith a dose of 2.33 Mrad and then analyzed for particle size, bupiva-

aine content, in vitro release characteristics and in vivo activity, therradiated formulations had similar parameters and in vivo perfor-

ance in the rat paw analgesia model to the non-irradiated controlsnd to bupivacaine HCl solution (Marcaine®). However, a moreareful analysis of the formulation ingredients should be performedince phospholipids may degrade during irradiation (Domb, 2006).nother concern when utilizing the �-irradiation sterilization is the

ormation of free radicals, which might undergo secondary reac-ions with the formulation ingredients, leading to their chemical

odification.

. In vitro characterization of lipospheres and PNL

Proper characterization of lipospheres and PNL is a serious chal-enge due to the colloidal size of the particles and the complexitynd dynamic nature of the delivery system (Mukherjee et al., 2009).he most important parameters which need to be evaluated areiscussed below:

.1. Particle size

The accurate estimation of the particle size is extremely impor-ant as it can affect the in vivo performance of the lipid basedncapsulation system. These effects will be detailed in the following

s of Lipids 165 (2012) 438– 453 441

paragraph. Particle size can be measured using a number of meth-ods: photon correlation spectroscopy (PCS), also known as dynamiclight scattering (Bohets et al., 2001; Kuo and Chen, 2009; Mehnertand Mader, 2001), laser diffraction (LD) (Muller et al., 2001), trans-mission electron microscopy (TEM) (Cardiello et al., 2003), scanningelectron microscopy (SEM) and atomic force microscopy (AFM).PCS and LD are the most widely used for particle size evaluationof dispersion systems. By the PCS technique particle size is mea-sured based on the fluctuation of the intensity of the scattered light,caused by the movement of the particle. LD measures the size ofa particle by the diffraction angle on the particle radius, as moreintense scattering is caused by smaller particles. LD covers a broadrange of particles size, from nano- to hundreds of microns. LD hasan advantage over PCS when evaluating particles larger than 3 �m,as PCS is a relatively accurate and sensitive method to character-ize particles in the nano range, but it fails to detect micron sizedparticles. Though an improvement in detecting smaller particlesby LD was achieved due to development of phase sensitive inten-sity difference scattering (PIDS), it is advised to use both of thesetechniques simultaneously (Mehnert and Mader, 2001).

The need for other methods for particle size measurement arisesfor several reasons. Firstly, in the case where the particle shape isnot spherical the particles may be wrongly measured as micropar-ticles though in reality they are in the nano-range (Seyfoddin et al.,2010). Secondly, it has been suggested that during lipid crystalliza-tion platelet structures can occur and result in the measurementof a sample with several populations of particle size (Mehnert andMader, 2001).

Electron microscopy provides information on the morphologicalshape of the particle as well as particles size. However, the biggestdrawback of this method is the preparation of the sample which canlead to possible artifacts (e.g. solvent removal) (Mehnert and Mader,2001). Thus, in addition to particle size and distribution evaluation,SEM, TEM and AFM are also very valuable in morphology assess-ment of lipid spheres. These techniques are further discussed inSection 3.4 of this review.

3.1.1. Particle size in oral administrationIn the case of peroral administration the particle size of lipid

based encapsulation systems is one of the key factors with a directimpact not only on the in vitro evaluated parameters (e.g. stabilityand release kinetics) but also on the in vivo performance (Mehnertand Mader, 2001). Orally administered lipid-based encapsulationsystems have a large surface area which results from the nano-range particle size and enables pancreatic lipase to efficientlyhydrolyze triglycerides forming mixed micelles, promoting solu-bilization of the lipophilic drug in the aqueous environment of theintestinal lumen (Markos et al., 1994). Several studies report thatsmaller droplet size, when measured in vitro, was found to have afavorable effect on the bioavailability of the drug incorporated intolipid-based encapsulation systems. Tarr and Yalkowsky assessedthe bioavailability of CsA in rats by administering the drug in var-ious microemulsions forming particles with different sizes. Theyreported enhanced oral absorption of CsA in rats by reducing thesize of the droplets (Tarr and Yalkowsky, 1989). It is important tonotice that in this study the formulations were prepared using thesame ingredients and differed only in the method by which theywere prepared. Thus allowing the researchers to rule out any otherinfluence on the bioavailability of the drug and to isolate solelythe effect of the size of the particles (Desai et al., 1996). Thoughin this described study a microemulsion was assessed, it demon-strates a very valuable principle which can be of a great importance

for the nano-dispersion systems. The findings of a study publishedin 2004 by Bekerman et al. corroborated the results describedby Tarr and Yalkowsky. This study evaluated several dispersiblePNLs incorporating CsA in vivo (in healthy volunteers). The range

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f the particle size formed by introducing the PNL into aqueoushase varied between 25 and 400 nm. According to several lead-

ng researchers in the field of lipid based formulations (e.g. Pouton,000, 2006), the key to lowering the particle size was the mix-

ng of hydrophilic and lipophilic surfactants (in this case Tween 20nd Span 80, respectively). Study findings demonstrated an inverseorrelation between the particle size of the tested PNL and theral bioavailability of the incorporated CsA (Bekerman et al., 2004).

similar tendency was observed and reported by several otheresearches investigating versatile lipid based formulations and lipidased encapsulation systems (Thomas et al., 2005; Sigfridsson et al.,009).Thus, it is reasonable to deduce that particle size distributionould have a substantial impact on the oral bioavailability of a drugncorporated into a lipid based encapsulation system.

.1.2. Particle size in parenteral administrationOne of the most important factors needed to be taken into

onsideration in lipid-based encapsulation systems for i.v. admin-stration is particle size. In fact, due to the possibility of capillarylockage which could lead to a fat embolism and result in death, theize of the lipospheres is a key factor for i.v. injections. The diam-ter of the fine capillaries is about 9 �m. Thus, for safety reasonst is recommended that the size of the particles should be in theubmicron range (Mehnert and Mader, 2001). However, there are

number of commercial microemulsions for parenteral nutritionith particle sizes exceeding 9 �m, i.e. in diameter larger than theiameter of the fine capillaries (Mehta et al., 1992; Puntis et al.,992) Clearly, this cutoff is more crucial for lipospheres composedf lipids with a melting point above human body temperature (i.e.37 ◦C) such as trilaurin. The reason for this is derived from theact that a solid lipid is not deformable as liquid oil. Therefore,n contrast to microemulsions, capillary blockage will occur if theiameter of the particles exceeds the diameter of the blood vesselMehnert and Mader, 2001). Accordingly, the maximal size of lipo-pheres is below 1 �m, this lipid-based encapsulation systems cane used for i.v. administration with minimal risk for blood clottingnd aggregation leading to embolism (Uner et al., 2004).

.2. � Potential

The � potential of a dispersion system is defined as the poten-ial between the tightly bound surface liquid layer of a dispersedarticle and the bulk phase of the solution. Therefore, the � poten-ial can determine the electrostatic interactions between dispersedarticles.

� Potential is an important parameter in characterizing the prop-rties of dispersion systems affected by this electrical phenomenonLi and Tian, 2007). The magnitude of the � potential gives anndication of the potential stability of the dispersion system. There-ore, if the particles have high � potential values, either positive oregative, the colloidal system will be stable. Physically stable nan-dispersions stabilized only by electrostatic repulsion should have

minimum zeta potential of ±30 mV in order for the dispersion toesist flocculation (Muller et al., 2001; Mishra et al., 2009).

� Potential can be measured by a traditional technique calledlectrophoretic light scattering, a technique which measures theelocity of the particles suspended in a fluid medium using a cellith electrodes at either side through which an electric field is

pplied. The movement of the particles is towards the electrodef opposite charge. The velocity of the particles moving through

fluid in the electrophoresis technique is measured using Laseroppler Velocimetry. The sample is irradiated with a laser beam at

certain angel and the scattered light emitted from the particless detected. The rate of fluctuations in the scattered light emittedrom the particles is proportional to the velocity of the particles andhe velocity is proportional to the amount of charge of the particles,

s of Lipids 165 (2012) 438– 453

and thus � potential can be estimated. A more modern techniqueis phase analysis light scattering (PALS) in which the same opti-cal setup as in conventional laser Doppler electrophoresis is used.However, a different signal processing method is employed. Thistechnique allows to apply low voltages, thus avoiding damage tohigh conductivity samples. Moreover, low particle mobility can beaccurately measured by using this technique (Washington, 2011).

3.2.1. � Potential and oral administration� Potential is an important parameter for measuring storage

stability, particularly in the case of lipid nanoparticles. Moreover,surface charge is not only involved in physical stability but can alsohave an impact on the interaction of the lipid encapsulation systemswith biological membranes. Thus, the surface charge of nanoen-capsulation systems can affect the in vivo performance, potentiallypromoting drug delivery (Harush-Frenkel et al., 2007; Gershaniket al., 1998). Findings of physiological studies indicate that, rela-tively to the intestinal fluids, the apical surface of the intestinalepithelium is negatively charged. Gershanik and Benita developedlipid based encapsulation systems and assessed the impact of sur-face charge on their interaction with the intestinal mucosa in vitroand ex vivo. The study findings indicated that positively chargedparticles exhibited electrostatic interaction with enteroctes (Caco-2) monolayer and the mucosal surface of the averted rat intestine.In comparison to the corresponding negatively charged formula-tion, positively charged particles enhanced the oral bioavailabilityof progesterone in young female rats and exhibited higher bloodlevels of CsA in perfused rats (Gershanik and Benita, 1996, 2000;Gershanik et al., 1998) These observations indicate that the chargeof the particles dictates their interaction with biological compo-nents in the intestinal milieu (Gershanik et al., 1998). However,it should be mentioned that for formulations designed for oraladministration, in order to achieve highest physical stability in GImedium, the combination of a steric stabilizer is recommended(e.g., Tween 80 or Poloxamer 188). Steric stabilization is impairedby the presence of electrolytes to lesser extent compared with elec-trostatic solubilization (high zeta potential) (Muchow et al., 2008).

3.2.2. � Potential and ocular administrationThe significance of � potential in affecting drug absorption is

not exclusive to oral administration. In fact, it was reported thatthe surface charge of lipid encapsulation systems is essential inthe mechanism of drug absorption in ocular delivery. Cationic lipidbased nanocarriers can potentially increase drug residence time inthe negatively charged corneal epithelial cells, which in turn couldultimately enhance drug penetration to its site of action (Seyfoddinet al., 2010).

3.3. Drug release studies

Drugs incorporated into the solid lipid core of the lipospheresare usually released by diffusion through the matrix, and/orbiodegradation and surface erosion of the particle (Sawant andDodiya, 2008). For release kinetics evaluation, especially when con-trolled release delivery systems are under investigation, in vitrodrug release studies can be conducted. However, it should beemphasized that the release kinetics depends on release conditions,e.g. sink/non-sink, release medium, etc. Due to the colloidal size ofthe lipospheres, release studies are not trivial, and can be performedunder several separation techniques (filtration, centrifugation, dial-ysis), each having its own advantages and drawbacks.

The use of simple aqueous media to assess dissolution profiles of

lipophilic drugs, which are the most suitable candidates for incor-poration into lipospheres, is limited by the low intrinsic aqueoussolubility of the drug, resulting in the difficulty to maintain sinkconditions. Taken together with the limited analytical sensitivity

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nd technical issues such as nonspecific drug adsorption to filtersnd other components of the dissolution apparatus, this may leado irreproducible and inaccurate dissolution data and release pro-le assessment. To overcome this issue, a non-aqueous dissolutionedia or simple surfactant solution can be utilized. It has to be

mphasized though, that similarity between such media and the GIilieu is limited. Thus, in-order to improve in vitro in vivo predic-

ion, a modified dissolution media that more accurately reflects theolubilization capacity of the GI fluids was developed. This medias generally based on the presence of bile salts and phospholipids,

imicking either fasted or fed GI conditions (Kohli et al., 2010).Studies performed with prednisolone demonstrated the suit-

bility of lipid base solid nano-particulates for prolonged drugelease. The release profile can be further modified by alteringhe lipid matrix, surfactants, and production parameters. Carefuldjustment of these parameters can result in particles with a pro-onged release profile of up to 5–7 weeks (zur Muhlen et al., 1998).

An immediate burst effect that releases a major portion ofhe drug in a short period of time is a major challenge in theevelopment of lipospheres. This phenomenon was observed byortesi et al., who demonstrated a biphasic release profile of sodiumromoglycate encapsulated in lipospheres. The first part was char-cterized by rapid drug release followed by a slower release rate,uring which the drug was released in a linear mode (Cortesi et al.,002). This can be explained by surface adsorption, rather thanncapsulation of the drug in the lipid matrix. A variety of factors,uch as the concentration of lipids, drug solubility inside the lipidore, drug-lipid interactions, production temperature, surfactantelection and particle size can promote surface adsorption (Sawantnd Dodiya, 2008). However, the particle structure and the shell-nriched model discussed here, particularly in Section 3.5, canrovide an additional explanation.

.4. Structure and morphology

Scanning electron microscopy, transmission electronicroscopy, and atomic force microscopy are very useful

echniques to determine the shape and morphology of lipidanoparticles. These techniques can also determine the particleize and size distribution. TEM utilizes electron transmissionhrough the sample, while the image is produced by interpretinghe interaction of the electrons passed through the specimen,hich is consequently visualized. SEM utilizes electron transmis-

ion from the sample surface and offers excellent resolution andasier sample preparation (Sawant and Dodiya, 2008). In contrasto PCS and LD, SEM and TEM provide direct information on thearticle shape and size. Several SEM (Varshosaz et al., 2009) andEM (Tsai et al., 2010) studies showed a spherical shape of theipid nanoparticles (Das and Chaudhury, 2011). TEM has smallerize limit detection, and is a good validation for other methods.owever, the statistically small sample size and the effect ofacuum on the particles must be taken into account.

Although normal SEM is not very sensitive to the nanometerize range, field emission SEM (FESEM) can detect nanometer sizeange. However, sample preparation (e.g., solvent removal) maynfluence the particle shape. Cryogenic FESEM might be helpful inhis case, where liquid dispersion is frozen by liquid nitrogen and

icrographs are taken in the frozen condition.The AFM technique (Drake et al., 1989) is also gaining popu-

arity for nanoparticle characterization (Shahgaldian et al., 2003).FM provides a three-dimensional surface profile, unlike elec-

ron microscopy which provides a two-dimensional image of a

ample. AFM directly provides structural, mechanical, functional,nd topographical information about surfaces with nanometer-o angstrom-scale resolution. In this technique, the force actingetween a surface and a probing tip results in a spatial resolution of

s of Lipids 165 (2012) 438– 453 443

up to 0.01 nm for imaging. Direct analysis of the originally hydrated,solvent-containing samples is possible as no vacuum is needed dur-ing operation and the sample does not need to be conductive. ZurMuhlen compared AFM with SEM and reported the same particlesize for the nanoparticles by both methods (Das and Chaudhury,2011; zur Muhlen et al., 1996).

NMR can be used to determine both the size and the qualita-tive nature of nanoparticles. The selectivity afforded by chemicalshift complements the sensitivity to molecular mobility to provideinformation on the physicochemical status of components withinthe nanoparticle (Mukherjee et al., 2009).

In the following studies, for instance, in order to determine thelamillarity of lipospheres obtained at different phospholipid-to-neutral lipids ratios, NMR spectroscopy was used in the presence ofparamagnetic ions. The phospholipid content on the surface of lipo-spheres was determined by 31P-NMR before and after manganese(Mn2+) or preseodimium (Pr3+) complexation and by trinitroben-zenesulfonic acid (TNBS) labeling using liposphere formulationscontaining phosphatidylethanolamine (PE) (Fenske, 1993). In bothmethods, the agents, Mn2+ or TNBS, interact specifically withthe exposed phosphate or amino groups of the phospholipid orphosphatidylethanolamine. Determination of the surface phos-pholipid using the TNBS method showed that 70–90% of thephospholipid polar heads are in the surface of the liposphereparticles prepared from triglyceride:phospholipid ratios of 1:0.5to 1:0.25 (w/w). Increasing the phospholipid content decreasesthe percent of surface phospholipid polar heads which indicatesthe formation of other phospholipid structures in the formula-tion, such as liposomes. Similar results were obtained by 31PNMR analysis using Mn2+ and Pr3+ ions for interaction with thephosphate polar heads. Lipospheres composed of tricaprin: phos-pholipid at a weight ratio of 2:1 and 5:1 showed 75% and 90%of surface phospholipid polar heads, respectively. For comparison,liposomes of the same composition and size but without tricaprinhad only 40% of their phospholipid polar heads in the surface(Fenske, 1993). This data suggests that the proposed structure ofa liposphere is a spherical particle with a monolayer of phos-pholipid molecules surrounding the internal solid fat core, wherethe hydrophobic chains of the phospholipids are embedded ontothe surface of the internal triglyceride core containing the activeagent.

3.5. Drug incorporation and loading

A very important point in the judgment of the suitability of alipid based drug carrier system is its loading capacity (Muller et al.,2000). First, we must differentiate between loading capacity (LC)and entrapment efficiency (EE). EE is defined as the percentageof drug incorporated into the lipid based particles, relative to thetotal drug added, i.e. percent of drug included in the particles vs.percent of drug remaining in the dispersion medium. LC is the per-centage of drug incorporated into the lipid particles, relative to thetotal weight of the lipidic phase (drug + lipid). Ultrafiltration andmicrodialysis are considered the most reliable techniques for EEquantification, while results obtained by ultracentrifugation, thefastest and easiest technique, are not always accurate (Liu et al.,2009). Entrapment efficiencies are usually high and might rangebetween 80% and 99%, depending on the incorporated drug, whileLC is the more important parameter for characterization and opti-mization of lipid based drug carriers. The LC mainly depends onthe solubility of the drug under investigation in the core lipid/lipidsblend, miscibility of drug melt and lipid melt, chemical and physical

structure of the solid lipid matrix and the polymorphic state of thelipid. The reported LC values range between 1% for prednisolone,20–25% for CsA and up to 50% for extremely lipophilic substancessuch as Vitamin E (Muchow et al., 2008).

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Mehnert et al. (zur Muhlen et al., 1998) proposed three incor-oration models for drugs in lipid based nano-particulate systems,ased on the preparation method, melting temperature and relativeolubility of the drug and the lipid core. In the first, solid solutionodel, the drug is molecularly dispersed in the lipid matrix. This

tructure is obtained when the particles are produced by the coldomogenization technique, without the use of lipid solubilizingurfactants. The second model, the drug enriched core structure,s obtained by production using the hot homogenization tech-ique; the drug precipitates first, before the lipid recrystallizes. Therug enriched shell structure is explained by a lipid precipitationechanism. After homogenization, the mixture of drug and lipid is

ooled. If the lipid precipitates before the drug, a drug free core, or core with reduced drug content is obtained. Another reason foruch structure formation could be improved drug solubility in theater–surfactant mixture at high temperatures. During the coolingrocess, the solubility in this outer phase decreases and the druge-enters the lipid core, resulting in a drug enriched shell, in thease where the particle core has already started to solidify (Mullert al., 2000). Such a particle structure might lead to a burst drugelease, which is a desirable effect when the drug must produceigh peaks of plasma concentrations in order to be effective, suchs CsA (Muchow et al., 2008).

The compatibility of the encapsulated drug with the solid coreaterial is a key issue for maintaining the drug in the liposphere

articles. When an incompatible solid core is used, the drug mayigrate out of the lipospheres and crystallize in the solution. Thisas demonstrated with bupivacaine free base incorporated in ethyl

tearate. Bupivacaine migrates out of the particles and crystallizeso form needle-like crystals. The migration process is a result ofradual dissolution of the drug by the aqueous medium to satura-ion where the drug molecules start to precipitate from the solutiono form crystals. To avoid this migration, which occurs in the pres-nce of water, the liposphere dispersion should be lyophilized andept dry until reconstituted shortly before use (Domb, 2006).

The viscosity of the liposphere formulation is dependent onhe drug properties, the ionic strength and pH of the continuousqueous solution, and the ratio and amount of phospholipids andriglycerides. An increase in the content of the insoluble ingredi-nts (drug and lipids) and in the salt concentration of the aqueousedium increases the viscosity of the formulation. In several stud-

es, the LC of drugs such as oxytetracycline, itraconazole andexamethasone into lipospheres was assessed and was reportedo be as high as 20%, maintaining the formulations fluid enough toe injected. However, for other agents, like bupivacaine, lidocaine,nd chloramphenicol, a LC of above 10% was possible, but produced

viscous lotion (Domb, 2006).

.6. Crystallinity and polymorphism

A liposphere is always referred to as a spherical liquid dropletcore) surrounded by an emulsifier shell. Since the core compo-ent is a solid lipid, a crystalline substance, the obtained particlesill also crystallize upon solidification and exhibit all the features

f crystalline materials. This includes a solid–liquid transition at certain temperature and the occurrence of various crystallineodifications when polymorphic raw materials such as triglyc-

rides are used (Bunjes, 2010). Polymorphic substances usuallyorm metastable modifications on crystallization and transitionnto more stable forms might occur upon storage (Freitas and

uller, 1999). Such polymorphic transitions may result in reduc-ion of drug load efficiency due to alterations in the core lipid

acking and thus in the whole particle structure (Jenning et al.,000). Both the crystallization behavior and the kinetics of poly-orphic transitions can be modified by the type of emulsifier

sed for the stabilization of the nanoparticles. In addition, the

s of Lipids 165 (2012) 438– 453

particle shape might be affected by the polymorphic transitions(Bunjes et al., 2003). Determination of the crystallinity of the com-ponents of liposphere formulations is also crucial since both thelipid matrix and the incorporated drug may undergo a polymorphictransition leading to possible undesirable drug expulsion duringstorage (Souto et al., 2006). Lipid crystallinity is also strongly cor-related with drug incorporation and release rates. Thermodynamicstability and lipid packing density increase, whereas drug incor-poration rates decrease in the following order: supercooled melt,�-modification, �′-modification, and �-modification. If in storagethe lipid forms a more stable and highly ordered crystalline matrix,the result can be undesirable expulsion of the incorporated drug.Triggering factors for the lipid transformation can be temperaturechanges and water loss of the SLN dispersion, e.g. after topicaladministration. However, lipid crystallization and the consequen-tial modifications might be delayed due to the small size of theparticles and the presence of emulsifiers (Muller et al., 2000). Differ-ential scanning calorimetry (DSC) and X-ray diffractometry (XRD)are two widely used techniques for the examination of the crys-tallinity and polymorphic behavior of the ingredients of the lipidbased nano-particulates. Information on the melting and crystal-lization behavior of all solid and liquid components of the particlesmight be obtained by the DSC technique, while XRD can identifyspecific crystalline compounds based on their crystal structure.The basis behind DSC is the fact that different lipid modificationshave different melting points and melting enthalpies. With increas-ing formation of the more stable ˇ′/ˇ′

i-modifications of the lipid

matrix, a higher melting peak will be obtained. On the other hand,a lower melting peak reflects an unstable �-modification. Usually,when the lipid core is comprised of more than one lipid, the ten-dency towards more stable modifications is reduced (Attama andMuller-Goymann, 2007). In XRD, the monochromatic beam of theX-ray is diffracted at angles determined by the spacing of the planesin the crystals and the type and arrangement of the atoms, whichis recorded by a detector as a pattern. Such a pattern is unique toeach type of crystalline material, and the pattern can predict themanner of arrangement of lipid molecules and phase behavior, andcharacterize and identify the structure of lipid and drug molecules(Bunjes et al., 2007). The X-rays reflected from the crystalline lipidswill generally appear above the amorphous background of the non-crystalline lipids (Attama and Muller-Goymann, 2007). It shouldbe noticed that the best results are observed when lipid basednanoparticulate dispersions are investigated directly, since sol-vent removal may affect the modification. Another two techniques,infrared and Raman spectroscopy, are also useful to investigatestructural properties of lipids (Das and Chaudhury, 2011).

3.7. In vitro lipolysis

Lipospheres and PLN involve the use of triglycerides as a phar-maceutically acceptable solid carrier comprising their core. Thus,lipospheres and PLN developed to be orally administered are sub-jected to the digestion process in the GI milieu, similarly to anyingested exogenous dietary fat. Once realizing that the performanceof lipid-based formulations is affected by digestion and the incor-poration of exogenous digestion products into endogenous micellarspecies, there has been an increasingly widespread use of lipiddigestion models for in vitro assessment of lipid-based formula-tions.

The primary mechanism by which lipid-based formulationsenhance drug solubilization within the GI tract is by deliveringthe entire dose as a solution, thus avoiding solid-state limita-

tions (Baker et al., 2007). The solubilized phase is not obtainedsolely from the administered lipid, but most likely from the intra-luminal processing to which lipids are subjected prior to absorption(Humberstone and Charman, 1997; Porter and Charman, 2001).

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Following lipid ingestion, several changes occur in the GI tracthich lead to increased secretion of endogenous biliary-derived

olubilizing components and digestion enzymes such as bile salts,hospholipids, pancreatic lipase and co-lipase from the pancreas.ubsequently, the digested lipids are hydrolyzed to more water sol-ble components, e.g. free fatty acids and monoglycerides which aremulsified with bile salts to form mixed micelles. The formation ofhe aqueous mixed micellar phase significantly expand the solubi-ization capacity of the small intestine for lipid digestion productsnd incorporated drugs (Baker et al., 2007; Porter and Charman,001).

The in vitro dynamic lipolysis model is being increasingly useds a tool to facilitate in vitro evaluation of lipid-based drug deliveryystems in terms of the ability to predict the potential to enhancehe bioavailability of poorly water soluble drugs. Dynamic lipolysis

odels are conducted at 37 ◦C and the reaction medium consists of mixture of bile salts, phospholipids, buffer and a lipid substratee.g. a dietary lipid or a lipid-based formulation incorporating therug substance). The lipolysis process is initiated by addition of

ipase solution to the system. The pH of the reaction mixture isaintained by a computer-controlled addition of sodium hydrox-

de, since with the initiation of the digestion of TG, free fatty acidsre liberated, consequently causing the pH to drop. By using theH stat it is possible to follow the progress or rate of FA liberation.ollowing the digestion process, the reaction medium undergoesltracentrifugation, resulting in separation into three layers: undis-ersed oil typically containing the drug, undigested TG and someG; an aqueous phase containing the drug solubilized in micel-

ar structures composed of BS and PL and lipid digestion productsFA, MG and DG); and a pellet that contains precipitated drugnd insoluble soaps of FA. The model allows obtaining informa-ion regarding the distribution of the drug between the floatingil phase, water phase and pellet phase formed by the digestionrocess, and to inspect which type of lipid will increase the concen-ration of the drug in the aqueous water phase, i.e. in the intestinaluids. The underlying hypothesis in this case is that the waterhase, containing mixed bile salt micelles, represents the pool ofrug readily available for intestinal absorption (Larsen et al., 2008).ubsequently, the final goal is to try to correlate the amount ofoorly water soluble drug in the aqueous phase with its absorp-ion in vivo. A number of research groups investigated the abilityf the model to predict the performance of lipid based formula-ion in terms of in vitro/in vivo correlation. Dahan and Hoffman2007a,b) reported a strong positive correlation between the per-ent of dose solubilized in the water phase generated during in vitroipolysis to the bioavailability in vivo after oral administration ofrogesterone when administered in triglycerides with differenthain lengths. In another study, they demonstrated the same cor-elation for two lipophilic drugs; dexamethasone and griseofulvinDahan and Hoffman, 2007a,b). Porter et al. reported a correla-ion between the fraction of halofantrine absorbed in dogs andhe amount solubilized in the aqueous phase in the lipolysis modelhen solubilized in medium or long-chain triglycerides (Balayssac

t al., 2005). These reports suggest that enhancement in absorptionould be explained by the increase in effective luminal drug concen-ration following digestion of the drug administered.Utilization ofhe in vitro dynamic lipolysis model can serve as a potential tool tonderstand the complex interaction between formulation derived

ipids, the GI milieu and a co-administered drug.

. Applications of lipospheres and PNL

.1. Oral delivery

The majority (∼85%) of the 50 most-sold pharmaceutical prod-cts in the North American and European markets are given orally.

s of Lipids 165 (2012) 438– 453 445

This route is the most preferred mode of drug administrationpresently because of its safety, comfort, low economic burden andimproved patient medication adherence in comparison to alterna-tive administration routes such as intramuscular, subcutaneous,rectal or pulmonary drug delivery (Lennernas et al., 2007). Theintroduction of combinatorial chemistry accompanied by advancesin in vitro high throughput screening methods resulted in the rapididentification of many highly potent but poor water soluble drugcandidates. Many of these compounds fail to proceed to theadvanced stages of research and development mainly due to theirlow bioavailability and high inter-patient variability in absorptionpattern upon oral administration. To date, more than 30% of topmarketed drugs in the USA and 70% of all new drug candidatesare lipophilic and consequently have poor aqueous solubility,accompanied by high membrane permeability (Srickley, 2007),thus falling into class 2 as defined by the biopharmaceutical classi-fication system (BCS) proposed by Amidon et al. (1995). The clinicaluse of such compounds is hampered by low oral bioavailability,high inter/intra subject variability and lack of dose proportionality(Hauss et al., 1998). Development of orally available drugs requiresa profound understanding of the physico-chemical properties ofthe substance, the physiology of the gastrointestinal (GI) tract andthe absorption mechanism of the drug (i.e. passive diffusion, activetransport, facilitated transport, etc.).

An initial rational for the use of lipid vehicles was the revela-tion that co-administration of poorly water soluble drugs with foodcan improve their bioavailability. For example, Gupta et al. (1990)reported in their in vivo studies with cyclosporine A, a BCS class2 drug, that a high fat meal markedly increased its bioavailabilityin healthy volunteers compared to individuals receiving the drugwith a low fat meal.

The three primary mechanisms by which lipids and lipophilicexcipients affect drug absorption, bioavailability and dispositionfollowing oral administration are:

• Pre-enterocyte level: Alteration of the composition and characterof the intestinal milieu and increased solubilization.

• Intra-enterocyte level: Interaction with enterocyte-based trans-port processes such as permeability through the cell membraneand effects on intra-enterocyte metabolism and efflux.

• Post-enterocyte level: Recruitment of intestinal lymphatic drugtransport.

Molecular solubilization of a drug within the gastrointestinaltract is a prerequisite for its absorption into and across the ente-rocyte following oral administration. Only after the drug moleculeis presented in its dissolved state it can enter into the enterocyteand finally cross it. Thus, the absorption of lipophilic drugs can belimited by both the dissolution rate and extent. Thermodynami-cally, solubilization requires reduced intermolecular forces in thesolid state and enhanced solute–solvent interactions in the bulksolution. The primary mechanisms by which lipid-based drug for-mulations enhance drug solubilization within the GI tract are bypresentation as a solubilized formulation (thereby avoiding solid-state limitations) and by inducing changes to the character of theGI environment such that solute–solvent interactions and drug sol-ubility are enhanced.

The solubilization capacity of a drug in the GI fluids can beregarded as the combined effects of the intrinsic aqueous solu-bility of the drug, the enhancements in solubility resulting fromthe presence of endogenous solubilizing components, and theenhancements in solubility resulting from the presence of the lipid-

based formulation derived components (Porter et al., 2007).

The presence of lipids in the GI tract stimulates gall bladdercontracts and biliary and pancreatic secretions, including bile salts,phospholipids, and cholesterol. These products, along with the

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astric shear movement, form a crude emulsion, which promoteshe solubilization of the co-administered lipophilic drug (Dahannd Hoffman, 2007b).

Concurrently, certain exogenous components might enhancerug solubilization. Typical examples of such components includeurfactants, co-solvents and complexation agents. The solubi-ization capacity of the GI tract is therefore determined by thenteraction of exogenous lipids with the GI environment, thehysiological changes that the lipid component stimulates andhe combined involvement of both exogenous and endogenousomponents in the colloidal species that support enhanced drugolubilization.

The colloidal structures formed during the digestion of lipidsrovide a lipophilic phase within which lipophilic drugs mighteside during GI transit, thereby preventing precipitation andnhancing absorption of the drugs. The nature of the colloidalpecies formed by the intercalation of formulation componentsnd their digestion products with endogenous bile salt, phospho-ipid and cholesterol species is a crucial determinant of the drugolubilization and drug absorption patterns. As the initial physico-hemical and colloidal properties of a lipid based formulation mighte expected to persist for only a limited period in vivo, it is the col-

oidal species that form after interaction of the formulation withhe GI environment that are the actual ‘carriers’ or solubilizingpecies for co-formulated poorly water-soluble drugs (Porter et al.,007).

In addition, lipids in the GI tract provoke delay in gastric emp-ying, i.e., gastric transit time is increased. As a result, the inputate of the coadministered lipophilic drug into the small intestineecreases. This enables better dissolution of the drug at the absorp-ive site, and thereby improves absorption (Dahan and Hoffman,007b). Additionally, this phenomenon is extremely beneficial forompounds with a narrow absorption window located at the upperIT (Kagan and Hoffman, 2008).

Further barrier to the effective uptake of lipophilic moleculesrom the intestinal lumen into enterocytes is diffusion across thenstirred water layer (UWL), which separates the bulk fluid phasef the small intestine lumen from the brush border membrane ofhe enterocytes. The UWL mixes poorly with the bulk fluid phasend together with intestinal mucus forms an acidic microclimatedjacent to the brush border membrane. Thus, the UWL represents

major diffusional barrier for lipids and lipophilic molecules asheir solubility in aqueous media is extremely low (Porter et al.,007).

Hydrophobic compounds permeate lipophilic membrane muchaster than they can be transported through the UWL. Under suchonditions, diffusion through the UWL becomes the rate-limitingtep in the absorption process. Solubilization of lipophilic drugs inicellar and mixed-micellar structures, such as colloidal species

ormed by the intercalation of formulation components and theirigestion products with endogenous bile salt, phospholipid andholesterol species can greatly enhance the mass transport ofolecules across the unstirred water layer, thereby enhancing

ipophilic compounds absorption. Upon reaching the enterocyterush border, lipophilic molecules dissociate from the mixed micel-

ar phase before partitioning into the enterocyte. Free lipophilicrug molecules can then be absorbed across the apical membraney passive diffusion or carrier-mediated transport.

Further barrier towards reaching the systemic circulation fromhe GI lumen is the enterocyte cell membrane. A variety of lipids andharmaceutical excipients commonly utilized in lipospheres andNL formulations have been shown to change the physical barrier

unction of the gut wall, and hence, to enhance permeability. It isnown that pharmaceutical non-ionic surfactants increase the per-eability of cell membranes in a concentration dependent manner

Dimitrijevic et al., 2000).

s of Lipids 165 (2012) 438– 453

Next barrier towards oral bioavailability of poorly water sol-uble compounds is the simultaneous activity of the multidrugefflux pumps and Phase I metabolism by the intestinal cytochromeP450s inside the enterocyte (Benet, 2010). In some cases, as shownrecently, excipients incorporated into lipid based formulations caninhibit both presystemic drug metabolism and intestinal effluxmediated by P-gp resulting in an increased oral absorption of BCSclass 2 compounds. The potential for lipidic excipients to attenu-ate the activity of P-gp efflux pumps was demonstrated in severalrecent studies. Vitamin E-TPGS (d-�-tocopheryl polyethylene gly-col 1000 succinate) is a good example, as it increases the absorptionflux of amprenavir (Yu et al., 1999), and has been characterized asan inhibitor of P-gp-mediated drug transport in the human intesti-nal Caco-2 cell monolayers and other cell lines (Dintaman andSilverman, 1999). It has been shown to enhance the bioavailabilityof CsA in human volunteers (Chang et al., 1996) and of colchicine inrats when the drugs were administered orally (Bittner et al., 2002;Cornaire et al., 2004).

Another mechanism by which lipospheres and PNLs compo-nents may impact the disposition of lipophilic compounds isthrough inhibiting the cytochrome P450 enzymes in cellular micro-somes of the enterocytes. In the study by Ren et al. (2008), 22pharmaceutical excipients were tested for their ability to inhibitthe activity of CYP3A4 enzymes. Fifteen of the tested excipientsinhibited the activity up to 50%. Similar results were obtained intheir in vivo midazolam (a CYP3A4 substrate) study upon single andchronic administration of the excipients under investigation. More-over, the inhibition of CYP450 activity was observed with additionalamphiphilic ingredients (Mountfield et al., 2000).

Bioavailability of lipophilic drugs may be enhanced also bythe stimulation of the intestinal lymphatic transport pathway.After absorption into the enterocyte, lipophilic drug either diffusesdirectly across the cell and enters the portal vein, which leads toaccess to the systemic circulation by the liver, or is trafficked intra-cellularly to the endoplasmic reticulum. Subsequently the drugconstitutes a core lipid component of intestinal lipoproteins thatfuse with the basolateral cell membrane of the enterocyte beforerelease into the interstitial space. Following exocytosis, the imper-meability of the vascular endothelium combined with the largeinter-endothelial gaps present in the lymphatic endothelium pref-erentially direct lymph lipoproteins towards selective uptake bythe intestinal lymphatic system rather than the blood capillaries.The unique anatomy and physiology of the intestinal lymphaticsystem provides a number of drug transport advantages when com-pared with portal blood transport. Mainly, drugs that enter themesenteric lymph are directly transported to the systemic circula-tion without first passing through the liver. As such, augmentationof drug uptake into the lymph system reduces the opportunityfor hepatic first-pass metabolism and might therefore be a usefulmechanism to enhance the bioavailability of drugs in which signif-icant hepatic first-pass metabolism is a limitation to oral bioavail-ability (Porter et al., 2007). Lipid base formulations share the poten-tial to enhance the lymphatic transport of highly lipophilic drugs.Various mechanisms of targeting drugs to intestinal lymphaticsinclude the paracellular mechanism, transport through M cells ofPeyer patches and the transcellular mechanism. Among these, tran-scellular mechanism is the most relevant for the transport of lipidcarriers. Accomplishment of lymphatic targeting can be achievedthrough lipid-based carrier systems such as microemulsions,nanoemulsions, liposomes, lipospheres, and SLN. Lipid based nanoparticulate systems can induce stimulation of chylomicron forma-tion by enterocytes which promote the absorption of a lipid matrix

through intestinal lymphatics (Hu et al., 2004). For example, SLNhave been reported to enhance the oral bioavailability of docetaxel(Nassar et al., 2011) and lopinavir (Aji Alex et al., 2011). Bargoniet al. (1998) demonstrated the role of the intestinal lymphatics in

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ptaking SLN after intraduodenal administration. Several consid-rations have to be taken into account upon designing lipid basedano carrier system for enhanced lymphatic absorption of a drug.oth the type and mass of co-administered lipid can alter the extentf lymphatic drug transport. Fatty acids (FA) with chain lengths of4 or greater are more lymphatically transported than shorter chainA (which are more water soluble and are primarily absorbed viahe portal blood). As such, long chain FA and triglycerides moreffectively support lymphatic drug transport than their mediumnd short chain counterparts. The degree of unsaturation of thedministered FA also influences the extent of lymphatic lipid andrug transport. In general, mono- and poly-unsaturated FA produce

arger sized lipoproteins and therefore enhance lymphatic drugransport more effectively when compared with the equivalent sat-rated FA. Phospholipids also enhance lymphatic lipid transportTrevaskis et al., 2008). Conversely, Hussain et al. (2001) had sev-ral reservations regarding the lymphatic absorption potential ofntact solid nano-particulate systems. He rightly claims that chy-omicrons are formed by the re-synthesis of fatty acids in the Golgiomplex of the enterocyte, upon adding multiple apolipoproteinsnd lipids which have a vital role in chylomicron transport. Hence, itemains unclear how drugs will remain attached to the lipid particlehrough the various stages of metabolism and modification.

Nano-sized particulates have several additional advantages iterms of improving oral bioavailability. First, a general property ofanoparticles is that they are adhesive (Tarr and Yalkowsky, 1989).fter adhesion to the gut wall, the drug can be released exactly at

he site of absorption. This adhesion of lipid based nanoparticlesf CsA was demonstrated by Muchow et al. (2008), and resulted ineduced variability of plasma CsA concentrations, and more pre-ictable absorption similar to that of the commercially availableandimunn® Neoral. An additional nanoparticulate feature that hasbsorption augmentation potential is the surface charge of the par-icles (Gershanik and Benita, 1996; Gershanik et al., 1998). In atudy carried out on perfused rats, the administration of CsA in aositively charged self-emulsifying drug delivery system resulted

n elevated drug plasma concentrations as compared to negativelyharged formulation (Gershanik et al., 2000). The reason behindhis phenomenon is probably the negatively charged surface com-onents of the GI mucosa that readily interact with the positivelyharged particles.

An additional advantage of oral delivery of BCS class 2 com-ounds as lipospheres or PNL is the ability to maintain controlledelease properties of the delivery system, when the lipid coreemains solid at body temperature. As opposed to microemulsions,hat release their contents instantaneously, in the case of lipo-pheres or PNL it is possible to control the rate of drug releaserom the delivery system by accurate selection of the formula-ion ingredients. This trend was demonstrated by Muller and Keck2004) in their bioavailability study of CsA. They were able to avoidhe undesired nephrotoxicity of Sundimmunn® Neoral caused by

high plasma CsA peak, while maintaining the low variability inlasma concentrations and high bioavailability, by loading CsA intoLNs.

Lipid based nano-particulates have been shown to improve theral bioavailability of several poorly water-soluble and lipophilicrugs (Hauss et al., 1998; Bekerman et al., 2004; Charman, 1998;orter et al., 2004; Nielsen et al., 2008; Mueller et al., 1994).acrolimus bioavailability studies following incorporation into PNLere recently performed by our group. Tacrolimus is a potent

mmunosuppressant (Spencer et al., 1997, Shapiro, 1999) forhich absorption barriers are responsible for its poor and variable

ystemic bioavailability following oral administration (Kagayamat al., 1993). Its clinical use, in spite of its proven efficacy andavorable adverse effect profile, is hampered by extremely highnter- and intra-patient variability and a poor dose-plasma

s of Lipids 165 (2012) 438– 453 447

drug concentration correlation caused by extensive presystemicmetabolism by intraenterocyte CYP 3A enzymes and efflux by P-gp pumps (Kino et al., 1987; Shimomura et al., 2002; Tamura et al.,2002). For tacrolimus bioavailability studies, tacrolimus PNL werefreshly prepared 30 min before each experiment, and administeredto the animals (1 mg/kg) by oral gavage (n = 6). The control groupreceived 1 mg/kg tacrolimus suspension prepared from the con-tent of Prograf® capsules suspended in water (n = 6). Blood sampleswere collected and analyzed for tacrolimus concentration. Based onthe AUC calculations, the relative bioavailability of tacrolimus-PNLwas increased 1.5 folds in comparison to tacrolimus alone (AUC val-ues of 69.4 ± 8.96 h × ng/ml vs. 40.8 ± 10.7 h × ng/ml, respectively).Similar results were obtained for the Cmax values (unpublisheddata). Furthermore, significant reduction of the variability intacrolimus plasma concentrations calculated using the coeffi-cient of variance parameter was evident following tacrolimus-PNLadministration.

In conclusion, lipospheres and PNL are especially advantageousin the case of oral administration of hydrophobic compounds, interms of stimulation of endogenous solubilization processes, adhe-sion to GI mucosa, increased transcellular transport, controlleddrug release, stimulation of lymphatic absorption and avoidanceof intestinal first pass metabolism. Careful and intelligent selec-tion of the lipids, surfactants an co-solvents composing the deliverysystem, combined with profound understanding of oral absorptionpathways and obstacles, will enable further achievements in thefield of improved oral absorption of lipid based nano-particulatedrug delivery systems.

4.2. Ocular drug delivery

The main challenge in ocular drug therapy is to provide andmaintain an adequate drug concentration at the action site. Themajor obstacles associated with currently used eye drop solutionsinclude poor ocular bioavailability, pulse drug release, systemicexposure due to nasolacrimal drainage and poor entrance tothe posterior segments of the eye (Eljarrat-Binstock and Domb,2007). The poor bioavailability results directly from the ocularanatomical and physiological structure. Blinking, dilution and naso-lacrimal drainage are responsible for clearance of approximately90% of a standard dose of eye drops within 2 min (Davies, 2000).Furthermore, the cornea is a highly efficient absorption barriercomposed of several layers: the lipophilic epithelium layers, con-nected by tight junctions and permeable only to small lipophilicdrug molecules, while the hydrophilic stroma consists of fibroustissue that restricts lipophilic compound penetration (Gershkovichet al., 2008). This unique combination of hydrophilic and lipophiliclayers presents a major obstacle for ocular drug absorption. Fur-thermore, the vast majority of a dose administered topically istaken up by the conjunctiva rather than cornea due to its largersurface area, rich blood flow and membrane leakiness. Addition-ally, systemic absorption is responsible for drug uptake throughthe embedded blood vessels prior to intraocular diffusion (Bourlaiset al., 1998). Therefore, frequent drug administration of high dosesis required, which often causes high drug concentration fluctua-tions and various systemic side effects.

It is important to notice that PNL are not a suitable delivery sys-tem for ocular drug administration, since the limited liquid volumeof the eye fluids and lack of agitation will not enable in situ emul-sification and nano-liposphere assembly. Nano-sized lipospheres,on the other hand, are a promising delivery system for this purposedue to several physicochemical properties of this unique carrier

system.

Lipospheres have the advantage in ocular drug delivery for itwas found that one of the criteria for a particle to enter ocularmucosa, apart from lipophilicity, is that it should be of submicron

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ize (Alonso, 2004). For an ocular drug delivery to be successful, ithould have a small particle size, with a narrow size range, shoulde nonirritant, compatible with ocular tissue, and cause no blurredision (Bourlais et al., 1998; Sahoo et al., 2008). The colloidal char-cter of a drug carrier such as nanosized lipospheres improves drugenetration by prolonging the ocular residence time, reducing theasolacrimal drainage and increasing interaction with corneal sur-

ace, combined with the advantage of being an easy-to-use liquidosage form. The small particle size is also advantageous in termsf patient comfort since they do not cause a “scratchy” feeling ormpaired vision following ocular administration (Eljarrat-Binstocknd Domb, 2007). Submicron sized particles have an extremely highurface to volume ratio, and therefore have an increased dissolutionate, advantageous for absorption of such poor water soluble com-ounds as paclitaxel, CsA and amphotericin B, which are frequentlysed in ophthalmic therapy (Wissing et al., 2004).

Several unique considerations have to be taken into accounthen dealing with drug carriers for ocular delivery.

For ophthalmic lipid based DDS, the oil concentration should beimited to 5% (w/w) to prevent blurry vision and eye irritation. Inddition, ocular preparations should be isotonic in order to be com-atible with ocular fluids. For this purpose, tonicity modifiers suchs glycerol must be added to the formulation. The irritation maylso arise due to emulsifiers used for the preparation of ocular lipo-pheres (Tamilvanan and Benita, 2004). Additionally, the deliveryystem should be easily sterilized, since sterility is a prerequisiteor all ocular preparations. The sterilization technique should avoidegradation or aggregation of the solid lipids, in order to preventoxicity or instability (Mehnert and Mader, 2001). Commonly usedechniques for the sterilization of lipid based nano-particulatesre filtration, aseptic production and �-radiation (Seyfoddin et al.,010).

SLN were assessed by Cavalli et al. (2002) as ocular lipid basedano-particulate delivery system for tobramycin. The aqueousumor concentration of tobramycin was determined for up to

h following topical administration of tobramycin-loaded SLN toabbits. When compared to tobramycin administered by standardye drops, tobramycin-SLN administration resulted in significantlyigher bioavailability. A promising lipid base nano-particulateelivery system was developed by Attama et al. (2008). The coreonsists of theobroma oil containing phospholipids that couldrove to be a good ocular drug delivery system considering the

ow particle size, particle size stability and in vivo tolerability ofhe component lipids. The obtained particles demonstrated a con-rolled drug release profile and negligible toxicity.

An additional example for the beneficial effect of lipospheresn ocular lipophilic drug delivery is the case of CsA. The commer-ially available product Restasis® ophthalmic emulsion (CsA 0.05%)s indicated to increase tear production in patients whose tear pro-uction is presumed to be suppressed due to ocular inflammationssociated with keratoconjunctivitis sicca. This condition is alsonown as chronic dry eye or dry eye syndrome. The most commondverse event was ocular burning (upon instillation) – 17% (PDR,012). Avoiding this unpleasant side effect is of extreme impor-ance, taking into consideration that the dry eye syndrome is ahronic condition that requires prolonged treatment. Therefore,everal liposphere-based delivery systems of CsA were designed,eveloped and assessed in vitro by our group (unpublished data).he optimized formulation resulted in a particle size of 40–50 nmith a narrow size distribution and was further assessed in vivo

n rabbits. Two major parameters were evaluated following top-cal administration of our PNL vs. Restasis®: CsA’s penetration to

arious eye segments and ocular burning. The ocular burning wasssumed to be directly proportional to the blinking frequency andBS was used in the control group. The results demonstrate awofold reduction in blinking frequency in the liposphere group as

s of Lipids 165 (2012) 438– 453

compared to the Restasis® group (3 vs. 7 blinks per minute, respec-tively). As for CsA distribution to various eye compartments, nosignificant difference was found between liposphere and Restasis®

groups, implying equally high distribution of the drug in the eye.Thus, our lipospheres are a promising formulation for ophthalmicCsA delivery that causes less eye irritation and burning in compar-ison to the commercially available formulation, probably due tosignificantly lower surfactant content.

4.3. Parenteral delivery and CNS targeting

One of the challenges in pharmaceutical science is parentaldelivery of lipophilic molecules. Their low water solubility raisesthe need to use solubilizing agents, some of which are possiblytoxic. Thus, there is a need for safe and efficient DDS for sys-temic and especially IV administration of lipophilic drugs. Lipidbased nano-encapsulation systems present an opportunity to sys-temically deliver hydrophobic drugs while avoiding the use ofpotentially toxic solubilizing agents. The use of lipid particlessuch as nano-lipospheres has a very desirable advantage, that is,its matrix is composed of biodegradable and well-tolerated lipidcompounds and excipients of an accepted regulatory status (e.g.GRAS; generally recognized as safe status) (Souto and Muller,2007). For instance, listed below are several surfactants whichhave been regulatory accepted for use in parenteral formulations;lecithins, polyssorbate 80 (Tween 80), poloxamer 188, sodium gly-cocholate, sorbitan esthers (Span 85), and low molecular weightpolyvinylpyrrilidone (PVP) (Schwarz and Mehnert, 1999; Morelet al., 1995).

Following IV administration, lipospheres are cleared from thecirculation by the reticuloendothelial system (RES) consisting ofphagocytic cells originating from the bone merrow, and are par-ticularly distributed into the liver (e.g. Kupffer cells of the liver)and the spleen, tissues enriched in these phagocytic cells. Thus, theparticles are highly up-taken by the RES system before reaching thetargeted organ, e.g. the brain (Uner et al., 2004). This feature can beutilized in the case of a tumor located in the liver, where nanopar-ticles can serve as passive targeting for the treatment of hepaticcancers (Chiannilkulchai et al., 1990). Otherwise, the application oflipid nanospheres, for instance, for CNS targeting, is rather limitedby the relatively intensive clearance from the circulation. Coatingthe nanoparticles with a hydrophilic coat of polyethylene oxide andblocking polyoxyethylene polypropylene co-polymers, a techniqueknown by the name “stealth technology” creates sterically stabi-lized lipid-based nanoparticulates which are not recognized by theRES system (Bocca et al., 1998). Avoiding the RES by applying stealthtechnology prolonged the circulation time of the particles in thesystemic blood and enabled to target the delivery of drugs moreprecisely and to increase drug concentrations in tumors, brain andthe cerebrospinal fluid (Fundaro et al., 2000; Chen et al., 2001). Fun-daro and coworkers assessed the pharmacokinetic parameters andtissue distribution of doxorubicin incorporated into a lipid basedencapsulation system after IV administration to rats. 24 h afteradministration of the lipid based encapsulation system doxorubicinand its metabolite were still present in the blood, while they wereundetectable after the injection of the commercial solution. More-over, significantly higher drug concentrations were found in thebrain of the rats treated with doxorubicin incorporated into thelipid based encapsulation system (Fundaro et al., 2000). This studydemonstrated the potential of the lipid based encapsulation sys-tem to achieve prolonged drug plasma levels (Charuk et al., 1995).

Prolonged circulation time creates a larger period of time for thedrugs to reach the CNS. Similar findings were reported by Chenet al. (2001), as their findings indicated that such a hydrophilic coat-ing increased the accumulation of paclitaxel incorporated into lipid

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ased encapsulation system in the tumor tissue. zur Muhlen et al.1998) reported that prednisolone loaded in a lipid based encap-ulation system showed a distinctively prolonged release over aonitored period of 5 weeks in vitro. Prolonging the circulation

ime of the drug in the systemic blood may enable the reductionf the dosing frequency, thus potentially reducing the side effectsf some drugs. Additionally, reducing dosing frequency may lessenhe economic burden associated with parenteral drug administra-ion.

In another study, Gasco and coworkers developed and assessedhe pharmacokinetic and tissue distribution of non-stealth andtealth lipid based encapsulation system incorporating doxorubicinith an increased concentration of stealth agent. The formula-

ions were administered IV to rabbits. The AUC of doxorubicin wasncreased while clearance rates were decreased as a function of themount of stealth agent, indicating that the AUC and the circulationime can be modulated by varying the amount of stealth agent used.

oreover, by increasing the amount of stealth agent employed, anncrease of doxorubicin concentration in the brain was achieved.his indicates that relatively small amounts of stealth agent notnly improved the circulation time but also increased the amountf doxorubicin in the brain. The authors attributed the brain target-ng of the stealth lipid based encapsulation system to the retentionf lipid nanoparticles in brain–blood capillaries with absorptiono capillary walls. This could create a higher concentration gra-ient leading to enhanced transport across the endothelial cells.nother explanation could be that lipid nanoparticles may be endo-ytosed or transcytosed through the endothelial cell barrier. Lipidanoparticles could also permeate the tight junctions between thendothelial cells (Zara et al., 2002a).

Another rather effective strategy to avoid the rapid clearancef the IV administered lipospheres from the blood circulation is byltering another physicochemical property of the particle, such as,article size. Fenestrations in the spleen typically do not exceed00–500 nm in width (Moghimi et al., 2001). Nevertheless, ideally,he size of the particles should not exceed 200 nm, with a preferableange of the particles ranging between 120 and 200 nm in diame-er, if particles are not deformable, in order to substantially avoidarticle trapping in the RES (Moghimi et al., 2001).

Since the in situ assembly of PNL involves the release of anrganic amphiphilic co-solvent at the site of formation, i.e. in thelood, the utilization of lipospheres as a delivery system for IVdministration is more appropriate, as its potential toxicity is muchower.

The nanosize range of the lipospheres (Mishra et al., 2009), theelatively slow and prolonged release of the drug from the lipo-phere system of up to 5 weeks in vitro (zur Muhlen et al., 1998),nd the use of biodegradable carrier lipids and excipients of anccepted regulatory status (Mishra et al., 2009) makes them therug delivery system of choice for IV administration and suitableelivery systems for CNS targeting.

. Current status of lipospheres and PNL’s in research andlinic

During the past two decades the potential of utilizing lipid-ased dispersion systems as efficient drug carriers for differentdministration routes have been recognized by many researchersnd have been extensively studied both in vitro and in vivo inhe attempt to develop commercial products. However, despitehe attractive advantages attributed to lipid dispersion systems

nd immense efforts put into research, currently, there are only

few commercially available systems based on solid fats andhospholipids such as lipospheres, PNLs and SLN. The relativelyigh manufacture costs combined with possible active ingredient

s of Lipids 165 (2012) 438– 453 449

instability are mainly responsible for this phenomenon. Herein, webring some examples of products which successfully passed all thestepping stones on the way from laboratory research to clinicalapplication.

Deximune® (Dexel Pharma Ltd.) is a PNL formulation approvedby the EMEA and is commercially available today. Deximune® con-tains the immunosuppressive compound cyclosporine A (CyA) at adosage strength of 25, 50 and 100 mg. It is indicated for prophylaxisof organ rejection in kidney, liver and heart allogenic transplants inconjunction with corticosteroids. It may also be used in the treat-ment of chronic rejection in patients previously treated with otherimmuno-suppressive agents, bone marrow transplantation andendogenous uveitis. CyA is a highly lipophilic molecule with limitedwater solubility and extensive intestinal first pass metabolism andefflux, resulting in poor and highly variable absorption from the GItract. The PNL formulation Deximune® contains CyA in a mixtureof Polysorbate 20, Sorbitan oleate, lecithin, tricaprine, macrogol-glycerol hydroxystearate and ethyl lactate, all approved for clinicaluse. This solution, which presents self-assembling PNL, is loadedinto soft gelatin capsules and is administered orally. When the con-tent is released into the GI milieu, a nanodispersion with a particlesize of 25 nm is spontaneously formed in situ. The bioavailabilityof CyA in the product Deximune is similar to the commercial prod-uct Sandimmune Neoral® (Novartis), which forms a microemulsionin the stomach, and is up to 50% greater than that of the originalSandimmune® formulation composed of oil and alcohol solutionof the drug {Mueller et al., 1994} (Bekerman et al., 2004). Possiblemechanisms responsible for the increased bioavailability of CyAPNL are inhibition of the intra-enterocyte P-gp efflux pumps andintra-enterocyte metabolism by CYP 3A enzymes (as described indetail in Section 4.1 of this review).

SkedaddleTM (Little Point Corp., Cambridge, MA) is anotherliposphere-based product which was approved for use by the Envi-ronmental Protection Agency (EPA) in the USA in 1992 to be sold asan insect repellant for children over the age of 2 years. SkedaddleTM

is a lotion for topical application containing 6.2% N,N-diethyl-meta-toluamide (DEET) in a liposphere microdispersion composed ofnatural solid triglyceride dispersed in a buffer solution with spher-ical particles in the range of 15 �m. This liposphere formulationenabled the reduction of the systemic bioavailability of DEET bythree fold as compared to ethanol solution, thus reducing the tox-icity of DEET related to its high skin absorption (up to 50%) (Dombet al., 1995).

Despite the low rates of success in the transfer from bench tobedside of lipospheres and PNL, there is a non-exhaustive researchcurrently taking place in many laboratories. Examples of such stud-ies in different stages of research are summarized in Table 1.

6. Summary and future perspectives

The rapidly growing pharmaceutical industry is stronglyfocused on the development of new active molecules. However,a successful delivery system is not of less importance to assure theprogress of a new chemical entity from the preclinical to clinicaldevelopment stage, and ultimately to the market. The drug deliverysystem should enable therapeutic drug concentrations at the drugtarget site for a sufficient time period, and avoid the occurrenceof as many adverse effects in other tissues and organs as possible.Lipid based nano-sized drug carriers, and lipospheres and PNL inparticular, have the potential to fulfill these requirements.

Lipospheres are solid, water insoluble nano- and microparticles

composed of a solid hydrophobic core containing a layer of a phos-pholipid embedded on the surface of the core. The hydrophobiccore is made of solid triglycerides or fatty acid esters containingthe active agent.

450 A. Elgart et al. / Chemistry and Physics of Lipids 165 (2012) 438– 453

Table 1Examples of outcomes of recent studies utilizing lipid based dispersion systems as carriers of drug substances, carried out by various research groups.

Composition of thelipid dispersionsystem

Activeingredient

Administrationroute

Drugassociatedproblems

Particle size Preparationmethod

Outcomes Reference

Compritol 888 ATO,soylecithin/PluronicF68 or soylecithin/Tween80

All-transretinoic acid(ATRA)

Oral to rats Poorly solubledrug

80–300 nm High-pressurehomogeniza-tionmethod

Significantlyimprovedbioavailabilityof ATRA

Hu et al. (2004)

Glycerylmonostearate,soya lecithin,Tween-80 andPEG 400

Quercetin Oral to rats Poorly solubledrug with verylow oralbioavailability∼1%

155.3 ± 22.1 nm Emulsificationandsolidification

5 fold increaseinbioavailabilityof quercetin

Li et al. (2009)

Glycerolmonostearate,Tween 80,lecithin,

Candesartancilexetil (CC)

Intragastric/intraduodenaland Oraladministrationto rats

Very poorsolubility atphysiologicalpH range, highfirst passmetabolism byCYP P450enzyme

24.3 ± 8.5 nm Film homoge-nization

Increased CCabsorptionafterintraduodenaladministration,12-foldincrease in AUCafter oraladministration

Zhang et al.(2011)

Trigleceride(tripalmitin/trimyristin/tristearin),phosphatidyl-choline andpoloxamer 188

Nitrendipine Intraduodenaladministrationto rats

Variable andpoorbioavailability(10–20%), highfirst passmetabolism byCYP P450

100–120 nm Hot homoge-nizationfollowed byultrasonication

2.81–5.35 foldincrease inbioavailability

Manjunath andVenkateswarlu(2006)

Stearic acid,lecithin andtaurocholatesodium salt

Doxorubicin IV to rabbits Poorpenetration tobrain tumorcells due topoorpermeationthrough theBBB and effluxby P-gpexpressed atthe brain

60–95 nm Dispersingwarm oil inwatermicroemul-sions in anaqueousmedium

Increaseddoxorubicinconcentrationin the brain,prolongedcirculationtime

Zara et al.(2002b)

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Herein, we have reviewed several existing and potential appli-ations for lipospheres and PNL. In oral delivery, highly lipophilicnd poorly water soluble compounds that also undergo extensiveresystemic intestinal metabolism are the ultimate candidates for

ncorporation into lipospheres to obtain increased and less variableioavailability. Lymphatic absorption was proposed as one of theossible mechanisms for bioavailability enhancement; however,dditional in vivo studies must be conducted in order to elucidatehese suggested mechanisms.

Lipospheres have the potential to be a major contributor to theearch for better ocular drug delivery systems due to their improvedorneal adsorption and penetration, combined with their suitabilityor ocular drug delivery.

The potential of lipid based nano particles for parenteral, andspecially CNS drug delivery is also successfully established, how-ver, more studies are needed to establish the CNS penetrationechanism and safety.In addition, from a practical point of view, lipid nanoparticulates

ulfill essential prerequisites for entering the market, such as lowost production, clinical and large-scale production facilities, andccepted status of excipients. Furthermore, their physical stabilitys a significant advantage as compared to other lipid base delivery

ystems, i.e. liposomes.

As the number of new drug candidates with poor bioavailabil-ty issues is constantly growing, while profound understandingnd clinical experience with lipid based solid nanoparticulates is

increasing, these drug delivery systems are expected to becomemore widely used in the near future.

Acknowledgments

The authors would like to thank Dr. Wahid Khan for his excellentassistance. Prof. A.J. Domd and Prof. A. Hoffman are affiliated withthe David R Bloom Center of Pharmacy.

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